DOI: 10.31038/EDMJ.20261041

Abstract

A 33-year-old woman presented to the emergency department after generalized seizure activity and was found to have profound hypotonic hyponatremia (serum sodium 107 mmol/L; serum osmolality 219 mOsm/kg). After initial stabilization, focused history-taking revealed approximately 10 years of secondary amenorrhea beginning after her second childbirth, together with chronic fatigue, weakness and reduced exercise tolerance. Morning cortisol was markedly low at 0.70 µg/dL; free T4 was low with a non-elevated TSH, and LH and FSH were low, indicating deficiencies across multiple anterior pituitary axes. MRI showed a partially empty sella without an acute intracranial lesion. The patient received controlled 3% hypertonic saline and intravenous hydrocortisone, followed by hydrocortisone and levothyroxine replacement. Neurological status improved, serum sodium normalized during hospitalization, and no further seizures were reported. The case highlights the emergency value of integrating electrolyte abnormalities with menstrual and postpartum history and early assessment of cortisol and pituitary function.

Keywords

Hyponatremia, Seizure, Hypopituitarism, Secondary adrenal insufficiency, Central hypothyroidism, Amenorrhea, Empty sella, Emergency medicine

Learning Points

• Severe symptomatic hypotonic hyponatremia requires immediate, controlled treatment while the underlying cause is investigated in parallel.
• Secondary adrenal insufficiency is an important reversible cause of severe hypotonic hyponatremia and may present with hypotension and nonspecific constitutional symptoms.
• A low free T4 with a low, normal or mildly elevated TSH can indicate central hypothyroidism when interpreted in the setting of pituitary disease.
• Long-standing secondary amenorrhea may provide an important historical clue to chronic gonadotropin deficiency and broader pituitary dysfunction.
• A partially empty sella is a radiological finding rather than, by itself, proof of hypopituitarism or its cause; imaging must be correlated with clinical and biochemical findings.
• When central adrenal insufficiency is suspected with central hypothyroidism, glucocorticoid replacement should be addressed before or together with thyroid hormone replacement.

Background

Severe hyponatremia is a time-critical emergency because marked reductions in plasma sodium can cause cerebral dysfunction, seizures and coma. In patients with neurological manifestations, stabilization and etiological evaluation should proceed simultaneously. Endocrine causes, particularly glucocorticoid deficiency related to hypothalamic or pituitary disease, are important reversible contributors and may be missed when chronic symptoms are nonspecific [1-3].

Pituitary dysfunction can evolve over years, with fatigue, weakness, menstrual disturbance and reduced exercise tolerance preceding recognition of the disorder. Empty or partially empty sella may be incidental, but clinically relevant pituitary hormone deficiencies occur in a subset of patients and require biochemical assessment [4,5]. This case is reported because an acute seizure-associated electrolyte emergency led to recognition of previously unintegrated chronic endocrine symptoms and abnormalities involving several anterior pituitary axes.

Case Presentation

Emergency Presentation

A 33-year-old woman presented following generalized seizure activity characterized by loss of consciousness, upward eye deviation, teeth clenching and stiffening of all four limbs, followed by postictal drowsiness. The available clinical documentation described recurrent vomiting and several seizure episodes over approximately two days. One radiology referral described an episode lasting approximately 30 minutes, whereas the clinical record described the principal generalized seizure as approximately three minutes; therefore, the event is not classified as status epilepticus in this report.

There was no documented history of head trauma, fever or central nervous system infection. The available record did not document headache, focal neurological deficit, sensory symptoms or other acute intracranial symptoms. She had a history of hypothyroidism and was taking levothyroxine 25 µg daily.

Initial Emergency Assessment

On arrival, she was postictal but arousable. Blood pressure was 92/60 mmHg, heart rate 96 beats/min, respiratory rate 18/min, temperature 36.7°C and oxygen saturation 98% on room air. No focal neurological deficit was documented.

Laboratory Evaluation

Initial testing demonstrated serum sodium of 107 mmol/L and serum osmolality of 219 mOsm/kg, confirming profound hypotonic hyponatremia. Serum potassium was 3.33 mmol/L and blood glucose was 86 mg/dL. Urine osmolality was 209 mOsm/kg and urine sodium was 67.6 mmol/L. Non-contrast CT of the brain showed no acute intracranial pathology (Table 1).

Table 1: Initial emergency laboratory investigations.

Endocrine Clues and Diagnostic Work-up

After immediate stabilization, a more detailed history changed the diagnostic direction. Menstrual cycles had previously been regular but stopped approximately 10 years earlier, after her second full-term vaginal delivery at 23 years of age. The prolonged amenorrhea had not previously been evaluated. She also reported longstanding generalized weakness, chronic fatigue, reduced exercise tolerance and intermittent dizziness.

Endocrine investigations demonstrated a morning cortisol concentration of 0.70 µg/dL, low free T4, TSH 1.37 mIU/L, and low LH and FSH. A morning cortisol below 3 µg/dL is strongly suggestive of central adrenal insufficiency in the appropriate clinical setting, and a low free T4 with a low, normal or mildly elevated TSH supports central hypothyroidism when pituitary disease is present [3]. The combination of marked cortisol deficiency, low free T4 with a non-elevated TSH, and low gonadotropins therefore supported multiple anterior pituitary-axis deficiencies. Growth hormone/IGF-1 and prolactin were not available in the supplied record; consequently, the term “panhypopituitarism” is not used as a definitive diagnosis (Table 2).

Table 2: Endocrine investigations demonstrating abnormalities in multiple anterior pituitary axes.

Imaging

Brain MRI performed on 8 April 2026 showed no obvious focal intracranial lesion, acute infarction or hemorrhage. The report described bilateral prominent preoptic cerebrospinal-fluid sleeves with a partially empty sella and recommended ophthalmic examination to assess for papilledema. That recommendation is reproduced from the radiology report; no diagnosis of papilledema or raised intracranial pressure is made because an ophthalmic examination result was not available in the supplied record (Figures 1-3).

Figure 1: De-identified MRI brain report dated 8 April 2026. The report describes a partially empty sella with prominent bilateral preoptic CSF sleeves and recommends ophthalmic assessment for papilledema. Patient and radiologist identifiers are obscured.

