Evaluation of alpha-tocopherol contained in plasma lipoproteins: How should the data be expressed?
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ABSTRACT: Triglyceride-rich lipoproteins that contain apolipoprotein CIII (apoCIII) are prominent in diabetic dyslipidemia. We hypothesized that these lipoproteins increase coronary disease risk in diabetic patients beyond that caused by standard lipid risk factors. Diabetic patients with previous myocardial infarction were followed for 5 years, and 121 who had a recurrent coronary event were matched to 121 who did not. VLDL and LDL that contained or did not contain apoCIII (CIII+ or CIII-) were prepared by immunoaffinity chromatography and ultracentrifugation. IDL was included in the LDL fraction. LDL CIII+, rich in cholesterol and triglyceride, was the strongest predictor of coronary events (relative risk [RR] 6.6, P<0.0001, for 4th versus 1st quartile). LDL CIII+ comprised 10% of total LDL. The main type of LDL, LDL CIII-, was less strongly predictive (RR 2.2, P=0.07). The increased risk associated with LDL CIII+ was unaffected by adjustment for plasma lipids, apoB, non-HDL cholesterol, or the other VLDL and LDL types. For VLDL CIII+, RR 0.5, P=0.07; for VLDL CIII-, RR 2.3, P=0.046. The presence of apolipoprotein E with CIII on VLDL and LDL did not affect risk. LDL with apoCIII strongly predicts coronary events in diabetic patients independently of other lipids and may be an atherogenic remnant of triglyceride-rich VLDL metabolism.Arteriosclerosis Thrombosis and Vascular Biology 05/2003; 23(5):853-8. · 6.34 Impact Factor
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ABSTRACT: Naturally occurring antioxidants like vitamin E, beta-carotene, and vitamin C can inhibit the oxidative modification of low-density lipoproteins. This action could positively influence the atherosclerotic process and, as a consequence, the progression of coronary heart disease. A wealth of experimental studies provide a sound biological rationale for the mechanisms of action of antioxidants, whereas epidemiological studies strongly sustain the 'antioxidant hypothesis'. To data, however, clinical trials with beta-carotene supplements have been disappointing and their use as a preventive intervention for cancer and coronary heart disease should be discouraged. Only scant data from clinical trials are available for vitamin C. As for vitamin E, discrepant results have been obtained by the Alpha-Tocopherol, Beta-Carotene Cancer Prevention Study with a low-dose vitamin E supplementation (50 mg daily) and the Cambridge Heart Antioxidant Study (400-800 mg daily). Currently ongoing are several large-scale clinical trials that will help in clarifying the role of vitamin E in the prevention of atherosclerotic coronary disease.Pharmacological Research 10/1999; 40(3):227-38. · 4.35 Impact Factor
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ABSTRACT: Oxidative modification of low density lipoprotein (LDL) has been implicated as a factor in the generation of macrophage-derived foam cells, the hallmark of atherosclerotic plaques. Because LDL consists of discrete subfractions with different physicochemical characteristics, the question arises as to whether these LDL subfractions differ in their susceptibility to oxidative modification. To answer this question, three LDL subfractions, LDL1, LDL2, and LDL3, were isolated from the plasmas of 11 healthy volunteers by density gradient ultracentrifugation. The LDL subfractions were oxidatively modified by incubation with copper ions. Differences in the subfractions' susceptibilities to lipid peroxidation were studied by measuring the formation of the 234-nm-absorbing oxidation products every 3 minutes on an ultraviolet spectrophotometer. A significant inverse linear relation was found between LDL subfractions and lag time (regression coefficient = -8.50, p less than 0.001), indicating that both the dense LDL3 and the light LDL2 were less well protected against oxidative modification than the very light LDL1. The LDL subfractions showed a positive linear relation with the rate of oxidation (regression coefficient = 0.46, p less than 0.001) and the amount of conjugated dienes formed in the LDL subfractions after 4 hours of oxidation (regression coefficient = 9.24, p less than 0.001). Thus, both LDL3 and LDL2 were more extensively modified in time than LDL1, which may be explained by the significantly higher concentration of polyunsaturated fatty acids in LDL3 (micromoles per gram LDL cholesterol) compared with LDL1 (Tukey's test, p less than 0.05). These results indicate that the more dense LDL subfractions, that is, LDL2 and LDL3, are more susceptible to oxidative modification and therefore may contribute more to foam cell formation than the less dense LDL subfraction LDL1.Arteriosclerosis and thrombosis: a journal of vascular biology / American Heart Association 11(2):298-306.
