The effect of diabetic control on very low-density lipoprotein--triglyceride metabolism in patients with type II diabetes mellitus and marked hypertriglyceridemia

We examined the effect of diabetic control on very low-density lipoprotein-triglyceride (VLDL-TG) metabolism in six patients with type II (noninsulin-dependent) diabetes mellitus and marked hypertriglyceridemia. VLDL-TG transport was determined using 3H-glycerol as an endogenous precursor of VLDL-TG, and the resultant kinetic data were evaluated by multicompartmental analysis. Studies were performed in the hypertriglyceridemic diabetic subjects during poor diabetic control and again after 3 months of diabetic treatment, and the results were compared to studies in nondiabetic normolipidemic subjects and nondiabetic subjects with familial forms of hypertriglyceridemia. In the poorly controlled diabetics, mean VLDL-TG synthesis was threefold higher than in the normolipidemic subjects, and the mean fractional catabolic rate (FCR) of VLDL-TG was only one-third of the normals. With diabetic treatment, plasma triglyceride levels fell by more than 50%, but remained fourfold higher than the normals. This was associated with a decrease in mean VLDL-TG synthesis to a level similar to that observed in the genetic hyperlipidemic subjects, but still 2.6-fold higher than the normals. In addition, the mean FCR rose after diabetic control to a level slightly above that of the genetic hyperlipidemic subjects, but remained less than one-half of the normal value. However, the response of VLDL-TG kinetics to diabetic treatment was not uniform. In four subjects, control of hyperglycemia ameliorated the hypertriglyceridemia primarily by decreasing VLDL-TG overproduction. In the other two subjects, diabetic treatment had a greater effect on the FCR than an overproduction of VLDL-TG. Thus, in this select group of diabetic, hypertriglyceridemic subjects, poor diabetic control contributed to both VLDL-TG overproduction and low FCRs. Failure of diabetic treatment to restore VLDL-TG kinetic parameters to normal suggests that the hypertriglyceridemia was due not only to diabetes mellitus but also to an additional abnormality affecting lipoprotein metabolism.(ABSTRACT TRUNCATED AT 250 WORDS)     

Pathways of insulin degradation in isolated adipocytes: evaluation by gel filtration and differential precipitation

Insulin degradation by isolated rat adipocytes was evaluated using gel filtration and a new technique of differential precipitation to fractionate the sample by molecular size using polyethylene glycol and trichloracetic acid. At 37 degrees C, 125I-insulin bound to adipocytes was rapidly degraded into small fragments or iodotyrosine. 125I-insulin in the medium was also degraded into iodotyrosine, as well as fragments intermediate in molecular weight between insulin and iodotyrosine. Lowering the temperature to 15 degrees C or adding bacitracin to the medium inhibited degradation in the medium but had little effect on cell-associated degradation. Methylamine, on the other hand, inhibited cell-associated degradation, but had little effect on the insulin degradation in the medium. Addition of methylamine or bacitracin or lowering of the temperature increased the amount of 125I-insulin bound to the cell and prolonged the steady-state of binding. Bacitracin also produced a slight shift to the left in the dose response curve for insulin-stimulated glucose oxidation. Methylamine increased basal glucose oxidation, but had no effect on insulin sensitivity as measured in the glucose oxidation bioassay. These data suggest that isolated adipocytes in vitro exhibit at least two distinct pathways of insulin degradation, a cell-associated pathway which can be inhibited by methylamine and a medium pathway which can be inhibited by bacitracin. Neither pathway, however, appears to be closely linked to insulin's ability to stimulate glucose metabolism in these cells.     

Insulin secretory dynamics after two consecutive intravenous stimulations with glucose and/or tolbutamide

Intravenous glucose and/or tolbutamide administered in two consecutive pulses 30 and 60 min apart to the same subjects using identical doses showed that insulin secretory responses was altered during a subsequent stimulation and that this was modulated by the time factor. Insulin response was more sustained after the second glucose pulses and the insulin peak response was delayed and diminished if the second glucose dose was given 30 min after the first, but not if given 60 min later. It is suggested that the beta-cell membrane might remain partially depolarized above a certain glucose level or that a postulated signal relay mechanism might become saturated. Responses to two tolbutamide pulses did not show these characteristics; however, the second insulin response was smaller than the first. When the first pulse was glucose and the second tolbutamide, or vice versa, the second responses were altogether different from those elicited by the double doses of either tolbutamide or glucose. To explain these characteristic patterns of insulin secretory dynamics, the existence of occult glucose receptors on the beta-cell that are opened up by tolbutamide was postulated. These studies do not support the two-pool theory, or at least restrict it to glucose-stimulated insulin response. The positive correlations between the first and the second insulin responses in all tests argue strongly against the existence of an insulin feedback mechanism in man.     