Figure 2: Representative non-contrast CT brain images from the presenting evaluation. No obvious acute intracranial hemorrhage, large territorial infarction or major mass effect is evident on the supplied images.

Figure 3: Emergency diagnostic pathway emphasizing simultaneous treatment of severe symptomatic hyponatremia and evaluation for endocrine causes.

Emergency Management

The immediate priority was treatment of acute symptomatic severe hyponatremia. Controlled administration of 3% hypertonic saline was undertaken with close monitoring of serum sodium and attention to avoiding overly rapid correction, consistent with guideline-based emergency management of severe symptomatic hyponatremia [1,2]. Given the hypotension and subsequently documented severe cortisol deficiency, intravenous hydrocortisone 100 mg was administered. After stabilization, hydrocortisone and levothyroxine replacement were continued. In suspected central adrenal insufficiency, glucocorticoid treatment should precede or accompany thyroid hormone replacement because thyroid hormone can increase cortisol requirements and may precipitate adrenal decompensation [3].

Clinical Outcome and Follow-up

The patient’s neurological status improved after correction of severe hyponatremia and corticosteroid therapy. Serum sodium gradually returned toward the normal range during hospitalization, and no further seizures were reported. She was discharged in stable condition with endocrine follow-up and hormone replacement therapy.

Discussion

This case illustrates an important emergency–endocrine overlap: the seizure was the acute event that brought the patient to medical attention, while the biochemical pattern pointed toward chronic pituitary dysfunction. At a serum sodium concentration of 107 mmol/L, severe hypotonic hyponatremia can produce major neurological manifestations. Current guidance emphasizes prompt hypertonic saline therapy for severe symptoms, together with close biochemical and clinical monitoring [1,2].

The markedly low morning cortisol is a central diagnostic finding. Glucocorticoid deficiency can promote non-osmotic antidiuretic hormone activity and reduce renal free-water excretion, thereby causing or aggravating hypotonic hyponatremia [1,3]. In this patient, the low cortisol occurred alongside hypotension and years of constitutional symptoms, strengthening the clinical relevance of the result.

The thyroid profile also required a pituitary-oriented interpretation. A TSH value within the laboratory reference interval does not exclude central hypothyroidism when free T4 is low. Endocrine Society guidance recommends interpreting free T4 and TSH together in patients with suspected pituitary disease [3]. Here, low free T4 was accompanied by cortisol and gonadotropin abnormalities, making a central process clinically plausible.

The decade-long secondary amenorrhea was the most useful historical clue to chronic gonadotropin-axis dysfunction. Its onset after childbirth raises the possibility of postpartum pituitary injury, including Sheehan syndrome. However, the supplied record does not document postpartum hemorrhage, severe postpartum hypotension, shock or failure of lactation. Because severe postpartum hemorrhage is a defining historical feature of classic Sheehan syndrome, a specific postpartum etiology cannot be established from the available evidence [6].

The partially empty sella provides structural support for a pituitary disorder but is not, by itself, diagnostic of hypopituitarism or its cause. Reviews emphasize that empty sella can be an incidental radiological finding, while a subset of patients has clinically significant endocrine dysfunction [4,5]. In this patient, the imaging finding becomes more relevant because it coincides with biochemical abnormalities in several anterior pituitary axes and a long history of endocrine symptoms.

From an emergency-medicine perspective, the key lesson is not to stop the diagnostic process once a seizure has been recognized. Severe hyponatremia requires immediate treatment, but its cause must be pursued in parallel. A brief review of menstrual history, postpartum events, chronic fatigue, orthostatic symptoms and previous endocrine treatment can substantially redirect the differential diagnosis. In this case, focused history-taking connected a decade of amenorrhea with the acute electrolyte emergency and prompted targeted pituitary evaluation.

Conclusion

A neurological emergency may be the first presentation of a chronic endocrine disorder. In this patient, profound hypotonic hyponatremia with seizure led to identification of marked cortisol deficiency, central thyroid-axis dysfunction and gonadotropin deficiency, with a partially empty sella on MRI. For emergency and endocrine clinicians, severe unexplained hyponatremia accompanied by hypotension, chronic fatigue or longstanding amenorrhea should prompt early assessment of the hypothalamic–pituitary–adrenal axis and other pituitary hormones. Careful history-taking remains a high-yield diagnostic intervention.

Declarations

Consent for Publication: Taken.

Ethics Approval: Not applicable for this case report, as stated in the supplied manuscript record.

Competing Interests: The authors declare that they have no competing interests.

Funding: No specific funding was received for this case report.

Author Contributions: Dr. Minakshee Patil: clinical care, data collection and manuscript preparation; Dr. Jagdish Patil: clinical interpretation; Dr. Sanyogita Naik: manuscript preparation; Dr. Sarbari Swaika: supervision and final approval.

Data Availability: Only de-identified information is presented in the manuscript. The data supporting this case are contained within the clinical record and are not reproduced in identifiable form.

AI-assisted Writing: Generative AI was used as a language and formatting aid during manuscript preparation. The authors remain responsible for verifying all clinical facts, interpretations, references and final wording before submission.

References

1. Sterns RH (2015) Disorders of plasma sodium—causes, consequences, and correction. N Engl J Med. [crossref]
2. Spasovski G, Vanholder R, Allolio B, et al. (2014) Clinical practice guideline on diagnosis and treatment of hyponatraemia. Eur J Endocrinol. [crossref]
3. Fleseriu M, Hashim IA, Karavitaki N, et al. (2016) Hormonal replacement in hypopituitarism in adults: an Endocrine Society clinical practice guideline. J Clin Endocrinol Metab. [crossref]
4. Lundholm MD, Yogi-Morren D (2024) A comprehensive review of empty sella and empty sella syndrome. Endocr Pract. [crossref]
5. Padovano Sorrentino F, Chiloiro S, Giampietro A, et al. (2024) Empty sella syndrome: an update. Pituitary. [crossref]
6. Diri H, Karaca Z, Tanriverdi F, Unluhizarci K, Kelestimur F (2016) Sheehan’s syndrome: new insights into an old disease. Endocrine. [crossref]

DOI: 10.31038/EDMJ.20261033

Introduction

Dementia occurrences increase significantly in older adult type 2 diabetes mellitus (T2DM) [1]., Epidemiologic studies indicate diabetes confers an ~ 1.5-fold increased risk of non-vascular dementia and a ~ 2-fold risk for vascular dementia occurrence compared to rates in age-matched non-diabetic populations [1] Because of the current world-wide epidemic of type 2 diabetes mellitus, diabetic dementia occurrences are expected to increase substantially in the coming decades. There is a need for better understanding of the underlying disease mechanisms including potentially modifiable risk factors to reduce the future medical, social and economic burdens of diabetic dementia.

