Monthly Archives: January 2019

Intervention Breathing Exercises and Their Effect on Breathing Stereotype and Vital Lung Capacity

DOI: 10.31038/AWHC.2019211

Abstract

Objective: Breathing difficulties are abundant throughout the whole population spectrum. Breathing pattern dysfunctions are frequent despite the fact that correct breathing is a necessary prerequisite for optimal functioning of the musculoskeletal apparatus, correct body posture and mental well-being. Furthermore, good breathing ventilation is immensely important in endurance sports. The quality of breathing stereotype also influences vital lung capacity. Incorrect breathing can be caused by blocked ribs and vertebra, dysfunction of breathing and stabilisation muscles, allergies, lung diseases, heart failure and above all excessive stress. When stimuli causing unsuitable breathing stereotype prevail too long, the dysfunction becomes fixated and must be eliminated and consciously corrected (using compensation exercises).

Methods: In this research, muscle dynamometer MD03 was utilised for examination of the breathing stereotype, followed by Spirometric measurements of forced exhalation of the vital capacity and exhalation volume values for 1 second. Two months intervention of targeted breathing exercises was applied on a group of adolescent athletes (19 individuals contributing to the intervention and 18 used as a control group) aged on average 17.26 ± 1.80.

Results: Factual as well as statistically significant influence on the involvement and strengthening of breathing muscles activated during diaphragmatic breathing at rest, as well as during deep breathing was shown. Improved values of FVC (forced expiratory vital capacity) by 6 % from 4.51 ± 1.13 L to 4.78 ± 1.12 L were obtained together with improved FEV1 (the forced expiratory volume in 1 second) by 6.1 % from 3.54 ± 0.71 L.s-1 to 3.76 ± 0.66 L.s-1. Both changes are both factually and statistically significant although the factual change displays only a slight effect. Only minimal change could be sees for the control group where FVC increased by 0.45 % and FEV1 by 0.86 %. The test group also showed significant increase in breathing volumes.

Conclusion: This study has confirmed that a two-month intervention of targeted breathing exercises has both factually and statistically significant influence on the activation and strengthening of breathing muscles.

Keywords

breathing pattern, breathing exercises, posture, spirometry, vital capacity

Introduction

Dysfunctions of the breathing pattern can be first indicators of a problem within the human body, be it of mechanical, physiological or mental kind [1]. Incorrect breathing can be caused by blocked ribs or vertebra, dysfunction of breathing and stabilising muscles, allergies, lung diseases, heart failures and above all excessive stress. Major role also belongs to a change in posture control [2] or changed dynamic spine stabilisation [3]. The breathing process is perfectly coordinated and controlled by the brainstem and medulla oblongata [4]. Breathing movements happen automatically, induced by the autonomous nerves, although the depth and rhythm of the breathing can be consciously regulated. This otherwise spontaneous activity can thus be influenced by will [5, 6]. Breathing and breathing movements maintain not only the fundamental metabolic processes connected to gas exchange but they also strongly influence body posture [7]. This implies that breathing influences posture stability while at the same time the quality of postural stability influences breathing. According to Kolář et al. [8] postural stability is defined as the ability to maintain upright body posture and at the same time have the ability to react on changed external and internal forces so as to prevent an unintentional or uncontrolled fall. Dylevský, Druda and Mrázková [9] describe postural stability as a balanced and coordinated posture of the body as a unit and as a highly specialised process of maintaining body balance and position of the body and its parts in a constantly changing environment. It is a musculoskeletal regulatory body mechanism that precedes every movement and afterwards seeks to maintain the reached position. It is therefore not a simple taking of a position but a continuous maintaining of its stability [8]. Furthermore, breathing movements are influenced not only by internal and external environment but also by the state of mind. While happiness undermines extensive body posture and increases breathing movements, sadness and depression are exhibited by flexed body posture and hindrance of breathing movements [10].

Breathing movements are divided into three areas, namely abdominal, lower thoracic or diaphragmal and upper thoracic or subclavian. The thoracic breathing movements differ by the distinctive activation of the ribs in the given areas. Breathing movements go through rhythmical interchanging activity of the breathing muscles in dependence on the physical activity and the stress condition of the organism, with simultaneous activation of the axial organ muscles. Breathing muscles are further divided according to their anatomic function into primary and accessory inhalation and primary and accessory exhalation muscles with the most important breathing muscle being m. diafragma (the diaphragm) [8, 10]. During inhalation both the diaphragm and mm. intercostales externi compress. Muscle fascicles of the diaphragm are concentrically contracted causing pressure on the content of the abdominal cavity from above. This pressure is further transferred to the pelvic area, and in order to avoid prolapse of the abdominal organs the muscles of the pelvic area start contracting concentrically together with the diaphragm. These two muscle units then work as a sort of pistons working against each other from below and above forcing the content of abdominal cavity toward the remaining directions – forward and to the sides, respectively backwards. The relatively large area of the diaphragm induces an increase of the internal abdominal pressure while at the same time the lumbar spine area is heavily compressed using the function of the transverse abdominal muscle that is eccentrically activated and thus hinders the content of the abdominal cavity to move forward and to the sides causing the circumference of the waist to enlarge. On the other hand, the tendency of the abdominal cavity to move backwards during inhalation is for the majority of postural situations minimal [8, 11].

Simultaneously, the diaphragm is an important muscle for the postural function [4], with up to 75% of the volume change of the intrathoracic space during quiet breathing being caused by diaphragm function while its function alone is sufficient for 2/3 of the lung vital capacity [8]. The range of diaphragm movement is 1–2 cm during quiet breathing and up to 10 cm during heavy breathing which activates even the external intercostal muscles. Furthermore, during extreme breathing also the auxiliary respiratory muscles get activated which help to lift the first and second ribs together with sternum [12].

Clifton-Smith and Rowley [1] define dysfunction of the breathing stereotype as disproportionate breathing which is sufficient for the utterance of given symptoms even with no organic causes. Without such structural causes the dysfunction stems from muscular dysbalances, functional disorders of the muscular-skeletal system, that lead to ineffective breathing. This in its turn then induces muscular adaptational mechanisms towards the outside influences. Muscular dysbalance usually evolves due to a disturbed muscular synergy when one antagonistic muscle gains dominance over its counterpart. The cause of muscular imbalance can be even a remote dysfunction, such as reflexive change in a muscle that causes pain or influence other connected muscles which in its turn causes the organism toward an antalgic body posture not ideal for movement stereotypes [10, 11]. Therefore when trying to influence breathing functions it must be taken into consideration that the breathing and muscular-skeletal systems must be approached as interconnected [8].

In order to assess breathing stereotype, several methods can be used such as palpation examination, whole-body plethysmography, chest skiagram, spirometry or usage of different instruments that are able to detect movement of individual segments of the respiratory system [13–15]. Involvement of the individual muscle segments can be determined for instance through a 3D system [16]. Kaneko and Horie [16] conducted such measurements using thirteen probes in different positions, during quiet and deep breathing and found connections between breathing movements and age, body posture and gender. Monitoring of breathing movements is enabled by measurements of chest circumference over meso-sternale and xipho-sternale junctions, evaluating and registering the difference between in- and exhalation [13]. Bockenhauer et al. [17] measured chest broadening using a measuring tape at two different levels (heights). The upper measurement of chest expansion was conducted at the level of the spinous process of the fifth thoracic vertebra and third intercostal space in the middle of the clavicle, while the lower measurement proceeded at the level of the spinous process of the tenth thoracic vertebra and the baseplate tip. Three different people measured maximal inhalation and maximal exhalation circumference at both levels and obtained thus a value of the chest expansion. This study confirmed the objectivity of examination chest movements in the middle and upper sectors of the rib cage using a tape measure [17]. Moll and Wright [18] used two separate techniques to measure the expansibility of the chest, using the calliper and using the tape measure at the fourth intercostal space with the arms of the patient raised above the head. They then concluded that circumference measurements alone should suffice. Burgos-Vargas et al. [19] also measured the expansiveness of the chest with a tape measure at the fourth intercostal space (arms raised). The activity of the breathing muscles during breathing cycle can also be investigated through polyelectromyography examination [14]. In order to learn about the strength of the breathing muscles even non-invasive methods of examination of maximum inhalation and exhalation mouth pressures can be used [20, 21]. The performance of breathing movements is denoted as the breathing wave. It begins with inhalation, proceeds from the lower over the medium to the upper sector. The exhalation wave on the other hand starts with lowering of the abdominal wall and simultaneous caudal lowering of the chest. Further, the sternum constricts and exhalation process is finished by activated abdominal muscles [10]. The importance of measurements of the chest expansion was confirmed by Bockenhauer et al. [17], and also Fisher, Cawley and Holgate [22].Their studies all showed the important connection between the chest expansion and vital capacity of the lungs. Effectivity of highly trained long distance runners is often limited by their breathing system. Spirometric examination of the static and dynamic ventilation parameters belong to basic methods for diagnosis of the respiratory system [23].

Change in the breathing stereotype is possible through active training of deep breathing, as agreed by Thomas and McIntosh [24] in their work. During such training it is necessary to monitor the breathing pattern of the examined individual and make him aware of its possible insufficiencies [4]. In order for the consequences of the training to start showing, the training must proceed for a certain time with a minimum of ten breathing exercises which evoke an important regular rhythm in the body. One such technique is isolated breathing, where three different types of breathing are practiced, namely diaphragmal, chest and subclavian breathing. The aim of diaphragm breathing is above all to realise its activity and learn to use it. The focus of chest breathing is then to gain increased flexibility of the rib cage, while subclavian breathing is meant to loosen the area of the neck and teach about the accessibility of the upper lobes of the lungs [6].Training exercise of at least six to eight weeks is necessary in order to be able to influence intermuscular coordination and improve intermuscular cooperation. Adaptational changes in the form of hypertrophy then appear after longer time, within months or years [25].

In both clinical and sporting practice vital capacity of the lungs is most often measured using the FVC test (basic forced volume vital capacity), where also the velocity of exhalation is determined, for instance for the time period of one second (FEV1), which also points to the strength of the exhalation muscles, an important and surveyed factors in endurance sports. Higher values of vital capacity (VC) can be reached through endurance training, although the values are also influenced by body constitution and size of the rib cage [26]. During long-term submaximal training inhalation muscles experience fatigue which is a limiting factor in endurance training. However, breathing exercises can enhance the efficiency of the breathing muscles. Endurance athletes usually have higher values of vital capacity, although their performance needs not be directly dependent on those. Highest values of VC have been determined for swimmers (up to 8 litres) which results from their breathing into the water and therefore against resistance. Adapted breathing is exhibited by a lower breathing work during same load in comparison to the unadapted. A certain amount of oxygen then remains for other purposes. As a consequence of endurance training the respiratory tract increases its ability to transport oxygen, which will also show during load. On the other hand, the maximum static and dynamic values remain basically unchanged. The aim of this work was to determine the influence of the intervention breathing exercises on the breathing stereotype and values of the forced volume vital capacity of the lungs [26].

Methodology

This study was conducted in the Laboratory of stress diagnosis, at the Department of Physical Education and Sports, Pedagogical faculty of the South Bohemian University. 37 individuals aged 17.26 ± 1.80 participated in this study, where each does endurance sport training six times a week; they are mostly mid-distance and long-distance runners. 19 individuals were involved in the intervention (ten females and nine males), while eighteen individuals functioned as a control group (nine females and nine males). The age of the females at the time of the study was 17.40 ± 2.01 years of age, with average weight 59.07 ± 6.70 kg and height 169.47 ± 4.04 cm. The age of the male group at the time of the research was 17.11 ± 1.52 years of age, average weight 60.47 ± 14.32 kg and height 176.40 ± 9.49 cm. Targeted selection was conducted based on the fact that endurance athletes are expected to have above-average breathing functions with strong connection to their performance. The aim of this research was to prove the influence of breathing exercises on the values of vital capacity of the lungs (VC) and on the quality of the breathing pattern. It was presumed that there will be a transfer of increased values of VC to values of breathing volume under load, which in its turn will influence endurance performance, which consecutive research will prove.

Vital capacity was measured using basic forced volume vital capacity test (FVC) as well as exhalation values under 1s (FEV1). Thereafter, the test group got instructions to conduct breathing exercises for two months, minimum five times a week for at least ten minutes. At the same time, once a week a common exercise session was organised at the end of running training session, where the execution of the exercises was checked and corrected. The tested individuals would each day take notes of the time of the exercises into a prepared table that they continuously checked in. After two months a re-test was conducted of all followed variables. Same measurements were simultaneously conducted for the control group. Test methodology was conducted according to the instrument instructions: after quiet inhalation and exhalation follows maximal inhalation and forced maximal exhalation. This procedure is repeated three times and the best result is registered. The instrument registers if sufficient force and necessary length of exhalation were used. If the parameters of the test were insufficient, the experiment was not registered and experiment needed to be repeated. The FVC test was conducted in upright position with Spirometer Otthon instrument, while the evaluation was conducted using ThorSoft program. Figure 1 shows the development of the breathing curve as registered by the spirometer instrumentation.

Muscle dynamometer was used for the examination of breathing stereotype [27–30]. Due to the ability of the muscle dynamometer to register movement dynamics, it is possible to use it to monitor the dynamics of the breathing function. Breathing analysis is based on the concept of three sectors (parts) of the chest and therefore three measuring sensors were also used. The exact positions for placing the sensors were chosen based on the kinematics of the mentioned chest sectors [9].The lower chest sector (abdominal) is found below apertura thoracis inferior. Anatomically the abdominal muscles and their initiations on the cartilaginous parts of the false ribs and the breast bone participate in the build-up of this part of the chest. The correct spot for the first sensor is then on the ventral side at level of L4–5. For the central sector of the chest the correct placement is on the thoracic spine limited by the area between Th6-Th12 and the fifth to twelfth rib. The sensor for this sector was placed on the level of 8–9 rib on the ventral side just below the sternum. The upper chest sector (apical) spreads from C4 to Th3–4 to the upper aperture and the fifth rib. The third sensor was placed on the level of 3 to 4 rib on the ventral side in the area of the sternum. Movements of the thoracic spine sector influence the dynamics of breathing, while breathing will influence dynamics of the spine [8].The test of breathing dynamometry is conducted in the upright body position as this position is physiological for breathing [4]. The dynamometer instrument can register activation of the breathing muscles, as it measures immediate values of force activity of the muscles as a function of time. Both the strength and dynamics of the muscle force can be evaluated. Through the sensors the lifting of individual segments during quiet breathing for one minute and during deep breathing for one minute was monitored. During data analysis ten inhalations and exhalations was averaged. The test group trained the breathing exercises for eight weeks. The intervention program was focused on isolated breathing in different positions, training for the breathing wave, full breathing and rhythmical breathing. Full breathing was also trained in accordance with movements during short dynamical sets of exercises [31–33]. After input measurements, the test group was introduced to breathing exercises and their training was conducted under expert supervision. The test group was asked to conduct the given exercises at home at least five times a week for at least ten minutes. Once a week a mutual training session was organised, after running training session, where the exercises were checked and corrected by expert supervisor. An output examination similar to the input one was conducted. The control group did not do any breathing exercises. For evaluation of the collected data these tests were used: the determination of the effective significance Cohen’s d test for determination of practical significance and Student‘s paired t-test for dependent samples. The level of significance was determined based on α = 0.05. Data analysis was conducted using Microsoft Excel 2016 and Statistica 12.

Results

The test group athletes did breathing exercises for two months for 13.2 ± 3.87 minutes a day on average. The FVC results before the intervention exercises (Figure 1) were calculated to 4.51 ± 1.13 L and those after the intervention time equalled 4.78 ± 1.12 L (see Figures 2 and 3). For the control group the results amounted to 4.39 ± 1.21 L before the intervention and 4.41 ± 1.23 L thereafter. For the test group the improvement of the breathing volume during FVC was 6.0 % while for the control group 0.45 %. Therefore for the test group the factually significant improvement of the FVC (Cohen’s d = 0.24, small effect), is therefore statistically significant (Figure 2, 3). No significant differences between male and female individuals were found.

AWHC-18-116 - Renata Malátová_Czech Republic_F1

Figure 1. Breathing curve during FVC test.

AWHC-18-116 - Renata Malátová_Czech Republic_F2

Figure 2. FVC value for tested individuals before the intervention of breathing exercises and after.

AWHC-18-116 - Renata Malátová_Czech Republic_F3

Figure 3. The distribution of values of FVC in tested individuals before interventions breathing exercises and after.

During the analysis of the breathing movements (Figure 4) in the individual sectors of the chest after the intervention program, an improvement particularly for the abdominal breathing was found, for both the quiet breathing, increase by 46.5 % (Cohen’s d=0.56, medium effect), and deep breathing with increase by 61.5 % (Cohen’s d = 0.82, large effect). For subclavian breathing both factually and statistically significant increase by 19.2 % of its contribution to deep breathing was found (Cohen’s d = 0.42, small effect). An increase for quiet subclavian breathing by 6.9 % was observed, although the change is neither factually or statistically significant (Cohen’s d = 0.11). For chest breathing a decrease in values by 20.5 % during quiet breathing was found, a change that is factually (Cohen’s d = 29.4, small effect) although not statistically significant. For deep chest breathing the activity of breathing muscles decreased by 21.8 %, a change that is factually (Cohen’s d = 35.9, small effect) and statistically significant. For the control group no factually or statistically significant change was observed, with change not larger than 1.5 % for any breathing muscle segment was found.

AWHC-18-116 - Renata Malátová_Czech Republic_F4

Figure 4. Involvement of the different segments of the thorax during quiet breathing and deep breathing before the intervention and after (breathing: BK – abdominal quiet, BH – abdominal deep, HK – chest quiet, HH – chest deep, PK – subclavian quiet, PH – subclavian deep).

Discussion

In order to be able to strengthen the necessary breathing muscles during the intervention, the three sections of deep breathing must be consciously acknowledged, trained separately and then reunited. The first most important section of breathing is the diaphragmal or abdominal breathing that is responsible for 60% of the full breathing efficiency. Chest breathing corresponds to 30% and subclavian to 10% of efficient breathing. The given percentage ratio applies to most of everyday activities. During physical exercise or some (pathological) changes in the organism this ratio is significantly changed [34]. As Kolář et al. [8] state the fact remains that the diaphragm is responsible for 2/3 of the gas exchange in the lungs.

The aim of the intervention program was to achieve controlled optimal stabilisation interplay of the breathing muscles and make the correct breathing stereotype automated. The emergence and development of overload on the musculoskeletal system can thus be prevented. However, success of the therapy is highly dependent on active participation of the test group. The output examination concluded improvement of both quiet and deep abdominal, as well as deep subclavian breathing. At the same time the chest breathing was shown to decrease for both the quiet and deep breathing types. Breathing movements that led to improved diaphragm breathing and simultaneous decrease of activity of chest breathing represents a positive change towards optimal activation of breathing muscles [8].The reality that needs to be respected is the fact that postural functions, positioning of the body in certain position or during a movement and the breathing functions are strongly interconnected and the keystone of this connection is the diaphragm, the major breathing muscle. A positive influence on the breathing pattern will also improve the stabilisation function of the diaphragm. Véle [10] states that quite frequently we meet patients with inspiratory positioning of the thorax (horizontal progression of the ribs) with activated accessory muscles (auxiliary inhalation muscles) during quiet breathing. These muscles have multiple connections onto the spine and for long-term inspirational positioning these muscles become central for the positioning of individual segments of the spine. This is the primary objective necessary to alter and teach the patient how to relax these muscles and at the same time activate the muscles of the lower respiratory sector so that the dysfunction of the breathing mechanism can be eliminated and as a feedback the postural functions can be influenced. This aim has been achieved by the application of the intervention program and the diaphragmal breathing was improved.

The chest and diaphragm breathing complement and compensate each other, which is why their function must be optimised. If the chest expansion is malfunctioning and the chest is rigid, the participation from the diaphragm increases and starts to dominate and vice versa. Provided that the extension of the chest is desired, the abdominal wall muscles need first to become relaxed in order for the diaphragm to be able to get as low as possible. During exhale, the abdominal muscles must contract to cause the compressed bowels to push the diaphragm up in the cranial direction. This causes a decrease of the chest volume and the exhale deepens. During influence of the external forces abdominal muscles are gradually activated, fixating the lower section of the chest. At the same time the spine is stabilised and hinders inspirational positioning of the chest [8]. Success of the therapy presumes an active participation of the test group. This was not always the case and for some tested individuals the improvement was minor, for some even negative. Once a week the exercises were conducted under supervision and their correct performance checked and if necessary corrected. The rest of the week, though, the exercises were conducted in home environment outside supervisor control where the effect of the exercises could be influenced by the quality of their performance. The results of the FVC tests are therefore not uniform, with half of the test group showing improvement by more than 0.3 L, while two individual did not show any improvement. After the two-month intervention exercises an improvement in FVC values was achieved for 95 % of the followed individuals. This change is furthermore both factually and statistically significant. No factually significant differences were observed between male and female individuals. For the FEV1 values an improvement was observed for 78 % of the tested individuals, for half of them by more than 0.3 Ls-1. One quarter of the individuals exhibited worsened, if by minimum, FEV1 values.

Conclusion

This study has confirmed that a two-month intervention of targeted breathing exercises has both factually and statistically significant influence on the activation and strengthening of breathing muscles through diaphragm breathing during both quiet and deep breathing and has at the same time factually and statistically significant influence on elevation of FVC and FEV1 values.

Acknowledgement

This research was supported by Grant agency of the University of South Bohemia within Team research project no. 034/2015/S.

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Surface Roughness Evaluation of Different Polishing Techniques on Dental Porcelains By Atomic Force Microscopy

DOI: 10.31038/JDMR.2018124

Abstract

Introduction: This study was conducted to find out different polishing techniques’ effects on different porcelain materials by using atomic force microscopy (AFM).

Material and Methods: Samples were made from four different porcelain materials (VMK 95, Ceramco III, Matchmaker, Vitablocs Mark II). Nine groups (n = 5) were randomly formed from the samples of each ceramic and nine different polishing methods were applied on them. AFM was used to evaluate the external topography and roughness of dental porcelains. To analyze the results of the study ANOVA and Tukey test were used statistically ( = 0.05).

Results: Within the porcelain groups Ceramco and Matchmaker had higher Ra values, however VMK 95 and Mark II had lower Ra values. However when the different polishing techniques were compared, Gl (.1110) and SlPg (.2295) were found to have the lowest Ra values and no statistical differences were found between these two techniques (p > .05). In addition, no differences were found between glazed samples (Gl) and after polishing with Sof-lex and Prisma Gloss (Sl-Pg). On the other hand polishing with Sl and Pg together (Sl-Pg) significantly decreases the surface area.

Conclusions:There were statistically significance between the glazed and the other specimens. Different polishing methods influenced the surface topography of different porcelains significantly ( p < .001).

Key words

AFM, surface roughness, polishing, dental porcelain

Introduction

The key factor of an esthetic fixed restorations is dental porcelains. Transparency, conveyance of light, and biocompatibility supply dental ceramics with exceptionally advantageous esthetic properties [1]. A smooth surface is important for the function, esthetic and biocompatibility of dental porcelains [2] and also it’s preferable to decrease bacterial holding and to obtain a clear appearance [3]. The dental porcelain surface finishing procedure is achieved by glazing since their surfaces are smooth. In the delivery of porcelain fixed prosthesis, external alterations are crucial to improve occlusal obstructions and poor contours, to finalize the boundary of porcelains, and to enhance final look of restorations [4]. Generally, a smooth surface is essential because surface modifications of the porcelain damage glazing, thus creating rough, unpolished, nutrient-enhancing surfaces.

In this situation to refinish the porcelain surfaces intraorally with different polishing methods are mandatory to restablish surface smoothnes [4, 5]. To create the surface smoothness of dental porcelains there are many different mechanical polishing methods using rotary instruments such as diamond bars and drills, different pastes, stones, rubber discs or different polishing kits.

Atomic force microscopy (AFM) is an important tool for qualitative and quantitative evaluation of surfaces which is presented in 1986 by Binning et al [6] . With the help of mechanical scanning, AFM directly inspects the exterior of the sample without using any lens or photon. With a sharp tip on, it scans over a surface, and measures the deflection, it is possible to get a topographic photograph of the surface. The deflection sensor may help to measure with enough sensitivity in disclose profiles with nanometer scale resolution [7]. With the contact, noncontact and tapping modes it’s possible to have three different measurement records from different samples. AFM topographs give quantitive and structural (3D) information about surface of a material nanometrically which allows a high accuracy in finding out the external roughness. AFM can analyze the external roughness of dental porcelains [5, 8–11]. The null hypothesis in this research was that there are no differences between surface roughness of dental porcelains after different polishing methods by means of AFM evaluation.

