Barry J. Gould

Effect of Vitamin C on Glycosylation of Proteins

Twelve nondiabetic subjects consumed 1 g/day vitamin C for 3 mo. A fasting blood sample was taken at the start of the study and at the end of each month for the measurement of plasma and intraerythrocyte glucose, vitamin C, glycosylated hemoglobin (affinity chromatography and electrophoresis), and glycosylated albumin (affinity chromatography). Although there were no significant changes in fasting glycemia, glycosylated hemoglobin (affinity chromatography) decreased 18%, from 6.18 ± 0.48% (mean ± SD) at the start to 5.05 ± 0.50% (P < 0.0001) after 3 mo, whereas, HbA1 measured by electrophoresis increased 16%, from 6.17 ± 0.61 to 7.16 ± 0.59% (P < 0.0001) in this period. Glycosylated albumin decreased 33%, from 1.56 ± 0.24 to 1.04 ± 1.01% (P < 0.0001) after 3 mo. This discrepancy between glycosylated hemoglobin measured by electrophoresis and affinity chromatography was due to methodological differences between the two techniques, with affinity chromatography measuring “true” glycosylated hemoglobin. The greater decrease found with glycosylated albumin was probably due to the different distribution of vitamin C between plasma and within the erythrocyte, levels after 1 mo of supplementation being 109 ± 19 and 59 ± 9 μM, respectively (P < 0.001). This indicates that administration of oral vitamin C may inhibit the glycosylation of proteins in vivo by a competitive mechanism.

Unexplained variability of glycated haemoglobin in non-diabetic subjects not related to glycaemia.

SummaryWe have studied levels of glycated haemoglobin in a sample of 223 people aged over 40 years without known diabetes mellitus screened in a community study. Each had a glucose tolerance test and glycated haemoglobin measured by four methods — agar gel electrophoresis with and without removal of Schiff base, affinity chromatography and isoelectric focusing. The correlation coefficients between 2 h blood glucose and levels of glycated haemoglobin were between 0.43 and 0.64. This poor correlation was not explained on the basis of assay or biological variability of either 2 h blood glucose or glycated haemoglobin. Multiple regression analysis showed that other assays of glycated haemoglobin contributed to the variance of any single glycated haemoglobin value by 0.1%–52.9% (median 12.8%) compared to the variance of 18.6%–41.4% (median 30.8%) explained by 2 h blood glucose alone, suggesting that in a non-diabetic population, the degree of glucose intolerance may explain only one third of the variance of glycated haemoglobin levels, but other factors operate to produce consistent changes in levels of glycated haemoglobin. Investigation of 42 subjects with consistently high (20 subjects) or low (22 subjects) levels of glycated haemoglobin relative to their 2 h blood glucose level showed no difference in age, gender, body mass index, haemoglobin levels or smoking, although 50% of low glycators had impaired glucose tolerance. Neither ambient bloodglucose levels, as estimated on two five-point blood-glucose profiles, nor dietary intake of carbohydrate, starch, sugars, fibre or alcohol, explained the difference between high and low glycators. The determinants of the consistent interindividual differences in levels of glycated haemoglobin in nondiabetic subjects remain to be determined.

Investigation of the mechanism underlying the variability of glycated haemoglobin in non-diabetic subjects not related to glycaemia

The Islington Diabetes Survey identified two groups of non-diabetic individuals, low and high glycators, who remained consistently classified 4.4 +/- 0.2 years after the original study. To investigate the mechanism for this grouping, 12 original subjects, 5 with low and 7 with high levels of glycated haemoglobin relative to their 2 h blood glucose, were studied. Glycated albumin and fructosamine measurements gave comparable classifications, with three individuals being misclassified for each measurement; in addition glycated albumin was positively correlated with mean blood-glucose concentration (r = 0.53; P < 0.05). Fasting plasma glucose concentration was greater than the intra-erythrocyte concentration (P < 0.05), but their ratio was reduced in low compared to high glycators (0.77 +/- 0.12 and 0.94 +/- 0.13, P < 0.0001). No differences between groups were found for plasma insulin, urea or non-esterified fatty acids; plasma or intra-erythrocyte inorganic phosphate or vitamin C; nor plasma, erythrocyte or urinary total amino acids. Erythrocyte 2,3-diphosphoglycerate, a catalyst of glycation, was elevated in high compared to low glycators (5.61 +/- 0.26 and 4.81 +/- 0.24 mmol/l, P < 0.001). Mean centile glycated haemoglobin was positively correlated with intra-erythrocyte pH (r = 0.55; P < 0.05) and negatively with plasma total amino acids (r = -0.57, P < 0.05). These data indicate that the intra-erythrocyte environment of high glycators favours glycation of haemoglobin. This could have important consequences for diabetic patients in terms of monitoring their glycaemic control and in the progression of those complications related to non-enzymic glycation of intracellular proteins.