Evaluation of α α α α-tocopherol Contained in Plasma Lipoproteins:
How Should the Data Be Expressed?
Fernando Britesa, Pablo Evelsonb, Guillermo Gambinoa, Marina Travaciob, Susana
Llesuyb, Regina Wikinskia.
a Laboratorio de Lípidos y Lipoproteínas, Departamento de Bioquímica Clínica, Facultad de
Farmacia y Bioquímica, Universidad de Buenos Aires, CONICET, Argentina.
b Cátedra de Química General e Inorgánica, Facultad de Farmacia y Bioquímica, Universidad de
Buenos Aires, CONICET, Argentina.
Short title: α-tocopherol in plasma lipoproteins.
Keywords: α-tocopherol, lipoproteins, atherosclerosis, antioxidant.
Corresponding author: Fernando Brites, Ph.D.
Departamento de Bioquímica Clínica
Facultad de Farmacia y Bioquímica
Junin 956. (1113). Buenos Aires. Argentina.
Fax: 54-11-4508 3645. e-mail: firstname.lastname@example.org
To the Editor,
α-tocopherol is a potent, lipid-soluble, chain breaking antioxidant, which has a great
impact in the prevention of chronic diseases believed to be associated with oxidative stress such
as cancer , cardiovascular disease and atherosclerosis [2,3]. Moreover, α-tocopherol may
also play an atheroprotective role through nonantioxidant processes .
The knowledge that α-tocopherol possesses several beneficial properties raised a great
interest in the study of its transport through the body fluids. It is well known that α-tocopherol,
as other liposoluble antioxidants, is transported through plasma by lipoprotein particles. Some
authors studied the distribution of α-tocopherol among the main lipoprototein classes. Behrens
et al.  found that although LDL and HDL were the main carriers of α-tocopherol in both
males and females, more tocopherol was detected in LDL than in HDL in males but the
opposite was true in females. Similar findings were reported by Carcelain et al.  who
validated their results employing two different methods for isolating lipoprotein fractions.
Nevertheless, no studies have evaluated the different ways to express α-tocopherol
content of plasma lipoproteins and the influence that this could have on possible conclusions
which can be drawn with clinical and experimental purposes. The aim of the present study was
to measure α-tocopherol contained in lipoprotein fractions isolated by ultracentrifugation and
to compare different forms of expressing the data.
Venous blood was collected from 30 healthy, non-smoker, fasting normolipemic male
subjects. Serum samples were separated by centrifugation at 1500g and 4º C for 15 minutes,
and immediately used for lipoprotein separation. Lipoprotein fractions were isolated by
sequential flotation in a Beckman XL-90 ultracentrifuge using a fixed-angle rotor type 90Ti
. Very low density lipoprotein (VLDL), intermediate density lipoproteins (IDL) plus LDL,
and HDL fractions were isolated at their characteristic densities (1.006, 1.063, and 1.210 g/ml,
respectively). The infranatant (density > 1.210 g/mL) was also recovered and the purity of each
lipoprotein fraction was tested by agarose gel electrophoresis . Each nondialized fraction
was immediately used for lipid and protein measurements. Aliquots were also supplemented
with butylated hydroxytoluene (0.048 % w/v) and stored at -70º C for α-tocopherol
determination. α-Tocopherol was quantified by reverse-phase high-performance liquid
chromatography with electrochemical detection using a Bioanalytical Systems (West Lafayatte,
IN) amperometric detector with a glassy-carbon working electrode at an applied oxidation
potential of 0.6 volts . Results were expressed in different ways and statistical analysis was
carried out employing the Mann-Whitney non-parametric test (U test). Differences were
considered significant at P < 0.05 in the bilateral situation.