Material and Methods

Four different commonly used dental porcelains (Vita VMK 95, Ceramco, Vitablocks Mark II and Matchmaker) and six different polishing systems glaze, finishing and polishing with polishing discs (Sof-Lex), polishing kit (Dialite II), polishing pastes (Sparkle, Zircate, Prisma Gloss) and combinations of them were investigated in this study (Table 1). One researcher prepared cylindrical samples (15x2mm) of four different dental porcelains by means of using polyvinylsiloxane mold to encapsulate the ceramics. All of the samples were mixed with a standart quantity of ceramic and liquid which were placed in the mold and condensed by using a plastic aparatus. A tissue (Selpak; Eczacıbaşı Holding, Sakarya, Turkey) was used to absorb excess moisture (Programat P80; Ivoclar Vivadent, Liechtenstein). The samples were taken away and putt into the oven in line with the producer’s instructions (roughly 920–960ºC ). The rectangular ceramic samples which were 12, 14, 18 mm in size were sliced into pieces of 2, 14, 18 mm in size with a Buehler Isomet Low Speed cutting machine (Lake Bluff,  Illinois,  60044–1699,  USA). For a period of ten seconds, 600 grit silicon carbide paper was used by using a 300-rpm grinding MetaServ polishing (MetaServ, Buehler, England) to wet-ground ceramic discs. The samples were grouped in nine subgroups (n = 5) and the following procedures were applied (Table 2):

Table 1. Materials used in the study.

Material

Manufacturer

Material

VMK 95

Vita Zahnfabrik, Germany

Feldspathic porcelain

Ceramco III

Degudent GmbH,USA

Feldspathic porcelain

Matchmaker MC

Schottlander,UK

Low fusing porcelain

Vitablocks Mark II

Vita Zahnfabrik, Germany

Machinable feldspathic porcelain

Sof-Lex

3M ESPE,USA

Finishing and polishing dics

NTI Cera Glaze

NTI-Kahla GmbH,Germany

Porcelain polishing kit

Dialite II

Brasseler,USA

Porcelain polishing kit

Sparkle

Pulpdent,USA

Diamond polishing paste

Zircate

Dentsply,USA

Zirconium silicate cleaning-prophy paste

Prisma Gloss

Dentsply,USA

Aluminium oxide polishing paste

Table 2. Different polishing groups.

Study Groups

Polishing Techniques

Group- Gl

Glaze

Group- Sl

Sof- lex discs

Group- Di

Dialite II polishing kit

Group- Sp

Sparkle diamond polishing paste

Group- Zr

Zircate polishing paste

Group- Pg

Prisma Gloss polishing paste

Group- SlSp

Sof- lex + Sparkle

Group- SlZr

Sof- lex + Zircate

Group- SlPg

Sof- lex + Prisma Gloss

Group Gl: with a predetermined glaze material, the samples were glazed.

Group Sl: the samples were polished with polishing discs of 12.7 mm diameter (Sof-Lex; 3M/ESPE, St. Paul, MN, USA) for ten seconds for fine and superfine discs at 30, 000 rpm and for ten seconds for coarse and medium discs at 10, 000 rpm by using an electric handpiece set by following the producer’s directions.

Group Di: Dialite II ceramic polishing kit which had pre, fine and high-shine wheels was used to polish the samples for ten seconds at 10, 000 rpm.

Groups Sp: Sparkle diamond polishing paste was applied for ten seconds to the samples with a prophylaxis rubber cup (Kenda Polishers, Kenda AG, Liechtenstein) mounted on an electric handpiece at 15, 000 rpm.

Groups Zr: Zircate zirconium silicate cleaning-prophy paste was applied as in the same method of Groups Sp above.

Groups Pg: Prisma Gloss aluminum oxide polishing paste was applied as in the same method of Groups Sp above.

Group SlSp: The samples were polished first by following the procedure in Group Sl and then by following the procedure in Group Sp.

Group SlZr: First by following the procedure in Group Sl, the samples were polished and then by following the procedure in Group Zr, zirconium silicate cleaning-prophy paste was applied.

Group SlPg: First polishing was done by following the procedure in Group Sl and then by following the procedure in group Pg aluminum oxide polishing paste was applied on the samples.

The same investigator performed all polishing procedures. Lastly, the specimens were cleaned ultrasonically for 10 min (Eurosonic Energy, Euronda, Italy) by using deionized water and then they were dried. Porcelain specimens were evaluated under an AFM (AFM, PSIA XE-100E, PSIA Inc, CA, USA) to obtain a quantitive and qualitative evaluation [5]. With a scan length of 20µm x 20µm and a scan rate of 0.5Hz [12]. AFM images were photographed (Figure 1a-4b). Following different external procedures, the average surface roughness (Ra) of the ceramic substrate was examined by a single operator and analyzed after different surface treatments. Three measurements were performed from three different areas all located in the centre of the specimens [13, 14]. Means and standart deviations of surface roughness measurements were determined. Two-way ANOVA was used to evaluate surface roughness data and SPSS (12.0.1; SPSS Inc, Chicago, IL, USA) was used for statistical analyses. Tukey was used to compare the mean values (p < 0.05).

Results

The null hypothesis of the study was rejected. Statistically significant differences occured due to the various polishing methods on the external topography of different porcelains (p < .001). In the figures (1a-4b) respectively three dimensional AFM images of different porcelain specimens subjected to different surface treatments are shown. There were statistically significance between the glazed and the other specimens (p < .05). On the other hand polishing with sof-lex and aluminum oxide polishing paste together (Sl-Pg) significantly decreases the surface area.

When the porcelain specimens are compared Mark II and VMK 95 were statistically different from Ceramco III and Matchmaker, and both of them presented similar result within as seen in (Table 3). When the polishing techniques are compared, group of Gl (.1110) and SlPg (.2295) were found to have the lowest Ra values and there were no statistically significant differences between the two groups (Table 4) (p > .05). The highest Ra value was obtained in the group of Sp (.8511) (p = .05).

Table 3. Mean surface roughness and differences between the groups of porcelains.

Porcelains

Ra

Difference*

VMK 95

,3895

A

Mark II

,3456

A

Ceramco

,4907

B

Matchmaker

,4967

B

* Mean Ra values for groups in non homogeneous subsets are displayed with different letters p= .05

Table 4.Mean surface roughness and differences between the groups of surface treatments.

Surface Treatment

Ra

Difference*

Gl

,1110

A

SIPg

,2295

Ab

SIZr

,3507

Bc

Zr

,4359

Cd

Pg

,4384

Cd

Di

,4609

Cd

SISp

,4727

Cd

SI

,5255

D

Sp

,8511

E

* Mean Ra values for groups in non homogeneous subsets are displayed with different letters p= .05

When all the porcelains’ surfaces were evaluated, the highest Ra value was in the group of Mark II Sp (1.147) (Figure 1a) and the lowest value was in Mark II Gl (0.0860) (Figure 1b) (Table 5). When the Ra value of VMK 95 porcelain evaluated the highest Ra was in the SlSp (Figure 2a) and the lowest was in Gl group (Figure 2b). There weren’t statistically significant differences between groups SlZr, Pg, SlPg, Zr, Sp and SlSp for VMK 95 porcelain (Table 6). When the Ra value of Mark II porcelain evaluated the highest Ra was in the Sp (Figure 1a) and the lowest was in Gl group (Figure 1b). There weren’t statistically significant differences between groups SlZr, Di, Pg, and Zr for Mark II porcelain (Table 5). When the Ra value of Matchmaker porcelain evaluated the highest Ra was in the Sl (Figure 3a) and the lowest was in Gl group (Figure 3b). No statistically significant differences were found between groups Gl, SlPg, SlZr and also the groups of Di, Pg, Zr, Pg, Sl ve Sp for Matchmaker porcelain (Table 7). When the Ra value of Ceramco porcelain evaluated the highest Ra was in the Sp (Figure 4a) and the lowest was in SlPg group (Figure 4b). No statistically significant differences were found between groups SlZr, Di, Pg, Zr, Pg, SlSp, Sl for Ceramco porcelain (Table 8).

JDMR-18-107-Goknil Ergun_Turkey_F1a

Figure 1a. Atomic force microscope image of Mark II Sp.

JDMR-18-107-Goknil Ergun_Turkey_F1b

Figure 1b. Atomic force microscope image of Mark II Glaze.

Table 5. Mean surface roughness and differences for the Mark II porcelain subjected to different surface treatments.

Surface Treatment

Ra

Difference *

Gl

,0860

A

SIPg

,2473

Ab

SIZr

,3660

Bc

Zr

,4293

Bcd

Pg

,4510

Bcd

Di

,4593

Cd

SISp

,5973

Bcd

SI

,6333

D

Sp

1,147

E

* Mean Ra values for groups in non homogeneous subsets are displayed with different letters p= .05

JDMR-18-107-Goknil Ergun_Turkey_F2a

Figure 2a. Atomic force microscope image of VMK SlSp.

JDMR-18-107-Goknil Ergun_Turkey_F2b

Figure 2b. Atomic force microscope image of VMK Glaze.

Table 6. Mean surface roughness and differences for the VMK 95 porcelain subjected to different surface treatments.

Surface Treatment

Ra

Difference *

Gl

,1163

A

SIPg

,2960

Ab

SIZr

,3303

Bc

Zr

,3680

Cd

Pg

,3703

Cd

Di

,3830

Cd

SISp

,4720

Cd

SI

,4837

D

Sp

,6860

E

* Mean Ra values for groups in non homogeneous subsets are displayed with different letters p= .05

JDMR-18-107-Goknil Ergun_Turkey_F3a

Figure 3a. Atomic force microscope image of Matchmaker Sl.

JDMR-18-107-Goknil Ergun_Turkey_F3b

Figure 3b. Atomic force microscope image of Matchmaker Glaze.

Table 7. Mean surface roughness and differences for the Matchmaker MC porcelain subjected to different surface treatments.

Surface Treatment

Ra

Difference*

Gl

,1353

A

SIPg

,1840

Ab

SIZr

,3817

Abc

Zr

,6187

Cde

Pg

,5360

Cde

Di

,6980

De

SISp

,4540

Bcd

SI

,7887

E

Sp

,6740

Cde

* Mean Ra values for groups in non homogeneous subsets are displayed with different letters p= .05

JDMR-18-107-Goknil Ergun_Turkey_F4a

Figure 4a. Atomic force microscope image of Ceramco Sp.

JDMR-18-107-Goknil Ergun_Turkey_F4b

Figure 4b. Atomic force microscope image of Ceramco SlPg.

Table 8. Mean surface roughness and differences for the Ceramco porcelain subjected to different surface treatments.

Surface Treatment

Ra

Difference *

Gl

,1063

a

SIPg

,1037

a

SIZr

,2870

ab

Zr

,2020

ab

Pg

,4180

b

Di

,2523

ab

SISp

,2913

ab

SI

,3497

ab

Sp

1,100

c

* Mean Ra values for groups in non homogeneous subsets are displayed with different letters p= .05

Discussion

The present study used AFM to examine the effect of various polishing techniques on four different conventional porcelain systems. Feldspathic VMK 95 and Mark II porcelain surfaces demonstrated significant lower Ra value than the other two porcelain systems after different polishing systems. The null hypothesis of the study was not accepted. There are several studies that report the effectiveness of porcelain polishing systems. Some authors report that there is no differences between the glaze and the polishing systems while others say that different polishing methods are not able to make a smooth surface as good as glazed [5, 15–24 ].

In the present study easy, useful and effective polishing systems were chosen. Sof-Lex is generally useful for the polishing of composites, and the manufacturer offers to use this system on the polishing of porcelains [16, 25]. NTI, CeraGlaze and Dialite II are two different polishing kits which are cheaper than diamond pastes. They are easy to use intraorally. Sparkle is a diamond paste which is indicated to use on porcelains, composites, gold and other alloys.

Zircate, is a zirconium silicate polishing paste which is indicated for tooth polishing [17, 26]. After periodontal threaphy polishing paste must not give damage to the restorative materials which are in the oral cavity. In this study we intend to observe the effect of zircate on porcelains. Prisma Gloss is an aluminium oxide polishing paste which is often used on composites [18, 27]. It’s also used in this study to observe its effectiveness on porcelains.

Flexural strength is affected by the material’surface roughness. Due to the decrease of the roughness of porcelain specimen surfaces, its flexural strength increases [19, 28 ].

The amount and the size of the abrasive fillers, and the shape of the polishing materials are the crictical issues for selection of the abrasive type for different contents of dental ceramics [18, 23]. It was observed that the surfaces of the ceramics were very rough and varied significantly for different polishing techniques and ceramic types. Vitablocs Mark II, which includes a glass matrix with nearly 30 vol% irregularly-shaped crystalline particles, is a modified feldspathic porcelain which is crystalline reinforced. It was stated by Yin et al [19] that surface finishing of Mark II which consisted of uniform and fine mica crystals was not succesful in decreasing surface roughness values. After mica crystals were cut with sharp Al2O3 abrasive particles, high peaks were left by Sof-lex disc. Leucite content of the dental ceramic seems to play an important role in surface roughness. Leucite porcelain involves fine leucite crystals which diffuse in glass matrix. Hence, when compared with porcelains with higher leucite content, lower leucite content were likely to show lower roughness after they were polished with rubber or disc followed by diamond pastes [18, 29]. In this study, lowest surface roughness was found in Glaze followed by Sof-Lex+Sparkle and Sof-Lex+ Prisma Gloss. However Sparkle polishing paste presented the highest Ra value with a mean of 1.147.

Different methods can be used to assess external roughness. In research on external roughness in dental materials, while qualitative methods such as SEM have been employed, quantitative methods such as surface profile analysis like profilometry and AFM have been employed. SEM has disadvantages in illustrating external topography, images allow only a two-dimensional view. In terms of both micron and nanometre scales, AFM has turned out to be a useful tool in examining material structure [20, 30]. To obtain the three-dimensional view and to evaluate the surface topography of various ceramics at nanometre scale, the surfaces were surveyed using AFM. SEM analysis confirmed the results obtained with profilometer in several studies [5, 15, 18, 22, 24].

In this study, polished ceramic types’ usual three diameter surfaces which were atained by AFM imaging (Figure 1b – 4b) showed a rougher surface for unglazed sample which included higher crystallites with pointed peaks coming out of the surface perpendicularly. In addition, when compared with glazed ceramics, this type was found to have deeper cracks. Glaze seals are known to crack and pore within the ceramic material. Three digital imaging of the glazed sample (Figure 1a) shows that glaze has smoothed the sharp edges. RMS values, Ra values and Z range were shown to be significantly (p < 0.01) higher in the unglazed samples as a result of statistical analysis.

In the study of Kakaboura et al [31], the surface characteristics of resin composites were illustrated through quantitative assessment of two-dimensional and three-dimensional profilometry and qualitative measurements by AFM and SEM. It was concluded that in distinguishing external roughness, AFM method was better when compared with two-dimensional profilometry and when compared with SEM, it defined external texture in more detail. Before this study Sarikaya and Guler [29] were evaluated surface roughness of the same materials subjected to the same polishing methods by profilometry. When we compared the results of the two study it’s obviously seen that AFM apperared to offer a powerful tool to directly evaluate the roughness of porcelain specimens. The differences between the two study may be due to the method of the surface roughness evaluation. In the results of their study it was seen that feldspathic ceramics (Mark II) had lower Ra values when compared with the others. In this study Mark II has lower Ra value (0, 3456) than feldspathic porcelain (VMK95) (0, 3895) but there wasn’t statistically differences between two materials. Also there were significantly differences among the polishing techniques for the Ra values of Mark II porcelain. Why the differences between the results of two study may be the effect of evaluation method of surface roughness measurements (profilometry and AFM).

Some authors have even recommended such polishing techniques as alternatives to glazing [15–24, 29, 32–34] but they concluded that it will be the object of further study. Also further studies are need with different composition of the particles and matrix substance, shape of the particles of abrasive ceramic polishing discs and wheels.

Conclusion

  1. The surface roughness of the glazed ceramic material was lower than of the other polishing treatments.
  2. The crystallites of the unglazed ceramic surfaces has higher and the pores were deeper compared to the glazed samples.
  3. Sof-Lex+Sparkle and Sof-Lex+ Prisma Gloss polishing techniques may be alternatives to glazing.
  4. AFM is a powerfull tool for the evaluation of surface roughness.

Conflicts of Interest: The authors declare that there is no potential conflicts of interest with respect to the authorship or publication of this article.

Funding Statement: This work is supported in part by Ondokuz Mayis University, PYO.DIS.1901.09.003

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Gender Dentistry A Systematic Review of Literature on Caries And Periodontitis

DOI: 10.31038/JDMR.2018123

Abstract

Already in the 1980s, the World Health Organization (WHO) has postulated to analyze in all medical fields differences between the sexes. But until now there is a disturbing lack of scientifically based and meaningful data over the whole area of gender dentistry – for prevention as well as for treatment. The aim of this study was to evaluate the actual situation described as an IS-analysis of literature review on the topic of gender dentistry with the sub-themes periodontal disease and tooth decay in the leading medical databases (Medline, Embase, BIOSIS Previews, Your Journals@Ovid, The Cochrane Library). We discuss these on the basis of an evidence-based guideline. For periodontal diseases we found no clear trend. Some studies showed no gender difference, others showed a higher prevalence for women or a higher prevalence for men. At the juvenile periodontitis and the occurring bacteria of periodontitis there were conflicting results as well. No simple statement can be made with respect to tooth decay, too. Respective studies merely show age as an important item. Totally it has to be noted that the scientific validity of some studies is not without any doubt as they are conducted with a too low number of clinical trials. Several studies are more than 15 years old, therefor providing a serious gender specific treatment in dentistry, future studies must increasingly incorporate and intensively discuss gender specific aspects.

Keywords

Caries, Dentistry, Gender, Men, Periodontitis, Women

Introduction

Already in the 1980s the World Health Organization (WHO) has postulated to analyze differences between sexes in all areas of medicine [1]. In the Vienna Declaration on investment in health of the WHO in 1992 was strongly admonished: “Maximum attention and urgency must be given to women’s health” [2]. Ten years later, in 2002, the WHO gender policy was written. The main objective of this policy is to enable a better health for women and men through modern health research, contributing programs and policies and to give due attention to the gender aspect [3]. And in the action plan 2000, “Gender Mainstreaming was mandatory prescribed in the medicine for all EU countries and decided in consequence” by the Austrian Federal Government as Univ.‐Prof. Dr. Margarethe Hochleitner [4]. The term “Gender Mainstrea-ming” was discussed for the first time in Nairobi already in 1985 on the 3rd UN World Conference of women and ten years later in 1995 the term has been further developed on the 4th UN World Conference of Women [5]. “Gender Mainstreaming” implies according to the BMFSFJ (German Federal Ministry for Family, Senior Citizens, Women and Youth): „… to take into account from the outset and periodically the different life situations and interests of women and men at all social projects, since there is no gender-neutral reality” (Bundesministerium für Familie, Senioren, Frauen und Jugend (Federal Ministry for Family Affairs, Senior Citizens, Women and Youth) 2012). And in the Basic Law was even expressly postulated: “To the actual enforcement of the equality between women and men the Government is expressly obliged by article 3, paragraph 2, sentence 2 Basic Law, it is an integral part of the political action of the Federal Government in all policy areas” [6]. The importance of a differentiated analysis with respect to gender-specific aspects is therefore obvious. Gender-specific considerations are nowadays no longer indispensable from dentistry. However the level of knowledge on this subject is until now surprisingly low. There is a disturbing lack of scientifically founded and reliable data in gender dentistry. “So far only little attention was paid to this topic in the German-speaking world, unlike in other countries. While gender research has found long ago entrance into scientific projects and teaching in medicine, the gender-specific dental, oral and maxillofacial surgery moves only slowly in the awareness of researchers and the practitioners, ladies and gentlemen. This topic is interesting and up to date, because just in dentistry in addition to biological differences, gender plays an important role“, so Priv.-Doz. Dr. Christiane Gleissner, 2011 [7]. To ensure a better dental care for male and female patients in the future, the present study delivers a systematic literature review on the topic of “Gender Dentistry”. The aim of this work is to offer a guideline-oriented, quality-based literature review, based on disease patterns, relevant for “Gender Dentistry”. Additionally all relevant studies have been analyzed, whether they meet the criteria and requirements of the WHO-Guidelines.

Material and Methodology

In the following we describe the preparation, the implementation of the literature review, the selected medicine database and search items as well as the handling of the results. The methodical approach is based on a guideline creation according to recommendation Rec(2001)13 of the Council of Europe (47 European States comprehending European international organizations), the AWMF (Work Community Association of scientific medical companies) [8] and the ZZQ Berlin (Center for dental quality) [9]. The main literature database was chosen to identify the current state of research regarding “gender” dentistry for evidence-based results. The selection was done on five databases: Embase, Your Journals@Ovid, The Cochrane Library, Medline, BIOSIS Previews. Access to these databases was given by the network of the University of Ulm. The search was as follows: for each clinical picture a search item has been set:

  • Periodontitis: gender periodontitis
  • caries susceptibility: gender caries

Then the specified keyword was given into the database and search started. The number of results was recorded. To narrow this large amount of results step by step, the ability of the program was used to divide the results by relevance. High relevance: 5 stars, low till no relevance: 4 till 0 stars. Then the option “five stars only” was made to observe only items with the highest relevance. The amount of search results, which now appeared, was also recorded. Depending on the number of results, the year of publication was still restricted. This and the new amount of search results were also recorded. Then the search results were read and the relevant publications were saved on RefWorks, a reference management software. The corresponding references, which are identified as possible matches, were downloaded as full text or ordered via interlibrary loan to check their contents in detail. The received information was translated, summarized and explained under results. Sequence and classification in reference 1, 2, are ordered alphabetically by the author’s name. So there is no ranking. To ensure the relevance of the results of the literature review, a further update was carried out in September 2013. Here, all five medicine databases were searched again in the period August 2012 – September 2013, as described above. The newly found references were saved and added to the already existing and edited.

Periodontitis

Keyword: gender periodontitis (Table 1)

Table 1:

Table 1. Gender Periodontitis. Results of the literature review in the five databases BIOSIS Previews, The Cochrane Library, Embase, Your Journals@Ovid und Medline from August 2012 and September 2013 and the total amount of stored references

Results, references = articles

Medical database

Basic Search

Restriction to five stars

Stored references August 2012

Stored references September 2013

Total references

BIOSIS Previews

8 491 results

335 results

16 references

1 result

17 references

The Cochrane Library

5 496 results

26 results

1 references

0 results

1 reference

Embase

10 882 results

824 results
(Restriction 2000 – today):

611 results

14 references

6 results

20 references

Your Journals @Ovid

16 955 results

17 results

2 references

0 results

2 references

Medline

10 164 results

673 results
(Restriction 2000 – today): 357 results

7 references

6 results

13 references

Caries

Keyword: gender caries (Table 2)

Table 2. Gender caries. Results of the literature review in the five databases BIOSIS Previews, The Cochrane Library, Embase, Your Journals@Ovid und Medline from August 2012 and September 2013 and the total amount of stored references

Results, references = articles

Medical database

Basic Search

Restriction to five stars

Stored references August 2012

Stored references September 2013

Total references

BIOSIS Previews

9001 results

541 results
(Restriction 2000 – today): 227 results

14 references

4 results

18 references

The Cochrane Library

5513 results

73 results

4 references

0 results

4 references

Embase

16 371 results

1179 results
(Restriction 2000– today):
773 results

6 references

3 results

14 references

Your Journals @Ovid

17 004 results

14 results

0 references

0 results

0 references

Medline

16 716 results

1125 results
(Restriction 2000 –  today);
500 results

4 references

11 results

15 references

Results

Periodontitis (Table 3, 4 & Figure 1)

Table 3. Results of the literature review on the topic of periodontitis. The total identified studies and the ultimately evaluated.

Amount of articles total

53

Amount of evaluated articles

21

Table 4. Results for the articles about periodontal diseases, which appeared before and right/after the year 2000, as well as a subject number higher or equal or lower 500 subjects or any information on the number of the person being tested.

Year of publication before 2000

5

Year of publication after/right 2000

16

Number of subjects higher/equal than 500

4

Number of subjects lower than 500

7

No information about the number of subjects

10

JDMR-18-108 -Jana Schwarz_Germany_F1

Figure 1. Gender Periodontitis. Gender-specific distribution of the edited articles about periodontal disease.

Caries

(Table 5, 6 & Figure 2)

Table 5. Results of the literature review on the topic of caries. The total identified studies and the ultimately evaluated.

Amount of articles total

51

Amount of evaluated articles

29

Table 6. Results for the articles about caries, which appeared before and right/after the year 2000, as well as a subject number higher or equal or lower 500 subjects or any information on the number of the person being tested.

Year of publication before 2000

4

Year of publication after/right 2000

25

Number of subjects higher/equal than 500

14

Number of subjects lower than 500

9

No information about the number of subjects

5

JDMR-18-108 -Jana Schwarz_Germany_F2

Figure 2. Gender Caries. Gender distribution of the edited articles about caries.

Discussion

Methodology

The current state of research in gender dentistry was collected with the help of a literature review, based on the creation of evidence-based guidelines. These guidelines summarize current knowledge, expert opinions and results of various working groups. Known and respected medical database were analyzed within the period of 10th – 20th of August 2012, and with another search from 12th -14th of September 2013.

Discussion related to the analyzed diseases

We have to notice that no clear result could be found and no simple conclusion for practice and teaching can be drawn. There are still too few studies, which offer enough reliability on gender-specific aspects. Some papers offer inconsistent results lacking of possible general evaluations. Further research seems to be indicated.

