QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) Purchase Article View Shopping Cart Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Manuel y Keenoy, B. Articles by De Leeuw, I. Search for Related Content PubMed PubMed Citation Articles by Manuel y Keenoy, B. Articles by De Leeuw, I.

---

Articles

Long-Term Pharmacologic Doses of Vitamin E Only Moderately Affect the Erythrocytes of Patients with Type 1 Diabetes Mellitus¹

Laboratory of Endocrinology and ^* Laboratory of Human and Pathological Biochemistry, University of Antwerp, B-2610 Wilrijk-Antwerp, Belgium

²To whom correspondence should be addressed. E-mail: begona{at}uia.ua.ac.be.

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
In erythrocytes from diabetic patients, increased membrane lipid peroxidation might lead to abnormalities in composition and function. To study this relationship, we investigated the effects of a moderate pharmacologic dose of vitamin E for 1 y on erythrocyte membrane peroxidation in vitro and on its fatty acid composition, antioxidant capacity and rheological function. In a random and double-blind manner, type 1 diabetic patients (n = 44) were assigned to the following two groups: Group S received 250 IU (168 mg) d- tocopherol 3 times daily for 1 y. Group P received placebo for 6 mo followed by d--tocopherol for an additional 6 mo. Variables were monitored every 3 mo. After 3 mo of supplementation, serum vitamin E doubled (P < 0.0005), thiobarbituric acid reactive substances in erythrocyte membranes incubated with tert-butyl hydroperoxide decreased by 25% (P = 0.006) and the lagtime of fluorescence increased from 28 ± 16 to 41 ± 28 min (P = 0.028). Patients who did not respond to supplementation (13 of 44) had lower serum lipids (P = 0.017) and body mass index (P = 0.024). We did not detect any significant effects of vitamin E supplementation on membrane lipid composition, antioxidant capacity or blood viscosity. Continuing supplementation for up to 1 y did not further affect serum vitamin E or membrane peroxidation. Stopping supplementation was followed by a return to inclusion values. These results show that the decrease in erythrocyte membrane peroxidation after vitamin E supplementation is moderate, saturable, reversible, restricted to some individuals and has no detectable effect on erythrocyte composition and function.

KEY WORDS: • vitamin E • type 1 diabetes mellitus • erythrocytes • lipid peroxidation • membranes • viscosity • humans

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
In diabetes mellitus, the high incidence of microvascular and atherosclerotic disease has been associated with abnormalities of erythrocyte composition and rheological function and with increased oxidative stress, among many other factors. The increased blood viscosity seen in diabetes (1) and more so in patients with established complications (2) has been ascribed to a decrease in erythrocyte deformability (3) and to changes in erythrocyte membrane fluidity (4 ,5) . The extent to which these changes are due solely to alterations in the lipid composition of the erythrocyte membrane is still controversial. Increased cholesterol content and cholesterol/phospholipid ratio, which correlated with the decrease in membrane fluidity in type 1 diabetes mellitus (T1DM),³ were identified as contributing factors (6) in addition to glycation of membrane proteins (7) . In contrast, other authors have found no alterations in erythrocyte membrane lipids of type 2 diabetes mellitus (T2DM) patients (8) or even opposite changes such as decreased cholesterol content (9) or increased phospholipid content in normolipemic T1DM and T2DM patients (10) . Concerning membrane fatty acid composition, again there is no consensus on the direction of changes in diabetes. The relative increase of polyunsaturated acids (PUFA; mainly 20:4 and/or 22:6) found by the three groups mentioned seems in contradiction to others (11 ,12) .

In T1DM, the unifying mechanism behind all of these changes has been postulated to be the deficiency in insulin because it has been observed that insulin treatment, which is known to improve membrane fluidity (4 ,5) and blood viscosity (13) , also increases conversion of dietary (n-6) fatty acids (mainly linoleic acid, 18:2) to PUFA (mainly arachidonic acid, 20:4) (14) .

Another possible mechanism, independent of the effect of insulin, is suggested by the higher levels of lipid peroxidation products such as lipofuchsin (15) and malondialdehyde (MDA) (16 ,17) found in erythrocytes from diabetic patients. The increased lipid peroxidation known to exist in diabetes (18) could target PUFA in membranes in addition to those in lipoproteins and in this way, decrease their PUFA content. The opposite hypothesis would be that the increased peroxidation of membranes from diabetics could be due either to their higher PUFA content as seen by some authors (8 9 10 ,16) or to a relative deficiency in membrane antioxidants, such as -tocopherol. Indeed, Horwitt et al. (19) already demonstrated in 1956 that erythrocyte membranes from vitamin E–depleted individuals were more easily peroxidized. In diabetes, the data concerning vitamin E status are not conclusive. With few exceptions (15 ,16 ,20) , most authors find no frank vitamin E deficiency in either plasma or tissue membranes in diabetes (21 22 23) . Nevertheless, the altered fatty acid composition (and thus the PUFA/vitamin E ratio) as well as the heightened oxidative conditions due to hyperglycemia (24) are both conducive to increased lipid peroxidation. Even if absolute vitamin E levels are not lower in diabetic patients than in healthy individuals, supplementation with lipophilic antioxidants such as vitamin E could serve to counteract this prooxidant state.

The evidence for the beneficial effects of vitamin E on lipoprotein peroxidation in vitro is substantial (25) , but the effects on membrane peroxidation in vitro have been less well investigated. For this reason we proposed to study the effects of moderate supplementation [750 IU (503 mg)/d] of vitamin E given for up to 1 y to T1DM patients. The effects on clinical variables and lipoprotein peroxidation have been published elsewhere (26) . In this study, we focused on the measurement of the peroxidation of erythrocyte membranes in vitro. This can be a useful tool with which to monitor the effects of antioxidants in tissues. We also investigated whether changes in erythrocyte membrane peroxidation were associated with changes in its lipid and fatty acid composition, in erythrocyte antioxidant capacity and in rheological function.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Study subjects.

