Clinical Chemistry 43: 285-289, 1997;
(Clinical Chemistry. 1999;43:285-289.)
© 1999 American Association for Clinical Chemistry, Inc.
Plasma and erythrocyte vitamin E content in asymptomatic hypercholesterolemic subjects
Emmanuelle Simon1,
Jean-Louis Paul1,3,a,
Théophile Soni1,
Alain Simon2 and
Nicole Moatti1,3
1
Laboratoire de Biochimie and
2
Centre de Médecine Préventive Cardiovasculaire, Hôpital Broussais, 96 rue Didot, 75674 Paris Cedex 14, France.
3
Laboratoire de Biochimie Appliquée, Faculté
des Sciences pharmaceutiques et biologiques, 92296
Châtenay-Malabry, France.
a Address correspondence to this author at: Laboratoire de Biochimie, Hôpital Broussais, 96 rue Didot, 75674 Paris Cedex 14, France. Fax 33 (0) 1 45 41 35 13.
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Abstract
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The present study was designed to assess plasma and erythrocyte vitamin
E concentrations in 57 asymptomatic hypercholesterolemic (HC) men
compared with 56 normocholesterolemic (NC) men. Vitamin E
concentrations were determined by using a reversed-phase HPLC method.
Compared with NC subjects, HC men had a significantly lower red blood
cell (RBC) vitamin E content in spite of their normal plasma vitamin E
concentration. This study demonstrates that total plasma vitamin E
concentration is not a suitable predictor of cell vitamin E status and
suggests an abnormal transfer of tocopherol between plasma and RBCs in
HC men. Moreover, the RBCs of HC men were more susceptible to a
peroxidative stress. The strong correlation between RBC susceptibility
to oxidation and RBC vitamin E content suggests that the low RBC
vitamin E content found in HC men has physiological consequences on the
RBC oxidation.
Key Words: indexing terms: 
-tocopherol • red blood cells • atherosclerosis • HPLC • antioxidants • peroxidative stress
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Introduction
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An increasing interest in the role of oxidative stress as a
potential initiating factor in atherosclerosis has been observed in
recent years (1)(2). A variety of antioxidant
defense systems in the human body are able to detoxify prooxidants and
scavenge oxygen free radicals. Among them, vitamin E is the major
chain-breaking lipophilic antioxidant in tissues and plasma; the most
biologically active form is -tocopherol. The results of human
studies of the potential antiatherogenic role of vitamin E are still
controversial. Some of them provide evidence of an association between
a high intake of vitamin E and a lower risk of coronary heart disease
(3) or show an inverse correlation between plasma vitamin
E (E-pl) and a cardiovascular disease
(4)(5).1
In contrast, several studies found no direct
associations between E-pl and mortality from coronary heart disease
(6)(7). The heterogeneity of the populations
recruited for a variety of cardiovascular risk factors could be
responsible for the controversial results reported from in vivo
studies. Otherwise, the value of E-pl concentration alone as an index
of vitamin E status is uncertain. Some authors suggested that the
tocopherol of red blood cells (RBCs) or platelets associated with the
tocopherol-to-lipid ratio of plasma could be more meaningful to
evaluate the vitamin E status in humans
(8)(9), but very few studies reported such
measurements.
Thus, the present study was designed to assess vitamin E status [not
only plasma, but also RBC vitamin E (E-RBC) concentrations] in
asymptomatic hypercholesterolemic (HC) men. We investigated also the
susceptibility of RBCs to an oxidative stress by determining the extent
of hemolysis induced by a water-soluble azo compound.
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Subjects and Methods
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Subjects were obtained from an ongoing risk factor screening
program conducted at their worksite for employees of several companies
within the Paris, France, area by a group of occupational health
physicians (PCVMETRA Group: Prévention Cardiovasculaire en
Médecine du Travail) (10)(11). After
their consent was obtained, 57 men with a total cholesterol (TC)
concentration >6.2 mmol/L (240 mg/dL) entered into the HC group of
this study, and were compared with a normocholesterolemic group (NC) of
56 men. All subjects were free of secondary hypercholesterolemia,
hypertriglyceridemia [triglycerides (TG) >2.0 mmol/L (175 mg/dL)],
renal failure, diabetes mellitus, or a history of myocardial
infarction, stroke, or intermittent claudication, and never took any
lipid-lowering drugs. Specific dietary histories were not available for
these subjects but there was no evidence in the medical record that any
subject had unusual dietary habits or took any antioxidant therapy.