Periodontitis

The available results of our investigation of the present studies on periodontal disease are not satisfying. There are eleven studies that have proven that men suffer more frequently from periodontitis than women [10–20] However, there are five studies showing no gender aspect at all [21–24].

There are four further studies that show that women would more often suffer from periodontal diseases than men [13, 23, 25–27] The latest studies show a higher incidence for men. Aggressive periodontitis is more common in women and chronic periodontal disease occurs more often in men [13]. The loss of attachment points towards men [12]. One study indicates juvenile periodontitis more often in women [25], but a different study suggests that the distribution of men and women is equal [21]. Furthermore there shown, that there were differences between men and women related to periodontal disease bacteria [16], another study, however, show no difference [26]. The majority of results indicates a higher incidence in men regarding periodontal disease.

When evaluating the aspect of periodontal disease, many other factors, such as age and hormone levels of women do play an important role, because the periodontal disease is known to be a multifactorial disease. All factors should be detected and examined. These aspects should be included in further studies, observed, presented and evaluated in order to get scientific based information, providing additional value for dental treatment. The evaluated studies are lacking to draw conclusions due to limited numbers of subjects and mixed timelines.

Caries

The examination of the studies on caries does not give a uniform result. There are six studies which show no gender-specific differences [28–33]. In contrary studies have proven that women have a greater rate of caries than men [33, 34–43].

The following studies however showed higher rates of tooth decay in men. Only one of these studies was performed in adults [44]. The other five studies have been conducted on children [37, 38, 45–51]. Therefore, there seems to be a tendency for higher caries rates in women. However, depending on the age of the subjects. There are studies demonstrating that boys have higher levels of tooth decay, but the tendency is expanding in adulthood towards women [37, 38].

Studies that examine a multicausal illness such as caries must include all ages and have to be divided into gender, age, eating habits, socioeconomic background, etc. to obtain useful information. Especially in countries with huge emphasis on prophylaxis and caries prevention, e.g. Germany, this study design is essential. On the subject of dental caries the actuality of the studies is satisfactory, the number of clinical trials however mediocre.

The authors declare that they have no competing interests.

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Prevalence of Nonreactive Non-Stress Test in Low Versus High Risk Pregnancy

DOI: 10.31038/IGOJ.2019211

Abstract

Objective: To determine the prevalence of nonreactive nonstress test (NST) in low risk and high risk pregnant women.

Materials and Methods: This prospective cohort study enrolled pregnant women with gestational age of 32 weeks or more who had been offered NST by their obstetricians at the antenatal clinic, King Chulalongkorn Memorial Hospital. High risk group were defined as having any maternal, fetal or placental risk factors. The NST result was interpreted by at least two obstetricians. All participants were followed until delivery and perinatal outcomes were recorded.

Results: A total number of 1,168 participants with 1,261 NST tests were included. 782 tests (62%) were offered to low risk and 479 tests (38%) to high risk group. Decrease in weight and maternal diabetes mellitus was the most common indication for low risk and high risk group, respectively. Overall prevalence of nonreactive NST was 0.32% (0.38% in low risk and 0.21% in high risk group). Only one newborn with non-reactive NST in high risk group was admitted in the NICU due to meconium aspiration syndrome. However, there was no significant association between nonreactive NST and obstetric risks or adverse perinatal outcomes.

Conclusion: The prevalence of nonreactive NST in this study was only 0.32%. NST is not routinely recommended in low risk pregnant women due to no association between non-reactive NST and perinatal morbidity.

Keywords

Antepartum Fetal Monitoring, Nonstress test, Pregnancy

Introduction

Various non-invasive antepartum fetal surveillance techniques are available including fetal movement assessment, non-stress test (NST), biophysical profile (BPP), contraction stress test (CST), and maternal uterine artery and fetal umbilical artery Doppler velocimetry [1,2]. The aim of antepartum fetal surveillance is to confirm the well-being of the fetus and detect early neonatal injury [3]. NST is currently and widely used in antenatal clinics as a continuous measurement of fetal heart rate (FHR) because it is simple and does not harm pregnant women or their fetuses.

Early detection in the abnormal change of FHR is useful to prevent neonatal injury [4]. NST aims to confirm whether the brain of the fetus is sufficiently oxygenated. [2] Non-reactive NST is significantly associated with fetal distress and low Apgar scores [5]. Testing is recommended for pregnant women who are at risk of fetal hypoxic injury or fetal death. Indications for NST can be divided into three groups as follows: (1) Maternal indications such as diabetes, hypertension, cardiovascular diseases, anemia, kidney disease; (2) Fetal indications such as decrease fetal movement, abnormal fetal growth, post-term pregnancy, abnormal amniotic fluid; and (3) Placental indications such as abnormal placentation, chronic abruption [6].

Due to the uncomplicated nature of the test, obstetricians in general practice often perform NST to pregnant women with minimal obstetric risks such as mothers with poor weight gain, decreased/static weight, or passed date [7]. No clear evidence exists to support the benefit of NST in this group. Even though NST is not an invasive testing method, it is not free of charge and the patient is required to spend at least 20 minutes in the examination room. Moreover, NST results can influence the decisions of the obstetricians. Based on a previous study, non-reactive NST results increased the incidence of labor induction by 90% and doubled the rate of cesarean delivery [8]; therefore, patients may be subjected to unnecessary obstetrics procedures.This study was conducted to determine the prevalence of non-reactive NST for each indication and also identify the necessity for the test in low risk group. We anticipate that this knowledge will be useful in making decisions whether to offer NST to pregnant women.

Materials and Methods

This prospective observational study was conducted at the antenatal clinic, King Chulalongkorn Memorial Hospital. Pregnant women with gestation age of 32 weeks or more who had been offered NST by their obstetricians were invited to participate. We excluded pregnancies with antepartum diagnosed fetal congenital anomalies and those who have had multiple pregnancies. After the participants gave their informed consent, the participants were interviewed. This study was approved by the Institutional Review Board, Faculty of Medicine, Chulalongkorn University (IRB351/55). The high risk group was defined as participants at risk for fetal hypoxia or fetal death according to the antepartum surveillance bulletin of the American College of Obstetricians and Gynaecologists as described previously [6]. While the low risk group was defined as participants who did not show any maternal, fetal or placental risks. Demographic data and indications of NST were recorded in well-designed individual case records.

NST examination requires at least 20 minutes. Participants were placed in a supine position and a fetal heart rate monitor was attached to an abdominal belt. The participants were asked to record any fetal movements by clicking a button. In cases where there was suspicion that the baby was asleep, vibroacoustic stimulation was performed. Results were interpreted by at least two obstetricians. Results are classified as reactive or non-reactive. Reactive NST is diagnosed if there are at least two times of FHR acceleration in 20 minutes, with each acceleration 15 beats per minute (bpm) or more above baseline and lasting for at least 15 seconds. The baseline FHR should be between 110–160 bpm with moderate variability of 6–25 bpm. If the FHR is elevated less than 15 bmp within a 20–40 minute period, the interpretation is non-reactive. [9] Two obstetricians were required to agree with the interpretation of each result. If their analyses differed, a third obstetrician was consulted. If the final results showed a non-reactive then further investigations including biophysical profile, CST or ultrasonography were immediately performed. If the participants were offered NST more than once, the worst pattern was analyzed and included in the research results. All participants were monitored until delivery, with data and perinatal outcome collected and recorded in detail. Participants who did not deliver their babies at the King Chulalongkorn Memorial Hospital or lost their medical data were excluded from the study. Statistical analysis was performed using the Statistical Package for the Social Sciences (SPSS) for Windows, version 22. Descriptive data were analyzed using frequency and percentage, while significant associations between the categorical data were assessed by Fisher’s exact test or Chi-square test. When the p-value was < 0.05, this was considered statistically significant.

Results

A total of 1,536 pregnant women were offered NST. After obtaining informed consent, 1,297 women participated in this study. A total of 129 were not included because their medical records after follow-up was incomplete or unavailable. The remaining participants in final analysis were 1,168 participants. Mean age was 29.7 ± 6.3 years with mean gestational age on the testing day was 36.6 ± 3.0 weeks. Basic clinical characteristics of the participants are shown in (Table 1).

Table 1. Demographic data.

Demographic data

Number of cases
(N= 1,168)

Mean age, years (SD)

29.7 (6.3)

Mean gestational age on the testing day, weeks (SD)

36.6 (3.0)

Primigravida

573 (49.1%)

Mean BMI, kg/m2 (SD)

22.8 (4.8)

Concomitant medical diseases

412 (35.3%)

History of previous surgery

278 (23.8%)

Smoking

12 (1.0%)

Illicit drug use

4 (0.3%)

Alcohol consumption

16 (1.4%)

Ninety-three participants were offered NST twice; therefore, a total of 1,261 tests were analyzed in our study. A total of 782 NSTs were offered to low risk participants and 479 tests were offered to high risk pregnant women. For women in the low risk group, decrease in weight was the most common reason for requesting NST (275 cases). Maternal indications, especially diabetes mellitus were the most common indication for high risk participants (Table 2).

Table 2. Indications for NST.

Indications

Number
(Total number = 1,261)

Low risk group

   Static weight gain

   Decrease in weight

   Poor weight gain

   Passed date (GA 40+1–41+6 weeks)

   Other

782 (62.0%)

193 (15.3%)

275 (21.8%)

84 (6.7%)

98 (7.8%)

132 (10.5%)

High risk group

   Maternal indications

   Fetal indications

   Placental indications

   Maternal and fetal indications

479 (38.0%)

346 (27.4%)

119 (9.4%)

8 (0.6%)

6 (0.5%)

Obstetrics and perinatal outcomes were shown in (Table 3). Mean gestational age at delivery was 38.7 ± 1.3 weeks. The rate of spontaneous vaginal delivery was 53.5% and cesarean delivery rate was 43.5%. Neonatal morbidity occurred in 11.9% of infants with 5.7% of them required admission. Four participants had non-reactive NST with overall prevalence at 0.32%; three out of 782 (0.38%) from the low risk group and one out of 479 (0.21%) from the high risk group had non-reactive NST. Only one new born with non-reactive NST in high risk group was admitted in the Neonatal Intensive Care Unit (NICU) for 5 days due to meconium aspiration syndrome. This newborn had complete recovery and discharged with mother. The other three non-reactive NST in low risk group, intrauterine resuscitation was given, and repeated tests became reactive. These three newborns had no perinatal morbidity (Table 4). Fisher’s exact test showed there was no association between NST result and pregnancy risk (P = 1.00). There were no associations between NST results and adverse perinatal outcomes such as Apgar scores, neonatal morbidity, NICU admission, perinatal ventilator requirement and fetal anomalies (Table 5).

Table 3. Obstetrics and perinatal outcomes.

Delivery data

Number of delivery (N=1,168)

Mean gestational age at delivery, weeks (SD)

38.7 (1.3)

Mean birth weight, grams (SD)

3,142.5 (455.5)

Delivery route

 Spontaneous vaginal delivery

 Cesarean delivery

 Forceps extraction

 Vacuum extraction

625 (53.5%)

507 (43.5%)

30 (2.6%)

6 (0.5%)

Sex of fetus

 Male

 Female

621 (53.2%)

547 (46.8%)

Fetal anomalies

 No

 Yes

1,127 (96.5%)

41 (3.5%)

Neonatal morbidity

 No

 Yes

1,029 (88.1%)

139 (11.9%)

NICU admission

 No

 Yes

1,120 (94.3%)

66 (5.7%)

Ventilator required

 No

 Yes

1,149 (98.4%)

19 (1.6%)

Table 4. Clinical characteristics of non-reactive NST cases.

Gravida

G4P1

G1P0

G2P1

G1P0

GA at testing day (weeks)

39

37

40

39

Indication for NST

Gestation diabetes

(High risk)

Decrease in weight

(Low risk )

Decrease in weight

(Low risk)

 Unspecified

(Low risk)

Delivery route

Emergency cesarean section

Cesarean section due to breech presentation

Vaginal delivery

Cesarean section due to CPD

Neonatal outcomes

Male fetus 3,325 grams

Male fetus 2,935 grams

Male fetus

2,800 grams

Male fetus

3,220 grams

APGAR scores

at 1 and 5 mins

9,9

9,10

9,10

9,10

Perinatal morbidity

Maconium aspiration syndrome

5 days of NICU admission

No

No

No

GA = Gestational age

Table 5. Association between NST results and perinatal outcomes including Apgar score, neonatal morbidity, NICU admission, perinatal ventilator requirement and fetal anomalies.

NST

P value

Perinatal outcomes

Reactive

Non-reactive

Apgar score at 1 min*

<7

25

0

1.00

7–10

1,138

4

Apgar score at 5 min*

<7

3

0

1.00

7–10

1,160

4

Neonatal morbidity

Yes

138

1

0.40

No

1,026

3

NICU admission

Yes

65

1

0.21

No

1,099

3

Perinatal ventilator need

Yes

19

0

1.00

No

1,145

4

Fetal anomalies

Yes

41

0

1.00

No

1,123

4

* one missing data

Discussion

This study showed the rate of non-reactive NSTs was low only 0.32%. Most NSTs (62.5%) were conducted in low risk participants. Prevalence of non-reactive testing was 0.38% and 0.21% in the low and high risk pregnant women, respectively. Overall prevalence of non-reactive NSTs in our study was very low compared to previous studies. Rayburn et al. conducted a prospective study of 315 pregnancies and determined 12% of NSTs had non-reactive patterns [10], while Abitbol et al. reported 10.9% of patients had non-reactive NSTs [11]. The results from these two studies were different compared to our study. These 2 studies were conducted before 1990, most patients had risk factors and different terminologies of abnormal NST result might be plausible explanation. The use of vibroacoustic stimulators in these two previous studies might be another possibility. There is clear evidence that vibroacoustic stimulation can reduce testing time but can contribute to higher rates of false non-reactive results [12].

Data from our study confirmed that offering NST to low risk pregnant women was pointless, waste of time and resources. Although, NST is simple and widespread use, there is poor evidence that it can reduce perinatal morbidity or mortality. One major drawback is high frequency of false positive rates. Pregnant women usually placed on supine position during the test. Compression of abdominal aorta results in reduction of uterine blood flow and associated with fetal heart rate change. [11] Repeated test in lateral decubitus position usually returns into normal results. Our study confirmed that non-reactive NST in low risk participants did not associated with perinatal morbidity. However, it may not conclude in high risk participants due to very low percentage of non-reactive results. Only 1 patients with maternal risk factors (gestational diabetes) showed non-reactive NST and meconium aspiration syndrome was diagnosed in this newborn. Despite low specificity to predict perinatal morbidity, antepartum NST is still recommended to use only in pregnant women with risk factors for adverse perinatal outcome [13].

To the best of our knowledge, this is the first and large study that looked at the results of NST in low risk pregnant women. The results indicated that NST is unnecessary for low risk pregnant women. However, there were some limitations in our study. Firstly, the prevalence of non-reactive NST was very low in both low and high risk pregnant women. As this result, it may not have enough power to significantly confirm our findings. Secondly, about 10% of the pregnant women who were lost to follow-up were excluded from the final analysis.

Conclusions

Overall, non-reactive NST was 0.32% (0.38% in low risk and 0.21% in high risk groups). NST is unnecessary for low risk pregnant women. There was no association between NST results and adverse perinatal outcomes including Apgar score, neonatal morbidity, NICU admission, perinatal ventilator requirements and fetal anomalies.

Declaration of interests: The authors declared no conflicts of interest.

Acknowledgement: This study was supported by Ratchadapiseksompoch Fund (RA55/69), Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand.

References

  1. Williams KP, Farquharson DF, Bebbington M, Dansereau J, Galerneau F, et al (2003) Screening for fetal well-being in a high-risk pregnant population comparing the nonstress test with umbilical artery Doppler velocimetry: a randomized controlled clinical trial. Am J Obstet Gynecol 188: 1366–1371.
  2. Liston R, Sawchuck D, Young D (2018) No. 197a-Fetal Health Surveillance: Antepartum Consensus Guideline. J Obstet Gynaecol Can 40: 251–271.
  3. Esin S (2014) Factors that increase reactivity during fetal nonstress testing. Curr Opin Obstet Gynecol 26: 61–66.
  4. Gobillot S, Fontecave-Jallon J, Equy V, Rivet B, Gumery PY, et al. (2018) Non-invasive fetal monitoring using electrocardiography and phonocardiography: A preliminary study. J Gynecol Obstet Hum Reprod 47: 455–459. [crossref]
  5. Flynn AM, Kelly J (1977) Evaluation of fetal wellbeing by antepartum fetal heart monitoring. Br Med J 1: 936–939. [crossref]
  6. [No authors listed] (2000) ACOG practice bulletin. Antepartum fetal surveillance. Number 9, October 1999 (replaces Technical Bulletin Number 188, January 1994). Clinical management guidelines for obstetrician-gynecologists. Int J Gynaecol Obstet 68: 175–185. [crossref]
  7. Mancuso A, De Vivo A, Fanara G, Denaro A, Lagana D, et al. (2008) Effects of antepartum electronic fetal monitoring on maternal emotional state. Acta Obstet Gynecol Scand 87: 184–189.
  8. Evertson LR, Gauthier RJ, Schifrin BS, Paul RH (1979) Antepartum fetal heart rate testing. I. Evolution of the nonstress test. Am J Obstet Gynecol 133: 29–33. [crossref]
  9. Preboth M (2000) ACOG guidelines on antepartum fetal surveillance. American College of Obstetricians and Gynecologists. Am Fam Physician 62: 1184, 1187–1188. [crossref]
  10. Rayburn WF, Motley ME, Zuspan FP (1982) Conditions affecting nonstress test results. Obstet Gynecol 59: 490–493. [crossref]
  11. Abitbol MM, Monheit AG, Poje J, Baker MA (1986) Nonstress test and maternal position. Obstet Gynecol 68: 310–316. [crossref]
  12. Smith CV, Phelan JP, Platt LD, Broussard P, Paul RH (1986) Fetal acoustic stimulation testing. II. A randomized clinical comparison with the nonstress test. Am J Obstet Gynecol 155: 131–134.
  13. Freeman RK, Anderson G, Dorchester W (1982) A prospective multi-institutional study of antepartum fetal heart rate monitoring. II. Contraction stress test versus nonstress test for primary surveillance. Am J Obstet Gynecol 143: 778–781.

Admission Potassium / Sodium Ratio Linearly Predicts Mortality Outcome Following an Emergency Medical Admission

DOI: 10.31038/IMROJ.2019411

Abstract

Background: Disturbance of sodium and potassium chemistry is commonly present during an emergency medical admission; we analyse the interaction of these ions and relate K+/ Na+ Ratios to hospital mortality outcomes.

Methods: All emergency medical admissions between 2002 and 2017 were studied. We log transformed sodium and potassium values and calculated their respective ratios as a predictor of 30-day mortality outcomes using a multivariable logistic model.

Results: There were 106,586 admissions in 54,928 patients. Patients with higher K+/ Na+ ratios at admission were older at 66.5 years (IQR 47.0–79.3) compared with 59.0 years (IQR 39.9–75.5) and more likely to be female (51.9% vs. 45.5%). They had a higher 30-day hospital mortality rate – 5.4% vs 3.5% (p<0.001). Across consecutive deciles of K+, rising admission levels linearly predicted outcomes unlike Na+ where falling levels linearly predicted an increased mortality. Disturbed K+/ Na+ balance (log ratio) was prognostic – 30-day mortality OR 1.12 (95%CI 1.11–1.13). Increasing age predicted an increased likelihood of an abnormal K+/ Na+ balance.

Conclusion: Disturbed K+/ Na+ is linearly predictive of 30-day hospital mortality and is strongly age associated; possibly it is a consequence of Na+ leaving the extracellular space (ECF) and K+ transit to the ECF due to impaired cellular membrane homeostatic function.

Keywords

Emergency Medical Admission, Potassium Sodium Balance

Introduction

An emergency hospital admission with an urgent clinical condition requiring immediate or specialist management 1] is a high risk period but has shown improved outcomes over time 2,3] particularly since the establishment of Acute Medical Admission Units [4,5]. Risk management of critical admissions requires reliable predictors and there has been debate regarding disturbance of sodium [6–10] and potassium homeostasis [11,12] in this regard.

In terms of sodium levels, abnormalities in the laboratory range are said to occur in about 15% of hospitalized patients [7]; a U-shaped curve is described with major morbidity and mortality at the extremes of the sodium distribution in many patient groups including general internal medicine patients (13–15). Hyponatraemia in particular is prognostic for specific groups of patients with cardiovascular disease, such as congestive heart failure [16,17] but also for patients with pulmonary hypertension [18]. In terms of potassium levels, a U-shaped curve for mortality is also described with lowest all-cause mortality for potassium values between 4.0 and <5.0 mEq/L. [19]; in a geographically diverse population (n = 911,698) 27.6% had a potassium <4.0 mEq/L, and 5.7% had a value ≥ 5.0 mEq/L. A further commentary on two large studies, of the relationship between cardiovascular outcomes and potassium levels, noted that there were not a significant association between hypokalaemia and mortality in the fully adjusted Cox model, but there was a trend toward higher mortality at lower serum potassium [20].

The problem with such analyses is that the conclusions drawn will not be identical if based on a unit versus a frequency based analysis. The unit based analysis (by K+ / Na+ interval) is unintentionally biased as some intervals may contain small numbers who are actually outliers in the distributions; the results in generalisation from the specific to the general population. However, a decile based analysis for sodium, indicated that, for emergency medical admissions, the highest quantile (decile) had the lowest mortality risk and that mortality progressively increased over every other decile [21]. As a generality, the same applies to potassium except that rising deciles determine worse outcomes. The exception, dictating bad outcomes for a few, should not confuse an overall understanding of the mortality relationship to the admission levels of sodium and potassium. We have previously shown that mortality is far more influenced by other factors such as the Acuity Illness Severity Score (AISS) [22,23], sepsis status [24] and the overall burden of disease as reflected by summative comorbidities – Charlson Comorbidity Index [25] or Chronic Disabling Disease Score [26]. In this paper we consider the relevance of the balance between potassium and sodium, at time of an emergency medical admission, in determining 30-day mortality in over 100,000 emergency medical admissions and draw inferences from these findings.

Methods

Background

St James’s Hospital, Dublin serves as a secondary care centre for emergency admissions in a catchment area with a population of 270,000 adults. All emergency medical admissions were admitted from the Emergency Department to an AMAU, the operation and outcome of which have been described elsewhere [2,3,27,28]. As a city centre hospital St James’s admits persons resident elsewhere but working in the capital in addition to visitors to Dublin who became acutely ill. The number of emergency medical admissions resident in the catchment area was 74.5%; this compares with a figure of 59% for ED presentations where the social influences on emergency department visitations on two London hospitals have been examined [29].

Data Collection

During any emergency hospital admission information on the clinical episode is entered into the Patient Administration System (PAS), admission metrics are determined and entered into the emergency room database and following admission data is recorded on the patient electronic record, different laboratory systems; abstracted data is subsequently processed by dedicated staff for downloading to the National Hospital In-Patient Enquiry (HIPE) scheme. HIPE is a national database of coded discharge summaries from acute public hospitals in Ireland [30,31]. International Classification of Diseases, Ninth Revision, Clinical Modification (ICD-9-CM) has been used for both diagnosis and procedure coding from 1990 to 2005 with ICD-10-CM used since then. Data included parameters such as the unique hospital number, admitting consultant, date of birth, gender, area of residence, principal and up to nine additional secondary diagnoses, principal and up to nine additional secondary procedures, and admission and discharge dates. We in 2002 designed programs to extract the HIPE data and admission biochemistry and haematological variables (linked via the Medical Record Number) to monitor the performance of the Acute Medical Admission Unit. These data were then anonymized using an ID sequence and the MRN to link the data that is held on our system in multiple separate files. These files are assembled into memory at time of statistical analysis (using Stata coding). The data does not exist as a discrete database; it is updated annually and is a closed system accessible only by the Principal Investigators. GDPR does not apply to anonymous datasets.

Acute Illness Severity Score

Derangement of biochemical parameters may be utilised to predict clinical outcome. We derived an AISS based on laboratory data – this is an age adjusted 30-day in-hospital mortality risk estimator, representing an aggregrate laboratory score based on the admission serum sodium, serum potassium, serum urea, red cell distribution width, white blood cell count, serum albumin and troponin values at admission (23, 32); the score predicts 30-day in-hospital mortality from the biochemical parameters recorded in the Emergency Department (33). The AISS can be enhanced with data from the ICD9/10 discharge codes to compute Co-Morbidity (as per the Charlson Index (25)) or Disability (26) status. This Risk Score is exponentially related to the 30-day mortality outcome with a range of mortality outcomes from 0.5% (0.40%–0.53%) to 39% (37.8%-40.2%). We have demonstrated using a nomogram that this laboratory model derives most of its predictive power from the admission values of albumin, urea and haemoglobin (34).