Use of manufactured foods enriched with fish oils as a means of increasing long-chain n −3 polyunsaturated fatty acid intake

The objectives of the present study were to determine the feasibility of using manufactured foods, enriched with eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) as a means of increasing the intake of these n-3 polyunsaturated fatty acids (PUFA), and to determine the effect of the consumption of these foods on postprandial lipaemia and other metabolic responses to a high-fat mixed test meal. Nine healthy, normotriacylglycerolaemic, free-living male volunteers (aged 35-60 years) completed the randomized, controlled, single-blind, crossover study. The study consisted of two periods (each of 22 d) of dietary intervention, separated by a 5-month washout period. During these two periods the subjects were provided with the manufactured foods enriched with EPA and DHA (n-3 enriched) or identical but unenriched foods (control). A mixed test meal containing 82 g fat was given to the fasted subjects on day 22 of each dietary intervention period. Two fasting, and thereafter hourly, blood samples were collected from the subjects for an 8 h period postprandially. Plasma triacylglycerol, total and HDL-cholesterol, non-esterified fatty acids (NEFA), glucose and immunoreactive insulin levels, post-heparin lipoprotein lipase (EC 3.1.1.34) activity and the plasma free fatty acid and phospholipid fatty acid compositions were measured. A mean daily intake of 1.4 g EPA+DHA (0.9 g EPA, 0.5 g DHA) was ingested during the n-3-enriched dietary period, which was significantly higher than the intake during the habitual and control periods (P < 0.001) assessed by a 3 d weighed food intake. A significantly higher level of EPA+DHA enrichment of the plasma fatty acids and phospholipids (P < 0.001) after the n-3-enriched compared with the control intervention periods was also found. The energy intake on both of the dietary intervention periods was found to be significantly higher than on the habitual diet (P < 0.001), with an increase in body weight of the subjects, which reached significance during the n-3 PUFA-enriched dietary intervention period (P < 0.04). The palatability of the enriched foods was not significantly different from that of the control foods. Significantly higher fasting plasma HDL-cholesterol and glucose concentrations were found after the n-3 PUFA-enriched compared with the control intervention period (P < 0.02 and P < 0.05 respectively). No significant differences were found for the postprandial lipid and hormone measurements, except for significantly lower levels of NEFA at 60 min after the n-3-enriched intervention period (P < 0.04). Enriched manufactured foods were a feasible vehicle for increasing n-3 PUFA intake. However the nature of the foods provided as the n-3 vehicle may have contributed to the increased body weight and higher energy intakes which were adverse consequences of the intervention. These factors, together with the short duration of the study may have been responsible for the failure to observe significant plasma triacylglycerol reductions in response to daily intakes of 1.4 g EPA+DHA.