Recent evidence (from autopsy studies) suggests that the underlying causes of ‘diabetic dementia’ likely differ from those in Alzheimer’s dementia (AD) [2] For example, the characteristic neuropathologic lesions found in AD brain (amyloid plaque, neurofibrillary tangles) were not evident at increased rate in brain autopsy specimens from diabetic dementia vs non-diabetic population [2]. Cerebrovascular burden was increased in diabetic dementia brain autopsy specimens including lacunar infarct, but not microinfarct or large vessel infarct [2]. Arteriolosclerosis of deep penetrating vessels was also a significant finding reported in diabetic dementia brain autopsy specimens [2].

Genome-wide association studies in dementia related to Alzheimer’s dementia have identified gene pathways involving immune function, extracellular matrix remodeling, lipoprotein metabolism and intracellular catabolism [3] as comprising several of the many (>100) genetic risk determinants. Several forms of dementia (including Alzheimer’s dementia) have a high genetic hereditability, but dementia is a complex trait and only a fraction of the overall hereditable variability could be accounted in GWAS studies [3]. Glaucoma is another late-onset neurodegenerative disease in which gene pathways involving extracellular matrix remodeling and vascular development contribute in part to overall genetic risk [4]. Yet only 9-17% of overall hereditability in late-onset glaucoma was accounted for in GWAS studies [4]. In addition, the inclusion in GWAS studies of glaucoma of multi-ethnic populations showed that different genes were significant risk determinants in different ethnic/racial populations [5].

Regarding the unknown mechanisms underlying diabetic dementia, the apoE4 gene isoform is associated with characteristic AD-like neuropathology and confers a large population attributable risk of AD [6]. Insulin resistance has been proposed to play a role in diabetic dementia, but the precise mechanisms are not well understood [7]. Taken together, novel ‘non-AD’ disease mechanisms are likely to be multifactorial and may overlap (in part) both with vascular dementia and Alzheimer’s dementia. The aim of the current review article is to summarize our prior findings from separate reports (largely from type 2 diabetic patients) having a late-onset neurodegenerative disorder, i.e. primary open angle glaucoma, or diabetic dementia, that support roles for dysregulated immunity, chronic inflammation and endothelial dysfunction in the mechanism of non-AD diabetic dementia.

One of the mediators of endothelial dysfunction and neurotoxicity in late-onset neurodegenerative diseases (occurring in subsets of diabetes) was a novel kind of anti-heparan sulfate proteoglycan autoantibody which not only interfered with the neurotrophic (and endothelial cell survival-promoting) effects of growth factors such as fibroblast growth factor 2 (FGF2) [8], but also appeared to bind to a G-protein coupled receptor, serotonin 2A receptor, causing long-lasting activation of PLC gamma/IP3R/Ca2+ intracellular signaling in neurons and endothelial cells [9]. Our studies support intracellular calcium dysregulation as another highly relevant mechanism in diabetes-associated neurodegenerative disease pathophysiology. G-protein receptor coupled signaling is pleiotropic and in electrically-excitable cells such as neurons [10], anti-HSPG autoantibodies evoked sustained depolarization as well as actin cytoskeleton rearrangement via RhoA/ROCK activation altering morphology in neurites (causing neurite retraction) and in glial cells (causing process withdrawal) [8].

In both type 1 and type 2 diabetes mellitus, loss of heparan sulfate proteoglycan (HSPG) is an early event underlying diffuse vascular injury, an idea formulated by the Steno Group and referred to as the ‘Steno hypothesis’ [11]. We later advanced the concept that chronic inflammation associated with obese type 2 diabetes mellitus or in systemic autoimmunity (type 1 DM) drives humoral immune response(s) to HSPG promoting formation of (endothelial and neuro-toxic) anti-HSPG IgG autoantibodies which we reported to be increased in association with a diverse array of diabetic microvascular complications including: retinopathy [12], nephropathy [13,14], and painful neuropathy [8]. In our prior study of anti-HSPG autoantibodies in painful diabetic neuropathy, we observed that patients suffering with painful neuropathy were significantly more likely to have co-morbid late-onset primary open angle glaucoma (POAG) [15], the latter also increases in type 2 DM. Both POAG and painful neuropathy involve damage to small unmyelinated axons, retinal ganglion cells in the case of POAG. Tezel et al. [16] reported increased circulating autoantibodies to optic nerve head heparan sulfate proteoglycans (HSPG) in subsets of normal tension or POAG. In our studies of diabetic microvascular complications, anti-HSPG AAB caused sustained activation of Rho A/Rho kinase signaling pathway in endothelial cells and neuronal cells [8,13]. Rho kinase activation affects endothelial-like trabecular meshwork cells which play a role [17] in mediating intraocular pressure changes which are an important determinant of glaucoma risk. Taken together, these findings, led us to undertake a small study in forty-four older adult type 2 diabetic patients (with and without POAG) to test for increased level of circulating anti-HSPG AAB [15].

Endothelial cell inhibitory activity in the protein A-eluate fraction was significantly increased in plasma from glaucoma or suspects (vs no glaucoma) (Table 1). There was a significant association between co-morbid painful neuropathy and glaucoma or glaucoma suspect (vs no glaucoma) (Table 1). Glaucoma patients were significantly more likely to have reported a family history of dementia in a first-degree relative compared to no glaucoma patients (Table 1). The latter observation led us to consider whether a component of the autoantibody encoded by a heavy chain, or light chain gene might contribute to a part of the unexplained hereditability in occurrences of late-onset POAG.