Comorbidity Instrument

Hospital HIPE codes [30,31] were interrogated to construct a measure of multi-morbidity. To devise the score, we searched ICD9 hospital episode discharge codes (back-mapping ICD10 codes to ICD9 as appropriate) based on the definition proposed by the US Department of Health and Human Services for chronic physical or mental health disorders, that limit people ‘in activities that they generally would be expected to be able to perform’. These ICD codes were similar to those proposed by the Canadian group for multi-morbidity (35) and the work of Quan [36,37]; they were grouped by system into the following ten groups: (i) cardiovascular, (ii) respiratory, (iii) neurological, (iv) gastrointestinal, (v) diabetes, (vi) renal, (vii) neoplastic disease, (viii) others (including rheumatological disabilities), (ix) ventilatory assistance required and (x) transfusion requirement. We have previously detailed the ICD9 codes for chronic physical or mental health disorders utilized as a supplementary Table [38]. In addition, we searched other hospital databases for evidence of diabetes (Diamond database), respiratory insufficiency (FEV1 < 2 L data pulmonary function laboratory), troponin status (high sensitivity troponin > 25 ng/L) [33], low albumin (<35 G/dL) and anaemia (haemoglobulin levels < 10 G/dL) or chronic renal insufficiency – MDRD < 60 mL/min*1.73 m2 [39]. The ‘morbidity score’ for each individual’s clinical episode during the study, was weighted by its relative important against the 30-day mortality outcome in the multivariable regression analysis.

Statistical Methods

Descriptive statistics were calculated for background demographic data, including means/standard deviations (SD), medians/interquartile ranges (IQR), or percentages. Comparisons between categorical variables and mortality were made using chi-square tests.

We employed a logistic model with robust estimate to allow for repeated admissions; the correlation matrix thereby reflected the average dependence among the specified correlated observations [23]. Logistic regression analysis identified potential mortality predictors and then tested those that proved to be significant univariate predictors (p<0.01 by Wald test). Mortality results have been presented either by admission (all admissions counted) or by patient (only one admission considered – last admission if > one). In an extended time series (16 yr.), 48.7% of patients were readmitted at least once, 9.3% > 5 times and 20 patients > 50 times each; whether per admission or per patient mortality analysis is optimal is uncertain. We used the margins command in Stata to estimate and interpret adjusted predictions for sub-groups, while controlling for other variables such as time, using computations of average marginal effects. Margins are statistics calculated from predictions of a previously fitted model at fixed values of some covariates and averaging or otherwise over the remaining covariates. In the multivariable model (logistic), we adjusted univariate estimates of effect, using the previously described outcome predictor variables. The model parameters were stored; post-estimation intra-model and cross-model hypotheses could thereby be tested.

Adjusted odds ratios (OR) and 95% Confidence Intervals (CI) were calculated for those predictors that significantly entered the model (p<0.10). Statistical significance at P<0.05 was assumed throughout. Stata v.15 (Stata Corporation, College Station, Texas) statistical software was used for analysis. This study had no interventional component, used anonymised routinely collected data, complied with data protection legislation and was undertaken as part of the Hospital Quality Improvement Programme to assess the overall impact of the Acute Medical Admission Initiative.

Results

Patient Demographics

During the 16-year study period (2002–2017), there were a total of 106,586 admissions in 54,928 unique patients. This represented all emergency medical admissions, including patients admitted directly into the Intensive Care Unit or High Dependency Unit. The proportion of males was 48.5%. The median (IQR) Length Of Stay (LOS) was 4.3 days (1.7–8.9). The median (IQR) age was 58.9 years (38.2–76.3) with the upper 10% boundary at 85.0 years.

The demographic characteristics (Table 1) are outlined with a cut at the midpoint of the calculated admission K+ / Na+ ratio. The K+ and Na+ data was log (natural) transformed prior to the analysis mainly to equalize the data spread of the two elements despite their numerical difference – anticipated to make the data easier to handle and interpret. Above and below that midpoint, the patient group characteristics are tabulated by Acute Illness Severity [23,32], Charlson Index [25], Co-Morbidity and Sepsis status [24]. Patient in the upper half of the K+ / Na+ ratio distribution were older 66.5 years (IQR 47.0–79.3) compared with 59.0 years (IQR 39.9–75.5) but had equivalent hospital LOS – 4.9 days (IQR 1.9–9.7) versus 5.1 days (IQR 2.3–9.7). Admissions with a K+ / Na+ ratio distribution above the median were more likely to be female (51.9% vs. 45.5%) and had a higher 30-day hospital mortality rate – 5.4% vs 3.5% (p<0.001). Those with a K+ / Na+ ratio distribution above the median had a higher Acute Illness Severity Score (top two groups 66.1% vs. 53.4%) and higher Charlson Comorbidity Index (top two groups 39.6% vs. 33.0%).

Mortality related to admission K+ / Na+ levels

The 30-day per patient mortality declined between 2002 and 2017 from 12.4% to 4.8%. This represented a relative risk reduction (RRR) of 61.3% with a number needed to treat (NNT) of 13.1. Lower admission sodium levels were associated with a linear increase in patient mortality rate across all deciles (OR 0.89 (95% CI 0.88–0.90); from decile 1 (lowest sodium) – 16.9% (95% CI 16.0%-17.8%) to decile 5 – 11.4% (95% CI 11.0%-11.8%) and decile 10 – 6.7% (95% CI 6.2%-7.2%). Higher admission potassium level predicted mortality – OR 1.07 (95%CI: 1.06, 1.09) and there was a progressive increase in 30-day in-hospital mortality across all deciles from decile 1 (lowest) – 8.9% (95%CI: 8.3%, 9.4%) to decile 5 – 11.0% (95%CI: 10.6%, 11.3%) and decile 10 – 14.2% (95%CI: 13.5%, 14.8%). This is a different interpretation to the conventional wisdom of a U shaped curve for both K+ and Na+ and mortality outcomes. However, an analysis by decile lacks numerical bias where very difference frequencies fall within discrete ranges. For Na+ for example, and taking decile 10 with the lowest overall mortality risk, the lower boundary is 142 mEq/l, with the median, 75% and 95% points at 144, 145 and 150 mEq/l. For K+ for example, and taking decile 1 with the lowest overall mortality risk, the upper boundary is 3.4 mEq/l, with the median, 75% and 95% points at 3.2, 3.0 and 2.6 mEq/l. Therefore the correct distribution mortality relationship is that 30-day mortality outcome, as determined by K+ and Na+ admission values, is that mortality increases with each decile of K+ decile, from lowest to highest with the opposite being true for admission Na+ levels.

Table 1. Characteristics of Emergency Admissions by Median K+ / Na+ Ratio.

Lower
(N= 45,135)

Upper
(N= 50, 726)

p-Value

Age (yr.)

 Mean (SD)

57.4 (20.78)

62.5 (20.29)

<0.001

 Median (Q1, Q3)

59.0 (39.9, 75.5)

66.5 (47.0, 79.3)

Length Stay (day)

 Mean (SD)

7.2 (6.45)

7.0 (6.51)

<0.001

 Median (Q1, Q3)

5.1 (2.3, 9.7)

4.9 (1.9, 9.7)

Gender

 Male

20530 (45.5%)

26321 (51.9%)

<0.001

 Female

24605 (54.5%)

24405 (48.1%)

30-day Hospital Mortality

 Alive

43542 (96.5%)

48000 (94.6%)

<0.001

 Dead

1593 (3.5%)

2726 (5.4%)

Acute Illness Severity

 1

1835 (4.3%)

1094 (2.5%)

<0.001

 2

3916 (9.2%)

2595 (5.8%)

 3

6234 (14.7%)

4630 (10.4%)

 4

7768 (18.3%)

6773 (15.2%)

 5

8638 (20.3%)

8329 (18.7%)

 6

14072 (33.1%)

21064 (47.4%)

Co-Morbidity Score

 < 6

11387 (38.3%)

11247 (32.4%)

<0.001

 >=6 < 8

8547 (28.7%)

9744 (28.0%)

 >=8 < 12

8181 (27.5%)

11098 (31.9%)

 >=12 < 20

1643 (5.5%)

2674 (7.7%)

Charlson Index

8638 (20.3%)

8330 (18.7%)

 0

22226 (49.3%)

21771 (43.0%)

<0.001

 1

12063 (26.8%)

13830 (27.3%)

 2

10768 (23.9%)

15042 (29.7%)

Sepsis Group

 1

33695 (74.7%)

39653 (78.2%)

<0.001

 2

9828 (21.8%)

9426 (18.6%)

 3

1612 (3.6%)

1647 (3.2%)

*LOS: length of stay, MDC: Major Disease Category, IQR: Inter-Quartile Range

Mortality related to admission K+ / Na+ Ratio (Figs 1 / 2)

The admission K+ / Na+ Ratio was predictive of episode 30-day hospital mortality – OR 1.12 (95% CI 1.11–1.13). This compared with the predictive capacity of the Acute Illness Severity Score of OR 3.28 (95% CI 3.03–3.56) , the Charlson Comorbidity Index of OR 1.20 (95% CI 1.18–1.23) and sepsis status OR 1.61 (95% CI 1.51–1.71). The admission K+ / Na+ Ratio was a linear predictor (Fig 1) of 30-day hospital mortality over the range of deciles from the lowest D1 with predicted mortality of 5.6% (95% CI: 5.2%, 5.9%), to D5 6.2% (95% CI: 6.0%, 6.4%) and D10 7.2% (95% CI: 6.9%, 7.5%), After adjustment for the above predictors, the predictive value of K+ / Na+ Ratio was reduced to 1.03 (95% CI 1.02–1.04).

IMROJ_2019-101-Bernard Silke_F1

Figure 1. The relationship between the admission K+ / Na+ ratio and 30-day in-hospital mortality. For each decile of the ratio, the 30-day in-hospital mortality rate was derived from the logistic regression model and adjusted for the Charlson Co-Morbidity, Co-Morbidity and Sepsis Scores.

IMROJ_2019-101-Bernard Silke_F2

Figure 2. The relationship between the admission K+ / Na+ ratio and patient age. The Decile of the Ratio, at time of admission, was regressed against the age group (Zero Poisson truncated) and adjusted for the Acute Illness Severity and Co-Morbidity and Sepsis Status. The Decile of K+ / Na+ ratio at time of admission was a linear function of age.

We also examined the likelihood that the admitted K+ / Na+ Ratio was age related – over the admitted age range by relating the likelihood that older admissions would fall into higher K+ / Na+ Decile Ratios. We regressed the age groups < 40, 40 + – 60, 60+ – 75, 75+ – 85 and >85 against counts of K+ / Na+ Ratio (zero truncated Poisson regression) by patient and adjusted for the Acute Illness Severity (22, 23), Charlson Comorbidty Index (25) and Sepsis Status (24). There was a linear relationship between the underlying age and the likelihood of being in a higher K+ / Na+ Decile Ratio.

Discussion

Our data shows that over a period of 16 years potassium and sodium levels at the time of an emergency medical admission were predictive of 30-day mortality outcomes. Although the mortality outcomes have improved over time, the predictive value consistent irrespective of such quality improvements. Falling deciles of sodium were associated with increasing mortality outcomes; the 30-day patient mortality comparing the highest and lowest decile was 6.7% vs 16.9%. For potassium the opposite was true; rising deciles were associated with increasing mortality from lowest decile rate of 8.9% to highest rate of 14.2%. Therefore in overall terms neither hypokalaemia nor hypernatraemia were prognostic risk factors of a 30-day hospital death. That is not to argue that specific patients with very disturbed admission hypokalaemia or hypernatraemia were not at risk. However, for high sodium levels (top decile > 142 mEq/l) only 25% of that decile were > 145 mEq/l. and 5% > 150 mEq/l. For the lowest potassium decile (<3.4 mEq/l), only 25% of that decile were < 3.0 mEq/l. and only 5% < 2.6 mEq/l. Thus taking what might occur in a small minority of any population distribution, and seeking to make statements regarding the overall population risk is an approach that may result in a misconceived view of the risk reality in the mind of the clinician.

If increasing potassium or decreasing admission levels are linearly prognostic, as our model relating deciles of each to 30-day mortality outcome, then a reasonable presumption might be that the disturbance in both would be linked. Sodium being the principal extracellular ion and potassium being intracellular might leak from their respective domains as the system aged; disorder of the pair might be thought of as reflecting impaired membrane function and the energetic process involved in maintaining the transmembrane electro-chemical gradient. The likelihood of being in a higher risk decile certain was strongly age dependent in our analysis. We wondered if the relationship of the two ions, as reflected in the K+ / Na+ Ratio, might be a better prognostic indicator that their individual ability to predict mortality outcome. The univariate prediction of the K+ / Na+ Ratio was considerably attenuated when other independent outcome predictors such as Acute Illness Severity Score, Charlson Comorbidity Index and sepsis status were accounted for in the multivariable model. This would suggest that in overall terms, disorders of these ions, whilst of clinical relevance in certain specific patient subsets, in general are part of more complex disorder of homeostasis, driven by illness severity and the overall burden of disease [40,41]. As linking the analysis to the combined behaviour, with the K+ / Na+ Ratio, did not improve the prognostic prediction over that from independent analysis of each, one cannot assume that the behaviour of the K+ and Na+ is linked in emergency medical admissions on average.

Of course, the practicing physician is rightly focused on clinical outcomes at the extremes of distribution due to the high mortality rates [42] and the need for urgent interventions to correct the disorder and mitigate the consequences [43]. Generally speaking disturbed biochemistry has been recognised to predict hospital mortality outcomes and been incorporated into laboratory score systems [44–46]. We must however distinguish a population based interpretation of the risk relating a predictor variable that is disturbed to mortality outcomes in general from that at the extremes of population or in certain specific high-risk subgroups, and as to whether patients die with or as a consequence of hyponatraemia [47] or if most deaths are caused by underlying diseases [42]. The widely quoted work of Gheorghiade et al. demonstrated a marked correlation between serum sodium < 135 mEq/l and both hospital LOS and mortality and an apparent “U shaped’ curve with increased mortality if serum sodium was greater than 140 mEq/l [16]. However, a recent large meta-analysis on dysnatraemia concluded that hyponatremia (HR 1.34; 95% CI: 1.15–1.57) but not hypernatremia (HR 1.12; 95%: CI 0.93–1.34), was independently associated with increased risk of all-cause mortality, when compared than the normonatremia category [48].

In respect of potassium levels and mortality outcomes, in 6515 patients prior to randomization in the MERLIN-TIMI trial, the lowest risk of cardiovascular death was observed in patients with admission potassium levels between 3.5 and 4.5 mEq/L. Both lower and higher levels of potassium were associated with tachyarrhythmias and bradyarrhythmias, suggesting a potential mechanistic explanation for the increased risk of cardiovascular death at the extremes of potassium homeostasis [49]. Krogager et al. [50] reported, in a cohort of nearly 2600 patients post myocardial infarction, a U-shaped relationship between serum potassium concentration on hospitalization and 90-day mortality; these observations of increased mortality also applied to low and high normal serum potassium levels. Hughes-Austin et al. [51]  found that high serum potassium concentration is significantly associated with a higher risk for all-cause mortality independent of kidney function or other cardiovascular disease risk factors; no significant association between hypokalaemia and mortality was found in the fully adjusted Cox model. We found that from lower to higher deciles of potassium a progressively rising risk of an adverse mortality outcome. Similarly, Loprinzi and Hall [12] investigated the potassium-mortality relationship in the general population of the United States, using data from the 1999–2006 National Health and Nutrition Examination Survey were employed, with follow-up through 2011. In a Cox proportional hazards model, after adjusting for confounding variables found that a 1-mmol/L increase in potassium was associated with a 40% increased risk of mortality (adjusted hazard ratio 1.40; 95% CI, 1.15–1.70).

Broadly speaking then we would conclude, that in an unselected population admitted with medical emergencies over 16 years that although disturbances of potassium or sodium were predictive of 30-day mortality outcomes in general, the lowest decile of admission potassium or the highest decile of sodium conferred the lowest risk, when adjusted for other major independent outcome predictors. Over all deciles of admitted potassium, rising levels were positive associated with increased mortality; the contrary applied to sodium where the greatest risk was for the lowest admitted levels, and all higher deciles demonstrated a falling mortality trend. Furthermore, although both rising potassium levels and falling sodium levels were independently predictive, in the full model with adjustment as described, the influence of each of the combined ratio was attenuated This implies that disorders of potassium and sodium and their ratio is a secondary phenomenon, reflecting system disorder and a ‘sick cell’ or unbalanced homeostasis due to major illness associated with comorbidity.

Our study utilises comprehensive data gathered over a prolonged period of time, however this information is in essence extracted from the medical record through coding and other standard procedures; therefore the robustness of our data is entirely dependent on the accuracy of the original recordings. The external validity of our results will need to be established by complementary studies in other institutions and settings. We do not collect information on the specific treatment of individual patients, therefore we were not able to analyse any potential effects of interventions in those with abnormalities of potassium or sodium. Finally, the design of our study by its nature assesses for associations rather than causation, while we adjust for multiple variables in our analyses we cannot comment on any potential causative role for electrolyte imbalance on mortality.

In conclusion this study demonstrates that the ratio of admission serum potassium to sodium is both strongly associated with age and linearly predictive of 30-day hospital mortality. The interpretation of sodium and potassium homeostasis as an interdependent ratio did not add significant material value to analysing the cations independently.

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Endothelial Cell Growth Promoting Activity in Graves’ Disease Sera is Neutralized by Anti-Basic Fibroblast Growth Factor Antibodies in Patients with Fat Expansive but Not Infiltrative Orbitopathy

DOI: 10.31038/EDMJ.2019313

Abstract

Purpose: To report a case of orbital fat expansion leading to globe prolapse in a Graves’ disease patient undergoing high-dose glucocorticoid therapy. To evaluate the growth factor receptor specificities of plasma autoantibodies in Graves’ disease patients who exhibited contrasting subtypes of thyroid-associated ophthalmopathy, i.e. orbital fat expansion-type vs. infiltrative.

Methods: Sera from Graves’ orbitopathy and control patients with or without Graves’ disease were subjected to protein-A affinity chromatography to obtain immunoglobulin G. A (1/50th to 1/1600th) range in dilutions of the protein-A eluate fraction was incubated for four days at 37 degrees C with bovine pulmonary artery endothelial cells to test for endothelial cell inhibition or stimulation. Growth stimulatory autoantibodies were co-incubated with specific neutralizing anti-insulin like growth factor 1 receptor antibodies or anti-basic fibroblast growth factor antibodies to assess autoantibody specificity in contrasting Graves’ orbitopathy subtypes.

Results: We observed increased mean endothelial cell growth promoting activity in the protein-A eluates of serum from eighteen patients with active Graves’ disease (117 ± 28%, n = 18) compared to mean endothelial cell activity (89 ± 10%, n = 13, P = 0.003) in thirteen adults without Graves’ disease. The protein-A eluate fraction in acute infiltrative-type Graves’ orbitopathy contained a high titer (> 1: 1000) of endothelial cell stimulatory activity which was significantly neutralized by specific monoclonal anti-human insulin-like growth factor 1 receptor antibodies. The protein-A eluate fraction in fat expansion-type Graves’ orbitopathy contained endothelial cell inhibitory activity (at low titers) and stimulatory activity (at high titers), and the latter stimulatory activity was completely neutralized by specific anti-basic fibroblast growth factor antibodies.

Conclusion: Graves’ disease suffering globe prolapse secondary to marked orbital fat-expansion had coexisting plasma fibroblast growth factor-inhibitory and -stimulatory autoantibodies. The latter was completely neutralized by anti-basic fibroblast growth factor antibodies.

Keywords

Graves’ disease, thyroid associated ophthalmopathy, autoantibodies, fibroblast growth factor

Introduction

Thyroid-associated ophthalmopathy (TAO) is a vision-threatening complication of Graves’ disease (GD) which can cause significant morbidity and impaired quality of life [1]. Extraocular muscle hypertrophy and local inflammation contribute to a severe infiltrative form of the disease whose underlying pathophysiology has been the focus of recent investigations [2]. Evidence suggests that increased local orbital glycosaminoglycan production (a hallmark in infiltrative- type Graves’ orbitopathy) results (in part) from circulating agonist insulin- like growth factor 1 receptor autoantibodies [2] and increased expression of insulin-like growth factor 1 receptor (IGF- 1R) in orbital fibroblasts [3] and in T and B lymphocytes [4].

Orbital fat expansion can accompany Graves’ extraocular muscle hypertrophy, but its underlying pathophysiology is less clearly defined. We report increased mean endothelial cell growth promoting activity in the protein-A eluates of serum from active Graves’ disease compared to adult controls without GD. We also report a patient with type 2 diabetes mellitus, active Graves’ disease, and focal segmental glomerulosclerosis (FSGS) who experienced acute globe prolapse secondary to severe orbital fat expansion in the setting of high-dose glucocorticoid therapy. The patient’s orbitopathy was characterized by a lack of extra-ocular muscle enlargement and his plasma contained novel growth stimulatory endothelial cell plasma autoantibodies whose activity (in vitro) was completely neutralized by specific anti-bovine basic fibroblast growth factor (FGF) antibodies. These data suggest a possible role for circulating fibroblast growth factor-like autoantibodies in rare cases of Grave’s orbitopathy characterized by pure orbital fat expansion.

Participants and Methods

Participants

Graves’ disease (GD) Pt 1: A 45-year-old man with hyperthyroidism who presented with infiltrative-type orbitopathy, diplopia and a triiodothyronine (T3) level of 663 ng/dL (59- 174). He was treated with radioactive iodine (RAI) ablation, methylprednisolone and underwent orbital decompression surgery. Thyroid stimulating immunoglobulin was elevated at 194% (0–130) and anti-microsomal antibody was 4128 IU/mL (0–34).

Graves’ disease (GD) Pt 2: A 53-year-old man with chronic Graves’ disease (treated with tapazole), type 2 diabetes mellitus, macular edema, nephropathy, fat expansion-type orbitopathy and idiopathic focal segmental glomerulosclerosis (FSGS) for which the patient was treated with high- dose glucocorticoids. Thyroid stimulating immunoglobulin was elevated at 199% (0–130) and anti-microsomal antibody was 2367 IU/mL (0–34). During steroid taper, the patient experienced prolapse of the right globe while driving his car. Magnetic resonance imaging of the orbits revealed markedly increased retrobulbar fat bilaterally with resulting proptosis. The extraocular muscles and optic nerves were normal. Doppler ultrasound of the thyroid revealed a‘thyroid inferno pattern’ suggestive of intrathyroidal angiogenesis. The patient progressed to end-stage-renal disease requiring dialysis and died suddenly of unknown causes six years later. Patient 3: A 72-year-old man with Graves’ disease treated with methimazole. He experienced progression in orbitopathy (over a three-year period) characterized by marked enlargement in extra-ocular muscles and moderate increase in retrobulbar fat.

Patient 4: A 64-year-old man with type 2 diabetes, macular edema, and nephropathy who experienced bilateral supero-temporal orbital fat prolapse (left> right) within 9 months of receiving high-dose oral prednisone followed by intra-tympanic injection of solumedrol to treat autoimmune sensorineural hearing loss.

Methods

Blood drawing/Protein-A Chromatography

Informed consent for the local Institutional Review Board-approved study was obtained from each participant prior to blood drawing which was performed in the morning. Protein-A affinity chromatography of serum was carried out as previously reported [5].

Cell culture

Bovine pulmonary artery (BPA) endothelial cells (EC) (Clonetics, Inc. San Diego, CA, USA) were maintained at 37°C in 5% CO2/95% air in Medium 199 (M199) plus 10% fetal calf serum (FCS). BPA endothelial cells were passaged continuously and used between passages 4 and 7. Bovine pulmonary artery endothelial cells are exquisitely sensitive to low concentrations of basic fibroblast growth factor and are an optimal test system for the detection of fibroblast growth factor-like inhibitory and -stimulatory substances [5].

Endothelial cell proliferation assays

Endothelial cell number assays were carried out as previously reported [5]. Confluent cells were trypsinized and plated at 1 to 10 × 103 cells/well in Medium 199 plus 10% fetal calf serum in 96- well plates. After 1 or 2 days incubation for cells to reach 60–80% confluency, test fractions (30 μg/mL of the protein- A eluates of serum) were added to wells in quadruplicate. After 4 days’ incubation in the presence of test fractions, cells were washed with phosphate- buffered saline and colorimetric estimation of cell number was determined as previously described [5]. Each point represents the mean of quadruplicate determinations.

Antibodies

Purified mouse monoclonal anti-human insulin-like growth factor-1 receptor antibodies were obtained from BD Biosciences (San Jose, CA). Polyclonal rabbit, anti-bovine basic fibroblast growth factor antibodies were obtained from R & D Systems, Inc. (Minneapolis, MN).

Protein determinations

Protein concentrations were determined by a bicinchoninic acid protein assay kit (Pierce Chemical Co., Rockford, IL, USA).