Serum TC and TG were determined by using enzymatic methods
(12). HDL cholesterol (HDL-C) was measured by an enzymatic
method after the precipitation of LDL and VLDL by a phosphotungstic
acid-MgCl2 reagent (13). LDL-C was calculated
according to the Friedewald formula (14), which is
accurate for TG concentrations <4.5 mmol/L: LDL-C = TC -
HDL-C - (TG/2.2). The results were expressed in mmol/L of plasma.
Blood collected into EDTA was centrifuged at 2000g for 10
min at 4 °C to separate plasma and RBCs. RBCs were washed three
times with NaCl solution (9 g/L) containing 0.5% pyrogallol as
antioxidant agent. The final hematocrit suspension was made up to about
50% with addition of distilled water containing 2 µmol/L butylated
hydroxytoluene (BHT). Vitamin E was determined with a reversed-phase
HPLC method by an adaptation of the method described by Bieri et al.
(15). Briefly, plasma or RBC suspensions were
deproteinized in the presence of ethanol-BHT containing
D--tocopherol acetate as internal standard. Vitamin E
was extracted with hexane and evaporated to dryness under a stream of
nitrogen. The residues were redissolved in methanol-HPLC containing 2
µmol/L BHT and injected onto the chromatographic column. The HPLC
system consisted of a Beckman Model 126 pump (San Roman, LA), a 50-µL
injection valve (Model 7151; Rheodyne, Cotati, CA), a C-18
reversed-phase column with RP-18 guard column (Merck, Darmstadt,
Germany), a UV-visible spectrophotometric detector (SPDA-6A; Shimadzu,
Tokyo, Japan), and an electronic integrator (Model HP 3396A;
Hewlett-Packard, Palo Alto, CA). Mobile phase consisted of
methanol-HPLC with a flow rate of 1 mL/min, and the detection was
accomplished by measurement of absorbance at 292 nm. E-pl was expressed
in µmol/L of plasma and E-RBC in µmol/L of packed cells.
For investigating the susceptibility of RBCs to an oxidative stress,
blood collected into heparin was centrifuged at 2000g for 10
min to separate plasma and RBCs. RBCs were washed three times in NaCl
(9 g/L). Supernatant and buffy coat were carefully removed by
aspiration after each wash. Washed RBCs were finally resuspended in
NaCl (9 g/L) at 22% hematocrit and used for free-radical-mediated
hemolysis determination. Hemolysis was induced by the thermal
decomposition of a water-soluble azo compound, the 2,2'-azo
bis-(2-amidinopropane) dihydrochloride (AAPH). The method of Miki et
al. (16) was applied to determine radical-mediated
hemolysis, with minor modifications. Briefly, 1 mL of RBC suspensions
was mixed with equal volumes of NaCl (9 g/L) containing AAPH (200
mmol/L). Suspensions were incubated at 37 °C for 4 h under
aerobic conditions and agitated gently throughout. The AAPH solution
was incubated for 1 h at 37 °C and the RBC suspensions were
incubated for 5 min at 37 °C before mixing. Aliquots were obtained
at times ranging from 0 (corresponding to the time of mixing RBC
suspensions with AAPH solution) to 240 min. Samples (50 µL) were
diluted in 2 mL of NaCl (9 g/L) and centrifuged. The extent of
hemolysis was measured spectrophotometrically at 540 nm, by comparing
the extracellular hemoglobin content of the aliquots with that of a
fully hemolyzed reference sample, which was prepared in the same way
except that the AAPH solution was replaced by distilled water.
Percentage of hemolysis was measured according to the equation: %
hemolysis = A/B x 100, where A is absorbance of the sample
aliquot at 540 nm and B is absorbance of the fully hemolyzed reference
at 540 nm.
Results are expressed as mean ± SD. The statistical analysis was
performed on an Apple Macintosh computer with the use of Statview
(Abacus Concepts, Berkeley, CA). The Student's t-test was
used to compare the HC and the NC subjects. Data were analyzed in a
univariate regression analysis. For all analyses, P <0.05
was considered significant.
