Basal Vacuolization in Renal Tubular Epithelial Cells at Autopsy and Their Relation to Ketoacidosis

Basal vacuolization of renal tubular epithelial cells is a useful postmortem marker for ketoacidosis. To investigate its incidence and relationship to the severity of ketoacidosis, 158 autopsy cases with elevated β‐hydroxybutyrate (>1 mmol/L) over a 7‐year‐period were retrospectively reviewed. Sixty‐eight cases (43%) exhibited basal vacuolizations (vitreous β‐hydroxybutyrate: 1.16–29.35 mmol/L, mean 10.28 mmol/L), and 90 cases (57%) did not (vitreous β‐hydroxybutyrate: 1.03–13.7 mmol/L, mean 2.84 mmol/L). Quantitative analysis revealed on average a fourfold elevation in β‐hydroxybutyrate in cases with basal vacuolizations compared to those without; 10.3% of cases with β‐hydroxybutyrate concentrations between 1.01 and 2.00 mmol/L had basal vacuolizations, and this incidence increased to 33.3% with concentrations between 4.01 and 6.00 mmol/L. A marked increase in incidence to >70% was observed with concentrations >6.00 mmol/L, and basal vacuoles were invariably present (100%) with concentrations >14.01 mmol/L. This study demonstrates that basal vacuolizations are a sensitive marker for significant ketoacidosis and reaffirms its use as an indicator for likely cases of fatal ketoacidosis at autopsy.     

Renal Cortical Pallor—A Useful Macroscopic Marker for Metabolic Derangements at Autopsy

Renal cortical pallor was studied as a potential marker at autopsy of diabetic ketoacidosis in 23 cases, hyperglycemic nonketotic coma in eight cases, and alcoholic ketoacidosis in five cases (vitreous humor glucose level ≥11.1 mM; β‐hydroxybutyrate level ≥5 mM). Renal cortical pallor was noted on macroscopic examination in 10 of 23 cases of lethal diabetic ketoacidosis (43.5%), three of eight cases of fatal hyperglycemic nonketotic coma (37.5%), and in two of five cases of alcoholic ketoacidosis (40%). Histologic examination revealed basal vacuolization of renal tubular epithelial cells in 12 cases, Armanni–Ebstein lesions in 10, and osmotic nephrosis in three. Although renal cortical pallor did not appear to be a particularly sensitive marker for hyperglycemia or ketoacidosis, and did not correlate with the severity of these parameters, it may still represent a useful macroscopic marker for underlying metabolic conditions at autopsy and should therefore prompt measurement of vitreous humor glucose and β‐hydroxybutyrate levels.     

How Useful is Basal Renal Tubular Epithelial Cell Vacuolization as a Marker for Significant Hyperglycemia at Autopsy?

Basal vacuolization of renal tubular epithelial cells (so-called Armanni-Ebstein phenomenon) has been attributed to hyperglycemia causing accumulation of cytoplasmic glycogen. Review of 34 autopsy cases with significant hyperglycemia (vitreous glucose ≥ 15 mmol/L/270 mg/dL) was undertaken to determine whether there was any significant association between the degree of hyperglycemia and the severity of this morphological change (graded as 0, 1+, 2+, and 3+). No association was demonstrated. Review of the subgroup of 14 cases with terminal hyperglycemia without ketoacidosis was then undertaken to assess the effect of hyperglycemia in isolation on renal tubular epithelial cells. Vitreous glucose levels in these 14 cases ranged from 17 to 49.7 mmol/L (306-894.6 mg/dL) with a mean of 26.25 mmol/L (472.5 mg/dL) and β-hydroxybutyrate levels ranged from 0.02 to 2.55 mmol/L (0.36-45.9 mg/dL) with a mean 0.79 mmol/L (14.22 mg/dL). Not one of the latter cases displayed basal vacuolization. No relationship between basal vacuolization of renal tubular epithelial cells at autopsy and terminal hyperglycemia could, therefore, be demonstrated.     