Middle-aged metabolically well-controlled T1DM patients (n = 44) with initial cardiovascular risk factors (e.g., lipidemia) and blood pressure within accepted limits were randomly assigned to two groups in a double-blind manner. Accepted limits were values <1 SD + the European guidelines [glycated hemoglobin (HbA_1c) 7.5%, total cholesterol 4.8 mmol/L, triglycerides 1.7 mmol/L and blood pressure 135/85 mm Hg] (27) . Exclusion criteria were intake of multivitamin preparations and drugs that interfere with the oxidant-antioxidant status and signs of neuropathy on the standard electromyogram. All patients were consuming a standard diet for diabetes that provided 7.5–8.5 MJ/d (50% of energy as carbohydrates, 20% as protein and 30% as fat). This diet ensures a daily intake of at least 3 mg vitamin E, 3000 mg vitamin A, 150 mg vitamin C and 26 mg flavonoids. Patient characteristics at inclusion are described in Table 1 .The first group (S) received d--tocopherol (OMEGA-PHARMA NV, Nazareth, Belgium) 250 IU (or 168 mg) 3 times per day at meals for 1 y. The second group (P) received placebo (consisting of 280 mg soybean oil, containing 0.25 mg tocopherol per capsule and identical in taste and appearance to the vitamin E capsules) for the first 6 mo and then d--tocopherol 250 IU (168 mg) 3 times per day for an additional 6 mo. After 12 mo, both groups stopped receiving supplements. Patients were monitored at inclusion and after 3, 6, 9, 12 and 15 mo. This partial crossover study design was chosen to study the effect of 3- and 6-mo supplementation in a greater number of patients (from groups S and P). Longer-term effects (9 and 12 mo) were studied in group S and the effects of stopping supplementation were also monitored in a total of 17 patients belonging to both groups.

Analytic methods.

At each of the six visits, antecubital venous blood was drawn from each patient and processed for the following measurements:

Routine blood tests including serum total cholesterol, HDL cholesterol and triglycerides were analyzed in the laboratory of the Antwerp University Hospital. Total analytical variability, expressed as the CV was 2, 1.9 and 0.9% respectively. LDL cholesterol was calculated according to the Friedewald equation (28) . HbA_1c was measured by using a HPLC cation exchange column (Modular Diabetic Monitoring System; Bio-Rad, Richmond, CA); the CV was 1.5%. Vitamin E in serum was measured by HPLC (Shimadzu, Kyoto, Japan) with a reversed-phase C18 column (Bio-Rad Laboratories, Hercules, CA) with 100% methanol mobile phase; detection was at 292 and 325 nm, respectively (29) with CV of 10 and 13%, respectively. Reduced glutathione (GSH) in whole blood was measured by using a colorimetric method with a CV of 7% (30) . Glutathione peroxidase activity in the hemolysate was measured with a commercial kit (Randox Laboratories, Crumlin, UK) with a CV of 6%.

The susceptibility of erythrocyte membranes to peroxidation in vitro was investigated by measuring the production of thiobarbituric acid reactive substances (TBARS), fluorescent products and the decrease in membrane thiols after incubation with tert-butyl hydroperoxide (t-BuOOH; Sigma, St Louis, MO). Packed erythrocytes were separated from whole blood anticoagulated with EDTA (4 mmol/L) by centrifugation (1000 x g for 15 min), washed three times with washing buffer (NaCl 150 mmol/L, NaH₂PO₄ 5 mmol/L, pH 8.0) and lysed in 10 volumes of hemolysis buffer (NaH₂PO₄ 5 mmol/L, pH 8.0). After washing in 5, 2.5 and 1.25 mmol/L NaH₂PO₄ and centrifugation at 24,000 x g for 15 min), the hemoglobin-free membranes were resuspended in washing buffer to a protein concentration of 1–2 g/L (31 32 33) and used immediately for the in vitro peroxidation assay (34) . After preincubation in washing buffer with gentle rotation for 30 min in an oven at 37°C, either t-BuOOH to a final concentration of 2 mmol/L or buffer was added in parallel to separate tubes containing 2.5 mL membrane suspension. After a further incubation of 30 min, the reaction was stopped by transferring 500-µL aliquots (in triplicate) to tubes containing 1 mL ice-cold 0.25 mol/L HCl containing 150 g/L trichloroacetic acid (TCA), 3.75 g/L thiobarbituric acid (Merck, Darmstad, Germany) and 0.2 g/L BHT (Sigma). After boiling for 15 min and cooling overnight at 4°C, TBARS formed were extracted in 1 mL 1-butanol and absorption at 532 nm was read against a blank (treated in the same way but containing no membranes). Results were expressed as nmol MDA equivalents by comparison of a calibration with 1,1,3,3-tetraethoxypropane (Sigma) treated in the same way as the samples. After correction for the protein concentration in the membrane suspension, t-BuOOH–induced formation of TBARS was expressed as the difference between the aliquots with and without t-BuOOH. Intra- and interassay CV were 3.5 and 12%, respectively. t-BuOOH–induced formation of fluorescent products was measured by incubating 3 mL membrane suspensions (60–120 µg membrane protein/mL) with and without 2 mmol/L t-BuOOH in separate fluorimetric cuvettes at 37°C. Fluorescence (excitation 366 nm/emission 440 nm), which was detectable only in the sample containing t-BuOOH, was monitored every 2 min for 2 h. The lagtime (in minutes) between the addition of t-BuOOH and the appearance of fluorescent products of peroxidation and the slope of the increase in fluorescence (in arbitrary fluorescent units/min) were calculated from the registered data. Membrane thiol concentration was measured according to Vissers et al. (35) . After the incubation with or without t-BuOOH, 100-µL aliquots of each membrane suspension were added to 100 µL SDS (100 g/L in buffer; Sigma); 790 µL of a solution containing Tris-HCl (400 mmol/L) and EDTA (20 mmol/L, pH 8.9) were added and absorption at 412 nm was read against a blank (treated in the same way but containing no membranes). The increase in absorption due to the addition of 5,5'-dithiobis-(2-nitrobenzoic acid) (to a final concentration of 0.2 mmol/L; Sigma) was expressed as nmol GSH-equivalents by comparison with a calibration with GSH treated in the same way as the samples. After correction for the protein concentration in the membrane suspension, the t-BuOOH–induced decrease in membrane thiol content was expressed as the difference between the aliquots with or without t-BuOOH.