Table 1: Association between diabetic glaucoma or suspect and co-morbid microvascular complications or family history of dementia.

Results are proportion; (), N number of patients tested; Glc, glaucoma, susp, suspect, *Fischer’s exact test, comparison of diabetics with or without glaucoma; ME, macular edema, AMD, age-related macular degeneration, FH, family history in a 1° relative of Alzheimer’s dementia; significant inhibitory endothelial cell (EC) activity, defined as ≤90% of basal cell number after 48 hours incubation at 37 deg C in the presence of test fractions, i.e. a ~30 ug/mL concentration of the protein-A eluate. Reproduced from [15].

We first reported an unusually potent EC-inhibitory fragment having peak apparent MW 12.5kD [8] (Figure 1D) in the protein-A eluate from two prostate cancer patients’ plasma. The activity appeared following long-term storage of protein-A eluates (at 0-4 deg C). We used a combination of heparin Sepharose, and hydroxyapatite affinity chromatography to obtain N-terminal amino acid microsequencing data showing that peak active EC-inhibitory activity had significant 87% amino acid homology to a human kappa V variable light chain [8].

Peak inhibitory activity in the protein-A eluates from both diabetic and prostate cancer patients (Figure 1A-C) had apparent MW corresponding to light chain 23kD or half-light chains 12 kD [8]. Peak prostate cancer IgG EC toxicity was substantially enhanced after heating at 56 deg x 30 minutes (Figure 1E) [8] consistent with aggregation (in light chain fragment).

Figure 1: Mass spectrometry of diabetic (A and B) or prostate cancer (C) endothelial cell inhibitory autoantibodies; (D) increase in potency of inhibitory prostate cancer autoantibodies after long-term storage of protein-A eluates at 0-4 deg C and (E) unusual stability of the long-lasting antibodies. (A–C) Samples were analyzed by mass spectrometry as previously reported [8]. (D and E) Endothelial cell activity (mean, SD) is expressed as % of basal cell number in quadruplicate assays as previously reported [8].

Since prostate cancer cells elaborate proteases such as furin [18] which activate other proteases [19,20] that can degrade extracellular matrix we next tested whether exposure of protein A-eluates to recombinant furin could reproduce the effects of long-term eluate storage. Furin is a proprotein convertase which cleaves proteins at specific multi-basic amino acid residues [21] including those present in the variable-constant switch region in a limited subclass of kappa light chains. In a small sample of five diabetic dementia patients (mean age 71 ± 5.8 years; mean diabetes duration 22.4 ± 12.6 years) long-term storage of diabetic dementia protein-A eluates (at 0-4 deg C) unmasked potent EC inhibitory activity (Table 2) [15], but not in the protein-A eluates from an age-matched subset of diabetes having macular edema and/or nephropathy without dementia (Table 2). Brief exposure of the (freshly-isolated) protein-A eluate of a diabetic dementia, POAG patient sample to recombinant furin caused similar unmasking of latent potent EC inhibitory activity (Table 2) [15] perhaps consistent with cleavage at a furin-susceptible multi-basic peptide region in the mechanism of generation of highly potent EC inhibitory activity.

Table 2: Effect of long-term storage of protein A eluates or furin digestion o EC activity.

Reproduced from [15]. Furin digestion of protein-A eluate fraction was carried out as previously described [15]. Endothelial cell (EC) survival (mean, SD) was determined in quadruplicate after 48 hours incubation at 37 deg C with test fractions and is compared to survival (in EC) without added test fractions (100%)

Light chain amyloidosis is a systemic disease caused by overproduction of a monoclonal light chain or fragment by a plasma cell clone. Peripheral nerve, heart and kidney are among the most common sites of deposition of monoclonal light chains in AL disease. We observed that a patient manifesting co-occurrence of atrial fibrillation, nephropathy and a family history of dementia harbored an IgG in the protein-A eluate fraction susceptible to furin digestion resulting in a highly EC toxic fragment (Table 3). In three age-matched type 2 DM patients with atrial fibrillation alone, exposure of the protein-A eluates to furin did not generate a toxic EC fragment (Table 3). These data suggest that in diabetic patients with a family history of dementia exposure of the protein-A eluate of plasma to recombinant furin might be a simple method to identify patients at high risk for harboring a latent highly EC toxic light chain fragment capable of contributing to the later development of diabetic dementia.

Table 3: Effect of furin treatment in diabetic depression protein A eluates on endothelial cell inhibitory activity.

Endothelial cell activity (%)- endothelial cell number was determined after 48 hours incubation (at 37 deg C) with a 30 ug/mL concentration of the protein-A eluate fraction of plasma: Results (mean, SD) is expressed as a percent of basal cell number in bovine pulmonary artery endothelial cells not exposed to test fractions.
*Comparing (mean, SD) EC activity in quadruplicate samples before and after exposure to recombinant furin as previously reported [15].

It is known that kappa light chains comprise functional antibodies [22] and that certain kappa light chain genes cluster in populations that have been exposed to similar kinds of evolutionary selection pressure. For example, subclasses of kappa II gene-encoded light chains are involved in functional antibodies providing protective immunity against the life-threatening childhood infection, Hemophilus influenza meningitis [23]. Yet because these light chains harbor a furin-targetable multi-basic amino acid sequence in the variable-joining switch region, they could contribute to late-onset neurodegenerative disease (which we hypothesize) may be due in part to unusually potent toxicity in a light chain fragment.

There have been several recent reports that plasma furin was increased significantly in adult type 2 DM and was associated with an increased risk of dysmetabolism and cardiovascular morbidity and mortality risk [24,25]. Among its many potential protein targets, furin is known to inactivate endothelial and hepatic lipoprotein lipase [26] which are responsible for normal metabolism of very low-density (triglyceride-rich) lipoproteins (vLDL). Diabetic dyslipidemia is characterized by increased plasma concentration of triglyceride TG-rich lipoproteins and it is an important risk factor accounting for substantial excess CVD morbidity in adult type 2 DM. Hepatic production of TG-rich very low-density lipoproteins (VLDL) increases as a result of either insulin deficiency and/or insulin resistance, and vLDL metabolism is affected by Apo C I a recently reported genetic risk determinant in Alzheimer’s related dementia [3]. It is of interest that the metabolism of TG-rich lipoprotein was reported to increase blood brain barrier BBB permeability and contribute to neuroinflammation which are hallmarks of Alzheimer’s related dementia [27]. Taken together, diabetes associated increases in plasma furin might have a dual role in generating toxic LC fragments which can circulate in VLDL particles and contribute to enhanced endothelial cell toxicity and neurotoxicity underlying dementia.