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Results
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There were no statistical differences between the HC and the NC
groups in age (HC = 47 ± 8 vs NC = 45 ± 10
years), systolic blood pressure (HC = 135 ± 16 vs NC =
140 ± 19 mmHg), diastolic blood pressure (HC = 86 ± 11
vs NC = 89 ± 14 mmHg), and proportion of smokers (HC =
29.8% vs NC = 33.9%). Compared with the control group, HC
subjects had by definition higher TC (7.16 ± 0.75 vs 5.23 ±
0.64 mmol/L, P <0.0001) and higher LDL-C (5.18 ± 0.76
vs 3.50 ± 0.56 mmol/L, P <0.0001), but also had
higher TG (1.35 ± 0.44 vs 0.98 ± 0.46 mmol/L, P
<0.0001), although hypertriglyceridemic subjects (TG >2 mmol/L) were
excluded. No differences existed between the two groups in HDL-C
(1.31 ± 0.28 vs 1.28 ± 0.27 mmol/L).
As shown in Table 1
, significantly higher E-pl was found in the HC group compared
with the NC group, when the vitamin concentration was expressed in
µmol/L of plasma. In agreement with previous reports
(17)(18), we found a significant positive
correlation between E-pl and TC (r = 0.59; P
<0.0001), suggesting that plasma lipid concentration passively
influences E-pl concentration. This correlation justified the
recommendation that plasma tocopherol concentrations should be
expressed relative to plasma lipid concentrations
(17)(18). Therefore, E-pl expressed relative
to both TC and TG was not different between the two groups (Table 1
).
To evaluate more accurately the vitamin E status associated with
hypercholesterolemia, we also determined the E-RBC. This parameter was
significantly lower in HC than in NC men (3.27 ± 0.70 vs
3.78 ± 1.10 µmol/L, P <0.004), as shown in Fig. 1
. Since there were smokers in the two populations, we verified
that there was no statistical difference in the vitamin E content
between smokers and nonsmokers in the HC group (3.42 ± 0.71 vs
3.20 ± 0.69 µmol/L) and in the NC group (3.69 ± 1.07 vs
3.63 ± 1.08 µmol/L). E-RBC was negatively correlated to TC
concentrations (r = 0.33; P <0.0005) and to
LDL-C concentrations (r = 0.36; P <0.0001).
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Figure 1. Comparison of E-RBC content between HC and NC men.
Graphic represents the mean (±SD) of E-RBC. Statistical analysis
(Student's t- test): **P <0.004.
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To verify if the low E-RBC of HC men had consequences on RBC oxidation,
we further studied the susceptibility of RBCs to an oxidative stress in
an additional group of 51 subjects (27 HC and 24 NC men). The clinical
and biological characteristics and the vitamin E status of these
subjects were the same as those described above (data not shown). An
example of the time-dependent rate of AAPH-induced RBC hemolysis is
shown in Fig. 2
. From the obtained sigmoid curves, we can calculate two
quantitative parameters: (a) the lag time (LT, min), defined
as the intercept between the linear least-square slope of the curve
with the axis of incubation time, which reflects the capacity of the
cell to buffer peroxyl radicals; and (b) the time required
to achieve 50% hemolysis (T50, min). These two
parameters were significantly decreased in HC men compared with NC
subjects (LT = 100.7 ± 16.6 vs 113.1 ± 17.8 min,
P <0.02 and T50 = 134.3 ± 16.8
vs 147.5 ± 23.6 min, P <0.03), expressing an
increased susceptibility of RBC of HC men to AAPH-induced oxidation.
Since there were smokers in the two populations, we verified that there
was no statistical difference in the susceptibility to oxidative stress
between smokers and nonsmokers in the HC group (LT = 101.94
± 13.15 vs 99.78 ± 18.93 min and T50 =
137.36 ± 15.20 vs 132.21 ± 17.95 min) and in the NC group
(LT = 115.23 ± 16.81 vs 112.36 ± 18.52 min and
T50 = 150.53 ± 20.27 vs 146.45 ±
24.96 min). The E-RBC concentration was positively correlated with LT
(r = 0.37, P <0.008) and with
T50 (r = 0.30, P
<0.03).
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Figure 2. Typical sigmoid curves obtained for the time-dependent
rate of AAPH-induced RBC hemolysis.
Graphic represents the typical sigmoid curves obtained for one HC man
and one NC man, and from which one can calculate the LT and the
T50 as described in Results.