Extraction of lipids from the erythrocyte membranes isolated from whole blood anticoagulated with heparin was done according to Bligh and Dyer (36) . Chloroform/methanol (7.5 mL; 1:2, v/v) containing 0.1 g/L BHT was added to 2 mL membrane suspension and then kept at -20°C until further analysis. Aliquots of this extract were taken for the measurement of phospholipid content, using lecithin as standard for the calibration of phosphorus (37) and cholesterol content (using the kit Merckotest CHOD-PAP enzymatic method, Merck). Phase separation was obtained by adding 2.5 mL water and 2.5 mL chloroform; the chloroform under-phase was washed, evaporated under nitrogen and resolubilized in 1 mL chloroform/methanol (2:1, v/v) containing 0.1 g/L BHT. Phospholipids were separated by passing through a silicic acid column (BIO-SIL A 200–400 mesh, Bio-Rad), washing with 10 mL chloroform to discard the cholesterol fraction and collecting the phospholipids in the 10 mL methanol-BHT eluate (38) . The percentage fatty acid composition of this phospholipid fraction was analyzed by methylesterification with 5% H₂SO₄/methanol (v/v) (39) and gas chromatography (Delsi gas chromatograph equipped with a 25 m x 0.25 mm WCOT fused silica column (Varian-Chrompack, Middelburg, The Netherlands) and a flame ionization detector, using nitrogen as carrier gas, oven temperature from 200–256°C, isotherm for 4 min and then increasing at a rate of 8°C/min. Peak identification was by comparison of retention times with standard fatty acid methyl esters (Alltech, Deerfield, IL).

Viscosity of blood anticoagulated with EDTA was measured according to international guidelines (40) using a Contraves Low-Shear 30 viscosimeter (Contraves AG, Zürich, Switzerland) at 37°C. Viscosity of whole blood (standardized to a hematocrit of 0.45) and packed erythrocyte (standardized to a hematocrit of 0.70) was measured at rates of 0.945/s (low shear) and 128.5/s (high shear). Plasma viscosity was measured at 69.5/s (13) .

Statistical methods.

Data were analyzed by using SPSS software, version 9 (SPSS, Chicago, IL). Results are expressed as means ± SD. In view of the non-Gaussian distribution of the data on membrane peroxidation in vitro, the effects of vitamin E or placebo were analyzed by paired comparison (values before vs. after the intervention) using a nonparametric test (Wilcoxon) and the relationship between parameters by Spearman correlation. Repeated-measures ANOVA also was used. Two-tailed P-values < 0.05 were considered significant.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Randomization at inclusion resulted in two groups of patients comparable in clinical characteristics (Table 1) , erythrocyte membrane composition (Table 2 ) and in vitro susceptibility to t-BuOOH (Table 3 ) and rheological variables (Table 4 ). In group S receiving vitamin E supplements, concentrations of serum vitamin E increased from 36.9 ± 10.9 µmol/L at inclusion to 66.4 ± 18.3 µmol/L after 3 mo (P < 0.0005). Continuing supplementation for up to 1 y did not result in any further changes (60.8 ± 19.0, 62.0 ± 15.8 and 66.2 ± 22.3 µmol/L after 6, 9 and 12 mo, respectively, P = 0.11–0.88) (Fig. 1 ), suggesting that the stores of vitamin E in serum were already saturated after 3 mo. In group P receiving placebo for 6 mo, serum vitamin E remained stable (from 33.9 ± 10.8 at inclusion to 34.4 ± 10.4 and 31.7 ± 6.6 µmol/L at 3 and 6 mo, respectively). When this second group received supplements for the following 6 mo, serum vitamin E concentrations also doubled (to 79.7 ± 37.2 µmol/L at 9 mo, P < 0.0005 compared with the period under placebo) and then also remained stable during supplementation (69.4 ± 29.3 µmol/L at 12 mo) (Fig. 1) . Three months after stopping supplementation, concentrations had returned to the inclusion values in both groups (P < 0.0005 for the change within each group) (Fig. 1) .

View larger version (31K): [in this window] [in a new window]   Figure 1. Evolution of serum vitamin E and erythrocyte membrane peroxidation in vitro [tert-butyl hydroperoxide (t-BuOOH)–induced thiobarbituric acid reactive substances (TBAR)S production, expressed as nmol malondialdehyde (MDA) equivalents/mg membrane protein] in type I diabetic patients receiving vitamin E (group S) or placebo (group P). Group S received vitamin E (250 IU d--tocopherol three times daily) for 12 mo, n = 22. Group P received placebo for the first 6 mo and then vitamin E for the following 6 mo, n = 22. Also shown are the values measured 3 mo after stopping supplementation (15 mo) in 9 patients from group S and 8 from group P. Values shown are the median, the 25th and 75th percentiles (interquartile range) and the highest and lowest nonoutlier values. *P < 0.05 and **P < 0.0005 paired comparison with the preceding visit.

In contrast, the susceptibility of these erythrocyte membranes to in vitro peroxidation decreased after vitamin E supplementation (Table 3) . When these membranes were incubated with t-BuOOH for 30 min, the t-BuOOH–induced production of TBARS was 25% less after 3 mo of vitamin E (P = 0.006, paired comparison before vs. after vitamin E) and only 10% less after 3 mo of placebo (P = 0.90, paired comparison before vs. after placebo). The lagtime before the formation of fluorescent products of peroxidation was prolonged by 13 min after 3 mo of vitamin E supplementation (P = 0.028).