Results

Graves’ disease autoantibodies induce endothelial cell proliferation

We observed increased mean endothelial cell growth promoting activity in the protein-A eluates of serum from eighteen patients with active Graves’ disease (117 ± 28%, n = 18) compared to mean endothelial cell activity (89 ± 10%, n = 13, P = 0.003) in an identical 30 µg/mL concentration of the protein-A eluate in thirteen adults without Graves’ disease (including six normal participants, three patients with rheumatoid arthritis, and one patient each with polycystic ovarian syndrome, systemic lupus erythematosus, sarcoidosis or Sjogrens’ syndrome) (Fig 1). Graves’ disease patients did not differ significantly in their mean age, or male: female ratio from the control group of 13 participants (Table 1). Mean endothelial cell activity (30 µg/mL concentration of the protein-A eluate) did not differ significantly in Graves orbitopathy (124 ± 23%, N = 9) compared to Graves without orbitopathy (115 ± 32%, N = 9, P = 0.53). The mean thyroid stimulating hormone, thyroid stimulating immunoglobulin, anti-thyroid peroxidase antibody, or anti-thyroglobulin antibody levels did not differ significantly in active Graves’ disease patients affected (GO+, N = 9) or not affected by orbitopathy (GO-, N = 9) (Table 1).

EDMJ - Mark B Zimering-F1

Figure 1. Endothelial cell growth-promoting activity in a one-fiftieth dilution (30 µg/mL) of the protein-A eluate fraction of serum from active Graves’ disease (inverted, open diamonds) and control patients without Graves or thyroid disease (closed diamonds). Mean endothelial cell growth promoting activity in GD sera significantly exceeded activity in control sera. Bovine endothelial cells were incubated for four days in the presence of test fractions and % of basal activity was determined as described in Methods.

Table 1. Baseline characteristics in Graves’ disease patients and control participants.

Risk factor

Graves’ disease (N=18)

Systemic autoimmunity and normals (N=13)

P-value*

Age

46.4(13.0)

40.9(11.2)

0.25

Gender (M/F)

7/11

5/8

1.0**

GO+(N=9) GO- (N=9)

TSH (μIU/mL)

2.28(3.19) 0.12(0.10)

NT

0.09

TSI AB (%)

216 (91) 241(110)

NT

0.65

Anti-TPO AB (IU/mL)

1374(1449) 726(778)

NT

0.32

Anti-TG AB (IU/mL)

42(50) 97(111)

NT

0.35

Results are mean (SD); GO Graves orbitopathy, + present, -absent; M-male; F-female

TSH- thyroid stimulating hormone; TSI- thyroid stimulating immunoglobulin; TPO- thyroid peroxidase; TG- thyroglobulin; AB-antibody.

^ N = 6 normal participants, 3 rheumatoid arthritis, 1 systemic lupus erythematosus, 1 sarcoidosis, 1 polycystic ovarian syndrome, 1 Sjogren’s syndrome patient.

*T-test; ** Fischer’s exact test

Lack of association between EC activity in GD protein-A eluates and thyroid autoantibodies

There was no significant correlation between thyroid stimulating immunoglobulin (TSI) level and endothelial cell growth activity in the protein-A eluates of all eighteen active GD sera (Fig 2A). There was no significant correlation between endothelial cell growth activity in the protein-A eluates of GD sera and either anti-thyroglobulin antibody (n = 13) or anti-thyroid peroxidase antibody (n = 15) level (Fig 2B, 2C). We next sought to characterize the endothelial cell growth stimulatory activity in the protein-A eluates of a subset of Graves’ disease orbitopathy patients presenting with contrasting subtypes of ophthalmopathy, i.e. infiltrative vs. fat- expansion type.

EDMJ - Mark B Zimering-F2

Figure 2. Correlation between endothelial cell growth promoting activity in the protein-A eluate of Graves’ disease serum and level of A) thyroid stimulating immunoglobulin, B) anti-thyroid peroxidase antibody or C) anti-thyroglobulin antibody. R2= [correlation coefficient].2

Endothelial cell activity in protein-A eluates of Graves’ disease having infiltrative vs. fat- expansion type orbitopathy

Active Graves’ disease manifesting with moderately-severe acute infiltrative TAO, i.e. GD1, was characterized by extraocular muscle enlargement with subtle fatty degeneration in the lateral rectus muscle (Fig 3A). The GD1 protein-A eluate fraction contained a high titer of endothelial cell stimulatory activity (dashed black line, Fig 3C). Active Graves’ disease presenting with globe prolapse, i.e. GD2, was characterized by severe orbital fat expansion (Fig 3B). The GD2 protein- A eluate contained a low-titer of endothelial cell inhibitory activity together with higher titer endothelial cell stimulatory IgG activity (GD Pt 2, dashed gray line, Fig 3C).

Patients who had experienced either long-standing infiltrative-type TAO (Pt 3) or orbital fat prolapse after receiving high-dose glucocorticoid therapy (Pt 4) were included as controls for the results in GD1 and GD2. The Pt 3, infiltrative-type TAO GD protein- A eluate contained a monophasic low titer of endothelial cell growth stimulatory activity (solid green line, Fig 3C), similar to GD1. The Pt 4, diabetic, orbital fat prolapse, protein- A eluate elicited biphasic endothelial cell- inhibitory and -stimulatory responses at low and higher titers, respectively (solid blue line, Fig 3C), similar to GD2.

A

EDMJ - Mark B Zimering-F3a

B

EDMJ - Mark B Zimering-F3b

C

EDMJ - Mark B Zimering-F3c

Figure 3. Contrasting infiltrative (A) and fat expansion (B) subtypes of thyroid associated ophthalmopathy and (C) corresponding dose-endothelial cell proliferation curves in protein-A eluates from patients having predominantly muscular hypertrophy or orbital fat expansion.

A) Computed tomography scan of the orbits (GD, Patient 1) showed bilateral proptosis, enlargement of the extraocular muscles sparing the tendons associated with fatty
degen­eration consistent with the clinical diagnosis of Graves ophthalmopathy. Optic nerves are within normal limits. B) Magnetic resonance imaging of the orbits (GD, Patient 2) revealed markedly increased retrobulbar fat bilaterally with resulting proptosis. The extraocular muscles and optic nerves were normal. Fat expansion caused complete loss of normal architecture of the orbit which together with shallow orbits predisposed to globe prolapse. C) Dose-endothelial cell proliferation curves of protein-A eluates in representative patients with Graves’ disease having infiltrative-type orbitopathy (GD 1), retrobulbar fat expansion-type (GD2), or mixed infiltrative and fat expansion type orbitopathy (Pt 3); and in a diabetic nephropathy patient who experienced bilateral retrobulbar fat enlargement and orbital fat prolapse (Pt 4). Each point represents the mean of quadruplicate determinations which varied by 5–15%. DM-diabetes mellitus, GD- Graves disease, TAO-thyroid associated ophthalmopathy, expans(ion).

Neutralization of EC bioactivity in GD with acute infiltrative TAO sera by anti-IGF1 receptor antibodies

Peak endothelial cell stimulatory activity in the protein-A eluate from GD1 infiltrative-type TAO patient was significantly neutralized (P< 0.01)) (~67%) by co-incubation endothelial cells with a 5 µg/mL concentration of monoclonal anti-human insulin-like growth factor 1 receptor (IGF1R) antibodies (e.g. Pt 1; Fig 4A). Seventy to one-hundred percent neutralization of endothelial cell activity (mean 123%) was observed in the protein -A eluates in two additional GD patients (one each having mild GD orbitopathy or no orbitopathy) following co-incubation with (5 µg/mL) monoclonal anti- human IGF1R antibodies (data not shown in Fig 4). The anti-IGF1R antibodies alone had no significant effect on EC proliferation (Fig 4A). Endothelial cell stimulatory activity in the protein- A eluate of GD1 was not significantly affected by co-incubation with a 5 µg/mL concentration of specific anti-bovine basic fibroblast growth factor (bFGF) antibodies (Fig 4B).

A

EDMJ - Mark B Zimering-F4a

B

EDMJ - Mark B Zimering-F4b

Figure 4. Neutralization of GD1 infiltrative-type ophthalmopathy autoantibodies by specific insulin-like growth factor 1 receptor antibodies.

Neutralization of endothelial cell proliferative activity in A) GD1 protein-A eluate fraction by specific anti-insulin like growth factor 1 receptor antibodies. Similar results were observed in three Graves’ disease patients tested. B) Lack of effect of specific anti-bFGF antibodies on endothelial cell growth promotion by Graves’ disease protein-A eluate autoantibodies. A, B) Ten micrograms/mL concentrations of the protein-A eluate from Pt 1, Graves’ disease serum was incubated in the presence (black bar) or absence (open bar) of 5 µg/mL concentrations of anti- IGF1R antibodies (A) or 5 µg/mL concentrations of anti-bFGF antibodies (B). Each point represents the mean (+ SD) of quadruplicate determinations as described in Methods. AA-autoantibodies, IGF1R-insulin-like growth factor 1 receptor, GD1- Graves’ disease patient 1, AB-antibodies, AntibFGF AB- anti-basic fibroblast growth factor antibodies.

Neutralization of EC bioactivity in GD with retrobulbar fat expansion TAO sera by specific anti- bFGF antibodies

Peak EC stimulatory activity in the protein-A eluate from GD Patient 2 suffering globe prolapse was completely neutralized by co-incubation with 5 µg/mL anti-bovine basic fibroblast growth factor antibodies (Fig 5A). The anti-bFGF antibodies alone had no significant effect on endothelial cell proliferation (Fig 5A). Co-incubation with a 5 µg/mL concentration of monoclonal anti-human insulin-like growth factor 1 receptor antibodies had no significant effect on endothelial stimulatory activity in the GD Patient 2 protein-A eluate fraction (data not shown in Fig 5). Peak EC stimulatory activity in the protein- A eluate fraction in Pt 4 suffering orbital fat prolapse was completely neutralized following co-incubation with 5 µg/mL specific anti- bFGF antibodies (Fig 5B).

A

EDMJ - Mark B Zimering-F5a

B

EDMJ - Mark B Zimering-F5b

Figure 5. Neutralization of fat-expansion type ophthalmopathy autoantibodies by anti-basic fibroblast growth factor antibodies.

Neutralization of endothelial cell proliferative activity in A) GD2 protein- A eluate fraction or B) Pt 4 diabetic nephropathy, orbital fat prolapse patient’s protein- A eluate fraction by specific anti-bFGF antibodies. B) Two micrograms/mL concentrations of the protein-A eluate from GD, Pt 2 or Pt 4 serum was incubated in the presence (black bar) or absence (open bar) of 5 µg/mL concentrations of specific anti-bovine bFGF antibodies as described in Methods. Complete neutralization of endothelial cell growth stimulatory activity was observed in the protein-A eluates from GD2 and Pt 4 diabetic nephropathy patient suffering with idiopathic retrobulbar fat expansion and fat prolapse. Each point represents the mean (±SD) of quadruplicate determinations. GD2-Graves’ disease, Patient 2, Diab Neph(ropathy), OFP AA- orbital fat prolapse autoantibodies.

Discussion

The present data are the first to suggest increased circulating endothelial cell stimulatory autoantibodies in active Graves’ disease. Angiogenesis is a characteristic feature of the thyroid gland in Graves’ disease as evidenced by a characteristic “thyroid inferno” color Doppler flow sonography pattern [6] in Graves’ disease patient 2 in the present study. Goiter formation in the rat was accompanied by increased basic fibroblast growth factor expression in the thyroid follicular cells, stroma and endothelial cells as well as neovascularization [7]. Basic fibroblast growth factor (basic FGF) is a highly potent autocrine endothelial cell mitogen which lacks an amino terminal signal sequence needed for efficient secretion from cells [8]. Basic fibroblast growth factor does not normally circulate in healthy, nonpregnant adults [9, 10], however, plasma basic fibroblast growth factor immunoreactivity was increased in certain pituitary tumors, e.g. sporadic acromegaly [9] and in micro- or albuminuric diabetic nephropathy [11]. The occurrence of fibroblast growth factor-like autoantibodies in subsets of sporadic acromegaly [12] and in diabetic nephropathy [13] suggested a role for elevated insulin-like growth factor 1 [14] or chronic inflammation in enhanced autoantibody production [14] to circulating basic fibroblast growth factor or its receptors. The present data are the first to suggest that fibroblast growth factor-like autoantibodies in diabetic nephropathy suffering with co-morbid Graves’ disease or a different systemic autoimmune disease may be a driver of orbital fat expansion-type orbitopathy.

Prior reports suggested a possible local role for angiogenic factors such as basic fibroblast growth in promoting Graves’ orbitopathy. For example, basic fibroblast growth factor expression was increased in the orbital fibroblasts and adipocytes of patients suffering with severe Graves’ orbitopathy [15]. In addition, basic FGF acted synergistically with platelet derived growth factor in promoting orbital fibroblast production of hyaluronan and interleukin-6 [16]. Macrophages present in chronically- inflamed Graves’ orbital tissues [15] express a number of different angiogenic factors including basic FGF and insulin-like growth factor 1 [17]. Increased expression of insulin-like growth factor 1 receptor on T and B lymphocytes reported in Graves’ disease [4] may provide a mechanism for enhanced systemic autoimmunity to locally- or systemically-elaborated basic FGF via activation of the insulin-like growth factor 1 receptor on immune cells by subsets of circulating Graves’ disease immunoglobulins [18]. Although endothelial cell stimulatory autoantibody activity was not preferentially increased in a subset of Graves’ orbitopathy vs. Graves’ without orbitopathy in the present study, the sample sizes were small precluding a definitive conclusion.

In acute severe infiltrative Graves’ orbitopathy (GD patient 1), autoantibody-mediated endothelial cell stimulatory activity was neutralized (~67%) by specific anti-insulin-like growth factor 1 receptor antibodies, but was unaffected by specific anti-basic fibroblast growth factor antibodies. The autoantibody-mediated endothelial cell stimulatory activity in two of two additional newly-diagnosed Graves’ disease sera tested was also substantially or completely blocked by specific anti-insulin-like growth factor antibodies suggesting that fibroblast growth factor-like autoantibodies are not likely to play a significant role in early, active Graves’ disease patients lacking co-morbid diabetic nephropathy. Endothelial cell inhibitory autoantibody activity was largely absent in the protein-A eluates in seventeen of eighteen active Graves’ disease patients (Fig 1) consistent with a prior report of a low prevalence of anti-endothelial cell autoantibodies in thyroid autoimmunity patients having suppressed TSH [19]. Anti-endothelial cell autoantibodies were reported to occur at a (four-fold) significantly higher prevalence in thyroid autoimmunity patients having elevated TSH [19]. This may explain in part an increased occurrence of low-titer anti-endothelial cell autoantibodies in previously-treated, chronic Graves’ disease patients compared to newly-diagnosed Graves’ disease. In subsets of diabetic macular edema [20] and/or diabetic nephropathy [13] anti-endothelial cell autoantibodies targeted heparan sulfate proteoglycan, the low affinity FGF receptor, which is required for FGF-mediated endothelial cell survival and proliferation [21].

A possible dual role for endothelial cell inhibitory and fibroblast growth factor like stimulatory autoantibodies in promoting orbital fat expansion (in the setting of high-dose glucocorticoid therapy) is suggested by several observations. First, abrogation of basic fibroblast growth factor signaling (via knockout of the FGF2 gene in mice), promoted differentiation of bone marrow stromal cells into adipocytes [22]. Second, dexamethasone is known to promote adipocyte differentiation in pluripotent mesenchymal stem cells which have the potential to develop into osteoblasts or fat cells [23]. Third, basic fibroblast growth factor is a mitogen abundantly expressed in osteoblasts [24] and in preadipocytes [25], and in the latter cell type, basic fibroblast growth factor prevented adipocyte differentiation [25]. Taken together, fibroblast growth factor-like autoantibodies might drive expansion in a pool of precursor cells which later differentiate into fat cells under the dual influences of high-dose glucocorticoids and FGF- inhibitory autoantibodies. This proposed mechanism requires spatially- and temporally- independent actions by FGF-inhibitory and -stimulatory autoantibodies. Since FGF-like stimulatory, but not inhibitory autoantibodies displayed uniquely high affinity for hydroxyapatite which is a normal constituent of bone matrix [5] stimulatory autoantibodies might localize to a different region in the orbital- retro-orbital connective tissue space than soluble FGF-inhibitory autoantibodies.

The etiology of muscle enlargement in thyroid-associated ophthalmopathy is still a subject of debate with evidence supporting involvement of either TSH receptor autoantibodies and/or insulin-like growth factor 1 receptor autoantibodies [18]. A ten nanomolar concentration of insulin-like growth factor 1 was reported to cause an 80% increase in basal DNA synthesis in microvascular endothelial cells [26] and phosphorylation of the insulin-like growth factor 1 receptor in macrovascular endothelial cells [27]. Thus a subset of Graves’ disease IgG autoantibodies having specificity for the insulin-like growth factor 1 receptor may have accounted (in part) for the significant endothelial cell proliferation we observed here in a subset of newly-diagnosed Graves’ disease patients with infiltrative-type orbitopathy.

In summary, these data are the first to suggest a possible association between fibroblast growth factor-like stimulatory autoantibodies and subsets of orbital fat-expansion/fat prolapse in patients with Graves’ disease, diabetes and focal segmental glomerulosclerosis or in diabetes having a different systemic autoimmune co-morbid condition which also required treatment with high-dose glucocorticoids. More study is needed to determine whether coexisting endothelial cell inhibitory and stimulatory autoantibodies in Graves’ disease serum may be a useful biomarker for an increased risk of fat expansion- type orbitopathy following high-dose glucocorticoid therapy.

Acknowledgement

Presented in part at the 92nd Annual Meeting of the Endocrine Society, Boston, MA. June 4, 2011. We thank Terry J. Smith, MD for his valuable contributions to an earlier draft of the manuscript.

The authors report no multiplicity of interest affecting the objectivity of the presented findings. Supported in part by a grant from the Veterans Biomedical Research Institute, East Orange, New Jersey, USA to MBZ.

Abbreviations

BPA – Bovine Pulmonary Artery

EC – Endothelial Cells

FCS – Fetal Calf Serum

FGF2 – Fibroblast Growth Factor 2

bFGF – basic Fibroblast Growth Factor

FSGS – Focal Segmental Glomerulosclerosis

GD – Graves’ Disease

IGF1 – Insulin–Like Growth Factor 1

IGF1R – Insulin–Like Growth Factor 1 Receptor

M199 – Medium 199

TAO – Thyroid Associated Ophthalmopathy

T3 – Triiodothyronine IgG-Immunoglobulin G

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Best and Effective Practices of Wound Care and Healing Among Patients with Diabetes Mellitus

DOI: 10.31038/EDMJ.2019312

Abstract

Aim: With the continuing trend in the incidence of diabetic ulcer, current researches from evidence-based practice augment best and effective practices on wound care and healing among patients suffering from diabetes mellitus.

Methods: This study utilized a systematic review of literatures in light of wound care through Pub Med database with the key words “Diabetes Mellitus,” “wound practices,” “wound healing,” and “systematic review.” The searched literatures were of case reports, interventional studies, and review papers whose main texts were in English.

Results: There were 7,988 published articles yielded from the keywords used and 16 were included in this study. The practiced management and techniques in these literatures focused on comfortable and cost efficient means of wound care and healing with the involvement of group participation than individual plan of care.

Conclusions: There are meager evidences for vigorous wound and healing options that needs further studies in resolving wound issues among patients with Diabetes Mellitus. This systematic review establishes the insufficiency of high level of evidence based studies on wound healing in DM patients and brings a track for continuity of rigid studies concerning this topic.

Implications for Nursing Practice and/or Health Policy: Understanding innovative wound care is the chance for nursing professionals to associate evidence, expert opinion and patients’ preference for a best and effective practices of wound care and healing and thus eventually make them a step above their skills. The evolution of different treatment regimens like hyperbaric O2 therapy, TCOT device, natural latex associated with the LED circuit, low-level laser therapy, PRP treatment, salt-based spray, Telemedicine follow up, cord platelet gel application, Human Reticular Acellular Dermal Matrix, and acellular reticular allogenic human dermis will not only aid in the management of wound care and healing but also in strengthening the comprehensive and practical skills among nurses.

Keywords

Diabetes Mellitus, Systematic Review, Wound Care and Practices, Wound Healing

Introduction

Wounds are commonly categorized as chronic or acute and could rise from any traumatic damage or through a collapse of an unharmed skin [1]. Healthy people have strong skin integrity with a significant ability for healing but can be exposed to outer and inner harm brought about by older age and changed in physiology [2]. This can be intrinsic or extrinsic in nature. In people suffering from diabetes, healing of lesions is slow but progresses faster. It is therefore essential to understand diabetes and appropriate wound care along with the complexities of wound management and understanding for a better life and longevity. The incidence of diabetes is escalating faster in the countries of middle and low revenues and in 2014; a global data of 8.5% came from people aging 18 years old and above [3]. But the occurrence of diabetic foot ulcers is within 4% and 10% with 1:4 threat of having diabetic foot ulcer in their life [4].

Although minor injuries like burns, abrasions and lesions are normal accidents; these can bring severe medical concerns to people with diabetes [5]. Local infection can extend fast to the internal parts of the body and can be fatal. Studies suggests that curing of wounds is affected by: weakening in producing hormones related with development and therapy, decreasing in producing and repairing of new blood vessels, weakening of skin protection, and decreasing in the production of collagen [5]. There are recent studies that can help in bringing effective management of wound care among patients with diabetes. These are helpful practices that can save limbs for amputation.

This review is an investigation on the efficacy of different practices of healthcare professionals in the conduct of wound care and healing among patients suffering from diabetes. It has unveiled comfortable management of diabetic wounds as well as hopes to patients with DM to salvage their limbs. It is with the same reason why the author conducted this review. Results stated different practices and effects and there is a solid reason for more evidence-based studies that will heighten the rate of wound healing and introduce patients with DM easy to carry out wound care and practices. This study commenced to explore the different practices in wound care for patients with diabetes using systematic review of published evidence-based studies on wound management in diabetes.

Methods

The research composition of this systematic review to unveil different practices on wound care and healing among patients with diabetes mellitus. Pub Med database was searched for relevant literatures through keywords “Diabetes Mellitus,” “wound practices,” “wound healing,” and “systematic review.” It was piloted on November 1, 2018 and focused the search on case reports, interventional studies, and review papers written in English language. The focus of the search is practices and wound care that would lead to wound healing. Yielded published articles were systematically evaluated based on the country of the author, year the article is published, site of the study, method and design of the study, management team, and the results. Prior synthesis, data is extracted via two reviewers and reviewed for accuracy by another reviewer. Inconsistencies are being identified for illegibility and consensus while recognizing ineligibles based on the criteria.

Results

With the use of keywords yielded 7,988 published articles on wound management among patients with DM but only 16 fulfilled the criteria set in this study for review. Relevant data is summarized in (Table 1).

Table 1.Physiognomies of Selected Studies: Best and Effective Practices of Wound Care and Healing Among Diabetic Patients.

Author; year published; title of the study

Sample; wound type

Study design

Team composition

Intervention and period of coverage

Primary Outcome

Major results

Chang (2018)

274, Diabetic foot

Single-center retrospective study

Surgeon

Patient evaluation, wound preparation, improving vascularity, surgery and dressing, and rehabilitation

2005–20017

Infection and arterial occlusion

Improved vascularity

Chen et al. (2017)

38, Chronic diabetic foot ulcer

Randomized controlled trial

Doctor, nurse

Intervention group receives standard care and HBOT while control group receives standard care only.

20 days

Wound closure

Improved wound healing

Crews & Candela (2018)

25, Diabetic foot ulcer

Randomized controlled trial

Doctor, PT

Patients at risk for DFU’s with removable cast walkers in 20 mins walking trials using 5 footwear conditions

Bilateral shoes brought better comfort

Ankle-high removable cast walker combined with contralateral limb lift increases offloading and improve healing

Driver (2017)

130, Foot ulcer

A prospective, randomized, blinded, multicenter, parallel study

Not mentioned

TCOT device given to intervention groups following assessment and standard care while the control group received the sham device following standard care.

12 weeks treatment

Good wound results in both groups

Beneficial to older population

Eraydin & Avsar (2018)

65, Diabetic foot ulcer

Randomized control trial

Nurses

Intervention group given standard wound care and foot exercises for 12 weeks while the control group had standard wound care with no exercise

12 weeks

Difference in the ulcer areas in the two groups

Significant decrease of ulcer area in the intervention group.

Health Quality Ontario (2017)

8; Ulcer

7 randomized controlled trials, 1 nonrandomized controlled trial

Experts, end users, and applicants

Review and assessment of studies concerning efficacy and cost effectiveness of HBOT utilizing the standpoint of the Ministry of Health and Long-Term Care and assessed the clinical evidence with the use of GRADE.

Mixed results on standard of care in rates of amputation while there is impact on standard care and HBOT and no difference on the adverse events on both.

Satisfaction on patients using HBOT and positive perceptions on healing

Lopez-Delis (2018)

15, Lower limb ulcer

Randomized controlled trial

Nurses, patients

Group 1: recipients of dressing system adhesive of the natural latex associated with the LED circuit.

Group 2: recipients of dressing at home with calcium alginate or silver foam

Group 3: recipients of dressing at home with adhesive derived from the natural latex associated with the LED circuit.

1 month

Significant decrease in ROS formation

Latex and phototherapy brought better debridement and healing process

Mathur et al. (2017)

30, Grade 1 foot ulcer

Randomized clinical trial

Doctors, Nurses

Intervention group received low-level laser therapy with conventional therapy while control group only received conventional therapy

6 weeks

absolute and relative wound size reduction at 2 weeks compared to the baseline parameter.

results suggest that LLLT is beneficial as an adjunct to conventional therapy in the treatment of diabetic foot ulcers.

