Robert J. Konrad

The potential mechanism of the diabetogenic action of streptozotocin: inhibition of pancreatic beta-cell O-GlcNAc-selective N-acetyl-beta-D-glucosaminidase.

Streptozotocin (STZ), an analogue of GlcNAc, inhibits purified rat spleen O-GlcNAc-selective N-acetyl-beta-D-glucosaminidase (O-GlcNAcase), the enzyme that removes O-GlcNAc from protein. We have shown previously that STZ increases pancreatic islet O-linked protein glycosylation. In light of these data, we investigated the possibility further that STZ causes beta-cell death by inhibiting O-GlcNAcase. In isolated islets, the time course and dose curve of STZ-induced O-glycosylation correlated with beta-cell toxicity. STZ inhibition of rat islet O-GlcNAcase activity also paralleled that of its beta-cell toxicity, with significant inhibition occurring at a concentration of 1 mM. In contrast, STZ inhibition of rat brain O-GlcNAcase and beta-TC3 insulinoma cell O-GlcNAcase was significantly right-shifted compared with islets, with STZ only significantly inhibiting activity at a concentration of 5 mM, the same concentration required for beta-TC3 cell toxicity. In comparison, N-methyl-N-nitrosourea, the nitric oxide-donating portion of STZ, did not cause increased islet O-glycosylation, beta-cell toxicity or inhibition of beta-cell O-GlcNAcase. Enhanced STZ sensitivity of islet O-GlcNAcase compared with O-GlcNAcase from other tissues or an insulinoma cell line suggests why actual islet beta-cells are particularly sensitive to STZ. Confirming this idea, STZ-induced islet beta-cell toxicity was completely blocked by GlcNAc, which also prevented STZ-induced O-GlcNAcase inhibition, but was not even partially blocked by glucose, glucosamine or GalNAc. Together, these data demonstrate that STZs inhibition of beta-cell O-GlcNAcase is the mechanism that accounts for its diabetogenic toxicity.

Streptozotocin, an O-GlcNAcase inhibitor, blunts insulin and growth hormone secretion

Type 2 diabetes mellitus results from a complex interaction between nutritional excess and multiple genes. Whereas pancreatic beta-cells normally respond to glucose challenge by rapid insulin release (first phase insulin secretion), there is a loss of this acute response in virtually all of the type 2 diabetes patients with significant fasting hyperglycemia. Our previous studies demonstrated that irreversible intracellular accumulation of a glucose metabolite, protein O-linked N-acetylglucosamine modification (O-GlcNAc), is associated with pancreatic beta-cell apoptosis. In the present study, we show that streptozotocin (STZ), a non-competitive chemical blocker of O-GlcNAcase, induces an insulin secretory defect in isolated rat islet cells. In contrast, transgenic mice with down-regulated glucose to glucosamine metabolism in beta-cells exhibited an enhanced insulin secretion capacity. Interestingly, the STZ blockade of O-GlcNAcase activity is also associated with a growth hormone secretory defect and impairment of intracellular secretory vesicle trafficking. These results provide evidence for the roles of O-GlcNAc in the insulin secretion and possible involvement of O-GlcNAc in general glucose-regulated hormone secretion pathways.

Postmortem Diagnosis of Unsuspected Diabetes Mellitus Established by Determination of Decedent's Hemoglobin A1c Level

Although approximately 15.7 million Americans have diabetes mellitus, with the vast majority having type 2 diabetes, it is estimated that as many as 5.4 million are undiagnosed. The present case illustrates that undiagnosed diabetes can be a factor in otherwise unexplained deaths. A 39-year-old white male with no significant past medical history other than alcohol abuse was found deceased at his residence. The manner of death appeared to be natural, but no anatomic cause was found. Toxicological analysis revealed a blood ethanol level of 0.02 g/dL and was negative for drugs of abuse. Analysis of the vitreous fluid revealed a glucose level of 502 mg/dL. The blood glucose level was 499 mg/dL, and the hemoglobin A1c (HbA1c) level was 10.6%. Only trace urine ketones were detected, suggesting that the death was the result of hyperglycemic hyperosmolar non-ketosis (HHNK) from unsuspected diabetes. The postmortem HbA1c value serves as a definitive indicator of prolonged hyperglycemia. In order to aid the interpretation of the clinical data, this case is discussed in conjunction with a similar case of a known diabetic patient.