However, there was a wide interindividual variability in the response to vitamin E. In 13 of the 44 patients (29%), erythrocyte membrane peroxidation in vitro did not decrease after 3 mo of vitamin E. This group of nonresponders was distinguished by having lower serum LDL cholesterol (2.68 ± 0.54 vs 3.50 ± 1.02 mmol/L, P = 0.029) and lower serum triglycerides (0.69 ± 0.22 vs 1.05 ± 0.43 mmol/L, P = 0.017). Body mass index (BMI) was also lower in this group (22.3 ± 0.8 vs. 24.9 ± 0.7 kg/m², P = 0.024) and, even within this nonobese range, there was a significant correlation between the decrease in TBARS production after 3 mo of vitamin E supplementation and BMI (Spearman r = -0.40, P = 0.019, n = 35). Surprisingly, erythrocyte membrane composition, clinical characteristics, serum biochemistry, vitamin E at inclusion and its increase after supplementation were the same as in the group of responders.

Continuing supplementation for a further 3 mo did not cause any additional decrease in membrane peroxidation in vitro and the proportion of nonresponders rose to 50%. This larger group of nonresponders still tended to have lower serum cholesterol (5.06 ± 0.97 vs. 5.57 ± 1.16 mmol/L, P = 0.15) and triglycerides (0.83 ± 0.40 vs. 0.99 ± 0.35 mmol/L, P = 0.22) and BMI was significantly lower (22.8 ± 2.3 vs. 25.2 ± 3.6 kg/m², P = 0.031). The increase in serum vitamin E after 6 mo of vitamin E supplementation was also lower in the nonresponders (20.5 ± 15.3 vs. 39.4 ± 27.2 µmol/L, P = 0.023). Although membrane peroxidation in vitro was not related to serum vitamin E either at baseline or after supplementation, the decrease in peroxidation in vitro after 6 mo was strongly related to the increase in serum vitamin E (Spearman r = -0.48, P = 0.007, n = 31).

Erythrocyte membrane peroxidation in vitro after 9 and 12 mo of vitamin E supplementation was monitored in group S (n = 22), and the effects of stopping vitamin E supplementation was also monitored in a small number of patients from both groups P and S (n = 17). As seen in Figure 1 , both serum vitamin E and membrane peroxidation in vitro were maintained at the levels reached after 3 mo vitamin E, but continuing supplementation for up to 1 y did not result in any further changes. Stopping supplementation was followed by a return to inclusion values regardless of the duration of the supplementation given beforehand. In group S, which had received vitamin E for 12 mo, serum vitamin E decreased from 66.3 ± 22.3 to 35.2 ± 8.58 µmol/L (P < 0.0005) and t-BuOOH–induced TBARS production increased from 1.43 ± 1.01 to 3.75 ± 3.55 nmol MDA equivalents/mg protein, P = 0.11). In group P, which had received vitamin E for 6 mo, serum vitamin E decreased from 69.4 ± 29.3 to 36.0 ± 12.2 µmol/L (P = 0.001) and t-BuOOH–induced TBARS production increased from 1.10 ± 0.73 to 3.97 ± 4.39 nmol MDA equivalents/mg protein, P = 0.035) (Fig. 1) .

The influence of other factors on membrane peroxidation was studied in a stepwise multiple regression model including all membrane composition variables and serum vitamin E. t-BuOOH–induced TBARS production was predicted mainly by the membrane content in thiols and PUFA (together explaining 72% of the variance, F-value 23.07, P < 0.0005).

Erythrocyte viscosity at high shear decreased in all patients during the course of the study, regardless of receiving placebo or vitamin E, again suggesting a time trend or a study participation effect as seen for membrane composition. All other viscosity variables were unchanged for the 15 mo of the study (Table 4) .

Levels of serum lipids, HbA_1c (26) and RBC antioxidants (GSH and glutathione peroxidase) were not affected by supplementation with either vitamin E or placebo and did not change significantly throughout the study (Table 4) .

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
The aim of this study was to investigate whether supplementing type 1 diabetic patients with a moderate pharmacologic dose of vitamin E would improve the resistance of their erythrocyte membranes to peroxidation and whether this improvement would have some repercussions on the membrane composition, the antioxidant capacity and the rheological properties of the erythrocytes.

In contrast to the clear decrease in LDL peroxidation in vitro observed by various authors in diabetic patients after receiving vitamin E supplements (25 ,26 ,41 ,42) , the decrease in peroxidation in erythrocyte membranes was only moderate. TBARS production after a 30-min incubation with t-BuOOH was 25% lower after 3 mo of vitamin E supplementation, whereas TBARS formation in LDL and VLDL incubated with copper was 50–60% lower in the same patients (26) . A similar discrepancy was also observed in nondiabetic rabbits receiving different amounts of vitamin E in their diet. The high vitamin E diet led to an increased resistance of LDL to oxidation but had no effect on erythrocyte membrane peroxidation monitored by the rate of decay of parinaric acid (43) . The discrepancy in the magnitude of the effect of vitamin E might be due to differences in the kinetics of the two peroxidation reactions. TBARS production started almost immediately in erythrocyte membranes (in the present study) but only after 90 min in the lipoproteins (26) . Similarly, the lagtime before the appearance of fluorescent products ranged from 20 to 40 min in membranes, whereas in lipoproteins this was 100 min (26) . According to Esterbauer et al. (25) , it is only after the vitamin E in the lipoprotein has been completely consumed during the lagtime phase that the peroxidation reactions will proceed so that end products such as TBARS and fluorescent substances will start to appear. Incubation of erythrocyte membranes with t-BuOOH also leads to -tocopherol oxidation (44) . However, -tocopherol concentrations in erythrocytes (for example, 0.64 nmol/µmol total lipids) are lower than in plasma (2.57 nmol vitamin E/µmol total lipids) (45) . This could explain the shorter lagtime phase in the in vitro peroxidation model of isolated membranes. The lower vitamin E content in membranes can also explain the relatively moderate effect of vitamin E supplementation on the membrane peroxidation in vitro. Because concentrations of vitamin E in erythrocytes correlate well with those in serum and all of the -tocopherol in the erythrocyte is localized to the membrane (46) , concentration in erythrocyte membranes would be expected to rise by a factor of at least 1.5 when concentrations in serum doubled (47) in this group of patients with relatively high vitamin E status at inclusion. Thus, the concentrations of vitamin E reached in the membranes after supplementation would still be insufficient to prevent the production of the 1.5–3 nmol of MDA equivalents/mg membrane protein during the 30-min peroxidation with t-BuOOH.

