- Email: [email protected]
Effects of postprandial hyperlipemia on the vitamin E content of lipoproteins
Clinica Chimica Acta 277 (1998) 141–152
Effects of postprandial hyperlipemia on the vitamin E content of lipoproteins ´ ` Cambillaud c ,f , Remy Couderc a ,f , *, Jacqueline Peynet b ,f , Michele d ,f e ,f ` a ,f , Frank Tallet , Claudine Cosson , Guillaume Lefevre ´ Veronique Atger c ,f a b
ˆ Service de Biochimie, Hopital Tenon, 4 rue de la Chine, 75020, Paris, France ˆ ` , Assistance Publique, Paris, France Lariboisiere Service de Biochimie, Hopital c ˆ Broussais, Assistance Publique, Paris, France Service de Biochimie, Hopital d ˆ Service de Biochimie, Hopital Cochin, Assistance Publique, Paris, France e ˆ ˆ , Assistance Publique, Paris, France Bicetre Service de Biochimie, Hopital f 1 ´ , Paris, France GERBAP , Section Lipoproteines
Received 23 February 1998; received in revised form 13 July 1998; accepted 15 July 1998
Abstract Delayed postprandial clearance of triglyceride-rich lipoproteins (TGRL) could induce a decrease of low-density lipoprotein (LDL) and high-density lipoprotein (HDL) vitamin E content through its transfer into TGRL. We thus studied lipoprotein vitamin E content during postprandial hypertriglyceridemia induced by a high fat meal without vitamin E supplement. Venous blood was drawn from five healthy male volunteers following a 12 h fast at (t 0 ) then 2 h (t 2 ), 4 h (t 4 ), 6 h (t 6 ) and 8 h (t 8 ) after a 80 g fat meal. In plasma, only TG significantly varied during the postprandial period with a large interindividual variability. Mean composition of lipoproteins in terms of mass was not significantly modified. The amount of vitamin E significantly increased in TGRL and decreased in LDL plus HDL at t 4 and t 6 relative to t 0 . Vitamin E content of TGRL and LDL but not of HDL decreased significantly at t 4 . The mean decrease was 20% (range 5%–54%) for the LDL. LDL– and HDL vitamin E content correlated inversely with plasma TGRL levels. Our data suggest that LDL from subjects with delayed chylomicron clearance could be less protected against oxidation. 1998 Elsevier Science B.V. All rights reserved. *Corresponding author. Tel.: 1 33-1-56017989; Fax: 1 33-1-56017840; E-mail: [email protected] 1 ˆ GERBAP (Groupe d’Evaluation et de Recherche de l’Assistance Publique des Hopitaux de Paris) Section ´ Lipoproteines; Members (other than the authors): Bailleul Sophie, Bonneau Christine, Davit-Sprault Anne, ` Marie Bernadette, Myara Isaac, Turpin Elisabeth. Erlich Daniele, 0009-8981 / 98 / $ – see front matter 1998 Elsevier Science B.V. All rights reserved. PII: S0009-8981( 98 )00121-1
142
R. Couderc et al. / Clinica Chimica Acta 277 (1998) 141 – 152
Keywords: Vitamin E (alpha-tocopherol); Lipoproteins; Postprandial
1. Introduction Reduced postprandial clearance of triglyceride (TG)-rich lipoproteins (TGRL) may be involved in the development of premature coronary atherosclerosis [1,2]. Indeed, postprandial hypertriglyceridemia is delayed in subjects with coronary atherosclerosis in comparison to control subjects [3] and postprandial plasma levels of small chylomicron remnants were found to relate to the progression of coronary atherosclerosis [4]. Reduced postprandial clearance of TGRL generates potentially atherogenic lipoproteins through mechanisms involving exchanges and transfers of lipids and apolipoproteins between TGRL and low-density lipoproteins (LDL) and high-density lipoproteins (HDL) [5]. These transfers also involve lipid-soluble molecules carried by lipoproteins and especially vitamin E: this exchange between lipoproteins is greatly facilitated by a specific phospholipid transfer protein [6]. Vitamin E is incorporated into chylomicrons and secreted