John J. Cunningham

REDUCED MONONUCLEAR LEUKOCYTE ASCORBIC-ACID CONTENT IN ADULTS WITH INSULIN-DEPENDENT DIABETES-MELLITUS CONSUMING ADEQUATE DIETARY VITAMIN-C

Several recent studies suggest that vitamin C (ascorbic acid [AA]) status may be altered in insulin-dependent diabetes mellitus (IDDM). We measured the AA content of mononuclear leukocytes (MN-AA) as an indicator of tissue vitamin C status in adults with IDDM and nondiabetic adults matched for age and sex. Dietary vitamin C intake and plasma AA were analyzed to ensure that vitamin C availability was adequate. Dietary vitamin C intakes were above recommendations and were not different between the groups. MN-AA was reduced by 33% on average (P less than .05) in adults with IDDM (1.75 microgram/mg total protein [TP]) when compared with nondiabetics (2.60 micrograms/mg TP). When MN-AA is indexed to the dietary vitamin C intake (MN-AA/100 mg diet C), the storage deficit in adults with IDDM averages 50% (P less than .05). This observation suggests an impaired tissue AA storage in adults with IDDM and supports the theory that intracellular scurvy contributes to the chronic degenerative complications of the disease.

Elevated plasma ceruloplasmin in insulin-dependent diabetes mellitus: Evidence for increased oxidative stress as a variable complication☆

Ceruloplasmin (Cp) is an acute-phase-responsive oxidase enzyme. Prior reports suggest that Cp is increased in diabetes mellitus, perhaps reflecting greater oxidant stress. However, the situation in insulin-dependent diabetes mellitus (IDDM) per se remains unclear. Furthermore, vitamin C can interfere with one indirect assay for Cp, and vitamin C metabolism is altered in IDDM. We measured Cp levels by both a direct radial immunodiffusion (RID) assay and an indirect oxidase assay in 10 subjects with IDDM and 10 nondiabetics, both at baseline and after 30 days of vitamin C supplementation (100 or 600 mg daily, five subjects per group). Plasma copper level was measured independently also. Our data show that circulating levels of Cp are significantly increased in IDDM subjects as a group, and specifically that Cp is abnormally high in a subset of IDDM individuals. Vitamin C supplementation at either dose interfered with the oxidase assay for Cp in both groups, but vitamin C did not alter the RID assay. The observed increase in plasma copper suggests that circulating holo-Cp is increased. The finding of increased Cp in some individuals with IDDM supports the hypothesis of increased oxidant stress as a variable factor in the spectrum of chronic complications in diabetes. Measurements of Cp level by the oxidase assay must be considered unreliable for subjects taking vitamin C supplements of > or = 100 mg/d.

Vitamin C: an aldose reductase inhibitor that normalizes erythrocyte sorbitol in insulin-dependent diabetes mellitus.

OBJECTIVE Diabetic hyperglycemia promotes sorbitol production from glucose via aldose reductase. Since the intracellular accumulation of sorbitol, or its sequelae, are postulated to contribute to the progression of chronic diabetic complications, aldose reductase inhibitors (ARI) offer therapeutic promise. Others have shown that vitamin C at pharmacologic doses decreases erythrocyte (RBC) sorbitol. We examined whether smaller, physiologic doses of vitamin C were also effective in individuals with insulin-dependent diabetes mellitus (IDDM) and whether vitamin C was an ARI in vitro. METHODS Vitamin C supplements (100 or 600 mg) were taken daily for 58 days by young adults with IDDM and nondiabetic adults in an otherwise free-living design. Diabetic control was monitored by fasting plasma glucose, glycosylated hemoglobin, and glycosuria and was moderate to poor throughout the study. RBC sorbitol was measured at baseline and again at 30 and 58 days. Three-day dietary records and 24-hour urine collections were performed for each sampling day. RESULTS RBC sorbitol levels were significantly elevated in IDDMs, on average doubled, despite their more than adequate dietary intakes of vitamin C and normal plasma concentrations. Vitamin C supplementation at either dose normalized the RBC sorbitol in IDDMs within 30 days. This correction of sorbitol accumulation was independent of changes in diabetic control. Furthermore, our in vitro studies show that ascorbic acid inhibited aldose reductase activity. CONCLUSIONS Vitamin C supplementation is effective in reducing sorbitol accumulation in the erythrocytes of diabetics. Given its tissue distribution and low toxicity, we suggest a superiority for vitamin C over pharmaceutic ARIs.

Characterization of glycated hemoglobin in diabetic patients: usefulness of electrospray mass spectrometry in monitoring the extent and distribution of glycation

A combination of chromatographic and mass spectrometric techniques was used to evaluate the extent and distribution of glycation within the glycated hemoglobin (GHb) molecule. Studies on quantification of hemoglobin (Hb) glycation by electrospray ionization mass spectrometry (ES-MS) of intact globins employed specimens from 10 diabetic individuals and five normal controls. Detailed structural analysis of the phenylboronate affinity chromatography/ion-exchange (IE) HPLC-separated sub-populations of GHb was performed on a specimen carrying 13.7% GHb. An efficient protocol for mapping glycation sites within alpha and beta globins was developed, e.g., Glu-C/Asp-N proteolytic digestion followed by LC-ES-MS. Relative site occupancy within discrete components of GHb was evaluated. A correlation between the degree of glycation measured at Hb level (by affinity chromatography) and at globin level (measured by ES-MS) was carried out. The above studies led us to conclude that during the process of phenylboronate chromatography GHb dimers, rather than tetramers, are bound to the affinity resin so a fraction of glycated dimers rather than tetramers is measured. This finding implies that a process of glycation affects a much higher number of native Hb tetramers than was previously contemplated. No glycation sites appear to be missed by phenylboronate affinity chromatography. We have found no evidence of the presence of multiple glycations within a single globin chain. While glycation of both globins within a dimer cannot be excluded, it is unlikely to be a significant phenomenon. According to ES-MS data, an equivalent of about one globin per alphabeta dimer of the affinity chromatography-isolated GHb carried glycation.