Insulin resistance and monoclonal gammopathy

Two maturity-onset diabetic patients developed severe insulin resistance during the course of monoclonal gammopathies. One patient had Waldenström macroglobulinemia and the other had multiple myeloma with IgA gammopathy. The maximum insulin binding capacity (MIBC) was 121 U/liter and 54.7 U/liter, respectively, during insulin resistance. The clinical courses of insulin resistance paralleled the activity of the monoclonal gammopathies (MG) with the insulin resistance disappearing after the monoclonal gammopathies were controlled. Six other diabetic patients with concurrent insulin resistance and monoclonal gammopathies are reviewed.     

Effect of desialylation of very low-density lipoproteins on their catabolism by lipoprotein lipase

Very low-density lipoproteins (VLDL) contain sialylated apolipoproteins (apo) (eg, apo CIII1-3) that inhibit apo CII activation of lipoprotein lipase (LPL) and also uptake of triglyceride (TG)-rich lipoproteins by the liver. Hypertriglyceridemic patients can have an excess of sialylated apo CIII (apo CIII1 or apo CIII2) in VLDL. These observations have prompted the notion that sialic acid in VLDL may impede LPL or receptor-mediated clearance of VLDL and thus result in hypertriglyceridemia. The aim of this study was to determine whether desialylation of VLDL altered their property as a substrate for LPL. VLDL isolated from five hypertriglyceridemic patients was desialylated with neuraminidase, labeled with a fluorescent probe, dansyl phosphatidylethanolamine and 600 micrograms of labeled VLDL TG were incubated with a constant amount of purified bovine LPL. The change in fluorescence against time was monitored on a recorder to yield curves representing continuous lipolysis of VLDL by LPL. Mean initial velocity of reaction (Vi) and extent of lipolysis measured as total increase in fluorescence over baseline at 30 minutes (F30/FO) were similar (Vi = 10.2 +/- 0.37 control v 10.2 +/- 0.42 u/min desialylated VLDL; F30/FO = 4.1 +/- 0.15, control v 4.1 +/- 0.07 desialylated VLDL; n = 5). Thus, sialic acid does not influence VLDL catabolism by LPL. Our study does not exclude a possible role of the sialic acid in receptor mediated uptake of remnants produced by initial catabolism of VLDL by LPL.     

The determination of glycosylated hemoglobins in rats using high pressure liquid chromatography

Glycosylated hemoglobins (GHb) or fast hemoglobins (FH) are minor components of hemoglobin that so far have been quantified in men, monkeys, and mice, and they are elevated in diabetic subjects of all these species. Since the rat is a useful model for experimental diabetic studies, hemolysates from streptozotocin-induced diabetic rats were analyzed for FH fractions using a high pressure liquid chromatography method. In a long-term study (3 mo), the maximal increment of the FH fractions was achieved after 5 wk of diabetes (from 5.67% ± 0.41% SD to 10.80% ± 0.74%) supporting the notion that the biosynthesis of these compounds occurs continuously during the lifespan of the red cell. In a short-term study. however, an elevation of the FH by 11% after 2 days and by 26% after 6 days of diabetes was noticed suggesting that a rapid increase of the FH may occur in relation to rapid changes of the glucose level.     

Regulation of proline biosynthesis: the inhibition of pyrroline-5-carboxylate synthase activity by ornithine

Mammalian cells have the capacity for proline biosynthesis from ornithine or glutamic acid. Using a radioisotopic assay, we have studied the regulation by ornithine of delta 1-pyrroline-5-carboxylate synthase, the enzyme that catalyzes the first step of proline biosynthesis from glutamic acid. In homogenates from Chinese hamster ovary cells, ornithine was found to be a potent inhibitor of pyrroline-5-carboxylate synthase activity(50% inhibition at 0.37 mM). The effect was reversible and did not occur with amino acids other than ornithine. Preliminary findings suggest that the inhibition does not result from altered requirements for the cofactors NADPH and ATP. Significant inhibition was observed in four different Chinese hamster cell lines. Ornithine was also shown to inhibit the conversion of 3H-glutamic acid to 3H-proline in intact human skin fibroblasts. Cells from patients with a rare ocular disease, gyrate atrophy of the choroid and retina, were used for these studies since they lack interfering ornithine aminotransferase activity. We conclude that ornithine may be a physiologic regulator of the rate of proline formation from glutamic acid. This information allows us to construct an hypothetical model for the overall regulation of proline biosynthesis and also to suggest a pathophysiologic mechanism for the disease gyrate atrophy.     