by intestinal cells and its delivery to tissues is closely linked to the lipoprotein metabolism [7]. Vitamin E, mainly a-tocopherol, by its antioxidant activity, protects lipoprotein polyunsaturated fatty acids against oxidation [8] and may reduce the development of arteriosclerosis [9–11]. Moreover, in vitro studies have shown that oxidatively modified LDL are taken up, via the scavenger receptor, by monocyte / macrophage cells. Oxidized HDL have a reduced efficiency in mediating the cholesterol reverse transport and are less effective in protecting LDL against oxidation [12]. Lipoprotein oxidation depends on the balance between the free radical generation rate and protection systems which include vitamin E. Thus it is of interest to explore situations in which LDL and HDL could be depleted in vitamin E. In vitro, LDL and HDL tocopherol readily exchange with other lipoproteins and only the tocopherols in TGRL do not readily exchange [13]. In humans, the peak in tocopherol secretion in chylomicrons occurs between 6 and 12 h following the oral administration of deuterated tocopherols [14]. The rapid hydrolysis of TGRL, with the production of excess surface, allows the transfer of tocopherol from TGRL to HDL and then to other lipoproteins. There is no study, to our knowledge, on the variations of LDL and HDL vitamin E content after a high fat meal without tocopherol supplement. Our study was based on the following physiopathological hypothesis that delayed postprandial clearance of TGRL could induce a decrease of LDL and HDL vitamin E content through its transfer into TGRL. We therefore studied
R. Couderc et al. / Clinica Chimica Acta 277 (1998) 141 – 152
143
lipoprotein vitamin E content during postprandial hypertriglyceridemia induced by a high fat meal in healthy volunteers.
2. Methods Venous blood was drawn from five healthy male volunteers following a 12 h fast (t 0 ) then 2 h (t 2 ), 4 h (t 4 ), 6 h (t 6 ) and 8 h after (t 8 ) an 80 g fat meal including 20 g butter, 60 g cheese, two eggs, 40 g mayonnaise, 100 g white bread and a glass of orange juice. This meal contained about 10 mg of vitamin E; that is, the mean daily intake. A second protocol performed on four of these five men was designed to estimate the reproducibility of the postprandial variation of LDL vitamin E content. Lipoproteins were isolated from plasma EDTA by sequential ultracentrifugation. The chylomicron fraction was isolated from 3 ml plasma layered under 9 ml 0.9% NaCl for 0.5 h at 20 000 RPM in a Beckman SW41 rotor. VLDL, LDL and HDL were ultracentrifugated in the Beckman vertical NVT rotor at the densities of 1.006, 1.063 and 1.210 g / ml respectively. Lipids and proteins were assayed by using classical techniques and vitamin E by HPLC adapted from Biery [15]. For red blood cell (RBC) vitamin E assay, blood collected onto EDTA was centrifuged at 2000 3 g for 10 min at 48C to separate plasma and RBC. The RBC were washed three times with a 0.9% NaCl solution containing 0.5% of pyrogallol (Merck, Darmstadt, Germany) as antioxidant agent. The final RBC suspension solution was made up to about 50% with addition of distilled water containing 0.1% of BHT (butylated hydroxytoluene, Sigma, St Louis, MO, USA). RBC samples for vitamin E determination were stored frozen at 2 808C until time analysis. We checked that no loss of erythrocyte a-tocopherol occurred at 2 808C during at least six months. Statistical analyses were performed by using STATVIEW 4 software (Abacus Concept, Berkeley, CA). Unless otherwise stated, data are presented as mean6standard deviation. Statistical significance of the variation between t 0 and the other times after the meal was assessed by using the Student’s t-test for paired values.