We had previously shown that highly neurotoxic IgG fragments from three different diabetic dyslipidemia patients with dementia (mean plasma cholesterol 181 ± 51 mg/dL: and mean plasma triglyceride concentration 392 ± 164 mg/dL) displayed increased affinity for a Liposorber (dextran sulfate) column useful in lowering plasma concentrations of LDL and vLDL apolipoprotein B-100-containing particles [28]. In one of the three patients tested (Patient 5, Table 4), in whom the peak toxicity was purified to near homogeneity, N terminal amino acid sequencing revealed significant homology to a restricted subclass of kappa II light chain [28]. Peak EC toxicity in the highly-purified DM k light chain (Patient 5) (induced 70% EC apoptosis after 48hrs incubation at 37 deg C) which was nearly as potent as toxicity in an amyloidogenic monoclonal lambda light chain secreted by lymphoblasts obtained from a patient who had died from amyloid light chain (ALL) disease [28]. Patient 5 suffered from a constellation of systemic and neurovascular complications including multiple small and medium-sized cerebrovascular infarctions and died from complications related to ESRD about ten years after study entry and blood drawing for autoantibody determination [28].

Table 4: Clinical Characteristics in Five Diabetic Dementia Patients Harboring Toxic LC.

NHW-non: Hispanic white; AD: Alzheimer’s dementia; OHT: Ocular hypertension, TBI: Traumatic brain injury; FT: Frontotemporal; Vasc: Vascular; DMT: Dementia

There is no data to indicate whether or not toxic kappa LC may form insoluble, amyloid precipitates in target organs as occurs in AL disease. Heating may have induced aggregation in kappa LC half chains to form toxic soluble oligomers. Whether toxic oligomers can form under physiological conditions is not known. Light chains are comprised of a high percentage of hydrophobic amino acid residues which normally form tightly-packed beta sheets. Since vLDL particles are amphipathic, it is possible that light chains or fragments coalesce around the lipid-rich microenvironment of the particle which favors hydrophobic residue-rich peptides. Evidence of a possible permissive role for the vLDL particle in enhancing LC toxicity comes from our study of neurotoxicity in protein A eluates from patients with Parkinsons disease and/or dementia [29]. Compared to neurotoxicity in the protein A eluates from three patients with Parkinsons disease Figure 2A), when the Liposorber-retained fractions from two dyslipidemic diabetic dementia patients plasmas was purified on protein-A, the resulting IgG displayed significantly higher, unusually potent neurotoxicity (Figure 2B) [29]. The purified IgG present in diabetic dementia and dyslipidemic plasma was active in the low-ten nanomolar range of concentrations (Figure 2B) (approaching potency reported for oligomeric Abeta). The purified diabetic dementia IgG-induced neurite retraction was significantly, but not completely prevented by co-incubation with a 200 nanomolar concentration of the selective 5HT2AR antagonist M100907 (Figure 3B). A similar concentration of M100907 completely prevented PD Ig-induced neurite retraction (Figure 3A). These data suggest that hypertriglyceridemic plasma enhances the diabetic dementia autoantibody toxicity through unknown mechanisms. It is possible that heavy chain-bearing IgG components in diabetic dementia target the 5HT2AR receptor and are blockable by selective 5HT2AR antagonist, but that more potent light chain components interact with a different receptor.

Figure 2: The highly purified protein-A eluate fraction of diabetic dementia plasma (solid diamond) caused significantly greater dose-dependent suppression of N2A mouse neuroblastoma neurite outgrowth compared to the Liposorber eluate fraction (open square) from the same patient (A). Dose-dependent N2A mouse neuroblastoma cell toxicity in the protein-A eluates from type 2 diabetic dementia (solid diamond) and PD/DMT (open triangle) significantly exceeded toxicity in the corresponding Liposorber DMT eluate fraction (open square) (B). **P < 0.001;* P < 0.05 comparing neurite length-shortening or N2 mouse neuroblastoma cell survival induced by Liposorber eluate vs protein-A eluate fractions. Reproduced from [29].
PD/DMT: Parkinson’s disease with dementia.

Figure 3: Mean N2A neurite length suppression induced by three different diabetic PD autoantibodies was nearly completely prevented by co-incubation with 200 nanomolar M100907, a highly selective 5-HT2AR receptor antagonist (A). Potent, time-dependent suppression of N2A neurite outgrowth by diabetic dementia autoantibodies was only partially prevented by co-incubation with M100907 (B). Reproduced from [29].

The implications of these findings for the prevention of diabetic dementia are several-fold. First, while the inheritance of genes encoding a furin-cleavable light chain is not easily modifiable, the prevention of diabetic dyslipidemia is in part modifiable by measures such as adherence to a low-fat diet, exercise, maintenance of ideal body weight, medications that correct insulin resistance and insulin deficiency, and avoidance of alcohol and other substances that inhibit lipoprotein lipase. The regulation of extracellular furin in diabetes and how it might cleave a subset of LC is not completely understood. Furin contains a heparin-binding domain [30] and since EC and neurotoxic diabetes AAB targeted HSPG [8], anti-HSPG AAB and furin may colocalize in spaces rich in HSPG. For example, HSPG acts as a co-receptor for lipoprotein lipase and for lipoprotein-related protein LRP which is involved in the catabolism of vLDL particles [31]. Several genes involved in protein catabolism were reported to be risk factors for dementia [3]. We cannot rule out the possibility that toxic LC fragments may be generated in part during the process of vLDL intracellular catabolism, e.g. in an acidic cellular compartments which might favor spontaneous dissolution of LC harboring multi-basic amino acid residues.