Altered vitamin C transport in diabetes mellitus

Tissues sequester vitamin C in concentrations exceeding that present in plasma. The transmembrane transport mechanisms have been shown to be influenced by the concentration of glucose in vitro. On this basis an impairment of tissue vitamin C status may be present in diabetes mellitus. Recent evidence in support of this hypothesis, first proposed by Mann in 1974, is reviewed.

Effects of red blood cell transfusion on resting energy expenditure in adolescents with sickle cell anemia.

BACKGROUND Previous studies indicate that resting energy expenditure is elevated in children with sickle cell anemia, possibly caused in part by hemolysis and increased erythropoietic activity. The purpose of the present investigation was to determine whether erythrocyte transfusion normalizes resting energy expenditure in sickle cell anemia. METHODS Five adolescents with sickle cell anemia (12-16 years old; 4 boys, 1 girl) were studied before and 1 week after erythrocyte transfusion before elective surgery or at the initial transfusion for growth failure. Resting energy expenditure was measured by indirect calorimetry, and laboratory measures were determined by routine, validated methods. Data comparisons were by nonparametric analysis. RESULTS After erythrocyte transfusion, total hemoglobin levels increased (difference (D) = 15 g/l; p < 0.05), whereas hemoglobin S (D = -0.36; p < 0.05) and reticulocyte count (D = -0.12; p < 0.05) decreased. Mean pretransfusion resting energy expenditure was elevated to 124% above predicted levels (p < 0.05) and increased further to 134% above prediction (p < 0.05 vs. pretransfusion levels). Plasma triiodothyronine (T3) levels increased (D = 0.17 nmol/l; p < 0.05), reverse T3 (rT3) levels tended to decline (D = -0.04 nmol/l; p = 0.14), and rT3/T3 decreased (D = -0.03; p < 0.05). Plasma insulin-like growth factor-I (IGF-I) levels were low-normal before transfusion and did not change, despite the change in resting energy expenditure. CONCLUSIONS The results confirm that resting energy expenditure is elevated in patients with sickle cell anemia. However, resting energy expenditure further increased after transfusion, despite decreased erythropoietic activity. A posttransfusion decrease in rT3/T3 may contribute to the increased resting energy expenditure. That there was no change in IGF-I implies that the growth hormone-IGF system is not involved in posttransfusion regulation of resting energy expenditure. Therefore, our data are not consistent with the hypothesis that increased resting energy expenditure in sickle cell anemia is directly related to erythropoietic activity. The mechanisms by which resting energy expenditure increases after transfusion in sickle cell anemia require additional investigation.

The Effect of Red Blood Cell (RBC) Transfusion Therapy on Resting Energy Expenditure (REE) in Patients with Sickle Cell Anemia (SCA). |[dagger]| 658

Growth retardation is common in children with SCA, in spite of adequate dietary intakes. Previous studies have demonstrated elevated REE in children with SCA. An increased metabolic rate may be secondary to rapid hemolysis of sickled cells and the compensatory erythropoietic activity. The purpose of the current investigation was to determine whether the elevated REE in children with SCA can be normalized by RBC transfusion therapy. Five children aged 12 to 16 years (4 males, 1 female) were studied before and one week after a scheduled RBC transfusion. Measurements included anthropometics (height, weight), REE by indirect calorimetry, hemoglobin, reticulocyte count, and percent HgS. Growth failure was seen in 3 of 5 subjects who were below the 25% ile by NCHS standards for height. Pre-transfusion REE was significantly elevated above predicted REE (28%, p≤0.04, Wilcoxon matched-pairs signed-ranks test) consistent with reports in the literature. Surprisingly, REE increased further after RBC transfusion to 37% on average above predicted REE (pre versus post REE, p≤0.08) despite a significant decrease in erythropoietic activity (reticulocyte count: Δ = -9.3%, p≤0.04). Four of 5 subjects showed an increase in REE after RBC transfusion. The subject who had no change in REE demonstrated an increase one week after receiving a second RBC transfusion. Anemia improved after RBC transfusion with an increase in hemoglobin (Δ=1.5 g/dl, p≤0.04), and decrease in Hgb S (Δ=-31.4%, p≤0.04). These results suggest that REE is not correlated with erythropoietic activity in children with SCA and may, in fact, increase when RBC transfusion decreases erythropoietic activity. A possible explanation for this increase includes the hypothesis that a relatively hypoxic catabolic hypermetabolism exists prior to RBC transfusion that transitions in children with SCA to a more hypermetabolic anabolic state after RBC transfusion. If proven, this might explain the catch-up growth observed in children with SCA who receive chronic RBC transfusion therapy.