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Discussion
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To clarify the potential antiatherogenic contribution of vitamin
E, we have determined the vitamin E status of subjects free from any
symptomatic cardiovascular diseases but with a well-known
cardiovascular risk factor, hypercholesterolemia, compared with NC men.
We did not find any statistical differences between the two groups in
the E-pl concentrations when expressed relative to plasma lipid
concentrations. In contrast, an original result of the present study
relates the lower E-RBC content in HC men, despite their normal plasma
tocopherol concentrations. This result demonstrates that the plasma
-tocopherol-to-lipid ratio is not a suitable predictor of cell
vitamin E status, which could explain in part the discrepancy in
results obtained between different human studies of the antiatherogenic
role of vitamin E (3)(4)(5)(6)(7). Moreover, with lipoproteins
being the physiological transporters of vitamin E in plasma, this
result could reflect a disturbance in the transfer of vitamin E between
lipoproteins and RBCs and more generally tissues. Such an abnormal
transfer of -tocopherol from plasma to RBCs has been previously
shown in chronic renal failure and in liver cirrhosis
(19), but, to our knowledge, never in
hypercholesterolemia. The hypothesis of an impaired transfer of vitamin
E between lipoproteins and cells in hypercholesterolemia is indirectly
sustained by the negative correlations obtained between E-RBC and
concentrations of TC or LDL-C in this study. Furthermore, adipose
tissue is one of the major stores of tocopherol in the body and the
tocopherol efflux from this tissue could be important to maintain
plasma and tissue concentrations during vitamin E deficiency (for
review see ref. 20). Therefore adipose tissue tocopherol
concentrations have been used as indicators of vitamin E status in
patients (21)(22)(23). Some studies have shown that in vitamin
E-deficient patients there are mechanisms for the mobilization of
tocopherol from adipose tissue (21)(22) and
that -tocopherol adipose tissue may be more readily available than
previously thought. It would be of interest to measure the adipocyte
vitamin E content in HC subjects to establish if the storage of vitamin
E is also altered, in this pathology, because of putative impaired
transfer.
To assess whether a decrease of E-RBC concentrations of HC subjects has
a physiological consequence, we compared the susceptibility to
peroxidation of RBCs from HC and NC men. Our results showed a
significantly increased susceptibility of RBCs of HC men to
AAPH-induced oxidation. Moreover, we observed a direct correlation
between the E-RBC concentration and susceptibility of cells to a
peroxidative stress. Previous studies have shown that vitamin E has a
protective effect on the RBC membrane against peroxidation and
hemolysis induced by an azo compound. Some of these studies have been
performed on isolated RBCs from rats (16) or humans
(24), and showed that the addition of an excess of vitamin
E in vitro reduced the susceptibility to a peroxidative stress. Other
studies carried out on RBCs from vitamin E-deficient or -supplemented
rabbits (25), rats (26), or humans
(27) led to the same conclusion. By contrast with these
studies, which modified experimentally the tocopherol RBC content, our
results emphasize the essential role of vitamin E under
physiopathological circumstances. Although the decrease of vitamin E in
RBC of HC men is weak and does not correspond to a real deficiency, it
is sufficient enough to impair the response to an oxidative stress. In
addition, it is noteworthy that these results obtained in asymptomatic
subjects are in good agreement with the hypothesis of Kritchevsky et
al. (28), suggesting that antioxidant substances are
likely to exert their effects at the earliest stages of the atherogenic
process. Thus, in hypercholesterolemia, the evaluation of antioxidants
might be taken into account as a risk marker with respect to the
development of preclinical disease.
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Acknowledgments
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We gratefully acknowledge Véronique Atger for helpful advice
in the preparation of this paper. We thank Catherine Dumas for her
technical assistance.
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Footnotes
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1 Nonstandard abbreviations: RBC, red blood cell; HC, hypercholesterolemic; NC, normocholesterolemic; TC, total cholesterol; TG, triglycerides; HDL-, LDL-C, high-, low-density lipoprotein cholesterol; BHT, butylated hydroxytoluene; E-pl, plasma vitamin E; E-RBC, red blood cell vitamin E; AAPH, 2,2'-azo bis-(2-amidinopropane) dihydrochloride; LT, lag time; and T50, time required to achieve 50% hemolysis. 
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Abstract
Introduction