Mohammadi,et al. (2017)

100 Diabetic foot ulcer

A single-arm clinical trial

Doctor,

nurses

Weekly PRP treatment following primary wound care

Significant decrease of the area of wound

Platelet-rich plasma gel as treatment for non-healing DFU

Park et al. (2018)

167 Diabetic foot ulcer

A phase III multicenter, double-blind, randomized, placebo-controlled trial

nurses

Routine wound care and topical or spray of saline with 0.005% rhEGF (n=82) or (n=85) twice a day

12 weeks

Comparable results between placebo groups and the rhGF groups

Faster healing velocity and higher complete healing rate regardless of HbA1c levels.

Pougatsch (2017)

10, Diabetic foot ulcer

Prospective, case-cohort study,

Pilot study

Not mentioned

Used acceptable methods of cleansing and the use of salt-based spray then gauze sponges soaked with the spray is used to dress the ulcer and then covered with dry foam or gauze and wrap with ACE.

12 weeks

Wound closure

Viable natural wound care therapy

Santema et al. (2018)

120 Ischemic wound

DAMO2CLES multicenter randomized clinical trial

Not specified

40 sessions of HBOT was used for five days weekly or till complete wound healing was reached

One month

Limb salvage and

wound healing

Freedom from any amputation

Smith-Strøm et al. (2018)

165, Diabetic foot ulcer

cluster-randomized controlled non-inferiority trial

Doctors,

Healthcare personnel,

Community nurses

Intervention group utilized TM follow-up care in the community while the control group received SOC.

2012–2016

Faster Healing time

Technology as relevant alternative to wound care

Volpe et al. (2017)

20 Diabetic wound

Non-blinded, consecutive series, randomized clinical trial

Nurses

Surgeons

Group A is managed with standard wound care while Group B is managed with topical application of CBPG consisted of platelet gel application 2 times a week in 4 weeks and then once a week for an additional 4 weeks.

2 months

Ulcer reduction

Rapid healing than standard technique

Zelen et al. (2017)

20, Diabetic foot

A retrospective crossover study

Not mentioned

Indolent DFUs are given acellular matrices

12 weeks

12 out of 20 were eligible for crossover treatment

Complete healing achieved

Zelen et al. (2017)

40, Chronic diabetic ulcer

A prospective, randomized, controlled, multi-centre

clinical study

Doctor

Nurse

SOC group receives dressing change daily combined with collagen alginate while

HR-ADM group received grafts.

12 weeks

Healing proportion of wounds in 6 weeks.

Clinical superiority of HR-ADM and considered cost effective

Country and Year of Publication

The selected studies were authored by researchers from Finland (2), Netherlands (2), Korea (2), USA (2), Italy (1), Norway (1), Iran (1), India (1), Brazil (1), Canada (1), Turkey (1), and Taiwan (1). These are written in English and were published from 2017–2018. Therefore, the papers used for review were recent and current as these are being published in a year time.

Sample

Only four of the 16 studies are of small sample (8, 10, 15, 20, and 20 cases). Most of the selected studies were of large sample (25, 30, 38, 40, 65, 100, 120, 130, 165, 167, and 274 cases correspondingly) resulting to a mean of 105 cases (SD: 77 cases). The kinds of wounds were specified in all cases and the leading type was diabetic foot ulcer.

Study Design

Five (5) studies used randomized control trials (RCTs). Others were combination of designs like: two (2) utilized retrospective study where one is a single centered while the other one is a cross-over; three (3) are prospective studies where one is randomized, blinded, multicenter, and parallel study, the second is prospective, case-cohort study, and a Pilot study, and the third is prospective, randomized, controlled, multi-center clinical study. One (1) study used 7 randomized controlled trials and 1 nonrandomized controlled trial. Others studies are: one (1) single-arm clinical trial; one (1) phase III multicenter, double-blind, randomized, placebo-controlled trial; one (1) DAMO2CLES multicenter randomized clinical trial; one (1) cluster-randomized controlled non-inferiority trial; and one (1) Non-blinded, consecutive series, randomized clinical trial.

Management Team

Included studies were mostly composed of nurses and doctors. One had collaboration with experts, end users, and applicants. Two of the studies did not identify the composition of the healthcare team while another two studies included surgeons. There is no mention of inclusion of podiatry and orthotic studies

Intervention

Selected studies utilized both intervention and control groups where the control groups are given standard wound care following evaluation and rigid assessment. Some studies incorporate exercises while other studies add follow up care. The intervention groups are also given standard wound care and additional management that includes like hyperbaric O2 therapy (HBOT), transdermal continuous oxygen therapy (TCOT) device, natural latex associated with the LED circuit, low-level laser therapy, PRP treatment, salt-based spray, Telemedicine follow up, cord platelet gel application, Human Reticular Acellular Dermal Matrix, and acellular reticular allogenic human dermis. The identified interventions are of usual units of weeks.

Primary Outcome

Primary outcomes are of diverse occurrence. There is infection and arterial occlusion in improving vascularity, surgery, and rehabilitation as resulted in the single-center retrospective study of Chang [6]. In the use of HBOT, there is wound closure in the RCT of Chen et al., [7] utilizing HBOT while satisfaction and positive insights on healing is the result of 7 randomized controlled trials, 1 nonrandomized controlled trial by Health Quality Ontario [8] with primary outcomes of mixed results on standard of care in rates of amputation whereas there is impact on standard care and HBOT and no difference on the adverse events on both. The use of 5 footwear conditions as brought about by RCT of Crews and Candela [9] resulted to comfort from bilateral shoes. Santema et al. [10] used 40 sessions of HBOT five days a week or until complete wound healing was reached for limb salvage and wound healing.

TCOT on the other hand is used in intervention group in the prospective, randomized, blinded, multicenter, parallel study of Driver [11] brought no difference with the controlled group utilizing standard care. But in the RCT of Eraydin & Avsar [12] in the RCT, there is difference in the ulcer areas in the two groups where the intervention group is given standard wound care and foot exercises for 12 weeks while the control group had standard wound care with no exercise. The first elicited better wound area. The RTC of Lopez-Delis [13] used three groups for dressing. Group 1 is with natural latex related with LED circuit, group 2 is with calcium alginate or silver foam, and group 3 is with adhesive derived from the natural latex associated with the LED circuit. The primary outcome is significant decrease in reactive oxygen species (ROS) formation. Regarding laser therapy, RTC of Mathur et al. [14] brought out absolute and relative wound size reduction at 2 weeks compared to the baseline parameter where the intervention group had low-level laser therapy along with conventional therapy while control group only had conventional therapy. Another literature used Platelet-rich plasma (PRP) gel in the management of diabetic foot ulcer and Mohammadi, et al. [15] used a single-arm trial through weekly treatment after primary wound care and resulted primarily with significant decrease of the area of wound.

Comparable results between placebo groups and the recombinant human epidermal growth factor (rhGF) groups in the phase III multicenter, double-blind, randomized, placebo-controlled trial of Park et al [16] where routine wound care and topical or spray of saline with 0.005% rhEGF is used two times a day. This means a faster healing velocity. Pougatsch [17] made a pilot study using acceptable methods of cleansing combined with salt-based spray then gauze sponges soaked with the spray to dress the ulcer and then covered with dry foam or gauze and wrap with ACE to bring a wound closure. Volpe et al. [18] used three groups for a non-blinded, consecutive series, randomized clinical trial where Group A is managed with standard wound care while Group B is managed with topical application of CBPG consisted of platelet gel application 2 times a week in 4 weeks and then once a week for an additional 4 weeks. The primary outcome resulted to ulcer area reduction. There is a faster healing time in the cluster-randomized controlled non-inferiority trial of Smith-Strøm et al. [19] using TM follow-up care in the community while control group received SOC. Zelen et al. [20, 21] made 2 studies. First is a retrospective crossover study where Indolent DFUs are given acellular matrices and 12 out of 20 were eligible for crossover treatment and a prospective, randomized, controlled, multi-centre clinical study using daily dressing change that is combined with collagen alginate while HR-ADM group received grafts for healing proportion of wounds. All these are suggestive of wound healing and limb improvement with regards to appearance and function.

Discussion

With the aim of the study to bring out evidence based practice on wound care and healing, systematic review was undertaken. The key words “Diabetes Mellitus,” “wound practices,” “wound healing,” and “systematic review” yielded articles focused on the comfort and efficiency of patients with the identified and utilized practices on wound care and healing. The researchers explored case reports, interventional studies, and review papers written in the English language. Extracted data includes name and country of the author, year the article is published, site of the study, method and design of the study, management team, and the results. The studies were reviewed by two reviewers and the third reviewer checked and identified inconsistencies based on the set criteria. Therefore, the search was carried out systematically well.

The studies were reviewed by two reviewers and the third reviewer checked and identified inconsistencies based on the set criteria. Therefore, the search was carried out systematically well. The setting of the studies selected for review is of diverse origin. Two studies came from Finland, Netherlands, Korea, and USA respectively while one study came from the countries; Italy, Norway, Iran, India, India, Brazil, Canada, Turkey, and Taiwan. These are published from 2017–2018. With the evolution of research in the medical arena today, evolution of treatment will come next. As diabetes is the medical condition, wound healing becomes a problem. Wound types among the selected literatures are of diabetic ulcer. The study consists of large sample with a mean of 105 cases (SD: 77). This suggests validity and reliability. There are 5 studies of randomized control trials while other studies mixed their designs.

There are 3 prospective studies; first, a randomized, blinded, multicenter, and parallel study, the second is prospective, case-cohort study, and a Pilot study, and the third is prospective, randomized, controlled, multi-center clinical study. One of the selected study utilized 7 randomized controlled trials and 1 nonrandomized controlled trial. Other methods and designs used: single-arm clinical trial; phase III multicenter, double-blind, randomized, placebo-controlled trial; DAMO2CLES multicenter randomized clinical trial; cluster-randomized controlled non-inferiority trial; and non-blinded, consecutive series, randomized clinical trial. The dates of publication considered for inclusion are from studies published from 2017–2018. This means that the current and newest practices on wound care and healing are collated for review and these were based from the past practices that gained the reputation of healing and care. Most of the studies are published in journals.

In the study, the compositions of the teams who carried out the intervention are mostly doctors and nurses. One study collaborated with experts, end users, and applicants while two of the studies did not pinpoint the composition of the team while another two studies included surgeons. Regarding identified management, there is an intervention group and the control group where the former use additional treatment regime after the standard wound assessment and care while the later receives the conventional or traditional standard care and assessment alone.

In the study, the compositions of the teams who carried out the intervention are mostly doctors and nurses. One study collaborated with experts, end users, and applicants while two of the studies did not pinpoint the composition of the team while another two studies included surgeons. Regarding identified management, there is an intervention group and the control group where the former use additional treatment regime after the standard wound assessment and care while the later receives the conventional or traditional standard care and assessment alone.

The said intervention group received HBOT, TCOT device, natural latex associated with the LED circuit, low-level laser therapy, PRP treatment, salt-based spray, Telemedicine follow up, cord platelet gel application, Human Reticular Acellular Dermal Matrix, and acellular reticular allogenic human dermis simultaneously. This brought relative results to wound healing. It also heightened healthcare practice to a more evolved and acceptable treatment level of comfort and healing. Also, the identified regimen is of faster time treatment with the usual unit, weeks. Although the identified practices are of varieties, these are of the same results. It brought comfort and it brought healing faster than the traditional care and assessment alone. The data suggests the same dilemma of patients in different places with regards to wound/ulcer healing. One researcher is as engaged as the other to promote skin integrity and wound healing. And since the studies are of recent evidences and breakthroughs, wound healing is empowered to bring patients with diabetes better physicality and grip of hope against diabetic ulcer. Therefore, as per data suggests there is relative reduction of wound size.

Implications for Nursing and Health Policy

Understanding innovative wound care is the chance for nursing professionals to associate evidence, expert opinion and patients’ preference for a best and effective practices of wound care and healing and thus eventually give them a step above their skills. The evolution of nurses’ role in wound care contributed to bridge the need for specialist wound care guidance and globally, this role evolved to bring varieties of labels [22]. With the continuum of evidence-based studies empowered with technologies and other breakthroughs, nurses can improve their skills and hasten their knowledge for effective diabetic wound care. The influence of evidence-based practice and technology resonated through nursing practice, learning, and teachings patients’ self-care.

 Duttob, Chiarella & Curtis [22] discussed that there are claims directing to nurses contributory services resulting to improvement of patient care although it was hard to produce data concerning their impact on wound care but some researches direct to that bearing. Being part of the multidisciplinary team, nurses gain better skills and efficacy. Wounds International [23] in the article entitled, “International Best Practice Guidelines: Wound Management in Diabetic Foot Ulcers appraised the recommendations of the International Diabetes Foundation concerning the expert foot care group in including physicians having distinct attention in diabetes, individuals with informative skills, and individuals having recognized preparation in foot care. Comprehensively, this will be augmented by other specialists in the medical arena and soon, there will be improvement in the nursing practice along with the policies that strengthen it.

Acknowledgment

Engaging inspiration from those who helped in this review and the great minds in the medical research that never stop to give light on better management…

References

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  2. Moncrieff G, Van Onselen J, and Young T (2015) The role of emollients in maintaining skin integrity. Wounds UK 11: 68–74.
  3. World Health Organization (2018) Diabetes. [online] Available at: http: //www.who.int/news-room/fact-sheets/detail/diabetes [Accessed 23 Nov. 2018].
  4. World Union of Wound Healing Societies, Florence Congress, Position Document. Management of Biofilm. Wounds International 2016.
  5. Villines Z (2018) Diabetes and wound healing: What to know [Internet]. Medical News Today. MediLexicon International; 2018 [cited 2018Nov17]. Available from: https: //www.medicalnewstoday.com/articles/320739.php
  6. Chang JW, Heo W, Choi MSS, Lee JH (2018) The appropriate management algorithm for diabetic foot: A single-center retrospective study over 12 years. Medicine (Baltimore) 97: 11454. [crossref]
  7. Chen CY (2017) Adjunctive Hyperbaric Oxygen Therapy for Healing of Chronic Diabetic Foot Ulcers: A Randomized Controlled Trial. J Wound Ostomy Continence Nurs 44: 536–554.
  8. Health Quality Ontario (2017) Hyperbaric oxygen therapy for the treatment of diabetic foot ulcers: a health technology assessment. Ont Health Technol Assess Ser 17: 1–142.
  9. Crews RT, Candela J (2018) Decreasing an Offloading Device’s Size and Offsetting Its Imposed Limb-Length Discrepancy Lead to Improved Comfort and Gait. Diabetes Care 41: 1400–1405. [crossref]
  10. Santema K (2018) Hyperbaric oxygen therapy in the treatment of ischemic lower-extremity ulcers in patients with diabetes: results of the DAMO2CLES Multicenter Randomized Clinical Trial. Diabetes Care 41: 112–119.
  11. Driver V (2017) A prospective, randomized, blinded, controlled trial comparing transdermal continuous oxygen delivery to moist wound therapy for the treatment of diabetic ulcers. Osteotomy Wound Management 63: 12–28.
  12. Eraydin S, Avsar G (2018) The effect of foot exercises on wound healing in type 2 diabetic patients with a foot ulcer: A randomized control study. J Wound Ostomy Continence Nurs 45: 123–130.
  13. Lopez-Delis A (2018) Characterization of the cicatrization process in diabetic foot ulcers based on the production of reactive oxygen species. J Diabetes Res 2018: 4641364.
  14. Mathur RK, Sahu K, Saraf S, Patheja P, Khan F, et al. (2017) Low-level laser therapy as an adjunct to conventional therapy in the treatment of diabetic foot ulcers. Lasers Med Sci 32: 275–282. [crossref]
  15. Mohammadi MH (2017) Evaluation of wound healing in diabetic foot ulcer using platelet-rich plasma gel: A single-arm clinical trial. Transfus Apher 56: 160–164.
  16. Park, KH (2018) Topical epidermal growth factor spray for the treatment of chronic diabetic foot ulcers: A phase III multicenter, double-blind, randomized, placebo-controlled trial. Diabetes Res Clin Prac 142: 335–344.
  17. Pougatsch DA, Rader A, Rogers LC (2017) The Use of a Sea Salt-based Spray for Diabetic Foot Ulcers: A Novel Concept. Wounds 29: E5–5E9. [crossref]
  18. Volpe P, Marcuccio D, Stilo G, Alberti A, Foti G, et al. (2017) Efficacy of cord blood platelet gel application for enhancing diabetic foot ulcer healing after lower limb revascularization. Semin Vasc Surg 30: 106–112. [crossref]
  19. Smith-Strøm H (2018) The effect of telemedicine follow-up care on diabetes-related foot ulcers: a cluster-randomized controlled noninferiority trial. Diabetes Care 41: 96–103.
  20. Zelen C (2017) Human Reticular Acellular Dermal Matrix in the Healing of Chronic Diabetic Foot Ulcerations that Failed Standard Conservative Treatment: A Retrospective Crossover Study. Wounds 29: 39–45.
  21. Zelen CM (2017) A prospective, randomised, controlled, multicentre clinical trial examining healing rates, safety and cost to closure of an acellular reticular allogenic human dermis versus standard of care in the treatment of chronic diabetic foot ulcers. Int Wound J 14: 307–315.
  22. Dutton M, Chiarella M, Curtis K (2014) The role of the wound care nurse: an integrative review. Br J Community Nurs Suppl 39–40, S42–47. [crossref]
  23. International Best Practice Guidelines: Wound Management in Diabetic Foot Ulcers. Wounds International, 2013. Available from: www.woundsinternational.com

Factors Associated with Black & Latina Women Meeting the US Federal Physical Activity Guidelines: Secondary Analysis of NHIS Data

DOI: 10.31038/AWHC.2018135

Abstract

Background: Not meeting physical activity guidelines is a major public health concern. Studies show that Black and Latina women have general lower levels of physical activity (PA) than White women and are therefore at higher risk for chronic diseases including cardiovascular disease, diabetes, and obesity. The purpose of this study is to analyze National Health Interview Survey (NHIS) data to determine what factors, if any may contribute to Black and Latina women between the ages of 18 and 47, adhering to the federal PA guidelines. Identifying these factors will help in the development of PA promotion programs aimed at increasing adherence to the federal PA guidelines among minority women.

Method: Secondary analysis of data from the National Health Interview Survey 2008–2014 was analyzed to determine factors which may be associated with PA adherence among Black (N = 23,162) and Latina (N = 37,103) women.

Results: Logit modeling examined mean differences between groups and related variables. Blacks are more likely to meet muscle-strength guidelines with increased education and increased income. Latinas are more likely to meet muscle-strength as education and US acculturation increases.

Conclusion: These findings highlight the importance of understanding and considering racial/ethnic differences when developing strategies for promotion of PA among minority women. Identifying strategies and factors for increasing PA is a major step toward reducing risk factors associated with inadequate PA. Practitioners should be aware of these results and trends when discussing PA recommendations with their patients.

Introduction

Not engaging in adequate physical activity is a major public health problem associated with increased morbidity and mortality, accounting for 3.2 million deaths each year [1]. The numbers of individuals not meeting recommended physical activity levels have risen significantly over the last decade, contributing to increased prevalence of heart disease, diabetes, colon cancer, and high blood pressure [2]. Engaging in regular physical activity as per physical activity guidelines has shown to be essential and beneficial in reducing risk factors for many of these diseases (cardiovascular diseases, type 2 diabetes, breast & colon cancers) thereby reducing premature all-cause mortality [3]. Minority women, such as Black and Latino, are more likely to be less physically active in comparison to White women [4].

In 2008, the federal government adopted physical activity guidelines for all Americans [5]; the second edition of these guidelines was recently released in the fall of 2018 [6]. These guidelines serves as the primary, authoritative voice of the federal government for evidence-based guidance on physical activity, fitness, and health for Americans that describe the amount, types, and intensity of physical activity needed to achieve many health benefits for Americans across the life span [6]. These guidelines are further broken down into aerobic and Muscle Strengthening (MS) activities aimed at improving one’s health. The minimal recommendations for adults living in the United States are:

  1. 2 hours and 30 minutes (150 minutes) of aerobic activity of moderate intensity, such as, brisk walking every week, along with 2 or more days a week of muscle-strengthening activity that work all major muscle groups (legs, hips, back, abdomen, chest, shoulders, and arms (p.56, 2018).

Unfortunately, the overwhelming majority of Black and Latina women in the United States are not consistently meeting these guidelines. The prevalence of meeting aerobic physical activity guidelines for Black women in the US is 35% and 41% for Latina women, in comparison to White women at 51% [4]. The lower level of physical activity noted in minority women may account for the significant health disparities noted between White women and Black. (Table 1) shows the significant differences noted between White and Black women regarding diseases and risk factors.

Table 1. Prevalence of Diseases & Risk Factors in Women by Race/Ethnicity (%)

Diseases & Risk Factors

White

Black

Hispanic or Latina

Physician-diagnosed DM

7.4

13.6

12.7

High Blood Pressure

32.3

46.3

30.7

Overweight (BMI ≥ 25kg/m2)

63.7

82.2

77.1

*Met Federal Physical Activity guidelines for Adults (2015); DM = Diabetes mellitus.
Source: Table created by W. Williams from data from Heart Disease and Stroke Statistics – Prevalence 2011–2014 (Benjamin et al., 2018)

Physical activity is defined as bodily movement that increases energy expenditure above the basal level [7]. Leisure-time physical activity is defined as physical activities performed during free time, other than work or school, such as recreation and/or exercise [8]. Healthy People 2020 have defined health disparity as “a particular type of health difference that is closely linked with social, economic, and/or environmental disadvantage that adversely affects specific population groups [9].” Common themes noted in the literature that may help to explain these differences are cultural attitudes (body image, hair maintenance, society norms) and family responsibilities. Therefore, the purpose of this study is to analyze National Health Interview Survey (NHIS) data to determine what factors, if any may contribute to Black and Latina women between the ages of 18 and 47 adhering to the federal physical activity guidelines. Identifying these factors will help in the development of physical activity programs aimed at promoting adherence to the federal physical activity guidelines among minority women. Improving adherence to physical activity guidelines can lead to health promotion and disease prevent of various chronic diseases (hypertension, heart disease, obesity, diabetes, and some form of cancers) among minority women.

The Literature Regarding Racial/Ethical Differences

Black Women

A person’s experiences and perceptions play a major role in determining her participation in regular physical activity. A person’s attitude is shaped by her experiences, beliefs, and cultural influences [10, 11]. Evidence suggests that racial/ethnic differences exist regarding Black women’s attitudes toward physical activity.[11, 12] These attitudes toward physical activity may help explain the significant decline noted in Black women in comparison to White women. In the Black community, women are less concerned with body size and are more accepting of fuller, curvaceous figures [12–16]. Therefore, Black women who are satisfied with their body size may perceive being physically active as a waste of time since they have no desire to lose weight. Also, Black women are more accepting of individuality and tend not to conform to society’s views of beauty. [17] Although having a positive image of one’s self is a generally a good thing, such an attitude may help contribute to some Black women being less interested in engaging in adequate physical activity.

One aim of a systematic review of qualitative studies conducted by Siddiqi, Tiro, and Shuval [18] was directed at understanding barriers to physical activity among Black adults. This review indicated that among Black women, hair maintenance, family responsibilities, and environmental condition were some of the barriers the women gave for not being physically active. Hair maintenance is a major concern among Black women due to the cost and time-consuming process involved. Hair maintenance among Black women tends to be a major deterrent for engaging in PA [19–22].

Another barrier is family responsibilities [20, 23]. In 2011, 67.8% of Black births in the United States were to single Black or African American women, which is a significant rise in the last decade [24]. Being a single mother juggling multiple responsibilities related to work and caring for children does not leave sufficient time to be physically active for many women. Research shows that women with children are more likely to have decreased physical activity [25, 26]. Such a significant rise in single Black mothers may be one explanation why physical activity levels are significant lower in Black women than in White women [19, 24].

Latina Women

Similar to Black women, Latinas regard fuller figures as more desirable [27]. Latinas and Blacks have reported more of a positive body self-image at higher BMIs than White women, suggesting that they have higher thresholds for what is considered healthy [27]. Personal hygiene issues such as desire not to sweat are also barriers to Latinas engaging in physical activity similar to the desire not to redo their hair for Black women [12, 17].

Latinas’ cultural emphasis on family and familial unity or familismo highlights their sense of family duties and responsibilities [28]. Latinas also tend to be the homemaker and caregiver, which might influence how they engage in physical activity because their energy and time is put into household duties instead of Leisure Time Physical Activity (LTPA) [29]. Marianismo and acculturation are also influencing factors on Latinas participation in PA [28]. Marianismo, or prioritization of family responsibilities over self-care, has been shown to affect physical activity among Latina women. D’Alonzo (2012) found that Latina women who immigrated to the United States were less likely to participate in LTPA due to family priorities and working outside the home. Many Latina women feel that family is a priority and taking time to go to the gym or engage in physical activity takes away time that could be spent with children, spouses, or other family members [28].