Differences in postprandial lipaemic response between Northern and Southern Europeans

Postprandial lipaemic responses to two test meals were investigated in 30 Northern (15 British and 15 Irish), and 30 Southern (Greeks from Crete) healthy male Europeans. The meals were a saturated fatty acid (SFA) meal, which resembled the fatty acid composition of an average UK diet, and a monounsaturated fatty acid (MUFA) meal in which the fat consisted of olive oil. Habitual diets of the two groups differed, with higher total fat, (P < 0.03) and MUFA (P < 0.0001) and lower polyunsaturated fatty acid (PUFA) (P < 0.0001) intakes in Southern than Northern Europeans. Levels of total MUFA (P < 0.02) and oleic acid (P < 0.004) were also higher in adipose tissue of Southern in comparison to Northern Europeans. In both European groups there were no significant differences in postprandial triglyceride response between the two meal types, SFA or MUFA. However, Northern and Southern Europeans showed significant differences in their patterns of postprandial response in plasma triglycerides (P < 0.0001), apolipoprotein B-48 (P < 0.0001), NEFA (P < 0.0001), insulin (P < 0.0007), and factor VII activity (P-0.03). In the case of NEFA, areas under the response curve were higher following the SFA than the MUFA meal for both groups, (P < 0.003) and were greater in Southern than Northern Europeans (P < 0.002) and apo B-48 responses were lower (P < 0.005). Some of these differences may reflect differences in fasting levels since fasting apolipoprotein B-48 levels were lower (P < 0.01) and fasting NEFA (P < 0.02) and insulin (P < 0.005) were higher in the Southern than in the Northern Europeans. In addition, 9 h postprandial post-heparin lipoprotein lipase activity was lower in the Southern than in the Northern Europeans (P < 0.0006). This is the first report of differences in postprandial lipid, factor VII and insulin responses in Southern and Northern Europeans which may be of importance in explaining the different susceptibilities of these two populations to risk of coronary artery disease.

A sensitive method for the measurement of glycosylated plasma proteins using affinity chromatography

We describe a simple, sensitive affinity technique for the routine measurement of glycosylated plasma proteins in clinical laboratories. The commercially available phenylboronic acid gel used for the chromatography has recently been marketed as a kit for this purpose (Glycogel Test Kit, Pierce Chemical Co). The manufacturers of this kit recommend loading 200 μl neat plasma to each 1 ml gel column. This high loading is to enable the direct measurement of protein in the bound and unbound fractions at 280 nm. This loading is consistent with 10–15 mg protein being added per ml gel. Our results show that protein levels greater than 2 mg per ml gel overload the column. Therefore we used a modification of the more sensitive Bradford procedure to measure protein. The method discriminates between normals (6·29 ± 1·87%) and diabetic patients (12·62 ± 3·36%) and has good precision (CV 4–6%). The results obtained correlate with the colorimetric method using thiobarbituric acid (r = 0·70) and with glycosylated haemoglobin (r = 0·82).

Quantitation of apolipoprotein B-48 in triacylglycerol-rich lipoproteins by a specific enzyme-linked immunosorbent assay

This paper describes the use of an antiserum, specific for apolipoprotein (apo) B-48, in a competitive, enzyme-linked immunosorbent assay (ELISA) for apo B-48 in triacylglycerol-rich lipoprotein (TRL) fractions prepared from fasting and post-prandial plasma samples. Previously we showed the antiserum to act as an effective immunoblotting agent following sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Its use in this ELISA indicates that the antiserum recognises the C-terminal region of the protein on the surface of lipoprotein particles. The ELISA had a sensitivity of less than 37 ng/ml and intra- and inter-assay coefficients of variation of 3.8% and 8.6%, respectively. There was no cross-reaction in the ELISA against serum albumin, ovalbumin, thyroglobulin, or apo B-100 (purified by immunoaffinity chromatography), and high lipid concentrations (as Intralipid) did not interfere. A low density lipoprotein fraction reacted in the ELISA but SDS-PAGE-Western blot analysis confirmed the presence, in the fraction, of a small amount of apo B-48, indicating the existence of low density dietary-derived lipoprotein particles. ELISA and SDS-PAGE-Western blot analysis were used to measure apo B-48 in 12 series of postprandial samples collected from 4 diabetic and 8 normal subjects, following test meals of varying fat content. The mean correlation between the two methods was r = 0.74. The mean fasting concentration of apo B-48 in the TRL fractions from 15 healthy men was 0.46 microgram/ml of plasma.