Metabolic and endocrine studies in a case of lipoatrophic diabetes

A 20-yr-old female with congenital lipoatrophic diabetes was studied, with the following findings: (1) Serum insulin levels increased after both oral glucose and intravenous arginine administration; there was no growth hormone response to the latter. (2) The infusion of insulin (0.1 units and 0.5 units/kg) during the fed state and following a 110-hr fast produced only minimal changes of various fuels measured, with the exception of a decrease in the branched-chain amino acids. (3) There was a minimal production of ketones during the 110-hr fast. (4) Metabolic expenditure was markedly increased during the postabsorptive state (65–75 kcal/hr/sq m); it fell into the normal range during the 110-hr fast (31–35 kcal/hr/sq m). (5) Following meals, the patient experienced complaints ranging from cold and shivering to feeling hot with gross diaphoresis. These findings were associated with intermittent lability of her skin temperature, which varied 1°–2°F during a 3-hr period. (6) Progressive increases in doses of regular insulin before each meal resulted in up to a total of 9000 units/day being required before normal blood glucose levels were achieved. (7) A 2-wk therapeutic trial of pimozide provided no significant changes in a variety of hormones and fuels in the basal state or following insulin perturbations. (8) A variety of pituitary hormones and pituitary target organ hormones were studied in both the hypothyroid (Hashimoto's thyroiditis) and euthyroid state (following thyroid replacement). All the hormone responses were normal except that growth hormone did not rise during the slow wave sleep in either thyroid state.     

Treatment of diabetes mellitus by devices

A very important aspect of diabetes mellitus is whether or not normalization or near-normalization of blood glucose and/or other metabolites and hormones may reduce or eliminate the chronic complications of this disease. To answer this question and to provide a more “physiologic” approach to insulin administration, a constellation of devices have reached the stage of clinical investigation. These include small portable pump systems that can provide variable rates of insulin infusion via the subcutaneous intravenous or ultraperitoneal routes. In addition, bedside artificial “beta cells” having the capability of providing insulin infusions, with the rate varying as a function of continuous glucose measurements, are available for short-term studies. Under development are implantable continuous infusion devices and implantable glucose sensors that could in the future lead to a miniaturized implantable glucose-controlled insulin administration system.     

Apolipoprotein AI and AII metabolism in patients with primary high-density lipoprotein deficiency associated with familial hypertriglyceridemia

Plasma high-density lipoproteins (HDL) and their major proteins--apolipoprotein (apo) AI and apo AII--are subnormal in most patients with familial hypertriglyceridemia. However, the pathophysiology of low-plasma apo AI and apo AII is unclear. The kinetic parameters (turnover) of HDL apo AI and apo AII were studied in six lean patients with primary HDL deficiency associated with familial hypertriglyceridemia and five normolipidemic controls. Autologous 125I labeled HDL were injected intravenously (IV; 25 microCi) and blood samples drawn ten minutes after the injection and periodically thereafter for 12 days. Urine samples were collected daily and their radioactivity measured. Kinetic parameters were calculated from the area under the decay curve using three exponentials. Mean plasma apo AI and apo AII were significantly lower (P less than 0.001) in patients than normals (70.4 +/- 2.7 v 106.9 +/- 7.0; 24.2 +/- 1.6 v 39.2 +/- 0.9 mg/dL, respectively). The mean fractional catabolic rates (FCR) obtained from plasma 125I-HDL, apo AI, apo AII radioactivity decay curves and by Berson and Yalow's method (urine/plasma radioactivity ratios) were significantly greater (P less than 0.05) in patients than in controls (0.387 v 0.299; 0.391 v 0.309; 0.361 v 0.275; 0.272 v 0.207/d; respectively). The mean synthetic rates (SR) of apo AI and apo AII were significantly lower in patients than in controls (11.12 v 14.17 mg/kg body weight/d, P less than 0.05; 3.53 v 4.68 mg/kg body weight/d, P less than 0.05, respectively). In vitro lipolysis of triglyceride (TG) rich lipoproteins by bovine lipoprotein lipase, and measurement of hepatic TG lipase and lipoprotein lipase in postheparin plasma were similar in patients and controls, indicating no abnormality in these factors that are linked to HDL and TG catabolism. However, a significant positive correlation between hepatic TG lipase and the FCR of apo AI and apo AII was found. The data suggest that in this series of patients with HDL deficiency the low plasma HDL-cholesterol, apo AI, and apo AII levels resulted from decreased synthesis and an increased fractional catabolic rate of apo AI and apo AII, the major proteins of HDL.     