Fatal Diabetic Ketoacidosis—A Potential Complication of MDMA (Ecstasy) Use

A 19‐year‐old woman with insulin‐dependent diabetes mellitus was found dead in bed having allegedly recently taken ecstasy and consumed alcohol. At autopsy, there were microhemorrhages in the brain with subnuclear vacuolization and Armanni–Ebstein changes in renal tubules. Biochemical analyses confirmed diabetic ketoacidosis (vitreous glucose—46.5 mmol/L; β‐OH butyrate—13.86 mmol/L.). Toxicological analyses of blood showed a low level of 3,4‐methylenedioxy‐methamphetamine (MDMA) (0.01 mg/L), with acetone but no alcohol or other common drugs. Death was attributed to diabetic ketoacidosis most likely provoked by mixed MDMA/alcohol ingestion. Although the use of illicit drugs by young individuals with diabetes mellitus is being increasingly recognized, it has been noted that there is minimal information about the relationship between drug use and acute diabetic complications. Toxicological screening of cases of lethal diabetic ketoacidosis in the young may clarify lethal mechanisms in individual cases and also help to determine the extent of this problem.     

Utility of Postmortem Vitreous Beta-Hydroxybutyrate Testing for Distinguishing Sudden from Prolonged Deaths and for Diagnosing Ketoacidosis

A retrospective, cross-sectional analysis of vitreous beta-hydroxybutyrate (BHB) on 967 forensic cases over a two-year period was conducted. Cases were sorted into six categories of death: (i) sudden traumatic/non-natural (ST), (ii) sudden natural (SN), (iii) prolonged traumatic/non-natural (PT), (iv) prolonged natural (PN), (v) diabetic ketoacidosis (DKA), and (vi) alcoholic ketoacidosis (AKA). The mean BHB for all cases was 1.67 mmol/L (17.4 mg/dL; range: 0.11–18.02 mmol/L). The numbers of DKA, AKA, PN, PT, SN, and ST deaths were 21, 5, 155, 258, 275, and 253, respectively. Their mean vitreous BHBs were as follows: 11.04 mmol/L (DKA), 8.88 mmol/L (AKA), 1.56 mmol/L (PN), 1.55 mmol/L (PT), 1.26 mmol/L (SN), and 1.38 mmol/L (ST). There was a statistically significant difference between the mean BHBs of the PN and SN death groups (p < 0.001), as well as between those of the PT and ST death groups (p = 0.004). Given the overlapping ranges seen between the prolonged and sudden death groups, the identified differences did not hold clinical significance. In addition, we sought to determine a threshold value for vitreous BHB to definitely diagnose cases of ketoacidosis. BHB threshold concentrations between 2.5 and 5 mmol/L produced sensitivities >92% and specificities >96%. A receiver operator characteristic curve found 3.43 mmol/L to be the optimal cutoff value, demonstrating a specificity of 98.3% and a sensitivity of 96.2%.     

Postmortem vitreous Beta-hydroxybutyrate: interpretation in a forensic setting*

Abstract: Vitreous beta‐hydroxybutyrate (BHB) was retrospectively analyzed in 1795 forensic cases using the Pointe Scientific method. Comparison of vitreous BHB with vitreous glucose in 1781 of the cases showed moderately good correlation r = 0.731. Comparison with blood alcohol levels in 1561 of the cases showed no correlation r = ?0.053. Vitreous BHB was a marker of diabetic ketoacidosis when above 6.0 mM with a vitreous glucose over 200 mg/dL. It was an indicator (>50%) for alcoholic ketoacidosis when above 6.0 mM with a vitreous glucose below 200 mg/dL. Recommendations for interpretation of vitreous BHB: <0.4 mM normal; 0.41–1.2 mM slightly elevated, rarely (<1%) of concern; 1.21–2.0 mM moderately elevated, less rarely (2.5%) of concern; 2.01–6.0 mM significantly elevated, frequently of concern (12–48%); >6.0 mM usually (100% in this study) indicated life‐threatening conditions. Vitreous BHB was helpful evaluating cases with ketogenic conditions, especially diabetes and alcoholism.     