3. Results Only plasma TG significantly varied during the postprandial period (Table 1). There was a large interindividual variability, with particularly wide differences in TG levels which occurred at the late postprandial hours, i.e., between 6 and 8 h after ingestion of the test meal (Fig. 1). Among the five subjects, only three had TG , 1.5 mmol / l after t 8 . This high interindividual variability was
144
R. Couderc et al. / Clinica Chimica Acta 277 (1998) 141 – 152
Table 1 Lipid and vitamin E levels in plasma from five healthy men following a 12 h fast (t 0 ) then after an 80 g fat meal Time after meal (h)
TC mmol / l
TG mmol / l
PL mmol / l
Vit E mg / l
t0 t2 t4 t6 t8
5.8260.66 5.9060.54 5.6460.56 5.6260.63 5.8960.73
1.0560.18 1.646O.24** 1.8860.65* 1.6960.46* 1.6760.86
3.0260.38 3.1660.34 3.0860.37 3.1260.27 3.2760.29
14.362.1 13.761.9* 13.762.1 14.362.1 15.461.8*
Significantly different from t 0 : *P # 0.05; **P # 0.01. Mean61 standard deviation (n 5 5). TC: total cholesterol; TG: triglycerides; PL: phospholipids; Vit E: vitamin E.
confirmed by the second protocol. The mean composition of lipoproteins in terms of mass was not significantly modified. However, the cholesterol to TG molar ratio in HDL was significantly decreased at t 4 relative to t 0 (9.8962.95 vs
Fig. 1. Plasma triglyceride levels in five healthy men following a 12 h fast then after a high fat meal. Subject No. 1: s; No. 2: h; No. 3: n; No. 4: 앳; No. 5: 1 .
R. Couderc et al. / Clinica Chimica Acta 277 (1998) 141 – 152
145
7.7462.05; P # 0.05). The value of this ratio correlated negatively with the TG concentration in the plasma (r 5 2 0.616; P # 0.001). This relationship was particularly strong in the patient No. 5 (r 5 2 0.935). After the high-fat meal intake, the distribution of vitamin E between the lipoprotein classes was modified (Table 2). The amount of vitamin E significantly increased in triglyceride rich lipoproteins (chylomicrons plus very-low density lipoproteins) and decreased in LDL plus HDL at t 4 and t 6 relative to t 0 . The vitamin E content of all lipoprotein classes decreased 4 h after the meal (Fig. 2). However, significance was not reached for HDL. The mean decrease was 20% (range: 5–54%) for the LDL. The most significant decrease in LDL vitamin E content was found in the subject whose TG were still rising at the end of t 8 . The second protocol was designed to estimate the reproducibility of the variation of LDL vitamin E content. Indeed, in experimental conditions identical to those of the first protocol, we again found a significant (P # 0.05) decrease of vitamin E in LDL at t 2 (1.2260.09 mg / g) and t 4 (1.2060.07 mg / g) relative to t 0 (1.3960.09 mg / g). Vitamin E content of red blood cells (RBC), which provides a good model for the study of vitamin E exchanges between lipoproteins and cells, slightly increased at t 2 and t 8 relative to t 0 (Fig. 3). LDL vitamin E content correlated highly with the HDL vitamin E content (Fig. 4), and the latter (x) correlated highly with the esterified cholesterol HDL content ( y 5 10.2x 1 5.5, r 5 0.842, P # 0.0002), and with esterified cholesterol-to-TG molar ratio ( y 5 5.35x 1 1.33, r 5 0.592, P # 0.002) in these lipoproteins. LDL-vitamin E content correlated significantly with plasma TGRL levels (Fig. 5). There was also a weak correlation between LDL–vitamin E content and TGRL–vitamin E content (data not shown). Table 2 Distribution of vitamin E (percent) between the lipoprotein classes from five healthy men following a 12 h fast (TO) then after an 80 g fat meal Time after the meal (h)
TGRL (%)
LDL (%)
HDL (%)
t0 t2 t4 t6 t8
1967 2565 2669** 25611* 20612
4667 4366 4264** a 4267* a 4665
3564 3263 3265** a 3366* a 3468
Significantly different from t 0 : *P # 0.05; **P # 0.01; a Significantly different from t 0 when considering the sum of LDL- and HDL vitamin E rates. Mean61 standard deviation (n 5 5). TGRL: triglyceride rich lipoproteins; the amount of vitamin E in TGRL is the sum of the amount of vitamin E in very low-density lipoproteins and in chylomicrons.