Figure 1 shows the results obtained expressed in different forms. When results were
expressed as mg of α-tocopherol / dl of plasma or as percentage, IDL+LDL fraction showed the
highest α-tocopherol content, followed by HDL. When we employed the ratio mg of α-
tocopherol / mg of lipoprotein proteins, HDL exhibited significantly lower values than the other
two fractions, while no differences were detected between VLDL and IDL+LDL fractions.
When data were shown as mg of α-tocopherol / mg of lipoprotein lipids, the three lipoprotein
classes seemed to transport similar α-tocopherol amounts. The expression as moles of α-
tocopherol / moles of neutral lipids revealed the lowest value for HDL, followed by VLDL and
then by IDL+LDL fraction. Finally, α-tocopherol contained in VLDL resulted to be
significantly higher than the antioxidant content of the other fractions when results were
expressed as moles of α-tocopherol / moles of lipoprotein particles. Therefore, the findings are
directly conditioned by the way of expression of the data.
The different forms that we selected to express the results are widely employed in the
literature to show α-tocopherol carried by lipoprotein particles [5,6,10,11]. A hazardous
decision on the way to express the data could lead to non-comparable results. Goulinet et al.
 realised about this fact and suggested that the discrepancy between his results on α-
tocopherol content of LDL subspecies and those reported by other authors [13,14] raised, in
part, from the expression of the data. Accordingly, Perugini et al.  found completely
different results when they normalised α-tocopherol concentration in lipoprotein fractions for
cholesterol, protein or phospholipid content or when calculating the porcentual distribution.
These authors also suggest that expressing the results in relation to the protein content of apo B
containing lipoproteins approximates well the amount of antioxidant molecules in each
lipoprotein particle. This conclusion would be appropriate if those lipoproteins only contained
apo B, which is not true for VLDL or for LDL isolated from patients with different pathologies
such as diabetes or nephropathy [16,17].
Even if it is very difficult to select the most appropriate form of expression, we think
that a mole per particle bases could constitute a good approach to reflect the capacity of an
individual lipoprotein particle to transport α-tocopherol molecules. Moreover, the other ways of
expression could be of choice when the aim is to show the α-tocopherol carried by the whole
lipoprotein fraction, thus reflecting a combination of the number of particles present and the
individual capacity of each particle. Although α-tocopherol is very frequently expressed in
relation to the protein content [5,10,11,15], it would be more accurate to employ a form of
expression which includes the lipid content of lipoproteins, mainly due to the fact that α-
tocopherol is a lipid-soluble compound. Taking into consideration that the incorporation of α-
tocopherol in lipoproteins could be primarily influenced by neutral lipid core size, results could
be satisfactorily expressed per mol of triglycerides and cholesteryl esters.
In conclusion, the selection of a determined form of expression for data describing α-
tocopherol contained in lipoproteins should depend on the specific objective of the study.
Nevertheless, unification of the criteria employed would allow to compare different studies.
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2. Stampfer M, Hennekens C, Manson J, Colditz G, Rosner B, Willett W (1993) Vitamin E
consumption and the risk of coronary disease in human. N Engl J Med 328:1444-1449.
3. Marchioli R (1999) Antioxidant vitamins and prevention of cardiovascular disease:
laboratory, epidemiological and clinical trial data. Pharmacol Res 40:227-238.
4. Pryor WA (2000) Vitamin E and heart disease: basic science to clinical intervention trials.
Free Rad Biol Med 28:141-164.
5. Behrens WA, Thompson JN, Madère R (1982) Distribution of α-tocopherol in human
plasma lipoproteins. Am J Clin Nutr 35:691-696.
6. Carcelain G, David F, Lepage S, Bonnefont-Rousselot D, Delattre J, Legrand A, et al.
(1992) Simple method for quantifying α-tocopherol in low-density + very-low-density
lipoproteins and in high-density lipoproteins. Clin Chem 38:1792-1795.