New automated chemiluminescent assay for erythropoietin.

Erythropoietin (EPO) is a polypeptide hormone produced by the kidney that regulates erythropoiesis by controlling the proliferation and differentiation of erythroid progenitors in bone marrow. Assays for EPO are used to monitor dosage and response to human recombinant erythropoietin also may have diagnostic utility in the differential diagnosis of anemia and polycythemia. We evaluated an automated, chemiluminescent immunoassay for EPO (DPC Immulite) in terms of precision, linearity, interference, and correlation with reference assays. The Immulite assay demonstrated acceptable correlation with the reference immunochemiluminometric method (slope = 1.087, y intercept = 0.567, R value = 0.990). Within‐run CVs ranged from 2.3% to 5.0%, while between‐run CVs ranged from 4.1% to 9.5%. Linearity extended beyond the manufacturer’s stated claims, and recovery ranged from 96.8% to 100.9% across the concentrations tested. No significant interference was noted with hemoglobin, bilirubin, or triglyceride. Overall, this method compares favorably with the existing immunochemiluminometric reference method and offers clinical laboratories an alternative for the analysis of erythropoietin. J. Clin. Lab. Anal. 14:271–233, 2000.

Detection of Ketosis in Vitreous at Autopsy after Embalming

Ketosis occurs in ketoacidosis or malnourishment. When either is suspected in relation to a death, it may be important to analyze for ketosis at autopsy. We encountered a case where starvation was suspected in a deceased nursing home resident, where the body had been embalmed prior to autopsy. Gas chromatography (GC) was unable to separate acetone from formaldehyde, a component of embalming fluid. The Acetest is a simple test that can detect acetone and acetoacetate in body fluids. We validated the Acetest with GC on vitreous. The Acetest and GC were consistent except at very low levels of acetone or acetoacetate. The sensitivity of the Acetest for acetoacetate in vitreous was 10 mg/dL, consistent with early starvation. Significant interference from embalming fluid did not occur. The Acetest was negative in the described case. The Acetest is a simple and useful test for the detection of ketosis in embalmed autopsies.

Evaluation of the Beckman Coulter LX20 Clinical Chemistry Analyzer

Our goal was to consolidate different methodologies in the clinical chemistry laboratory and replace aging analyzers; therefore, we evaluated the newly available Beckman Coulter LX20 (Brea, CA). Results were obtained for linearity, within- and between-day precision, correlation, interference, and serum-vs-plasma studies. Satisfactory precision results were obtained, with most assays demonstrating within-day coefficients of variation less than 2% and between-day coefficients of variation less than 5%. The linearity for all assays was acceptable over the range tested. Correlation results were adequate. The major difference in serum-vs-plasma studies was potassium. The only significant interferences noted were that lipemia decreased uric acid and bilirubin results, while hemolysis increased potassium results. We conclude that the LX20 demonstrates good performance capabilities, making this instrument suitable for a medium- to high-volume laboratory.

Automated chemiluminescent assay for C‐peptide

C‐peptide is secreted in equimolar concentrations with insulin, and is often measured to assess pancreatic β‐cell function. C‐peptide analysis is most often performed by radioimmunoassay (RIA) which has several disadvantages. We evaluated an automated, chemiluminescent immunoassay for C‐peptide in terms of precision, linearity, interference, and correlation with a RIA method. The chemiluminescent assay demonstrated acceptable correlation with the RIA method (slope = 0.82, y‐intercept = 0.88 ng/ml, r‐value = 0.97). Between‐run Cvs ranged from 8 to 9%, which compared well with the RIA method. Linearity extended beyond the manufacturers recommendations and recovery ranged from 87 to 112% across the concentrations tested, with a slope of 1.007. No significant interference was noted with hemoglobin, bilirubin, or triglyceride. Overall this method compared favorably with the RIA method and offers an alternative to RIA for the analysis of C‐peptide. J. Clin. Lab. Anal. 14:17–19, 2000.