An Isolated Perfused Rat Kidney Model for the Evaluation of the Effect of Glucose on Renal Tubular Epithelial Morphology

An isolated perfused kidney model was used to evaluate the effect of hyperglycemia on renal tubular epithelial cell morphology. Ten Sprague–Dawley rat kidneys were perfused with Krebs–Henseleit buffer containing 70 mmol/L of glucose (five for 1 h and five for 2 h). Two control groups consisted of 10 kidneys perfused with Krebs–Henseleit buffer without hyperglycemia (five for 1 h and five for 2 h), and 10 nonperfused contralateral kidneys placed in the same environment for the same duration. The hyperglycemia group had significantly increased renal tubular vacuolization (p < 0.001) compared to both control groups at 1 and 2 h. The isolated perfused kidney model recapitulates the renal tubular vacuolization phenotype found in hyperglycemia and may be a potential tool for the investigation into causal factors in renal histology. The full pattern of the Armanni–Ebstein phenomenon was not, however, reproduced, suggesting that this change requires more time or involves more complex factors.     

Investigation of markers to indicate and distinguish death due to alcoholic ketoacidosis, diabetic ketoacidosis and hyperosmolar hyperglycemic state using post-mortem samples

Data from 191 post-mortem cases where post-mortem blood beta-hydroxybutyrate (βHB) and acetone concentrations and vitreous humor glucose concentrations (where available) had been measured were retrospectively investigated to determine the markers required to identify and distinguish between Alcoholic Ketoacidosis (AKA), Diabetic Ketoacidosis (DKA) and Hyperosmolar Hyperglycemic State (HHS). Blood βHB concentrations above 250 μg/mL were considered significant and it was shown to be the preferred marker of ketoacidosis. All cases with significant βHB detected also had acetone present (greater than 2mg/dL) demonstrating that acetone can be used as a marker to identify ketoacidosis and can be used to indicate when βHB measurement is necessary. Vitreous humor glucose concentrations above 6.9 mmol/L were considered high and indicative of hyperglycemia prior to death. Vitreous humor glucose concentrations can be used to distinguish between DKA and ketoacidosis from other causes and to identify deaths due to HHS. The data showed that ketoacidosis can occur without a history of alcoholism or diabetes. Many diabetics are undiagnosed for many years. Therefore, DKA or HHS should be considered in sudden or unexplained deaths and glucose should be routinely measured especially in cases with risk factors for diabetes including obesity, old age, a history of mental health problems or treatment with atypical antipsychotic drugs including clozapine, olanzapine, quetiapine and risperidone.     

Subnuclear renal tubular vacuoles in alcohol use disorder

Subnuclear vacuoles in the proximal renal tubules have been reported as a histologic sign of ketoacidosis. Originally described in diabetic ketoacidosis, renal vacuoles can be found in other ketogenic states such as alcoholic ketoacidosis (AKA), starvation, and hypothermia, underpinned by deranged fatty acid metabolism. A retrospective analysis of 133 deaths associated with alcohol use disorder (AUD) examined at autopsy between 2017 and 2020 was undertaken. This study aimed to determine the prevalence of subnuclear vacuoles in deaths of those with AUD and their specificity for deaths from AKA, and to elucidate what demographic, biochemical, and pathologic findings are associated with subnuclear vacuoles. In each case, vitreous humor biochemistry including electrolytes, glucose, and beta-hydroxybutyrate (BHB) was analyzed alongside postmortem hemoglobin A1c and renal and liver histology. Renal histology was graded for the presence of vacuoles as absent (0), scanty (1), or easily identifiable (2). Liver histology was graded for steatosis and for fibrosis if Masson trichrome staining was available. Vacuoles were commonly seen in the deaths of those with AUD. They were seen in deaths due to AKA but were not specific to that cause of death. With vacuoles present, lower vitreous sodium (139 vs. 142 mmol/L; p = 0.005), higher vitreous BHB (1.50 vs. 1.39 mmol/L; p = 0.04), severe hepatic steatosis, and severe hepatic fibrosis were seen, compared with those without renal vacuoles.     