Immigration to the United States and subsequent acculturation also plays a role in leisure time physical activity among Latina women. D’Alonzo (2012) showed that decrease leisured time physical activity among Latina women who had recently immigrated to the United States due to “role overload” was associated with lifestyle changes and acculturation (p. 138). These changes include working outside the home, increased number of hours worked in a day, socioeconomic pressures, decreased social support, learning English, and transitioning from a collective culture to an individualistic one [28].

Latinas also have a higher fertility rate than White women, and which several researchers [29–31] have found the increasing number of children to be negatively associated with reported physical activity levels. Having more children would presumably increase the duties and therefore hours associated with childcare and household tasks, thus reducing the number of hours available to engage in physical activity participation for Latinas as their family commitments would be more time consuming [32].

Methods and Materials

Design

Secondary analysis of data from the National Health Interview Survey (NHIS) 2008–2014 was analyzed to explore factors which may be associated with physical activity adherence among Black and Latina women. The NHIS is an annual in-person household survey conducted by the Centers for Disease Control and Prevention (CDC), National Center for Health Statistics (NCHS) and administered by the US Census Bureau [33]. The data is publicly accessible with no identifiable information of the participants. Adults aged 18 years or older are randomly selected through a complex stratified clustered process that oversamples Hispanics and non-Hispanic Blacks from the noninstitutionalized civilian population [33]. Oversampling of minorities allows for more precise estimation of health characteristics in these segments of the populations. The conditional response rate for this time frame was approximately 80% [33].

To be included in this study, NHIS responders for the years 2008–2014 had to be female, between the ages of 18 and 47, and identify race as Black or Latino. Sample size consisted of N=23,162 Blacks and N=37,103 Latinas women aged 18 to 47 years. This is 4.1% of the total population of respondents regardless of sex, age, or race.

Measures

Co-habitation status (partner present vs. no partner), education [less than high school (LHS); High School/General Education Diploma (HS/GED); some college, Associates Degree (AD), Bachelor’s, greater than Bachelor’s)] and family income which was categorized as multiples of the federal poverty level (FPL) [not poverty, below poverty limit, 150% above FPL, 200% above FPL, 250% above FPL, 300% above FPL, 400% FPL and above] were the primary variables that were explored to identify association to physical activity adherence related to the federal physical activity guidelines. Also, an acculturation measure was created, combining language spoken most often (Spanish or English) and place of birth (US born or foreign born). These categories are (a) English/US born, (b) English/ foreign born, (c) English and Spanish/ US born, (d) English and Spanish/ foreign born, (e) Spanish/ US born, and (f) Spanish/foreign born. Based on the federal physical activity guidelines recommendations and the available data, 2008–2014 IHIS participants were grouped as follows: one variable capturing adherence to only moderate aerobic activity recommendations was dichotomized into “meets recommendations” or “does not meet recommendations;” a second variable capturing adherence to only vigorous aerobic activity recommendations was dichotomized into “meets recommendations” or “does not meet recommendations;” and a third variable capturing adherence to muscle-strengthening activities was dichotomized into “meets recommendations” or “does not meet recommendations.”

Data Analysis

The analysis was conducted with the Statistical Package for the Social Sciences (SPSS) [34]. The primary outcome for this analysis was identification of factors associated with adherence to meeting the federal physical activity guidelines among Black and Latina women.

Bivariate analysis of the independent variables partner presence, educational level and income were performed to determine impact on meeting moderate aerobic, vigorous aerobic, and muscle-strengthening activities as outlined above. Logistic regression models were used to identify a relationship with the variables associated with higher odds of engaging in moderate or vigorous aerobic activity and adherence to muscle-strengthening recommendations. Results are reported using odds ratios adjusted for all listed associated variables, significant effects (SEs), and p values.

Results

Due to the difference between Black and Latina women, the results will be reported based on race. Results are reported using adjusted odds ratios for all listed variables, significant effects (SEs), and p values. For Black women, none of the associated variables were significant for meeting moderate or vigorous aerobic activity guidelines. However, bivariate analysis revealed partner presence, educational level and income significantly impacted adherence to muscle-strengthening (MS) guidelines (p<0.05) and engaging in MS are a significant part of the federal physical activity guidelines. Multivariable logistic regression was then performed to ascertain the effects the independent variables of partner presence, educational level and income had on the likelihood that Black women age 18 to 47 met the recommended muscle-strengthening guidelines. These independent variables were chosen based on their p value (p < 0.05) from the bivariate analysis. For Black females, the logistic regression model including combined effects of partner status (cohabitating or non-cohabitating), education and income level was statistically significant, χ2(30) = 77.70, p < .05. Results are reported using adjusted odds ratios for all listed variables, significant effects (SEs), and p values.

The multivariable logistic regression showed income and education to significantly impact the likelihood of meeting recommended muscle-strengthening guidelines for cohabitating Black women aged 18 to 47. Results were not significant for women who were non-cohabitating. Income significantly impacted those with a high school diploma or General Education Degree (GED), while education significantly impacted women who identified as having an income below the federal poverty level (FPL). The comparison group was Black women age 18 to 47 with less than a high school diploma, no partner present, and identified as “not poverty”.

(Table 2) shows Black women age 18 to 47 with a high school diploma or GED are significantly less likely to meet muscle-strengthening guidelines as income increases from Federal Poverty Level (FPL) to above FPL. Specifically, women with a family income identified as below the Federal Poverty Level (FPL) were 0.51 times less likely to meet muscle-strengthening act (p<0.01), while those with an income of 150% above the FPL were 0.67 times less likely to meet muscle-strengthening guidelines (p<0.01). However, there was an increase in likelihood of meeting muscle-strengthening guidelines as income rose from 150% above FPL. While still less likely to meet muscle-strengthening guidelines than women with an income below the FPL, those who made 200% and 300% above FPL were only 0.55 (p<0.05) and 0.59 (p<0.05) times less likely to meet muscle-strengthening guidelines.

Table 2. Effects of Income in Cohabitating Black Women Age 18–47 with a High School Diploma or General Education Degree on Meeting Muscle Strengthening Guidelines.

Meets MS Guidelines

Confidence Interval (CI)

O.R.

S.E.

P value

Lower CI

Upper CI

Reference Category: Not Poverty, less than high school diploma, non-cohabitating

Below FPL

0.49

0.24

0.004**

0.31

0.79

150% above

0.33

0.38

0.004**

0.16

0.70

200% above

0.45

0.36

0.026*

0.22

0.91

250% above

0.60

0.29

0.072

0.34

1.05

300% above

0.416

0.36

0.013*

0.20

0.82

400% + above

0.74

0.33

0.360

0.39

1.41

*P < .05.  ** P < .01
O. R = Adjusted Odd Ratio; S.E. = Significant effects; FPL = Federal Poverty Level; MS = muscle strengthening
Source: Table created by MacLean.

Among cohabitating Black women age 18 to 47 who identified a family income of below FPL, education appears to increase the likelihood of meeting muscle-strengthening guidelines (see Table 3). Women with a high school diploma or GED were 0.51 times less likely to meet muscle-strengthening guidelines (p<0.01), while those with some college were only 0.48 times less likely (p<0.05). However, women who had a partner present, identified family income of FPL, and had an associate degree were 0.74 times less likely to meet muscle-strengthening guidelines (p<0.05).

Table 3. Effects of Education in Cohabitating Black Women Age 18–47 with a Family Income of Below Federal Poverty Level on Meeting Muscle Strengthening.

Meets MS Guidelines

Confidence Interval (CI)

O.R.

S.E.

P value

Lower CI

Upper CI

Reference Category: Not Poverty, less than high school diploma, non-cohabitating

HS/GED

0.49

0.24

0.004**

0.31

0.80

Some college

0.52

0.30

0.029*

0.29

0.94

AD

0.26

0.59

0.023*

0.08

0.83

Bachelor’s

0.15

1.01

0.061

0.02

1.09

>Bachelor’s

1.31

0.75

0.718

0.30

5.74

*P < .05.  ** P < .01
All variables were included in the model to obtain the adjusted odds ratios.
OR=Adjusted Odd Ratio; S.E.=Significant effects; HS=High School; GED=Graduate Equivalency Degree; AD=Associates Degree; > Bachelor’s =greater than a Bachelor’s degree; MS=muscle strengthening
Source: Table created by MacLean

For Latina women, none of the associated variables were significant for Latinas in meeting moderate aerobic activity guidelines. However, trends in the data showed that Latinas were more likely to meet moderate aerobic activity guidelines as education and acculturation increased. They were less likely to meet moderate aerobic activity guidelines with increasing income. These trends are displayed in (Table 4) None of the associated variables were significant for vigorous aerobic activity for Latinas, nor were any trends present in the data.

Table 4. Latina Women Age 18–47 Moderate Aerobic Activity Guidelines.

Meets Moderate Aerobic Guidelines

O.R.

S.E.

P value

Reference Category: Less than HS

HS/GED

1.95

0.5

0.18

Some college

1.10

0.61

0.87

AD

2.36

0.59

0.15

Bachelor’s

2.87

0.62

0.09**

Greater than Bachelor’s

2.70

0.91

0.27

Reference Category: FPL

Below FPL

6.57

1.04

0.07

150% above

7.96

1.08

0.06

200% above

3.28

1.24

0.34

250% above

8.50

1.10

0.05

300% above

4.00

1.15

0.23

400% + above

2.86

1.20

0.38

Reference Category: English/US born

English/FB

0.91

0.40

0.80

S+E/US

0

5304.70

1.00

S+E/FB

0.63

0.62

0.46

S/US

0

6458.79

1.00

S/FB

0.39

0.62

0.13

*P < .05.  ** P < .01
AD (Associates Degree); HS (High School)/GED (General Education Diploma); FPL (Federal Poverty Level); FB (Foreign Born): S+E (Spanish and English); US (United States Born); S (Spanish)

However, just like with Black women, many of the associated variables for meeting muscle-strengthening guidelines were significant (p <.05) for Latinas as evidenced in (Table 5) As education increased, odds of meeting muscle-strengthening guidelines also increased in Latinas who had a bachelor’s degree; they were 87.9% [OR=1.88, CI=1.18–2.99, p=.008] more likely to meet muscle-strengthening guidelines than were Latinas with no high school degree. Data also showed that as income increased, Latinas were more likely to meet muscle-strengthening guidelines. Latinas who earn 400% or more above FPL were 215% [OR=2.15, CI=1.26–3.65, p=.005] more likely to meet muscle-strengthening guidelines that Latinas who earned income equivalent to the FPL. Latinas were 89.3% [OR=.11, CI=0.03–0.44, p=.002] less likely to meet muscle-strengthening guidelines if they were currently pregnant. Also, data showed that the odds of meeting muscle-strengthening guidelines increased as acculturation increased. Latinas born outside of the U.S. who predominantly spoke English were 22.5% more likely to meet muscle-strengthening guidelines than those who predominantly spoke Spanish [Eng/FB (OR=.68, CI=.50-.93, p=.014); Sp/FB (OR=.46, CI=.3-.70, p=.000)]. Trends in the data also showed that as BMI increased, the likelihood of a Latina meeting muscle-strengthening guidelines decreased. Latinas in the Obese III category were 68.6% [OR=.31, CI=.17-.58, p=.000] less likely to meet muscle-strengthening guidelines than a Latina of normal BMI.

Table 5. Latina Women Age 18–47 Muscle-Strengthening Guidelines.

Meets MS Guidelines

O.R.

S.E.

P Value

Reference Category: Less than HS

HS/GED

0.97

0.13

0.80

Some college

1.57

0.12

0**

AD

1.45

0.14

0.01**

Bachelor’s

2.01

0.13

0**

Greater than Bachelor’s

1.92

0.15

0**

Reference Category: FPL

Below FPL

0.84

0.11

0.09

150% above

1.17

0.13

0.25

200% above

1.33

0.14

0.04*

250% above

1.37

0.13

0.02*

300% above

1.57

0.12

0**

400% + above

1.88

0.12

0**

Reference Category: Not pregnant

Pregnant

0.48

0.18

0**

Reference Category: English/US born

English/FB

0.71

0.11

0.002**

S+E/US

0.71

0.45

0.44

S+E/FB

0.44

0.25

0.001**

S/US

0.68

0.50

0.43

S/FB

0.45

0.2

0.001**

Reference Category: Normal BMI

Underweight

0.72

0.15

0.03*

Overweight

0.78

0.07

0**

Obese I

0.48

0.1

0**

Obese II

0.50

0.15

0**

Obese III

0.32

0.14

0**

*P < .05.  ** P < .01
AD (Associated Degree); HS (High School)/GED (General Education Diploma); FPL (Federal Poverty Level); FB (Foreign Born); S+E (Spanish and English); US (United States Born); S (Spanish); muscle-strengthening (MS)

Limitations

The data utilized in this study was obtained from secondary analysis of NHIS data, which should be considered a limitation due to the inability to consider other variables that may be associated with physical activity. NHIS represents a cross-sectional research design that obtained household interviews of a sample clusters of addresses from each state. Therefore, because participants were not randomized the sample may not be representative of the population, contributing to selection bias. In addition, cause and effect cannot be determined with a cross-sectional design and the data may only capture what is occurring at that particular time and moment, which may not be transferable to other times or situations. NHIS relies on self-reported data, therefore, recall bias is a major issue and can potentially increases the risk of participants’ bias. Participants may also over or under report certain information to look good or to please the interviewer (socially acceptable bias). These findings may not be generalizable to other populations that are not women of Black or Latina descent.

Discussion

The findings and trends presented here are significant to understanding and developing effective strategies that could improve physical activity among Black and Latina women. Strategies to address physical activity participation in minority women must occur at multiple levels that consider cultural differences.

Although, no variables were found to be significant for meeting moderate or vigorous aerobic activity guidelines in either Black or Latina women. Adherences to muscle-strengthening activities were found to be associated with a few variables in both groups. Although, muscle-strengthening activities are only a partial requirement of the federal physical activity guidelines, it still represents some level of activity in these women. And current studies support that some activities are better than none [6].

For Black women, partner presence was associated with Black women maintaining muscle-strengthening activities, which stands to reason that Black women with a partner present, that is supportive may show higher levels of commitment due to encouragement and interest in them being active. A qualitative study by Mama et al. (2015) revealed that Black women did view support of a spouse or friend as important factor in engaging in regular physical activity [35]. The support can be in the form of working-out together, prompting, to offering to pay for a gym membership.

Higher educational attainment was associated with muscle-strengthening activities in both groups, which is consistent with other studies indicating that higher educational attainment is associated with being more physically active [36]. A study by Ainsworth et al. (2003) that looked at African-American women in South Carolina showed that educational attainment had a strong relationship for Black women to be physically active, with college educated women revealing a two-times higher likelihood of being physically active over high school educated Black women [37].

Also, income was associated with muscle-strengthening activities; these findings of income are consistent with other studies that have shown that women from lower socioeconomic status tend to be less physically active. [38, 39] In the Black community there is a higher prevalence of single family homes, headed by women, where they are the sole source of income for their families [24]. Therefore, being the head-of-the household may place financial burdens on single Black mothers that prevent them from being physically active (i.e., no money for gym fees, or a babysitter that would allow her to go out to a gym). Black women from a lower socioeconomic background may also have greater difficulty being physically active compared to their higher income counterparts due to environmental barriers as well (no local gym or nearby park, unsafe neighborhood and heavy traffic patterns) [40]. More research is needed that considers the economic context of physical activity among low-income Black women. A study by Harley et al. (2013) emphasizes the need for better understanding of how significant, if any, income is regarding being physically active. A recent study by Sun and colleagues (2016) revealed that higher socioeconomic status was positively associated with young Black women being physically active [36].

For Latina women, factors associated with lower levels of engagement in sufficient physical activity levels include lower income, lower education levels, and greater number of children [29–31]. This study found that Latinas were less likely to meet moderate aerobic activity guidelines with increasing income. The difference could be explained by different definition of physical activity or the use of leisure time physical activity versus total physical activity.

With the increase of reported education beyond high school and income, the odds of Latinas meeting muscle-strengthening guidelines increased. This study’s findings were consistent with the literature regarding the negative association of pregnancy and likelihood of meeting muscle-strengthening guidelines with Latinas who reported being currently pregnant were less likely to meet muscle-strengthening guidelines [41]. Similarly, as acculturation to the US increases, odds of meeting muscle-strengthening guidelines increased for Latinas. This is consistent with literature reporting on total leisure time physical activity associated with meeting physical activity guidelines [41]. Our data demonstrated similar trends regarding the association with increasing BMI and decreasing likelihood of meeting muscle-strengthening guidelines [41]. This study found racial/ethnic differences among Black and Latina women as pertains to their level of physical activity; these differences must be taken into consideration if we hope to reduce the growing trend of insufficient physical activity among minority women. Working together, providers, patients, and policy makers can reduce health care disparities associated with diseases related to engaging in insufficient physical activity.

Acknowledgment: Brian Rathmell for his review and assistance with the statistical analysis.

Funding: This work was supported by the Dean’s Summer Research Fund, School of Nursing, Camden, New Jersey, USA.

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Endoscopic Removal of Eroded Gastric Bands Using Lithotripsy Devices: A Case Series

DOI: 10.31038/IMCI.2018114

Abstract

Bariatric surgical procedures are being performed at an exponential rate. Gastric banding is a commonly performed Bariatric surgical procedure with band erosion being a known complication. Until recently eroded Gastric Bands have undergone surgical removal. Recent reports have described endoscopic removal using a biliary guide-wire and biliary mechanical lithotripter.

Aim: To determine the safety and feasibility of endoscopic removal of eroded gastric bands using lithotripsy devices following Bariatric surgery.

Methods: Thirteen patients (9w, 3m), age 33–59 (mean 45) presented with a diagnosis of eroded gastric band. These include 9 adjustable lap bands. Presenting symptoms included pain 10/13, N/V 7/13, fever/port infection 1/13. All patients had confirmation of gastric band erosion with endoscopy and CT scan 13/13.

Results: All patients with non-adjustable gastric bands were removed successfully, endoscopically. Of the patients with Adjustable Lap Bands, endoscopic removal was successful in 8/9 patients. The failed attempt was in a patient whose Band had significant fibrotic component and non-visualization of the buckle. This patient required laparoscopic removal. All patients underwent post-op contrast upper GI imaging to exclude gastric leak.

Conclusion: Patients with Gastric Band erosion present most often with abdominal pain, followed by nausea and vomiting. Diagnosis is established by endoscopy and CT imaging. Endoscopic removal is highly but not universally successful and requires coordination with a Bariatric Surgeon for the removal of the adjustable port and removal of the band in the setting of endoscopic failure. The endoscopist needs to familiarize him with Bariatric Surgical procedures and their inherent complications and eventually assist in their care when called upon.

Introduction

Obesity is defined as abnormal or excessive fat accumulation that presents as a risk to health [1]. Despite obesity being a well identified and acknowledged problem, it continues to grow as a global epidemic [2]. Obesity has emerged as a major problem, the primary reason being an imbalance between the consumption and expenditure of calories. Since 1975, the number of obese people has increased by three times across the globe. According to the 2016 World Health Organization statistics, 650 million people (13%) 18 years and above have been identified as obese [3]. 36.5% of the total population of United States is Obese [4]. Obese people can have a significantly negative impact on the quality of their lives in terms of an increased risk of having diabetes mellitus type 2, infertility, cardiovascular diseases, respiratory disorders, malignancies, as well as social and functional limitations [5].

Bariatric care has expanded over the period to be identified as a growing field for consideration as a treatment option for obesity [6,7]. Bariatric procedures cause weight loss by limiting the amount of food the stomach can hold or malabsorption of nutrients or a combination of both. The commonly performed bariatric procedures include Roux-en-Y Gastric Bypass, Sleeve Gastrectomy, Adjustable Gastric Band, and Biliopancreatic Diversion with Duodenal Switch [8].

In the United States, trends have been shifting towards Laparoscopic Gastric bands as means of attaining weight loss [9]. Where lap bands have proven as an effective alternative to the conventional weight loss methods, they come with a cost of complications ranging from mild to serious [10].

The pathophysiology of gastric band erosion has yet to be understood [11]. The established understanding of gastric band erosion reveals that acute and chronic tissue damage could be a potential trigger to initiate band erosion [12]. Undiagnosed gastric perforations that may occur during band placements and early infections can serve as the possible causes of early erosions. Late erosions are caused by gastric band induced high pressure leading to chronic ischemia [13,14]. This prolonged ischemia gives way to band erosion through the mucosal layer and hence a perception of pain.

Gastric band erosion has been identified as a common complication with its incidence varying from 0.23% to 32.65% [15]. Symptoms of gastric band erosion include abdominal pain, excessive vomiting, weight loss cessation or unexplained weight regain and the inability to regulate the stoma [16]. Endoscopic removal of eroded gastric bands has been identified as a potent and safe technique to address this complication [17].

We report a unique approach towards the removal of eroded gastric bands using a biliary guided wire and biliary mechanical lithotripter. This method was applied in 19 patients with eroded gastric bands and represents one of the largest case series in this respect. Literature review reveals that our technique is one of its kinds and can potentially contribute to determining the safety and feasibility of endoscopic removal of eroded gastric bands following Bariatric Surgery.

Materials and Methods

Thirteen patients (9w, 3m), age 33–59 (mean 45) presented to the University of Texas, and Memorial Hermann Hospital with symptoms leading to a diagnosis of eroded gastric lap band. This included 4 non-adjustable and 9 adjustable (attached injection port) lap bands. Presenting symptoms included; pain 10/13, nausea/vomiting 7/13, fever/port infection 1/13. Patients presented 5–24 weeks from start of symptoms (mean 16 weeks). All patients had confirmation of gastric band erosion with upper endoscopy and CT scan imaging.

Procedure technique; a flexible biliary hydrophilic guide-wire with flexible tip (0.025 in x 450cm) is advanced through the band while the endoscope is withdrawn (Figure 1). The endoscope is re-advanced alongside the wire and the distal tip is captured with forceps and withdrawn through the mouth (Figure 2). With the two ends of the wire now outside the patient’s mouth a flexible lithotripter cable, the Emergency Lithotripter System BML-110A-1 (Olympus America) (Figure 3) is advanced over the two ends of the wire until the tip rests against the gastric band (Figure 4). The lithotripter handle is then attached to the cable and progressively ratcheted down until the wire cuts across the band (Figure 5). The surgeon detaches and removes the port in those with adjustable bands while the endoscopist removes the cut band with a stiff, braided snare (Figure 6).

IRCI 18 - 104_F1

Figure 1. Guide-wire across the eroded lap band.

IRCI 18 - 104_F2

Figure 2. Guide-wire across the eroded lap band and both ends outside the mouth.

IRCI 18 - 104_F3

Figure 3. Emergency Lithotripter System.

IRCI 18 - 104_F4

Figure 4. Lithotripter advanced through the guide-wire now rests against the eroded lap band.

IRCI 18 - 104_F5

Figure 5.Wire cutting across the band.

IRCI 18 - 104_F6

Figure 6. Removed eroded lap band.

Results

All patients with non-adjustable gastric bands were removed successfully, endoscopically using the above technique. Of the patients with Adjustable Lap Bands, endoscopic removal was successful in 8/9 patients. The failed attempt was in a patient whose Band had significant fibrotic component and non-visualization of the buckle. This patient required laparoscopic removal at the same time as port removal. Procedure time recorded ranged from 18 minutes to 32 minutes (mean 24 minutes). All patients underwent post-op contrast upper GI imaging using dilute barium to exclude gastric leak within 12 hours of band removal. No leaks or perforations were identified. Minor bleeding occurred in one patient which resolved spontaneously. Patients were discharged 1–4 days post-op (mean 1.4 days). One patient with port infection required prolonged hospitalization for IV antibiotics and fever resolution.

Discussion

Obesity has been recognized as an important contributive factor towards premature deaths [18]. Laparoscopic gastric banding has evolved as an effective strategy for weight loss that has been proven to decrease total body weight by up to 20% [19]. Where gastric bands are now recognized widely as an effective tool for weight loss and significant contributions towards comorbidities of obesity, its complications like port or tubing malfunction, band slippage, and obstruction of the stoma, band erosion, pouch dilation, and infection cannot be ignored [20].

Where a definite cause of gastric band erosion has yet to be established, it is imperative to apply certain techniques at the time of insertion of gastric band to minimize the risk of gastric band erosion [12,21]. External as well as internal pressure applied to the gastric wall has been hypothesized as a key etiologic factor of band erosion [22]. External pressure may be induced by band over-filling, band rapid filling or band infection [23,24]. Binge eating after the band placement surgery is a contributive factor towards internal pressure [25,20]. One of the important pathophysiologic causes of band erosion stems from the recognition of gastric band as a foreign body by the immune system [26–28]. This results in the formation of an encapsulating fibrotic shell around the eroded lap band that leads to tissue contraction. This shrinked tissue protects the integrity of the fistulous tract [29]. As lap band erosion through the stomach wall occurs over a period of several years, gastric perforation is a rare occurrence [30]. This pathophysiology reflects that active perforation is absent in eroded lap bands. Endoscopic removal of lap bands is therefore a safe yet effective procedure for the removal of eroded lap bands [31–36].