Nevertheless, the results suggest that the increased capacity of the membrane to retard the lipid peroxidation process seen after vitamin E supplementation, although only moderate, was clearly due to an improvement in membrane antioxidant content rather than to any changes in its lipid content or fatty acid composition. This conclusion is reinforced by the direct relationship with the increase in serum vitamin E as well as by the reversibility of the effect. Decreased peroxidation in vitro was observed only with vitamin E supplementation and rebounded to presupplementation values after stopping supplementation. However, this effect of vitamin E was also saturable because peroxidation did not decrease any further when supplements were continued for 6, 9 and 12 mo. Thus, the time course of changes in membrane peroxidation was a mirror image of the time course of serum vitamin E levels. This suggests that during supplementation, an equilibrium is maintained with higher steady-state levels in both plasma and erythrocytes.

The lower concentration of vitamin E in membranes and the moderate effect of vitamin E supplementation as well as the high interindividual variability and proportion of nonresponders are all observations that suggest that other factors also play an essential role in membrane peroxidation. In this group of diabetic patients, membrane peroxidation in vitro at the moment of inclusion in the study was determined more by its content in thiols and PUFA than by vitamin E levels. This surprising observation can be explained in part by the more than adequate vitamin E status in these patients at the start of the study (35 µmol/L), with no patient having levels of biochemical vitamin E deficiency (<11 µmol/L), which leads to pathologically increased membrane peroxidation (19) . The important role played by other antioxidants cannot be overlooked, however. For example, it has been suggested that in the in vivo situation, the low concentration of membrane vitamin E is compensated by the presence of other antioxidants (mainly ascorbate and GSH), which would continuously regenerate the vitamin E being oxidized (48) . On the other hand, in vitamin E–deficient rats, incubation with ascorbate paradoxically promoted erythrocyte peroxidation (49) . The role played by blood lipids is illustrated by the surprising observation that the decrease in membrane peroxidation in vitro was more pronounced in the patients with higher BMI (although still within the nonobese range) and that this decrease occurred in the patients (responders) with higher serum triglycerides and LDL cholesterol and with a higher increase in serum vitamin E. In contrast, it was not related to erythrocyte membrane composition or to HDL cholesterol, which is known to mediate transport from the plasma to the membrane (50) . This result emphasizes the overriding importance of the capacity of plasma to transport vitamin E, already suggested by the good correlation of serum vitamin E with blood lipids found by some authors (51) and by the influence of dietary fat on vitamin E absorption (52) . Thus, individuals more likely to benefit from vitamin E supplementation are those with higher blood lipids and body fat.

When interpreting lipid peroxidation, one should also bear in mind that MDA formation accounts for only a fraction of the PUFA consumed during peroxidation processes (53) and that vitamin E in the membrane serves to stabilize the hydroperoxides being formed and thus reduce their further decomposition (54) . This means that MDA production does not explain the totality of the peroxidation process and that vitamin E supplementation might have had some effect (for example, on earlier phases of the lipid peroxidation process), which cannot be monitored by the analytical methods used in this study.

The clinical implications of a decrease in membrane peroxidation in vitro are not clear. In diabetes, hyperglycemia leads to heightened production of free radicals (24) , and the antioxidant defenses seem insufficient to prevent lipid peroxidation, consumption of vitamin E and membrane damage leading to hemolysis (55 ,56) . In such a situation, increasing membrane vitamin E and improving its resistance to peroxidation, even if only moderately, might serve to decrease the extent of erythrocyte peroxidation occurring in vivo. This possible effect of vitamin E in vivo is supported by the observation that vitamin E–deficient pigs have higher circulating erythrocyte TBARS (57) and that vitamin E supplementation restored MDA to normal concentrations in erythrocytes of T1DM children (58) .

In this study, the decrease in erythrocyte peroxidation in vitro was not accompanied by any significant change in erythrocyte GSH and glutathione peroxidase. In a similar human intervention study, vitamin E supplementation (280 mg/d for 10 wk given to smokers and nonsmokers) was followed by a decrease in erythrocyte peroxidation in vitro, a decrease in GSH and superoxide dismutase and an increase in catalase and in glutathione peroxidase only in nonsmokers (59) . More disturbingly, these same authors found that high supplementation dosages of vitamin E (1050 mg d- tocopherol given for 20 wk to nonsmokers) were followed by an increased peroxidation of erythrocyte in vitro (60) . These apparently contradictory results show that the effect of supplementation with vitamin E on erythrocyte antioxidant capacity and thus on the important function of the erythrocyte which is to "mop up" excess free radicals released into blood remains an unresolved issue.

In examining the effect of supplements on erythrocyte rheological function, no significant improvement in blood viscosity was found. This result, seen together with the lack of any effect on membrane lipid composition and fatty acid content, shows that the moderate improvement in membrane peroxidation as a result of vitamin E supplementation is not sufficient to reverse some of the abnormalities observed in erythrocytes from diabetic patients (1 2 3 4 5) . Other observations also indicate that lipid peroxidation is not always the mediating mechanism behind these erythrocyte alterations. For example, it has been demonstrated that high glucose–induced inactivation of erythrocyte ATPases is not mediated by increased lipid peroxidation but by the formation of noncovalent interactions between glucose and these enzymes (61) . Changes in blood viscosity in T1DM patients have been associated with the lower selenium and glutathione peroxidase levels in the erythrocytes of these patients, but not with vitamin E levels (23) . Nevertheless, both the micro- and macrovascular complications of diabetes are the result of a complex interaction between blood and vessel walls, and the effect of membrane peroxidation on the appearance of atherosclerosis remains to be studied. For example, a decrease in erythrocyte membrane peroxidation and thus also in erythrocyte aging might have an effect on other pathologic processes such as the altered adhesion to endothelial cells (62) and the release of catalytic iron (56 ,63) . Both processes contribute to LDL oxidation and thus promote atheroma formation (64) .