Evidence for coordinate genetic control of Na,K pump density in erythrocytes and lymphocytes

The erythrocyte is widely used as a model cell for studies of the Na,K pump in health and disease. However, little is known about the factors that control the number of Na,K pumps expressed on the erythrocytes of a given individual, nor about the extent to which erythrocytes can be used to validly assess the pump density on other cell types. In this report, we have compared the interindividual variance of Na,K pump density in erythrocytes of unrelated individuals to that seen with identical twins. Unlike unrelated individuals, in whom pump parameters, ie, ouabain binding sites. 86Rb uptake, cell Na concentration vary widely, identical twin pairs showed no significant intrapair variation for these values. Thus, a role for genetic factors is suggested. In addition, we established and validated a method for determining Na,K pump density and pump-mediated 86Rb uptake in human peripheral lymphocytes. Using this method, we show that whereas Na,K pump density differs markedly between erythrocytes (mean of 285 sites per cell) and lymphocytes (mean 40,600 sites per cell), there is a strong and highly significant correlation (r = 0.79, P less than 0.001) between the pump density in these cell types in any given individual. Taken together, these studies suggest that genetic factors are important determinants of Na,K pump expression, and that pump density appears to be coordinately regulated in two cell types in healthy individuals.     

Hormone levels and fuel flow in patients with weight loss and lung cancer. Evidence for excessive metabolic expenditure and for an adaptive response mediated by a reduced level of 3,5,3′-triiodothyronine

Hormone levels, fuel flow, and basal metabolic rate were studied in five men with weight loss and non-oat cell carcinoma of the lung and eight normal men during a brief fast (96 hours). The patients with cancer had lost approximately 11.0 ± 3.3 kg (16.6 ± 5.1% of ideal body weight) before participation in the study (mean ± SEM). They exhibited many of the characteristic clinical and laboratory features of this syndrome but, at 94.2 ± 6.9% of ideal body weight at the onset of the fast, were not wasted. Their estimated mean daily caloric intake before admission was 1832 ± 408 kcal. All subjects received a balanced weight maintenance diet for 62 hours before fasting. Compared with the normal subjects, the patients with weight loss and cancer demonstrated a diminished increase in the plasma free fatty acid level during the fast, a decreased blood alanine level at all times before and during the fast, a decreased total urine nitrogen excretion rate before and during the fast (in grams per 24 hours and grams per square meter of body surface area, but not in grams per gram of creatinine), and a decreased serum 3,5,3′-triiodothyronine (total T₃) level before and during the fast. The urine total ketone excretion rate was low in three of the five cancer patients. A comparison of the circulating levels of glucose, ketones, lactate, pyruvate, insulin, glucagon, 3,3′,5′-triiodothyronine (reverse T₃), norepinephrine, epinephrine, cortisol, and growth hormone in the cancer patients with those of the normal subjects before and during the fast revealed no important difference. The basal metabolic rate was significantly higher in the cancer patients than in the normal subjects on the last day of the fast, despite the reduced total T₃ level and the normal catecholamine levels. Our findings indicate that patients with weight loss and non-oat cell carcinoma of the lung exhibit excessive metabolic expenditure. In addition, they respond adaptively to total caloric deprivation, to the maladaptive elevation in metabolic expenditure, or to both, by conserving lipid and nitrogen stores; they do not waste nitrogen. This adaptive conservation of fuels may be mediated by the reduced total T₃ level, which can account for the decrease in lipolysis and which appears to decrease alanine release from the periphery. These findings cannot be attributed to recent deprivation of calories or carbohydrate, in view of the daily intake before admission and while on a balanced weight-maintenance diet before fasting. The pathogenesis of the reduced total T₃ level is unknown; it may reflect the loss of weight before the study or chronic illness. In cancer patients, the rate of weight loss reflects the level of caloric intake, the extent of excessive metabolic expenditure, and the effectiveness of the adaptive conservation of fuels mediated by the reduced total T₃ level.     