While patients with gastric band erosions can be completely asymptomatic, symptoms if present include repeated port infections, bowel obstruction, abdominal pain and rarely as serious as sepsis with peritonitis [37]. These being mentioned, band erosions can also lead to serious consequences like massive GI hemorrhage and circulatory collapse [38,39], pyelophlebitis of the portal vein [40] and intra-abdominal abscess [41]. Therefore, it is crucial to remove the eroded gastric band [8].

Various techniques have evolved over the course of time for the removal of eroded gastric bands. Unfortunately, literature review is lacking with respect to the comparison of different techniques used for the retrieval of eroded gastric bands [42]. However, the efficacy and design specific model of each technique varies from case to case. For instance, the hybrid technique considers the simultaneous use of laparoscopic and endoscopic methods to complement and facilitate each other [3]. Where some studies advocate the safety of removal of eroded gastric bands using a Single Incision Laparoscopic Surgery (SILS) [43], others urge on the use of standard endoscopy equipment for the removal of eroded gastric bands [44]. A study by Kohn GP explains the superiority of laparoscopic removal in terms of time duration from band erosion to band removal [45]. Nevertheless, physicians have yet to establish certain set guidelines identifying the superiority of one technique over the other in different situations. Due to the limitations of endoscopic and laparoscopic procedures in this regard, the role of a Bariatric surgeon becomes crucial. In certain cases, laparoscopic and endoscopic techniques may both fail in removing the gastric band in which case the role of bariatric surgeon emerges as one of utmost importance [46–48].

In our series band removal was successfully performed using our technique in 12/13 patients. CT scan and endoscopy was performed to confirm the diagnosis. We had 4 patients with adjustable gastric bands and 9 patients with non-adjustable gastric bands. Contrast upper Gastrointestinal imaging was performed in all patients after the procedure to exclude gastric leaks. Success rate with our technique was 92%. We had a failed attempt in a single patient. In this case the band had undergone significant fibrosis obscuring the buckle. In this patient laparoscopic removal was performed.

Conclusion

Abdominal pains followed by nausea and vomiting are the most common symptoms of gastric band erosion. CAT scan and endoscopy are used as the primary tools for establishment of a diagnosis. Bariatric surgeons and endoscopists should work in a closely coordinated team to address the complications of gastric band removal. Lithotripsy devices can be effectively and safely used in the retrieval of eroded gastric bands.

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Chorionic Villi Expression of Two Vascular Endothelial Growth Factor Proteins in Normal Human Pregnancy

DOI: 10.31038/IGOJ.2018132

Abstract

Introduction: Alternate splicing of vascular endothelial growth factor (VEGF)-A gene yields a number of proteins of varying amino acid lengths ranging from 121 to 206 amino acids. Recently, other isoforms of VEGF have been recognized that form from alternate splicing of the C-terminal region of exon 8 of VEGF gene. These sister isoforms are known as VEGFxxxb, where xxx denotes the number of amino acids present in the protein. During human pregnancy, VEGF165 and its receptors are recognized as master regulator of placental angiogenesis; and both VEGF165 and VEGF165b proteins are secreted by cytotrophoblasts. The present study compares the temporal expression of both isoforms of VEGF in chorionic villi tissues, throughout gestation, in normal human pregnancy.

Methods: Placentas were obtained from elective termination of pregnancy or term delivery. Tissues collected were dissected in saline to identify chorionic villi without associated decidua. VEGF protein expressions were determined by ELISA using monoclonal antibody to human VEGF165 and VEGF165b proteins as capture antibody, DY293B and DY3045, respectively (R&D Systems, Minneapolis, MN). Non-parametric test considered p<0.05 as significant.

Results: Both isoforms of VEGF were detected in 166 chorionic villi samples analyzed. The expression patterns of the two proteins differed markedly throughout gestation. While VEGF165 showed a minor dip in the second trimester, VEGF165b showed a peak during that time. The two isoforms were positively and significantly correlated in second and third trimesters of pregnancy.

Conclusions: The data reveal that both isoforms of VEGF play key roles in regulating the angiogenic balance throughout gestation in normal human pregnancy. We hypothesize that VEGF165b protein expression in placental tissues could be a physiological phenomenon to restrain overexpression of VEGF165 during placental development, which if left unchecked, could lead to pregnancy-related complications as pregnancy advanced.

Keywords

ELISA, Throughout Gestation, Uncomplicated Human Pregnancy, VEGF165, VEGF165b

Introduction

Placental angiogenesis plays a pivotal role in instituting a fetomaternal circulation and in establishing the placental villous tree that contributes to the development of the placenta throughout human pregnancy. Of the many angiogenic factors that were investigated e.g., five members of VEGF family, four members of the angiopoitin family, and one member of large ephrin family; vascular endothelial growth factor (VEGF) was recognized as the one specific for blood vessel formation [1–3]. Gene knockout studies have provided convincing evidence for a central role of VEGF in fetal and placental angiogenesis. Targeted homozygous null mutations of VEGF receptors in mice demonstrated failure in hematopoiesis, formation of blood islands and blood vessels, resulting in embryonic death by day 8 of pregnancy [4]. Carmeliet et al. further demonstrated that loss of a single VEGF allele in a mouse model led to gross developmental deformities in vessel formation that resulted in embryonic death between day 11 and 12 of mouse pregnancy. The authors concluded that not only fetal and placental angiogenesis were dependent on VEGF, but a threshold level of VEGF had to be achieved for normal vascular development to occur [5].

The VEGF A gene has eight exons. Alternate splicing of VEGF mRNA accounts for five isoforms of VEGF proteins: VEGF121, VEGF145, VEGF165, VEGF189 and VEGF206, of which VEGF165 isoform is most abundant in vivo [6]. In the past decade, the complexity of VEGF165 biology further intensified when it was found that alternate splicing of exon 8 at the C-terminal region of VEGF gene could yield yet other isoforms of VEGF. While the number of amino acids in the sister isoforms remained the same, there were however alternate open reading frames of six amino acids at the C-terminal region of the sister isoforms. The sister isoforms were identified as VEGFxxxb, xxx referring to the number of amino acids present in the protein [7]. The splicing event modifies the C-terminal region of the VEGF protein from CDKPRR (VEGFxxx) to SLTRKD (VEGFXXXb). The replacement of the six amino acids alters the tertiary structure of the newly formed protein, as the disulfide bond that was previously formed between cysteine 160 at the C-terminal region with cysteine 146 of exon 7, could no longer be formed. The terminal two arginine molecules (RR) being replaced with lysine and aspartic acid (KD) further modifies the overall charge of the protein; and replacement of proline (P) residue with arginine (R) additionally transforms the structure of the C-terminal domain [7].

In a previous study, placental expressions of VEGF165 and VEGF165b were examined in which the comparison was made between uncomplicated and complicated pregnancies at term [8]. In an earlier study, we have examined placental expression of VEGF165b throughout gestation in normal human pregnancy [9]. The two growth factors of VEGF are secreted by cytotrophoblasts; and are implicated in placental angiogenesis in human pregnancy. We, therefore, undertook the present study to simultaneously investigate the expressions of both VEGF165 and VEGF165b in placental tissues, throughout gestation, in women with uncomplicated pregnancy. Understanding the simultaneous temporal changes in these two growth factors of VEGF as placenta develops, we consider, would be extremely valuable.

Materials and Methods

The investigative protocol for the study was approved by the Human Subject Ethics Committee of the BronxCare Health System, New York, protocol # 10101304. Discarded placental tissue samples were collected within approximately 30 minutes of the procedures from normal pregnant women who underwent elective termination of pregnancy at 6 weeks to 23 weeks and 6 days; and from normal women who delivered uncomplicated singleton pregnancies at term. Placentas from missed abortion or from pregnancies complicated with diabetes, hypertension, chronic renal disease, and chronic peripheral vascular disease or with major fetal anomalies were excluded. The approved protocol allowed the collection of the following clinical information regarding the women from whom placental tissues were obtained. These included: maternal age, parity, race/ethnicity (self-reported), gestational age (as determined by ultrasound or by initial date of the last menstrual period), reason for pregnancy termination (whether elective, for maternal medical reasons or for fetal indications), and medicine(s) administered to induce termination of pregnancy. Placentas from pregnancies 7- 23 weeks and 6 days were collected from the elective termination group, and those from 37 to 42 weeks of gestation were collected from term delivery group. Placentas delivered below 37 weeks of gestation were not included because these placentas are considered as preterm.

Soon after collection, the placental tissues were processed to obtain chorionic villi samples by a method described earlier [10]. Briefly, portions of each placenta were first thoroughly washed in cold saline to remove maternal blood and were then dissected in saline to collect free floating chorionic villi that were not anchored to the basal plate nor were emerging from the chorionic plate surface vessels. Pieces of these chorionic villi samples were placed in separate tubes, each tube bearing the same ID# designated for that placenta. Tissues and clinical information were de-identified before exiting the delivery suites. The sample collection tubes were then transported to the laboratory on ice and stored at -800C until assay.

Chorionic villi VEGF165 and VEGF165b protein expressions were simultaneously determined by enzyme-linked immunoassay (ELISA) methods. The capture antibodies for the ELISA kits were monoclonal antibodies to human proteins VEGF165 (DY293B) and VEGF165b (DY3045), respectively (R&D Systems, Minneapolis, MN). Chorionic villi samples were homogenized in Reagent Diluent 2 (R&D Systems, Minneapolis, MN), and supernatants following centrifugation of the homogenate at 13000 rpm for 2 minutes were used to determine the free forms of the isoforms based on the manufacturer’s protocols. Simultaneous analysis of both VEGF isoforms meant that we carried out both VEGF165 and VEGF165b proteins assays using chorionic villi tissues samples isolated from the same placentas, the assays were not carried out using the same aliquots. A Tecan infinite 200 Pro microplate reader (Tecan Systems Inc., San Jose, CA) set at 450 nm with wavelength correction set at 540 nm was used to measure absorbance. The sensitivity of VEGF165 ELISA was 31.3 pg/ml and that of VEGF165b ELISA was 62.5 pg/ml. Intra-assay and inter-assay variations for both assay kits were between 7–10%.

Statistical Analyses

The statistical software package SPSS, version 25 (IBM Corporation, Armonk, NY) was used for statistical analyses. First, the normality of the data was statistically analyzed which showed that the data was not normally distributed. Hence, non-parametric statistics were applied. The data was grouped by trimesters and the following non-parametric tests were performed: 1) Kruskal Wallis test (an alternative test to One-Way-ANOVA) was used to explore the differences in VEGF isoform expressions among the trimester groups. (It is important to note that KW test does not provide post–hoc data the way one-way-ANOVA does). 2) The non-parametric Mann Whitney U test was applied to compare two trimester groups at a time against each other. 3) Spearman Rank correlation coefficient test was applied to summarize the strength and direction of a relationship between the variables as well as between the variables and gestational age in days. A p<0.05 was considered statistically significant.

Results

The demographic characteristics of women from whom placental samples were obtained showed comparable maternal age among the three trimester groups (28.2 ± 6.5, 28.2 ± 6.1 and 30.0 ± 6.8 years, for first, second and third trimester groups, respectively). Race/ethnicity was self-reported, and the distribution pattern was 30% Black, 60% Hispanic, 2% Caucasian and 8% of other ethnic origin. When 166 placentas were grouped by trimester the data showed that 71 placentas collected from elective termination of pregnancy were in the first trimester, with an average gestational age of 8 weeks; 33 placentas collected also from elective termination of pregnancy were in the second trimester, with an average gestational age of 16 weeks; and 62 placentas collected from term delivery were from third trimester, with an average gestational age of 39 weeks and 2 days.

In this study, non-parametric statistics were applied because the data were skewed. Kruskal Wallis test results revealed that VEGF165 protein expression was not statistically different among the three trimester groups, the expression of VEGF165b protein, however, was significantly different (p=0.0001). In (Table 1) the 25th, 50th and 75th percentile values of the two VEGF isoforms are shown. The expression patterns of the two proteins are shown graphically in (Figure 1), which depicts two entirely different expression patterns for the two isoforms of VEGF throughout gestation in normal human pregnancy. While VEGF165 protein showed a minor dip in the second trimester of pregnancy, the expression patterns of VEGF165b showed a significant peak during that time. Pairwise comparison of trimester groups using Mann Whitney U test further revealed that the expression pattern of VEGF165 was not significantly different, but VEGF165b protein expression in each trimester was significantly different from the other (p=0.0001).

Table 1. Chorionic villi VEGF Protein Expressions throughout Gestation in Normal Pregnancy

Groups

N

VEGF165 (pg/ 100 mg tissue)

VEGF165b (pg/100 mg tissue)

25th Percentile

50th Percentile

75th Percentile

25th Percentile

50th Percentile

75th Percentile

1st Trimester

71

68.00

87.45

147.30

122.06

166.51

240.00

2nd Trimester

33

50.80

68.40

148.10

297.42

428.97

529.66

3rd Trimester

62

77.25

110.95

212.80

175.09

256.52

344.98

VEGF165 : Vascular Endothelial Growth Factor165 ; VEGF165b: Vascular Endothelial Growth Factor165b;   GA: gestational age in days. The first trimester placental chorionic villi samples were from 70/7–120/7 weeks gestation, the average GA was 81/7 weeks; second trimester were from 121/7 to 236/7 weeks, the average GA was 160/7 weeks; and the third trimester term were from 370/7 to 414/7 weeks of gestation, the average GA 392/7 weeks.  Homogenized human placental chorionic villi samples were analyzed using assay kit from R&D Systems, Minneapolis, MN; to determine VEGF165 and VEGF165b protein expressions.  Data were not normally distributed hence non-parametric statistics were used and 25th, 50th and 75th percentile values of VEGF165 and VEGF165b are shown.

IGOJ 2018-113 - Jayasri Basu USA_F1

Figure 1. GA: gestational age.

The first trimester placental chorionic villi samples were from 70/7-120/7 weeks gestation, the average GA was 81/7 weeks; second trimester were from 121/7 to 236/7 weeks, the average GA was 160/7 weeks; and the third trimester term were from 370/7 to 414/7 weeks of gestation, the average GA 392/7 weeks.

Correlation between the two proteins of VEGF as well as between the isoforms of VEGF and gestational age were examined in this study. Spearman’s correlation test results revealed that expression of VEGF165b protein was significantly and positively correlated with gestational age in days in the first trimester of normal pregnancy (r= +0.299, p=0.011, (Table 2). The table also depicts the test results of Spearman’s correlation between the two isoforms. The data reveal a significant positive correlation in the second (r = +0.376, p=0.031) and third trimester (r=+0.271, p=0.033), of normal pregnancy.

Table 2. Correlation between VEGF165, VEGF165b and Gestational Age in Days throughout gestation

VEGF165

VEGF165b

First Trimester
(N=71)

VEGF165

Correlation
Coefficient
Sig. (2-tailed)

1.000

0.070
0.564

GA

Correlation
Coefficient
Sig. (2-tailed)

0.029
0.159

0.299*
0.011

SecondTrimester
(N=33)

VEGF165

Correlation
Coefficient
Sig. (2-tailed)

1.000

0.376*
0.031

GA

Correlation
Coefficient
Sig. (2-tailed)

-0.149
0.409

0.013
0.944

Third Trimester
(N=62)

VEGF165

Correlation
Coefficient
Sig. (2-tailed)

1.000
0.916

0.271*
0.033

GA

Correlation
Coefficient
Sig. (2-tailed)

-0.027
0.833

-0.112
0.385

VEGF165 : Vascular Endothelial Growth Factor165 ; VEGF165b: Vascular Endothelial Growth Factor165b; GA: gestational age in days. Data were not normally distributed hence Spearman’s correlation was applied. Significant positive correlation was seen between VEGF165b and GA in the first trimester of pregnancy.  The two VEGF proteins were significantly and positively correlated in the second and third trimester of normal pregnancy.

Conclusion

In this study, chorionic villi samples were isolated from each placenta, and VEGF165 and VEGF165b protein expressions were determined by ELISA that used monoclonal antibody to human VEGF165 or VEGF165b protein as the capture antibody in each case. VEGF protein expression and that of its receptors are closely regulated to times of vasculogenesis and angiogenesis [11]. In this study, both isoforms of VEGF proteins were identified in all 166 chorionic villi samples that were analyzed suggesting that both proteins of VEGF may exert regulatory roles in pregnancy-linked angiogenesis in normal human pregnancy.

The angiogenic potential of VEGF165 has been confirmed by several bioassays that measured VEGF receptor expression during embryogenesis and tissue repair [11], capillary growth in vivo in developing chick chorioallantonic membrane [12], and a temporal and spatial correlation between VEGF and ocular angiogenesis in a primate model [13]. These studies affirmed VEGF signaling pathways to be the master regulator of angiogenesis [11–14]. The pro-angiogenic potential of VEGF165 results from six amino acids at its C-terminus e.g., CDKPRR [7, 15]. Involvement of VEGF165 in human pregnancy, particularly in placental development has also been recognized. Reports on the placental expressions of VEGF165 protein state that the protein increases in first 10 weeks of normal pregnancy, but as pregnancy advances, VEGF and its receptor VEGFR-2 concentrations decline. In these reports, VEGF was suggested to increase vascular permeability, vasodilation and angiogenesis [16–18]. It is also suggested to be involved in the formation and maintenance of the trophoblastic plugs that block the spiral arteries in the first trimester of normal pregnancy [19]. Figure 1 depicts that VEGF165 protein expression is somewhat lower in the second trimester of normal pregnancy. This data is consistent with the finding of an immunohistochemical study that reported VEGF165 antigen staining to be weaker in mid-gestational placental tissues, compared to the intensity of staining for the protein in the first or third trimester placental samples [20].

The isoform VEGF165b has not been investigated in as much detail as VEGF165. VEGF165b protein is expressed in normal tissues of lung, pancreas, colon, skin and brain [7, 21, 22]. Reports reveal that VEGF165b constitute 50% or more of total VEGF expressed in most non-angiogenic tissue [23]. Investigators have alleged that VEGF165b is not pro-angiogenic in vivo [23] and it actively inhibits VEGF165 mediated endothelial cell proliferation and migration in vitro [4, 7, 21–23]. VEGF165b is further reported to inhibit physiological angiogenesis in developing mammary tissue, in transgenic animals, overexpressing VEGF165b [24]. Moreover, VEGF165b is reported to inhibit angiogenesis in vivo in six other different tumor models [21–25]. The results of the present study on chorionic villi VEGF165b protein expression throughout gestation in normal human pregnancy are similar to the results we have reported earlier [9]. Our studies underscore the importance of VEGF165b antigen in normal human pregnancy. Identification of VEGF165b protein in this study, in all 166 chorionic villi samples that were collected throughout all trimesters of human pregnancy validates this notion. This temporal variation in VEGF165b protein in human pregnancy perhaps reflects that VEGF165b protein expression is more stringently controlled at each phase of human gestation, and that the isoform may play a more active role in placental development. The spatial activation of VEGF165b protein at various phases of human gestation as seen in the study emphasizes that placental angiogenesis could be more dependent on VEGF165b isoform.

VEGFR-2 is the predominant signaling receptor for VEGF-mediated angiogenesis [26]. Phosphorylation of VEGFR-2 on tyrosine residue at 1052 position of the molecule generates a highly dynamic and complex signaling system that triggers angiogenic response [4, 21, 26]. The binding affinities of VEGFR-2 are identical between VEGF165 and VEGF165b isoforms. However, VEGF165b is less efficient than VEGF165 in inducing phosphorylation on Y1052 residue [27]. The lower ability of VEGF165b to induce VEGFR2 phosphorylation on Y1052 is due to its incapacity to induce optimal rotation of the intracellular domain of the receptor molecule that is required for phosphorylation to occur [27]. Reduced Y1052 phosphorylation induces rapid inactivation of the kinase domains of the receptors resulting in weak activation of the signaling pathways [23]. Recent studies have shown that VEGF165b can stimulate VEGFR-2, ERK1/2 and Akt phosphorylation in endothelial cells, however, the induced phosphorylation is weaker than that promoted by VEGF165 [21, 28]. VEGF165b is currently not considered as anti-angiogenic but as a weakly angiogenic form of VEGF [28].

Establishment of functional fetal and placental circulations is some of the earliest strategic events during embryonic/placental development [29, 30]. The significant positive correlation seen between chorionic villi VEGF165b protein expression and gestational age in first trimester of pregnancy in this study (Table 2) suggests that VEGF165b may contribute to the development of villous vascular network in early pregnancy. An increase in expression of plasma VEGF165b protein in first trimester of normal pregnancy has been reported by other investigators, and the authors suggest that failure in the upregulation of VEGF165b protein in the plasma in the first trimester of pregnancy was a predictive marker of preeclampsia [31]. Simultaneous increase of both isoforms of VEGF in human placental tissues in the third trimester of pregnancy has also been reported by other investigators [8]. The large increase in transplacental exchange which supports the exponential increase in fetal growth and uterine blood flow during the last half of gestation is suggested to depend primarily on the dramatic growth of placental vascular beds [32]. Vascular density of the placental cotyledons remains relatively constant throughout mid-gestation and increases dramatically during the last third of gestation in association with dramatic fetal growth [32, 33]. In third trimester of normal pregnancy, both fetal development and demands are at its peak; and our data show a positive correlation between VEGF165 and VEGF165b during this time. Hence, it may be suggested that angiogenic modification of placental vasculature that allows maximum blood to flow through may depend on the synergistic action of both isoforms of VEGF.

Our present findings suggest a notable difference between tumor and placental angiogenesis. It is widely reported that in human tumors, up-regulation of VEGF165 protein occurs with a proportional drop in VEGF165b levels; indicating that in tumor angiogenesis the balance between the two isoforms of VEGF is lost [34]. However, the findings of the present study suggest that gestational age-specific expression of both VEGF isoforms is necessary for optimal placental angiogenesis and growth to results in a successful viable outcome.

That hypoxic environment favors VEGF165-induced angiogenesis and tumorigenic growth is well known [35]. In an earlier study we have demonstrated that placental environment switches from a hypoxic to a normoxic environment beyond the first trimester of normal human pregnancy [36]. We hypothesize that perhaps during this transitional period from first to second trimester of normal pregnancy, placental oxidative environment per se regulate the alternate splicing of exon 8 of the VEGF gene. In hypoxic condition proximal splicing of exon 8 may be favored, which up-regulates the expression of VEGF165 protein. In a normoxic state, distal splicing of exon 8 may be favored, whereby the expression of VEGF165b protein gets up-regulated. That switch in the partial pressure of oxygen at the end of first trimester of normal pregnancy can modify the expression pattern of two proteins has been reported earlier, between placental derived growth factor and VEGF [37]. It is feasible that this switch in the spliceosome at the end of the first trimester may be the contributing factor that may have resulted in the peak VEGF165b protein expression seen in the second trimester of this study. It is likely that this dependence of placental growth on VEGF165b beyond the first trimester could be a physiological phenomenon to restrain any overexpression of VEGF165 which if left un-checked, could perhaps lead to pregnancy-related complications as pregnancy advanced.

The limitation of our study is that we have not confirmed our data by western blot, polymerase chain reaction or by immunohistochemical methods. We acknowledge that cells respond to extracellular stimuli through a series of signaling cascades. When a receptor is activated, varieties of proteins are recruited to interact with each other to generate a cascade of sequential steps that result in a biological effect [38]. Signaling molecules are common to several pathways and often form a complex intracellular network [38]. Yet, in this study we have not examined other isoforms of VEGF, their receptors or other factors that may have also been involved in placental angiogenesis. We believe that addition of all these factors would have made the study more complex and any conclusions drawn from the study would have been extremely difficult to interpret. The strength of our study is our relatively larger sample size in each of the trimester groups. Simultaneous expressions of these two important VEGF proteins in human chorionic villi tissue throughout gestation in normal human pregnancy have not been reported before. In this study, stringent ELISA methods using monoclonal antibodies to human VEGF165 and VEGF165b human proteins were used as capture antibodies. The manufacturer claims that DY293B capture antibody that was used does not cross react with placental growth factor (PIGF), VEGF-B167, VEGF-B186, VEGF-C, VEGF-D, recombinant (rh) human VEGF R1(Flt-1)/Fc Chimera or with rhVEGF R2(KDR)/Fc Chimeras. Similarly, the monoclonal capture antibody DY3045 does not cross react with rhhuman VEGF121, VEGF162, VEGF165, rhVEGF R3/Fc, VEGF R1/Fc or VEGF R-2/Fc Chimeras. Furthermore, the detection methods used were sensitive, allowing the detection of VEGF165 as low as 31.2 and VEGF165b as low as 62.5 pg/ml, respectively. The study underscores the importance of VEGF165b in placental angiogenesis in normal human pregnancy. We speculate that placental oxidative environment might influence the C-terminal VEGF angiogenic switch, whereby different VEGF variants could be formed from a single VEGF gene; that play key roles in regulating angiogenic balance during normal human pregnancy. It may be suggested that gestational age-specific expression of both VEGF165 and VEGF165b isoforms may be necessary for a successful viable outcome.

Acknowledgement

The authors would like to thank Dr. CM Salafia for her guidance for the study, and the staff members of the Department of Obstetrics & Gynecology at BronxCare Health System for their support. For this research, outside grants were not sought. The study was funded by the Residency Program of the BronxCare Health System.

Declaration of Interest: The authors declare no conflicts of interest with respect to the research, authorship and/or publication of this article.

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