7. Schumaker VN, Puppione DL (1986) Sequential flotation ultracentrifugation. Methods in
8. Noble RP (1968) Electrophoretic separation of plasma lipoproteins in agarose gel. J Lipid
9. Lang JK, Gohil K, Packer L (1986) Simultaneous determination of tocopherols, ubiquinols,
and ubiquinones in blood, plasma, tissue homogenates, and subcellular fractions. Anal
10. Vieira OV, Laranjinha JAN, Madeira VMC, Almeida LM (1996) Rapid isolation of low
density lipoproteins in a concentrated fraction free from water-soluble plasma antioxidants.
J Lipid Res 37:2715-2721.
11. Chappey B, Myara I, Benoit MO, Mazière C, Mazière JC, Moatti N (1995) Characteristics
of ten charge-differing subfractions isolated from human native low-density lipoproteins
(LDL). No evidence of peroxidative modifications. Biochim Biophys Acta 1259:261-270.
12. Goulinet S, Chapman MJ (1997) Plasma LDL and HDL subspecies are heterogeneous in
particle content of tocopherols and oxygenated and hydrocarbon carotenoids. Relevance to
oxidative resistance and atherogenesis. Arterioscler Thromb Vasc Biol 17:786-796.
13. de Graff J, Hak-Lemmers HLM, Hectors MPC, Demacker PNM, Hendricks JCM,
Stalenhoef AFH (1991) Enhanced susceptibility to in vitro oxidation of the dense low
density lipoprotein subfraction in healthy subjects. Arteriosclerosis 11:298-306.
14. Mackness MI, Arrol S, Turrington PN (1991) Paraoxonase prevents accumulation of
lipoperoxides in low-density lipoprotein. FEBS Lett 286:152-154.
15. Perugini C, Bagnati M, Cau C, Bordone R, Paffoni P, Re R, Zoppis E, et al. (2000)
Distribution of lipid-soluble antioxidants in lipoproteins from healthy subjects. I.
Correlation with plasma antioxidant levels and composition or lipoproteins. Pharmacol Res
16. Lee SJ, Campos H, Moye LA, Sacks FM (2003) LDL containing apolipoprotein CIII is an
independent risk factor for coronary events in diabetic patients. Arterioscler Thromb Vasc
17. Moberly JB, Attman PO, Samuelsson O, Johansson AC, Knight-Gibson C, Alaupovic P
(2002) Alterations in lipoprotein composition in peritoneal dialysis patients. Perit Dial Int
Acknowledgements: This work was supported by grants from the University of Buenos Aires
(B048, B089, and B124).
Fig. 1: α-tocopherol content of lipoproteins expressed in different ways: a) mg of α-tocopherol
/ dl of plasma (Panel A); b) percentage of α-tocopherol among the three lipoprotein classes
(Panel B); c) mg of α-tocopherol / mg of lipoprotein proteins (Panel C); d) mg of α-tocopherol /
mg of lipoprotein lipids (free cholesterol + cholesteryl esters + phospholipids + triglycerides)
(Panel D); e) moles of α-tocopherol / moles of neutral lipids (triglycerides + cholesteryl esters)
(Panel E); and f) moles of α-tocopherol / moles of lipoprotein particles (Panel F).
a P < 0.0001 vs VLDL; b P < 0.001 vs IDL+LDL; c P < 0.0001 vs VLDL.
VLDL, very low density lipoprotein; IDL, intermediate density lipoprotein; LDL, low density
lipoprotein; HDL, high density lipoprotein; TG, triglycerides; CE, cholesteryl esters.
mg of α−
α−tocopherol/dl of plasma
-tocopherol Concentration (mg/dl)
-tocopherol Concentration (%)
mg of α α α α-tocopherol/mg of proteins
mg of α α α α-tocopherol/mg of lipids
moles of α α α α-tocopherol/moles of TG+CE
moles of α α α α-tocopherol/moles of Lp