Measurement of chemical analytes in vitreous humor: stability and precision studies

The analytic accuracy and precision for measurement of chemical analytes in vitreous humor (VH) are critical if results are to be used in forensic pathology. The purpose of our study was to demonstrate the stability and the reproducibility of VH sodium, potassium, chloride, glucose, urea nitrogen, acetone, and beta-hydroxybutyrate in specimens obtained from both eyes in medical examiner cases. We also compared with calculated VH osmolalities. Small but significant increases were observed in VH electrolyte concentrations in specimens refrigerated 6-12 months: sodium pre 144 mmol/L, post 151 mmol/L; potassium pre 12.0 mmol/L, post 12.8 mmol/L; chloride pre 121 mmol/L, post 123 mmol/L. No differences were observed between eyes, and within-day precision for all electrolyte measurements were excellent, (<1%). Frozen specimens showed significantly higher measured (439 mOsmol/kg) as compared with calculated osmolality (305 mOsmol/kg), with 1% within-day precision and no significance between eye variation for glucose and urea nitrogen. In 20 of 24 medical cases selected for possible ketoacidosis, measurement of beta-hydroxybutyrate concentrations appears to be a promising diagnostic biomarker for confirming suspected ketosis in medical examiner cases by means of VH analysis.     

Septic Ketoacidosis—A Potentially Lethal Entity with Renal Tubular Epithelial Vacuolization

Fatal ketoacidosis due to diabetes mellitus, alcoholism, and starvation may produce characteristic basal vacuolization of renal tubular epithelial cells (RTEC). Septic ketoacidosis has recently been recognized clinically as a distinct condition in which septicemia can lead to elevation of ketones and various anions unrelated to diabetes mellitus, alcoholism, or caloric deprivation. We report four lethal cases with significantly elevated vitreous ketones secondary to sepsis and/or severe localized infection in individuals with no history of diabetes mellitus, alcoholism, or starvation. Three of four cases exhibited typical basal vacuolization of RTEC. We suggest that septic ketoacidosis is an appropriate cause of death in the forensic setting where sepsis or severe localized infection is found with significant ketoacidosis (β‐hydroxybutyrate > 5 mmol/L)—in the absence of diabetes mellitus, alcoholism, starvation, or other states associated with accelerated ketogenesis. The finding of basal vacuolization of RTEC in such cases provides morphological support for the underlying metabolic derangement.     

The Etiology of Basal Vacuolizations in Renal Tubular Epithelial Cells Evaluated in an Isolated Perfused Kidney Model

To determine whether basal lipid vacuolization characteristic of ketoacidosis could be induced with short‐term hypertriglyceridemia, adult Sprague Dawley rat kidneys were perfused in an isolated perfused kidney model with, and without, 11.3 mM (10 g/L) of triglycerides in Krebs‐Henseleit buffer, for 1 and 2 h (n = 5/group). Additional treatments included perfusion with triglycerides with 20 mM of β‐hydroxybutyrate and 2 mM of acetoacetate (n = 5) and perfusion with triglycerides with 70 mM of glucose (n = 1). Basal vacuolization was produced in all groups, but differed in morphology to that reported in postmortem studies. There was no further increase in vacuolization after 2 h of perfusion compared to 1 h (p = 0.24), and the addition of ketones did not alter the morphology or extent of vacuolization. This study using an ex vivo model has confirmed that isolated hypertriglyceridemia is sufficient to cause basal lipid vacuolization in renal tubular epithelial cells, but with different morphology to vacuoles observed in lethal ketoacidosis at autopsy.     

Measurement of Postmortem 1,5‐anhydroglucitol in Vitreous Humor for Forensic Diagnosis

In forensic diagnosis, postmortem blood glucose is known to be susceptible to change after death. However, the 1,5‐anhydroglucitol (1,5‐AG) concentrations in plasma and cerebrospinal fluid (CSF) reflect the mean blood glucose level for a short period of time. In this study, we compared the postmortem 1,5‐AG concentrations in vitreous humor and CSF in 47 subjects to evaluate the utility of this concentration in the vitreous humor for forensic diagnosis. The postmortem 1,5‐AG concentrations in vitreous humor (mean±SD: 20.2 ± 8.7 μg/mL) and CSF (16.8 ± 8.7 μg/mL) did not differ significantly and showed a strong correlation (r² = 0.87, p < 0.01). These results suggest that the vitreous humor 1,5‐AG concentration provides useful information on the antemortem blood glucose level, in addition to the HbA1c value and the CSF 1,5‐AG concentration.     