Supplementing T1DM individuals with moderate pharmacologic doses of vitamin E was followed by a moderate decrease in the susceptibility of their erythrocytes to in vitro peroxidation by t-BuOOH. This improvement was saturable and was observed only during the period of supplementation. It was not accompanied by any changes in cholesterol/phospholipid or in fatty acid composition or by any significant improvement in erythrocyte function variables such as GSH, and glutathione peroxidase and viscosity. These results suggest that vitamin E supplementation has only a limited clinical effect on erythrocyte function in T1DM patients, in clear contrast to the obvious beneficial effects on LDL and VLDL oxidation.

	ACKNOWLEDGMENTS

 
The authors would like to thank the patients who participated in the study, OMEGA-PHARMA NV (Nazareth, Belgium) for supplying the capsules of vitamin E (E-MED FORTE) and placebo, S. Schrans, P. Aerts and M. Vinckx for skilful technical assistance and M. Van Acker and W. Van Dongen for help and advice over the fatty acid analyses.

	FOOTNOTES

 
¹ Data concerning the initial 6 mo of this study were presented in poster form at the Meeting of the European Society for the Study of Diabetes (EASD), September 1998, Barcelona, Spain [Manuel y Keenoy, B. Shen, H, L., Engelen, W., Vertommen, J. & De Leeuw, I. (1998) Effect of vitamin E supplementation on the peroxidability of red blood cell membranes from type 1 diabetic patients. Diabetologia 41 (suppl. I): A350 (abs.)].

³ Abbreviations used: BMI, body mass index; GSH, reduced glutathione; HbA_1c, glycated hemoglobin; MDA, malondialdehyde; TBARS, thiobarbituric acid reactive substances; t-BuOOH, tert-butyl hydroperoxide; TCA, trichloroacetic acid; T1DM, type 1 diabetes mellitus; T2DM, type 2 diabetes mellitus.

Manuscript received December 5, 2000. Initial review completed January 12, 2001. Revision accepted March 14, 2001.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 

1. McMillan D. E. Plasma protein changes, blood viscosity and diabetic microangiopathy. Diabetes 1976;25(suppl. 2):858-864

2. Schut N. H., Van Arkel E. C., Hardeman M. R., Bilo H.J.G., Michels R.P.J., Vreeken J. Blood and plasma viscosity in diabetes: possible contribution to late organ complications?. Diabetes Res 1992;19:31-35[Medline]

3. McMillan D. E., Utterback N. G., La Puma J. Reduced erythrocyte deformability in diabetes. Diabetes 1978;27:895-901[Medline]

4. Bryszewska M., Leyko W. Effect of insulin on human erythrocyte membrane fluidity in diabetes mellitus. Diabetologia 1983;24:311-313[Medline]

5. Juhan-Vague I., Rahmani-Jourdheuil D., Mishal Z., Roul C., Mourayre Y., Aillaud M. F., Vague P. Correction by insulin added in vitro of abnormal membrane fluidity of the erythrocyte from type 1 (insulin-dependent) diabetic patients. Diabetologia 1986;29:417-420[Medline]

6. Bryszewska M., Watala C., Torzecka W. Changes in fluidity and composition of erythrocyte membranes and in composition of plasma lipids in type 1 diabetes. Br. J. Haematol. 1986;62:111-116[Medline]

7. Bryszewska M., Szosland K. Association between the glycation of erythrocyte membrane proteins and membrane fluidity. Clin. Biochem. 1988;21:49-51[Medline]

8. Urano S., Hoshi-Hashizume M., Tochigi N., Matsuo M., Shiraki M., Ito H. Vitamin E and the susceptibility of erythrocytes and reconstituted liposomes to oxidative stress in aged diabetics. Lipids 1991;26:58-61[Medline]

9. Baldini P., Incerpi S., Lambert-Gardini S., Spinedi A., Luly P. Membrane lipid alterations and Na⁺-pumping activity in erythrocytes from IDDM and NIDDM subjects. Diabetes 1989;38:825-831[Abstract]

10. Rabini R. A., Fumelli P., Galassi R., Dousset N., Taus M., Ferretti G., Mazzanti L., Curatola G., Solera M. L., Valdiguié P. Increased susceptibility to lipid oxidation of low-density lipoproteins and erythrocyte membranes from diabetic patients. Metabolism 1994;43:1470-1474[Medline]

11. Tilvis R. S., Miettinen T. A. Fatty acid composition of serum lipids, red cells and platelets in insulin-dependent diabetic women. J. Clin. Endocrinol. Metab. 1985;61:741-745[Abstract]

12. Ruiz-Gutierrez V., Stiefel P., Villar J., Garcia-Donas M. A., Acosta D., Carneado J. Cell membrane fatty acid composition in type 1 (insulin-dependent) diabetic patients: relationship with sodium transport abnormalities and metabolic control. Diabetologia 1993;36:850-856[Medline]

13. Van Acker K., Xiang D. Z., Rillaerts E., Van Gaal L., De Leeuw I. Blood rheology during intensified conventional treatment (ICIT) in insulin-dependent diabetes. Diabetes Res. Clin. Pract. 1989;6:259-264[Medline]

14. Tilvis R. S., Helve E., Miettinen TA Improvement of diabetic control by continuous subcutaneous insulin infusion therapy changes fatty acid composition of serum lipids and erythrocytes in type 1 (insulin-dependent) diabetes. Diabetologia 1986;29:690-694[Medline]

15. Jain S. K., Levine S. N., Duett J., Hollier B. Reduced vitamin E and increased lipofuscin products in erythrocyte of diabetic rats. Diabetes 1991;40:1241-1244[Abstract]

16. Peuchant E., Delmas-Beauvieux M. C., Couchouron A., Dubourg L., Thomas M.-J., Perromat A., Clerc M., Gin H. Short-term insulin therapy and normoglycemia. Effects on erythrocyte lipid peroxidation in NIDDM patients. Diabetes Care 1997;20:202-207[Abstract]

17. Jain S., McVie R., Duett J., Herbst J. J Erythrocyte membrane lipid peroxidation and glycosylated hemoglobin in diabetes. Diabetes 1989;38:1539-1543[Abstract]