Protein metabolism during total parenteral nutrition (TPN) in injured rats using medium-chain triglycerides

There are metabolic limitations to the infusion of large quantities of dextrose in critically ill patients receiving total parenteral nutrition. Of the alternative, nonprotein lipid sources, medium chain triglycerides (carbon chain length 8 and 10) are more rapidly oxidized and are deposited in the adipose tissue at rates much less than long chain triglycerides. In rats with burn injury receiving hypocaloric (dextrose and amino acids) parenteral feeding, we studied the changes in protein metabolism as a result of increasing the caloric intake by 33% by the addition of either dextrose, a soybean oil emulsion, a medium chain triglyceride emulsion, or a structured lipid emulsion of triglycerides synthesized from safflower oil (40%) and medium chain triglycerides (60%). Changes in body weight, blood glucose concentration, beta-hydroxybutyrate, lactate, amino acids, insulin, albumin, liver protein content, and nitrogen balance were measured during three days of feeding. Whole body leucine kinetics and muscle protein fractional synthetic rate were evaluated using a constant intravenous infusion of L-[1-14C]leucine. The addition of dextrose or soybean oil emulsion produced a significant increase in body weight and liver nitrogen but did not change albumin concentrations or leucine kinetics compared to those of the hypocaloric feeding group. Rats receiving medium chain triglycerides and structured lipid emulsions showed a reduction in branched chain amino acid concentrations and an improvement in serum albumin levels. However, the rats receiving the structured lipid emulsion also showed increased body weight, had a significant decrease in leucine oxidation, and showed a three day cumulative nitrogen balance significantly greater than zero.(ABSTRACT TRUNCATED AT 250 WORDS)     

Further development and automation of a high pressure liquid chromatography method for the determination of glycosylated hemoglobins.

A previously described high pressure liquid chromatography system for the determination of glycosylated hemoglobin concentrations has been automated and simplified. With this methodology it is possible to perform up to 60 analyses per day for hemoglobin A1a+b% and hemoglobin A1c%. Where an estimate of the total fast hemoglobin alone is required, then a considerably greater number of analyses can be performed. The individual values are calculated directly with an electronic integrator. The mean coefficient of variation of the duplicate determinations of 48 samples was 0.63 +/- 0.83% (mean +/- SD). Aliquots of pooled hemolysates have been maintained in liquid nitrogen at -90 degrees C and run at the beginning and end of each daily analytical run over an 18-mo period. Both the inter- and intra-run coefficients of variation of these values have remained consistently less than 3%. Therefore, the methodology offers a reliable and accurate method of containing glycosylated hemoglobin values for clinical use.     

Chylomicron and very low-density lipoprotein apolipoprotein B metabolism: mechanism of the response to stanozolol in a patient with severe hypertriglyceridemia

Studies of simultaneous autologous 131I-chylomicron (Sf greater than 400) and 125I-very low density lipoprotein (VLDL) (Sf 20 to 400) apolipoprotein B (apo B) were performed both before (triglyceride level c 1500 mg/dL) and during treatment with stanozolol, a 17 alpha-methyl anabolic androgenic steroid (triglyceride level c 750 mg/dL) in a 74-year-old woman with a past history of recurrent chylomicronemic pancreatitis. Both before and during stanozolol treatment chylomicron apo B disappeared rapidly and directly, little appearing in VLDL and virtually none in intermediate (IDL) or low density lipoproteins (LDL). Multicompartmental analysis indicated that the great majority of chylomicron apo B was removed via an extremely rapid compartment (estimated fractional catabolic rate [FCR], 5.0/h), accounting for 66% before and 88% during stanozolol treatment. The remaining 131I-apo B decayed biphasically, with total Sf greater than 400 residence times of 8.6 hours before and 3.7 hours during stanozolol treatment. Hence, despite a moderately depressed adipose tissue lipoprotein lipase activity, the subject's hypertriglyceridemia did not appear to proceed solely from retarded chylomicron removal, nor was the dramatic decrease in triglyceride in response to stanozolol a function only of the acceleration of such removal. VLDL apo B kinetics were analyzed by a multicompartmental model featuring a rapid, stepwise delipidation chain which proceeds either rapidly to IDL and LDL or to a slowly turning over compartment within VLDL. While VLDL. apo B synthesis remained essentially constant, the major effect of stanozolol was a substantial reduction in the fraction of VLDL apo B diverted to this slowly turning over compartment, which decreased from 5.0% before to 1.2% during treatment.(ABSTRACT TRUNCATED AT 250 WORDS)     