Glycosylated Hemoglobins and Glycosylated Plasma Proteins in the Diagnosis of Diabetes Mellitus and Impaired Glucose Tolerance

Total and stable glycosylated hemoglobins and glycosylated plasma proteins were determined on 53 patients referred for a glucose tolerance test. Significant correlations were found with fasting blood glucose (r > 0.89), 2-h glucose (r > 0.69), and area under the glucose tolerance curve (r > 0.75), but the correlations with labile glycosylated proteins were not significant. Thirty-one of the patients were normal, five had impaired glucose tolerance (IGT), and seventeen diabetes mellitus (DM) according to the WHO criteria. Comparison of the glycosylated protein values showed that, in all cases, the values for those with IGT and DM were significantly (P < 0.001) > the values for normals. The range of values of stable glycosylated hemoglobins for those with DM (9.4–24.4%), those with IGT (8.6–10.0%), and normals (5.0–8.5%) shows that there was no overlap between overt diabetic subjects and normal subjects. This was also found for total glycosylated hemoglobins. The results for glycosylated plasma proteins, total and stable, were comparable, but one patient with overt DM and two with IGT had values within the normal range. The measurement of glycosylated hemoglobins and glycosylated plasma proteins by the simple, precise, affinity-chromatography method is potentially a quick, accurate, and simple screening test for patients with DM and IGT and deserves consideration as criteria for their diagnosis.

Stereoselective pharmacokinetics of perhexiline

Blood plasma and urine excretion pharmacokinetics of the (+) and (-) enantiomers of perhexiline have been determined in oral single-dose studies in eight human volunteers, and compared with the pharmacokinetics of the racemate drug in the same subjects. The (-) enantiomer is more rapidly metabolized and eliminated, and is stereoselectively hydroxylated to the cis-monohydroxy-perhexiline. The peak plasma concn of unchanged perhexiline is greater, while that of the cis-monohydroxy-perhexiline metabolite is lower, after administration of the (+) enantiomer than after the (-) enantiomer or the racemate. Similarly, the AUC values for unchanged perhexiline and for the trans-monohydroxy-perhexiline metabolite are greatest and the AUC value for the cis-monohydroxy-perhexiline metabolite is lowest for the (+) enantiomer. The three stereoisomeric forms of perhexiline all had the same times to peak plasma concn of the unchanged drug or of the cis-metabolite, and all three forms had a similar plasma elimination half-life for unchanged perhexiline. Metabolism of racemic perhexiline to the cis-monohydroxy metabolite is the major mechanism of elimination of the drug in man and has been shown to be polymorphic in human populations. The (-) enantiomer which shows stereoselective metabolism to the cis metabolite might therefore show a greater polymorphic effect. Studies with rat-liver microsomal preparations in vitro showed that, in contrast to the human studies in vivo, hydroxylation of perhexiline yields mostly the trans-monohydroxy metabolite. The DA strain of rats exhibited slower rates of hydroxylation in vitro than Wistar or Lewis strains of rats.

Further studies on the pharmacokinetics of perhexiline maleate in humans

We have performed single-dose pharmacokinetic studies on perhexiline in eight young volunteers, each given 300 mg of Pexid orally, using an h.p.l.c. method for the separation and quantification of the drug and its monohydroxy metabolites in plasma and urine. The plasma concentration of the cis-monohydroxyperhexiline (peak of 473 +/- 43 ng/ml at 7.5 +/- 2.0 h) was always higher than for unchanged perhexiline (peak of 112 +/- 20 ng/ml at 6.5 +/- 2.0 h) whereas the concentration of the transmetabolite was either low or undetectable in plasma. These findings indicate the occurrence of stereospecific pre-systemic metabolism of perhexiline which reduces the bioavailability of the parent drug. The plasma elimination half-life of perhexiline was 12.4 +/- 6.1 h (range 7-23 h) while that for cis-monohydroxyperhexiline was 19.9 +/- 7.7 h (range 10-29 h). Not more than 0.3% of unchanged perhexiline was excreted in the urine over five days in eight subjects. Between 3 and 23% of the orally administered drug was excreted as the cis- or trans-monohydroxy metabolites, the ratio of trans to cis metabolites being 0.52 +/- 0.20.