In summary, the presented (published) data support our novel hypothesis that a subset of diabetic dementia may result (in part) from inheritance of specific genes encoding subclasses of kappa light chain which harbor a furin-cleavage site in the variable-constant switch region which can serve as a substrate for increased plasma furin reported in diabetes. Cleavage generates light chain autoantibody fragments highly toxic in both endothelial and neuronal cells. The LC fragments might aggregate to form soluble oligomers having enhanced cell neurotoxicity in the low-ten nanomolar range of toxic concentrations as reported for amyloid beta oligomers. More study is needed to substantiate this theory and to test whether furin cleavability in the protein-A eluates obtained from diabetic patients having other microvascular complications and a family history of dementia might be a significant predictor of subsequent occurrence of dementia compared to patients without similar family history of dementia.

The author declares no competing interests that would affect the objectivity of the presented results.

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DOI: 10.31038/EDMJ.20261032

Abstract

Background: Type 2 Diabetes Mellitus (T2D) impairs peripheral microvascular reactivity and skeletal muscle oxygen consumption during locomotion causing impaired exercise tolerance in a poorly understood manner. While linear spectral measures of NIRS capture total oxygen signal power, they fail to capture the complex and chaotic dynamics of muscle oxygen control.

Aim: Using bispectral analysis to quantify non-linear harmonic interactions and quadratic phase coupling (QPC) of muscle oxygenation in T2DM participants with and without DM complications.

Methods: Participants were stratified into two distinct cohorts ( n =12 each) based on T2DM severity: Group 1 (T2D with complications: elevated HbA1c, extended disease duration, clinical neuropathy/vasculopathy) and Group 2 (Well-controlled/no complications). Time-series continuous tissue oxygenation (StO₂) data (N = 1,826 frames per series at f_s = 10 HZ ) were collected from participants performing a standardized treadmill walking task at self-selected usual walking pace. Higher-order spectral analysis—specifically bispectrum magnitude |B (f₁ , f₂)|  and normalized bicoherence b (f₁ , f₂) —was computed across the principal non-linear domain ( f₂ ≤ f₁ ,  f₁+ f₂ ≤ 5.0).

Results: Group 1 exhibited an 84.1-fold increase in mean total bispectral energy ( 1.28 x 10¹¹ vs. 1.52 x 10⁹ arbitrary units p < 0.001 ) and an 8.7-fold higher peak bispectral magnitude ( 2.42 x 10⁵ vs. 2.79 x 10⁴ p < 0.001 ) compared to Group 2. Quadratic phase coupling concentrated along the fundamental diagonal (f₁= f₂), with Group 1 peaking at 0.94 Hz ( f₃= 1.88 Hz second harmonic) and Group 2 peaking at  ( second harmonic). Peak normalized bicoherence reached 0.886 in Group 1 versus 0.785 in Group 2. Group1 generated severe non-linear distortion and exaggerated low-frequency harmonic coupling in active muscle oxygenation during locomotion.

Conclusions: More advanced diabetes, or complications of T2DM, not only have direct impact on exercise capacity but also disrupt the poorly understood complex, dynamic local control of muscle oxygenation. Bispectral analysis of NIRS signals serves as a sensitive, non-invasive biomarker for evaluating the deterioration in oxygen dynamics, seen in more advanced diabetes, which is not detectable by conventional NIRS techniques and shows promise for early detection of clinical deterioration.

Introduction

Type 2 Diabetes Mellitus (T2D) is characterized by exercise intolerance (as characterised by a lower muscle oxygen consumption at VO2max compared with non-diabetes [1] although the precise mechanisms are not fully understood due to the complex interactions of local mechanisms controlling the balance of oxygen consumption and supply in local muscle tissue. During physical activity, skeletal muscle requires precise, real-time matching of local microvascular oxygen delivery to dynamic metabolic demand. In healthy individuals, local vasomotion, autonomic sympathetic tone, and endothelial nitric oxide release regulate local perfusion in a balanced, quasi-linear oscillatory manner, and each with distinct frequencies. Significant skeletal muscle deoxygenation is known to occur during exercise in type 2 diabetes [2]. Local recruitment of microvascular blood volume/hematocrit, blood flow, and VO_2peak are normally all mutually correlated but uncouple in T2DM [3]. In advanced T2D—marked by prolonged disease duration, poor glycaemic control (HbA1c > 8.0%) and microvascular structural remodeling, vasodilatory responsiveness becomes severely compromised [4].

When analysing oxygen levels in muscle (often measured via Near-Infrared Spectroscopy (NIRS) as Oxygenated Haemoglobin (HbO) or tissue oxygenation index (TSI), traditional linear signal processing techniques (e.g., Fourier Transform, Power Spectral Density) quantify energy distribution across isolated frequency bands. But this only indicates which frequencies are present, not the interactions and coupling effects between (e.g., myogenic, neurogenic, endothelial rhythms). As a result, overall NIRS oxygen dynamics in exercise often appear normal in T2DM [5,6]. These linear spectrums inherently assume phase independence between frequency components and mask higher-order non-linear dynamics. In contrast, the mean bispectrum magnitude reveals how these frequencies interact with each other and therefore serves as a powerful tool to detect nonlinear physiological control mechanisms and microvascular regulation [7]. The bispectrum |B (f₁ , f₂)| evaluates higher-order statistics by identifying quadratic phase coupling (QPC)—a mechanism where non-linear interactions between two frequencies f₁ and f₂ and  generate a coupled harmonic component at f₃= f₁ + f₂ . It extends the traditional power spectrum (which uses two-point correlations) to a three-point frequency correlation or third order statistic. It evaluates how three frequency components—f₁, f₂, and their sum f₁ + f₂—relate to one another in terms of both amplitude and phase. Because it is derived from a triple product of Fourier transforms, the bispectrum is a complex number possessing both a phase (biphase) and a magnitude. The 3 practical benefits of Bispectral compared to traditional power spectra are; Phase information to determine whether frequency components are interdependent. Quadratic phase coupling to reveal when two frequencies combine to create a third frequency. Gaussian noise suppression, making non-Gaussian or hidden signals easier to analyse.