18. Griesmacher A., Kindhauser M., Andert S. E., Schreiner W., Toma C., Knoebl P., Pietschmann P., Prager R., Snack C., Schernthaner G., Mueller M. M. Enhanced serum levels of thiobarbituric acid reactive substances in diabetes mellitus. Am. J. Med. 1995;98:469-475[Medline]

19. Horwitt M. K., Harvey C. C., Duncan G. D., Wilxon W. C. Effects of limited tocopherol intake in man with relationships to erythrocyte hemolysis and lipid oxidations. Am. J. Clin. Nutr. 1956;4:408-419[Abstract]

20. Karpen C. W., Cataland S., O’Dorisio T. M., Panganamala R. V. Production of 12-hydroxyeicosatetraenoic acid and vitamin E status in platelets from type 1 human diabetic subjects. Diabetes 1985;34:526-531[Abstract]

21. Vatassery G. T., Morley J. E., Kuskowski M. A. Vitamin E in plasma and platelets of human diabetic patients and control subjects. Am. J. Clin. Nutr. 1983;37:641-644[Abstract/Free Full Text]

22. Martinoli L., Di-Felice M., Seghieri G., Ciuti M., De-Giorgio L. A., Fazzini A., Gori R., Anichini R., Franconi F. Plasma retinol and alpha-tocopherol concentrations in insulin-dependent diabetes mellitus: their relationship to microvascular complications. Int. J. Vitam. Nutr. Res. 1993;63:87-92[Medline]

23. Osterode W., Holler C., Ulberth F. Nutritional antioxidants, red cell membrane fluidity and blood viscosity in type 1 (insulin dependent) diabetes mellitus. Diabetic Med 1996;13:1044-1050[Medline]

24. Baynes J. W. Role of oxidative stress in the development of complications in diabetes. Diabetes 1991;40:405-412[Abstract]

25. Esterbauer H., Rotheneder M., Striegl G., Waeg G., Sattler W., Jürgens G. Vitamin E and other lipophilic antioxidants protect LDL against oxidation. Fat Sci. Technol. 1989;91:316-324

26. Engelen W., Manuel y Keenoy B., Vertommen J., De Leeuw I. Effects of long-term supplementation with moderate pharmacologic doses of vitamin E are saturable and reversible in patients with type 1 diabetes. Am. J. Clin. Nutr. 2000;72:1142-1149[Abstract/Free Full Text]

27. European Diabetes Policy Group (1998) Guidelines for Diabetes Care. A Desktop Guide to Type 1 (Insulin-Dependent) Diabetes Mellitus (Home, P., ed.). Walter Wirtz Druck & Verlag, Speyer, Germany.

28. Friedewald W. T., Levy R. I., Frederickson D. S. Estimation of the concentration of low density lipoprotein cholesterol in plasma without the use of the preparative ultracentrifuge. Clin. Chem. 1972;18:499-502[Abstract]

29. Caye-Vaugien C., Krempf M., Lamarche P., Charbonnel B., Pieri J. Determination of -tocopherol in plasma, platelets and erythrocytes of type I and II diabetic patients by HPLC. Int. J. Vitam. Nutr. Res. 1990;60:324-330[Medline]

30. Beutler E. Red Cell Metabolism; a Manual of Biochemical Methods 1975 Grune & Stratton New York, NY.

31. Dodge J. T., Mitchell C., Hanahan D. J. The preparation and chemical characteristics of hemoglobin-free ghosts of human erythrocytes. Arch. Biochem. Biophys. 1963;100:119-130[Medline]

32. Burton G. W., Ingold K. U., Thompson K. E. An improved procedure for the isolation of ghost membranes from human red blood cells. Lipids 1981;16:946[letter][Medline]

33. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilising the principle of protein-dye binding. Anal. Biochem. 1976;72:248-254[Medline]

34. Sandhu I. S., Ware K., Grisham M. B. Peroxyl radical-mediated hemolysis: role of lipid, protein and sulfhydril oxidation. Free Radic. Res. Commun. 1992;16:111-122[Medline]

35. Vissers M.G.M., Stern A., Kuypers F., Vand den Berg J., Winterbourn C. C. Membrane changes associated with lysis of red blood cells by hypochlorous acid. Free Radic. Biol. Med. 1994;16:703-712[Medline]

36. Bligh E. G., Dyer W. J. A rapid method of total lipid extraction and purification. Can. J. Biochem. Physiol. 1959;37:911-917

37. Siakotos A. N., Rouser G., Fleisher S. Phospholipid composition of human, bovine and frog myelin isolated on a large scale from brain and spinal cord. Lipids 1969;4:239-242[Medline]

38. Rouser G., Kritchevsky G., Yamamoto A. Lipid Chromatographic Analysis 1967;1:99-162 M. Dekker New York, NY.

39. Popp-Snijders C. -3 Polyunsaturated Fatty Acids and Erythrocyte Membranes. A Study in Healthy and Diabetic Subjects. Doctoral thesis 1985 Vrije Universiteit Amsterdam, The Netherlands.

40. Expert Panel on Blood Rheology, International Committee for Standardisation in Hematology Guidelines for the measurement of blood viscosity and erythrocyte deformability. Clin. Hemorheol. 1986;6:439-453

41. Reaven P. D., Khouw A., Beltz W., Parthasarathy S., Witzum J. L. Effect of dietary antioxidant combinations in humans: protection of LDL by vitamin E, but not by ß-carotene. Arterioscler. Thromb. 1993;13:590-600[Abstract/Free Full Text]

42. Fuller C. J., Candalia M., Garg A., Grundy S. M., Jialal I. RRR- Tocopheryl acetate supplementation at pharmacologic doses decreases low-density-lipoprotein oxidative susceptibility but not protein glycation in patients with diabetes mellitus. Am. J. Clin. Nutr. 1996;63:753-759[Abstract/Free Full Text]

43. Wiseman S. A., Van den Boom M. A., De Fouw N. J., Wassink M. G., Op den Kamp J. A., Tijburg L. B. Comparison of the effects of dietary vitamin E on in vivo and in vitro parameters of lipid peroxidation in the rabbit. Free Radic. Biol. Med. 1995;19:617-626[Medline]