Fatal Overdose of Gamma‐hydroxybutyrate Acid After Ingestion of 1,4‐Butanediol

We report a case of fatal intoxication from 1,4‐butanediol (1,4‐BD), which was ingested by a young and “naïve” gamma‐hydroxybutyrate (GHB) consumer during a party with the co‐ingestion of alcohol, cannabis, and methylene‐dioxy‐methamphetamine. The following drug concentrations were found using gas chromatography coupled with mass spectrometry on autopsy samples and on a cup and a glass found at the scene: 20,350 mg/L (bottle) for 1,4‐BD; 1020 mg/L (femoral blood), 3380 mg/L (cardiac blood), 47,280 mg/L (gastric content), and 570 mg/L (vitreous humor) for GHB. The concentration of GHB is difficult to interpret in forensic cases due to the possibility of an endogenous production of GHB. The variable tolerance of the user may also modify the peri‐ and postmortem GHB concentrations. This case underscores the need to have many different sources of toxicology samples analyzed to avoid the hypothesis of endogenous production of GHB.     

Fatality from olanzapine induced hyperglycemia

A case history of a 31-year-old male schizophrenic patient is presented. The man was treated with olanzapine for three weeks before he died. After one week on a 10 mg daily dose of olanzapine, his fasting blood glucose was elevated to 11.3 mmol/L (203 mg/dL). In order to treat more aggressively his psychosis, the olanzapine dose was raised to 20 mg daily resulting in his fasting blood glucose climbing to 15.8 mmol/l (284 mg/dL). On the days preceding his death, he became progressively weaker, and developed polydipsia with polyuria. He had no personal or family history of diabetes mellitus and he was on no other medication at the time of his death. Postmortem blood, vitreous humor, and urine glucose concentrations were 53 mmol/L (954 mg/dL), 49 mmol/L (882 mg/dL), and 329 mmol/L (5922 mg/dL), respectively. Drug screen on urine and blood indicated only a small amount or olanzapine and no alcohols. Peripheral blood olanzapine concentration was within therapeutic limits, 45 ng/mL. Analysis of vitreous humor and urine revealed severe dehydration with small amounts of ketones. Death was attributed to hyperosmolar nonketotic diabetic coma, and olanzapine was felt most likely to be the cause. Another atypical neuroleptic, clozapine, has also been associated with the development and exacerbation of diabetes mellitus or diabetic ketoacidosis. We recommend including vitreous glucose and beta-hydroxybutyrate analysis as part of postmortem toxicology work up when the drug screen reveals the presence of either olanzapine or clozapine.     

Renal Tubular Epithelial Vacuoles—A Marker for Both Hyperlipidemia and Ketoacidosis at Autopsy

Review of 15 cases of nephrotic syndrome found that eight had significant hyperlipidemia with serum cholesterol levels ranging between 10.59 and 18.60 mmol/L (mean 12.88) and serum triglyceride levels between 2.30 and 9.92 mmol/L (mean 4.58); all of these cases displayed basal lipid vacuolization. Seven of the 15 study cases had normal–mild hyperlipidemia with serum cholesterol levels ranging between 4.71 and 7.54 mmol/L (mean 6.02) and serum triglyceride levels between 0.65 and 4.1 mmol/L (mean 1.57). Six of the seven cases had basal lipid vacuoles (86%). Of these, five cases were hyperlipidemic and one case had borderline hyperlipidemia with a serum cholesterol level of 4.71 mmol/L. Although hyperlipidemia was associated with renal tubular epithelial vacuolization, the vacuoles appeared morphologically different to those found in ketoacidosis. This study has shown that while hyperlipidemia in isolation may result in basal lipid vacuolization within renal tubular epithelial cells, the phenotype differs from that observed in ketoacidosis.     