Because the raw bispectrum magnitude is dependent on the amplitude of the underlying signal, a large peak can simply mean the oxygen level fluctuations have increased in volume, not necessarily that their coordination increased. To separate pure coordinate coupling from raw amplitude changes during muscle contraction or rest, the analysis is paired with bicoherence b (f₁ , f₂),. Bicoherence normalizes the mean bispectrum magnitude to a scale of 0 to 1, indicating the true strength of the phase coupling.

This study therefore applies bispectrum analysis to 24 recordings of skeletal muscle oxygenation (StO₂) recorded during treadmill walking in participants with T2DM. We tested the hypothesis that advanced diabetic complications (neuropathy, vasculopathy, extended duration, high HbA1c) reduce oxygenation dynamical complexity and instead induce structural non-linear distortions manifesting as significantly higher bispectral energy and altered quadratic phase coupling compared to spectra of no complications diabetes as the control.

Materials and Methods

Study Design and Dataset

Study sample size was based on previous studies [8] where DM affected oxygenation HbO by 11%. To detect a difference between complications and no complications T2DM groups with 80% power and 5% significance requires 24 individuals (12 in each group). To allow for missing data of 10%, sample size was set at 24.

Ethical Considerations

The study was conducted in accordance with ethical guidelines for observational research within a larger research program: IRAS Project ID: 67269. An amendment for further recruitment and data collection was approved on 5 March 2024. (10002.NSA6.5.03.2024) (Appendix 1) Patient confidentiality was maintained throughout the study, and all data were fully anonymised for analysis. Only data relevant to the statistical analysis was extracted from records. (Appendix 1)

Inclusion and Exclusion Criteria

Inclusion criteria included confirmed diagnosis of type 2 diabetes mellitus and age 18 years or older. The diabetes with complications group included T2DM adults with coded diagnoses of confirmed CVD, PAD, DPN in medical records or T2DM >12years, while the comparison group comprised T2DM adults with no such codes in the records.

Exclusion criteria were applied to ensure participant safety and data quality. Patients with mobility problems that would prevent gait assessment on a treadmill were excluded, as were individuals lacking mental capacity to provide informed consent or follow testing instructions.

Group Assignment

• Group 1 (Advanced T2D/Complications):  n = 12 participants corresponding clinically to elevated HbA1c levels, longer diabetic duration, and high prevalence of diabetic peripheral neuropathy and peripheral artery disease.
• Group 2 (Controlled T2D/no complications): n = 12 participants corresponding clinically to well-managed glycemia and minimal vascular/neuropathic complications.

Treadmill Testing

Participants were priorly provided with a participant information sheet and if in agreement signed consent forms. Participants familiarised themselves with treadmill walking starting at a slow pace and self-adjusting the speed control up to self-perceived natural walking pace. NIRS measurements of walking at steady pace for 2minutes were recorded.

Muscle Oxygenation Testing

Throughout the exercise, continuous non-invasive measurements of muscle oxygenation/deoxygenation status were measured with near-infrared spectroscopy (NIRS) (PortaMon; Artinis Medical Systems, Gelderland, the Netherlands) at 10 Hz. The portaMon was placed on the lateral head of gastrocnemius muscle of the dominant limb, secured using a Velcro strap, and covered with a cloth bandage to exclude ambient light. The NIRS monitor emits two wavelengths (780 and 850 nm) which interact with oxyhaemoglobin and deoxyhaemoglobin. Using the Beer–Lambert law to account for scattering we determined changes of relative concentrations of oxy (HbO) and deoxygenated (HHb)

Datasets

Continuous tissue oxygenation time-series signals were acquired from N = 24 adult participants diagnosed with T2D during a standardized treadmill walking protocol (TMv3). Each participant dataset comprised 1,826 sequential temporal data points recorded at a uniform sampling frequency f_s = 10.0 Hz (Δt=0.1 s), yielding a total continuous measurement window of  182.6 seconds.

Analysis

Analysis was performed using a dedicated script coded in python (Appendix 1).

Higher-Order Spectral Analysis Formulation

For a discrete zero-mean time series x(t), segment-averaged Fourier transforms X(f) were calculated using Welch’s modified periodogram approach with Hann windowing ( N_perseg = 128 points,  50% overlap). The discrete bispectrum B ( f_{1 ,} f₂) measures the complex third-order correlation between three spectral components satisfying the resonant condition f₁ + f₂= f₃ :

where IE[·] denotes the expectation operator across time segments and X^*(·) represents the complex conjugate. To eliminate magnitude dependence and quantify true non-linear phase coupling independent of signal amplitude, the normalized bicoherence index b ( f_{1 ,} f₂)  was calculated:

Total Bispectral Energy (E_B) was evaluated over the alternating region ( f_{1 ,} f₂ ≥ 0.15 Hz):

Results

Individual Participant Bispectral Profiles

Rank-ordering the 24 diabetic participants by total bispectral energy revealed a strong, multi-order-of-magnitude separation between the advanced disease cohort (Group 1) and the controlled cohort (Group 2) (Table 1) (Appendix 2).

Table 1: Demographics of the patients and of the healthy controls.

Group Comparative Analysis

Non-Linear Energy Disparity: Group 1 exhibits over 84 times greater total bispectral energy ( 1.28 x 10¹¹ vs. 1.52 x 10⁹ ) than Group 2. Harmonic Phase Coupling: Both groups display focused quadratic phase coupling along the diagonal ( f₁= f₂), generating strong second-harmonic interactions ( f₃=f₁ + f₂ ≈ 2f₁). (Table 2). Crucially, while the normalized bicoherence magnitude is comparable between groups ( b_max ≈ 0.52), the absolute bispectral magnitude |B| is 8.7 times higher in Group 1. The Bispectrum magnitude reflects the degree of quadratic phase coupling or nonlinear interactions between triads of frequencies of controllers of oxygenation in response to exercise. This demonstrates that while both groups display intrinsic non-linear phase coupling, the magnitude of non-linear wave interaction and dynamic amplitude distortion is vastly magnified in the group with more advanced diabetes disease.

Table 2: Comparative Group Statistics (Mean  SD).

The mean bispectrum magnitude (6.6x higher in Group 1), taken over multiple epochs, ensures that random, incoherent noise and phase fluctuations cancel out toward zero, isolating stable, phase-coupled harmonic components. The higher value indicates that fluctuations are more driven by deterministic, nonlinear physiological systems rather than random variations (Figures 1 and 2).