Structured medium-chain and long-chain triglyceride emulsions are superior to physical mixtures in sparing body protein in the burned rat

The present study was undertaken to compare the effectiveness of a physical mixture of long-chain and medium-chain triglycerides with an emulsion consisting of chemically synthesized triglycerides composed of medium-chain and long-chain fatty acids in similar proportions. Sprague-Dawley rats received a 25% body surface area full-thickness scald burn on the dorsum. For the next three days, all rats received 300 kcal/kg/day as 160 kcal/kg/day glucose, 50 kcal/kg/day amino acid, and an additional 90 kcal/kg/day lipid emulsion as either long-chain triglyceride, medium-chain triglyceride, a 1:1 physcial mix of medium-chain and long-chain triglycerides or a chemically structured triglyceride made up of 60% medium-chain fatty acid and a 40% safflower oil. Rats receiving the chemically structured lipid emulsion showed the greatest gain in body weight, the greatest positive nitrogen balance, and the highest serum albumin concentration, outstripping rats receiving the long-chain triglyceride, medium-chain triglyceride, and even the physical mixture long-chain and medium-chain trigylcerides (P < 0.01). A 30% increase in oxygen consumption and 35% increase in energy expenditure in rats given the medium-chain triglyceride emulsion alone (P < 0.01) was observed. This study confirms that the metabolism of chemically structured triglycerides composed of medium-chain and long-chain fatty acids markedly differs from similar physical mixtures. For these reasons, the new structured lipid emulsions may prove advantageous in feeding the severely injured patient.     

Metabolic effects of carbohydrate in low-calorie diets

Fasting plasma glucose turnover, urinary 3-methylhistidine excretion, and fasting plasma protein profiles were compared in a 4-week randomized clinical trial of two very low-calorie weight-reduction diets. Diet A (360 kcal) provided 1.5 g egg protein per kg ideal body weight (IBW) but no carbohydrate. Diet B (340 kcal) provided 0.8 g egg protein per kg IBW plus 0.7 g carbohydrate per kg IBW. Eleven moderately obese healthy young women were studied. After 3 weeks of dieting, fasting plasma glucose appearance and oxidation decreased by equal amounts (20% and 30%, respectively) for both diets. 3-methylhistidine excretion remained at control rates for the first week on the diets, then fell by equal amounts (25% to 30%) with both diets. Similar declines were observed for both diets in serum prealbumin and retinol-binding protein concentrations. Mean serum transferrin declined with both diets, but the changes were not statistically significant. Serum albumin was unchanged by either diet. Thus, there were no significant differences between the two diets with regard to any of the measured parameters.     

A rapid method for the determination of glycosylated hemoglobins using high pressure liquid chromatography

Hemoglobin (Hb) A_Ic is a minor component of Hb found in normal individuals but elevated two or threefold in patients with diabetes mellitus. Limited studies have suggested that the level of Hb A_Ic is proportional to the integrated concentration of glucose over time. Thus it could serve as an index of hyperglycemia. Its measurement may enable a more objective approach to assessing whether or not the control of hyperglycemia can be correlated with the severity of complications of diabetes. Large scale clinical studies of Hb A_Ic have not been undertaken for lack of a rapid assay system. This article describes a method of high pressure liquid chromatography (HPLC) which enables the isolation of Hb A_Ic in 27 min using only 12 μg of Hb (100 μl of blood) and a second method for the isolation of total fast Hb components (also elevated in diabetes) in 11 min. Using the first method, a total of 36 assays were performed on the blood of a single normal volunteer over a one month period. The mean level of Hb A_Ic was 4.95 ± 0.12% (SD) ± 0.02% (SEM), while the coefficient of variation (C.V.) was 2.4%. The mean Hb A_Ia&b level was 1.65 ± 0.06% ± 0.01% (C.V. = 3.6%). Values for Hb A_Ic in 10 normal individuals were 5.06 (mean) ± 0.32% (SD) ± 0.10% (SEM). Hb A_Ic values in 15 patients with diabetes mellitus ranged from 6.8 to 20.0%. The second method was designed to assay Hb A_Ia, Hb A_Ib, and Hb A_Ic as a single peak and yielded results identical to the sum of these components as determined by the first method (r = 0.98; p < 0.001).