Sudden infant death and liver phosphoenolpyruvate carboxykinase analysis

In an effort to substantiate the impaired gluconeogenesis-terminal hypoglycemia hypothesis of sudden infant death syndrome (SIDS), 52 infants ranging from 3 weeks to 7 months of age which had been brought to autopsy were studied. The stomach contents, vitreous humor glucose concentrations, hepatic glycogen content and hepatic phospho-enolpyruvate carboxykinase (PEPCK) activity were measured as part of the laboratory component of the postmortem investigation. The stomach contents, vitreous humor glucose concentrations and liver glycogen content were similar in SIDS/and non-SIDS victims. PEPCK activity was, however, significantly lower in SIDS (p < 0.001) victims and in SIDS with other findings (p < 0.01) victims when compared to non-SIDS victims. Despite the fact that SIDS victims had lower hepatic PEPCK activity and hence potentially lower gluconeogenic capacity, terminal hypoglycemia could not be demonstrated in this group as compared to the SIDS with other findings and the non-SIDS infants. The impaired gluconeogenesis-terminal hypoglycemia hypothesis thus could not be substantiated.     

Basal renal tubular epithelial cell vacuolization and alcoholic ketoacidosis

Subnuclear renal tubular epithelial cell vacuolization is a marker for diabetic ketoacidosis. Whether it is because of hyperglycemia or of ketoacidosis is unclear. To examine the effect of ketoacidosis on renal cells in isolation, five cases of lethal alcoholic ketoacidosis without hyperglycemia were examined (vitreous humor β-hydroxybutyrate: 6.42-8.75 mM, mean 7.66 mM; and glucose: 0.1-4.2 mM, mean 1.46 mM). Microscopic examination of the kidneys revealed basal vacuoles in three cases (60%). Seven control cases with acute alcohol toxicity without ketoacidosis (blood alcohol: 0.18-0.43%, mean 0.31%; and β-hydroxybutyrate: 0.12-0.42 mM, mean 0.21 mM) did not have these changes. In this study, basal epithelial vacuolization was found only in cases with significant ketoacidosis. Although the numbers are small, the finding of basal renal tubular epithelial vacuolization in normoglycemic cases with elevated β-hydroxybutyrate levels provide further evidence that disordered lipid metabolism may be involved in the pathogenesis of this phenomenon.     

双眼交替微量取样检测兔玻璃体液钾镁离子浓度推测死亡时间

目的研究死后眼玻璃体液钾镁离子浓度变化及其与死亡时间的关系。方法取健康家兔处死,死后不同时间于双眼玻璃体液交替微量取样,采用全自动生化分析仪检测样本中[K+]、[Mg2+],对所得数据进行统计学处理。结果死后12~120h,家兔玻璃体液中[K+]由11.01mmol/L上升至32.97mmol/L,[Mg2+]1.12mmol/L上升至2.22mmol/L,两种离子浓度与PMI进行回归分析自变量为[K+]的二次多项式回归方程为y=0.1756x2-3.3188x+29.804(R2=0.9868);自变量为[Mg2+]的二次多项式回归方程为y=-46.916x2+248.01x-213.81(R2=0.9340);自变量为[K+]和[Mg2+]的二次多项式回归方程为y=-17.55-4.82[K+]+0.20[K+]2+75.77[Mg2+]-19.47[Mg2+](R2=0.9881)。用[K+]的方程推测PMI,当[K+]<26.0mmol/L(PMI<60h)时,平均偏离时间为2.7~9.4h,当[K+]>28mmol/L(PMI>72h)时,平均偏离时间>15h;用[Mg2+]的方程推测PMI,平均偏离时间几乎均在10h以上;用二元方程推测PMI,平均偏离时间较前两者小。结论死后眼玻璃体液[K+]、[Mg2+]随时间延长而升高,且浓度变化与时间呈强相关性。但从实际运用角度考虑,R2<0.98,或平均预测偏离时间过大的回归方程,不宜用于PMI推测;应用多指标建立多元回归方程是提高推测PMI准确性的可行方法。