Figure 1: Log-Bispectrum and bicoherence contour plots averaged for Groups 1 and 2. Log scaling reveals underlying harmonically coupled structures across 0.5 Hz ≤ f₁ ≤ 2.5 Hz. Group 1 shows a higher baseline magnitude ( log₁₀ ≈ 4.5 to 7.5). Group 2 shows similar coupling but lower overall magnitude intensity ( log₁₀ ≈ 0.75 to 6.15) indicating higher diversity/complexity in oxygenation signals.

Figure 2: 3D contour maps of the bispectrum magnitude (vertical axis) and the geometric distribution of quadratic phase coupling for a) participant in Group 1 T2DM with diabetic complications and b) participant in group 2 without complications. Note the reduction and clustering of spectral energy and peaks in (a) reflecting muscle oxygen changing in a highly predictable, inflexible and restricted pattern, compared to (b). (b) shows a wider energy dispersal, and a more complex peak signature reflecting the complex, fluid, and non-linear variations seen in healthy tissues.

Discussion

The primary finding of this study is the high increase in total bispectral energy in participants with advanced/complicated T2D (Group 1) relative to those with T2D without complications (Group 2). High bispectral energy reflects muscle oxygen levels changing to a highly predictable, rigid and strictly coupled harmonic pattern, rather than showing the complex, fluid, and chaotic variations seen in healthy tissues. This shift in frequencies is quantified by the mean bispectrum magnitude. Chaotic systems have the advantage of being rapidly adaptable to changes in local conditions without the need for central control. Therefore, while a more inflexible pattern of oxygenation appears more organised (hence higher spectral energy/lower entropy) it loses the dynamic ability to adapt to changes in local oxygen demand. As a result, oxygenation not meeting the demands of muscle, over time, leads to muscle degenerative changes and sarcopenia [9]. The results accord with those only other bispectral NIRs muscle study by Molinari, where spectral energy reduced in muscle after 1 year of exercise training in diabetes [8]. The physiological and metabolic reasons behind why more advanced T2DM exhibits high bispectral energy (and thus lower signal entropy) in muscle oxygenation patterns results from three main factors:

Microvascular Stiffness

In healthy microvasculature, vasomotion is mediated by smooth muscle cell electromechanical coupling, endothelial nitric oxide release, and sympathetic innervation operating within a flexible, linear regulation envelope. The microvasculature supplying the muscle is incredibly dynamic. It constantly adjusts to minor local changes via endothelial nitric oxide release, neural inputs, and metabolic demands. This creates a highly complex, chaotic, and “organic” oxygenation signal (low bispectral energy, high entropy). Capillary basement membrane and advanced glycation end-product (AGE) cross-linking reduce arterial compliance. When subjected to the mechanical pumping of treadmill locomotion, stiffened microvessels generate non-linear pressure-flow wave interactions The capillaries lose their ability to dynamically fluctuate. The oxygenation signal becomes strictly bound to macro-hemodynamic forces—like the mechanical, repetitive pumping of the heart or rigid breathing rates. This strong phase-locking to external cyclic rhythms drives up bispectral quadratic phase coupling (QPC), creating high bispectral energy. Loss of sympathetic nerve fibre density impairs smooth vasomotor control, transforming smooth sinusoidal vasomotion into abrupt, asymmetric flux variations that generate strong higher-order harmonics [10].

Mitochondrial Dysregulation

Healthy muscle tissue rapidly and flexibly switches between metabolic substrates (carbohydrates and fatty acids) and continuously adjusts oxygen extraction based on immediate cellular needs [11]. Diabetic myocytes become metabolically inflexible and unable to dynamically “demand” and extract oxygen in a rapid and responsive manner. [12,13]. The oxygen extraction rate becomes severely impaired and fixed. The oxygenation signal loses its internal biological complexity and flattens into a simple, repetitive cycle dictated purely by systemic delivery.

De-conditioning and Structural Muscle Changes (Sarcopenia)

T2DM causes structural changes in the muscle, including muscle wasting (sarcopenia), fatty infiltration, and a shift in muscle fibre types (often a loss of capillaries wrapping around the fibres) [14]. When muscle is structurally simplified, alongside reduced capillary density, the diffusion path for oxygen into the tissue becomes uniform and restricted [15]. Localized oxygen pools that create complex signals are replaced by homogenous tissue saturations which translate mathematically into cleaner, highly coupled harmonic waves and high bispectral energy.

Clinical Implications as a Diagnostic Biomarker

Bispectral energy analysis, combining both phase alignment and third-order amplitude variance, acts as a highly sensitive non-linear biomarker that can detect loss of microvasculature complex dynamics, not visible by standard spectral analysis of NIRS signals and likely before clinical signs of deterioration. The separation observed between Group 1 and Group 2 suggests that bispectral profiling during walking tests could provide a non-invasive diagnostic tool to screen for:

• Subclinical diabetic peripheral neuropathy before sensory loss manifest.
• Microvascular diabetic vasculopathy and peripheral artery risk.
• Objective tracking of therapeutic interventions aimed at restoring vascular flexibility.
• Early diabetic myopathy prior to diabetic sarcopenia.

NIRS already has advantages over other methods of assessing muscle oxygen content (portable, low cost, non-invasive) and research also shows that NIRS provides reliable measures of exercise-induced oxygen responses regardless of the protocol used [16].

Conclusions

Bispectral analysis of skeletal muscle tissue oxygenation during locomotion provides novel insights into muscle oxygenation dysfunction in Type 2 Diabetes. Advanced diabetic complications—including neuropathy, vasculopathy, prolonged disease duration, and poor glycemic control—are characterized by an 84-fold surge in total non-linear bispectral energy and significant phase coupling, overall indicating a loss of physiological adaptability. Higher-order spectral analysis represents a promising, non-invasive approach for identifying muscle oxygenation dysfunction in T2DM and the potential for earlier detection of disease progression.

Funding

No funding was provided for this study.

Conflict of Interest

RB, SH and LC declare no conflicts of interest.

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