44. Vatassery G. T. Oxidation of vitamin E in red cell membranes by fatty acids, hydroperoxides and selected oxidants. Lipids 1989;24:299-304[Medline]

45. Jain S. K., Wise R., Bocchini J. J. Vitamin E and vitamin E-quinone levels in red blood cells and plasma of newborn infants and their mothers. J. Am. Coll. Nutr. 1996;15:44-48[Abstract]

46. Chow C. K. Distribution of tocopherols in human plasma and red blood cells. Am. J. Clin. Nutr. 1996;28:756-760[Abstract/Free Full Text]

47. Lehmann J., Rao D. D., Canary J. J., Judd J. T. Vitamin E and relationships among tocopherols in human plasma, platelets, lymphocytes, and red blood cells. Am. J. Clin. Nutr. 1988;47:470-474[Abstract/Free Full Text]

48. Constantinescu A., Han D., Packer L. Vitamin E recycling in human erythrocyte membranes. J. Biol. Chem. 1993;268:10906-10913[Abstract/Free Full Text]

49. Wang J. M., Huang C. J., Chow C. K. Red cell vitamin E and oxidative damage-a dual role of reducing agents. Free Radic. Res. 1996;24:291-298[Medline]

50. Kayden H. J., Bjornson L. The dynamics of vitamin E transport in the human erythrocyte. Ann. N.Y. Acad. Sci. 1972;203:127-140[Medline]

51. Horvitt M. K., Harvey C. C., Dahm D. H., Searcy M. T. Relationship between tocopherol and serum lipid levels for determination of nutritional adequacy. Ann. N.Y. Acad. Sci. 1972;203:223-236[Medline]

52. Dimitrov N. V., Meyer C., Gilliland D., Ruppenthal M., Chenoweth W., Malone W. Plasma tocopherol concentrations in response to supplemental vitamin E. Am. J. Clin. Nutr. 1991;53:723-729[Abstract/Free Full Text]

53. Esterbauer H., Scaur J. S., Zollner H. Chemistry and biochemistry of 4-hydroxynonenal, malondialdehyde and related aldehydes. Free Radic. Biol. Med. 1991;11:81-128[Medline]

54. Therond P., Coutourier M., Demelier J. F., Lemonnier F. Hydroperoxides of erythrocyte phospholipid molecular species formed by lipoxygenase correlate with tocopherol levels. Lipids 1996;31:703-708[Medline]

55. Jain S. K. Hyperglycemia can cause lipid peroxidation and osmotic fragility in human red cells. J. Biol. Chem. 1989;264:21340-21345[Abstract/Free Full Text]

56. Virgili F., Battistini N., Canali R., Vannini V., Tomasi A. High glucose-induced membrane lipid peroxidation on intact erythrocytes and on isolated erythrocyte membrane (ghosts). J. Nutr. Biochem. 1996;7:156-161

57. Wang Y. H., Leibholz J., Bryden W. L., Fraser D. R. Lipid peroxidation status as an index to evaluate the influence of dietary fats on vitamin E requirements of young pigs. Br. J. Nutr. 1996;75:81-95[Medline]

58. Jain S. K., McVie R., Smith T. Vitamin E supplementation restores glutathione and malondialdehyde to normal concentrations in erythrocytes of type 1 diabetic children. Diabetes Care 2000;23:1389-1394[Abstract/Free Full Text]

59. Brown K. M., Morrice P. C., Arthur J. R., Duthie G. G. Effects of vitamin E supplementation on erythrocyte antioxidant defense mechanisms of smoking and nonsmoking men. Clin. Sci. (Lond.) 1996;91:107-111[Medline]

60. Brown K. M., Morrice P. C., Duthie G. G. Erythrocyte vitamin E and plasma ascorbate concentrations in relation to erythrocyte peroxidation in smokers and nonsmokers. Dose response to vitamin E supplementation. Am. J. Clin. Nutr. 1997;65:496-502[Abstract/Free Full Text]

61. Rajeswari P., Natarajan R., Nadler J. L., Kumar D., Kalra V. K. Glucose induces lipid peroxidation and inactivation of membrane-associated ion-transport enzymes in human erythrocytes in vivo and in vitro. J. Cell. Physiol. 1991;149:100-109[Medline]

62. Wali R. K., Jaffe S., Kumar D., Kalra V. K. Alterations in organization of phospholipids in erythrocytes as factors in adherence to endothelial cells in diabetes mellitus. Diabetes 1988;37:104-111[Abstract]

63. Yuan X. M., Li W., Olsson A. G., Brunk U. T. Iron in human atheroma and LDL oxidation by macrophages following erythrophagocytosis. Atherosclerosis 1996;124:61-73[Medline]

64. Berliner J. A., Heinecke J. W. The role of oxidized lipoproteins in atherosclerosis. Free Radic. Biol. Med. 1996;20:707-727[Medline]

This article has been cited by other articles:

				J. A. Beckman, A. B. Goldfine, M. B. Gordon, L. A. Garrett, J. F. Keaney Jr., and M. A. Creager Oral antioxidant therapy improves endothelial function in Type 1 but not Type 2 diabetes mellitus Am J Physiol Heart Circ Physiol, December 1, 2003; 285(6): H2392 - H2398. [Abstract] [Full Text] [PDF]

				I. Ruffini, G. Belcaro, M.R. Cesarone, G. Geroulakos, A. Di Renzo, M. Milani, L. Coen, A. Ricci, R. Brandolini, M. Dugall, et al. Evaluation of the Local Effects of Vitamin E ( E-Mousse(R)) on Free Radicals in Diabetic Microangiopathy: A Randomized, Controlled Trial Angiology, July 1, 2003; 54(4): 415 - 421. [Abstract] [PDF]

This Article Abstract Full Text (PDF) Purchase Article View Shopping Cart Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Manuel y Keenoy, B. Articles by De Leeuw, I. Search for Related Content PubMed PubMed Citation Articles by Manuel y Keenoy, B. Articles by De Leeuw, I.