Lack of correlation between the a-tocopherol content of plasma and LDL , but high correlations for y-tocopherol and carotenoids

In 59 healthy human subjects (37 male and 22 female) the concentrations of the lipid-soluble antioxidants aand ytocopherol, aand pcarotene, lycopene, cryptoxanthin, canthaxanthin, and lutein + zeaxanthin were determined in plasma (pmol/L) and in isolated low density lipoproteins (LDL) (pmol/mmol cholesterol). Plasma a-tocopherol concentrations were significantly correlated with plasma total cholesterol concentrations (9 = 0.51, P < 0.0001) yet not with the LDL a-tocopherol content (? 0.05, ns). Plasma ytocopherol concentrations were weakly correlated with plasma total cholesterol (? = 0.12, P < 0.003) and both absolute and cholesterol standardized plasma ytocopherol concentrations correlated strongly with the LDL y-tocopherol content (9 = 0.58 and ? = 0.72, respectively). In contrast, carotenoid concentrations did not correlate with cholesterol concentrations, but their LDL content correlated significantly with the respective plasma concentrations (9 = 0.67 to 0.92, all P < 0.0001). In a subgroup of study subjects (n = 13) the distribution of vitamin E and carotenoids among LDL was calculated. The proportion of plasma aand y-tocopherol found in LDL was 48 t 7 (range, 36-61%) and 41 &? %, respectively, suggesting that LDL was in most of these subjects not the main carrier for these antioxidants. The lipophilic carotenoids, however, were predominantly carried by LDL (e.g., pcarotene: 87 k lo%), whereas the proportion of the more polar ones carried by LDL was much smaller (e.g., lutein + zeaxanthin: 36 k 6%). The results of this study show that plasma a-tocopherol concentrations are not predictive for the a-tocopherol content of LDL in nonsupplemented individuals. This finding could have implications in interpreting the cause of the inverse relationship between plasma a-tocopherol and risk of atherosclerosis. -Ziouzenkova, O. , B. M. Winklhofer-Roob, H. Puhl, J. M. Roob, and H. Esterbauer. Lack of correlation between the a-tocopherol content of plasma and LDL, but high correlations for y-tocopherol and carotenoids. J. Lipid Res. 1996.37: 1936-1946. in the dietary of antioxidants and fat, their absorption, and other factors (1-4). Similarly, the LDL content of vitamin E and carotenoids shows a substantial inter-individual variation (5-7). For instance, the LDL a-tocopherol content may range from about 3 to 16 molecules per LDL particle and that of p-carotene can range from 0.03 to about 2 molecules per LDL (5). It is widely believed that the variability in the antioxidant content of LDL is related to that in plasma antioxidant levels. This assumption is supported by several supplementation studies with vitamin E (7-9) or pcarotene (10, l l), and the reported ease of spontaneous exchange of a-tocopherol or p-carotene between different lipopro tein classes observed in in vitro experiments (12-15). However, in healthy individuals not taking antioxidant supplements, the putative relationship between the antioxidant content of LDL and plasma has so far not been examined. The question whether plasma levels of antioxidants indeed predict the antioxidant content of LDL is of importance in the context of the LDL-oxidation hypothesis (16-18) and the epidemiologic observations of an inverse relationship between the risk of atherosclerosis and plasma levels or dietary intake of vitamin E or p-carotene measured directly or estimated from dietary questionnaires (19-22). We have tested the hypothesis that the variability of the LDL antioxidant content is related to differences in plasma levels using a large number of data collected in the course of our studies on factors affecting the resistance to oxidation Supplementary key words min E m a-tocopherol * fkarotene * carotenoids plasma low density lipoprotein vitaPlasma concentrations of the lipid-soluble antioxidants vitamin E and carotenoids can vary considerably among healthy individuals, probably due to differences Abbreviations: VLDL, very low density lipoprotein; IDL, intermediate density lipoprotein; LDL, low density lipoprotein; HDL, high density lipoprotein; LP, lipoproteins; EDTA, ethylenediaminetetraacetic acid; BHT, butylated hydroxytoluene; HPLC, high performance liquid chromatography; ns, not significant; SD, standard deviation. ’To whom correspondence should be addressed. 1936 Journal of Lipid Research Volume 37, 1996 by gest, on N ovem er 6, 2017 w w w . j.org D ow nladed fom of LDL. The unexpected result, which we report here, is that in healthy subjects not taking supplements, the a-tocopherol content of LDL does not correlate with plasma a-tocopherol concentrations, whereas those of y-tocopherol and major carotenoids show a very high correlation. Preparation of plasma and LDL Fasting blood samples were obtained by venipuncture and collected by free flow into plastic tubes containing an aqueous solution of 10% EDTA. The final concentra. tion of EDTA was 1 mg/ml blood. The blood was centrifuged within 1 h at 10°C at 1000 g for 10 mir followed by 3000 g for 15 min. For measurement ol plasma content of vitamin E, carotenoids and lipids, an aliquot of the EDTA plasma was frozen immediately and stored at -70°C for not longer than 1 week. For preparation of LDL, the freshly prepared plasma was used and LDL was isolated by ultracentrifugation as previously described (25). LDL samples were filtered through a 0.45-pm filter into sterile evacuated glass vials and stored under argon at 4°C. The antioxidant content of LDL was determined within 1 week. Plasma and LDL containing EDTA can be stored under such condition for up to 2 weeks without a measurable loss of antioxidants (25). Measurement of the vitamin E and carotenoid content of plasma and LDL Plasma was thawed and 0.2 ml was diluted with 0.2 ml aqueous EDTA solution (1 mg/ml) followed by 0.4 ml ice-cold ethanol containing 0.05 mum1 butylated hydroxy toluene (BHT). Then 1 ml n-hexane was added and the mixture was vortexed for 1 min. After centrifugation (1000 g, 5 min) 0.8 ml of the upper hexane phase was collected and dried under nitrogen. The residue was dissolved in 0.1 ml ethanol-ethyl acetate 1O:l (v/v) and separated by HPLC on a Lichrospher 100 RP-18 (5 pm) column. Isocratic separation was performed with a mixture containing o.ol% ammonium acetate; flow rate was 1.2 ml/min. The effluent was monitored with two detectors fluorescence detector set at 292/335 nm; carotenoids quantification was done with a standard mkture separated under identical conditions. Lutein and zeaxanthin METHODS