146
R. Couderc et al. / Clinica Chimica Acta 277 (1998) 141 – 152
Fig. 2. Vitamin E content of plasma lipoproteins in five healthy men following a 12 h fast then after a high fat meal. Mean6standard deviation; *significantly different from t 0 (P # 0.05; Student’s t-test for paired values). TGRL (triglyceride-rich lipoprotein) vitamin E content is the sum of chylomicron- and very low-density lipoprotein vitamin E contents.
R. Couderc et al. / Clinica Chimica Acta 277 (1998) 141 – 152
147
Fig. 3. Vitamin E content of red blood cells in five healthy men following a 12 h fast then after a high fat meal. Mean6standard deviation; *significantly different from t 0 (P # 0.05; Student’s t-test for paired values). Hb: haemoglobin.
4. Discussion This study shows that LDL– and HDL–vitamin E contents decrease after ingestion of a high fat test meal and reach a minimum at 4 h after the meal; especially in the alteration in LDL vitamin E correlated to the plasma triglyceride-rich lipoprotein level. TGRL vitamin E content was also decreased 4 h after the meal but these lipoproteins carried a larger fraction of total plasma vitamin E than in the fasting state. Composition and metabolism of LDL and HDL depend on chylomicron clearance by lipases [16]. Moreover, many epidemiologic studies have highlighted the significance of triglycerides as a coronary atherosclerosis risk factor, and several of them have been realized in non-fasting subjects [17,18]. Nevertheless, protocol for testing fat tolerance on a small number of subjects must be standardized in terms of quality and quantity of the meal ingested. We chose to give a test meal consisting of commercially available food and providing 1 g fat per kg body weight and almost 3 g of fiber because preparations of liquid fat emulsions do not provide fiber which can attenuate the
148
R. Couderc et al. / Clinica Chimica Acta 277 (1998) 141 – 152
Fig. 4. Scattergram illustrating the relationship between the HDL-( y) and LDL (x) vitamin E content in five healthy men following a 12 h fast then after a high fat meal. Subject No. 1: s; No. 2: h; No. 3: n; No. 4: 앳; No. 5: 1 . The r-value for all subjects and all times (n 5 25) obtained by linear regression was 0.79 (P # 0.001); the equation of the linear regression was: y 5 0.768x 2 0.019.
postprandial triglyceride response [19] and may not induce the same oronasal stimulation as a solid meal [20]. The meals were ingested over 20–30 min and no subjects suffered from gastrointestinal pain. The alterations of lipoprotein composition in terms of cholesterol, TG, and phospholipids were in accord with previous studies, particularly given the presence of two peaks of TG in the same subjects [21]. However, in our study there was no significant alteration in postprandial plasma LDL-cholesterol concentration (data not shown). These results conflict with those from previous studies [21–23], in which LDL-cholesterol concentration, as estimated by Friedewald formula, decreased after the fat-rich meal. That could be due to the difference in meal composition, and to the fact that we have measured LDLcholesterol in LDL isolated by ultracentrifugation. The large inter-individual variations explain the fact that no significant alteration of the mass / mass lipoprotein composition was found.
R. Couderc et al. / Clinica Chimica Acta 277 (1998) 141 – 152
149
Fig. 5. Scattergram illustrating the relationship between the LDL-vitamin E contents and TGRL (triglyceride-rich lipoprotein) plasma levels in five healthy men following a 12 h fast then after a high fat meal. The r-value for all subjects and all times (n 5 25) obtained by linear regression was 0.53 (P # 0.01); the equation of the linear regression was: y 5 2 0.757x 1 1.935. TGRL levels were calculated by adding chylomicron and very low-density lipoprotein plasma levels expressed in g / l.
Plasma vitamin E levels in the subjects we studied were in the normal range before the meal (Table 1). In fasting sera, vitamin E distributed between VLDL, LDL and HDL with ratios of 1.0:2.4:1.8 in our five volunteers. These ratios are nearly the same as those published by Kostner et al who found ratios of 1.0:1.9:1.4 [6]. They slightly decreased at t 2 and slightly increased at t 8 . The fact that plasma vitamin E levels were not altered to a large extent may be the consequence of the high clearance of chylomicron vitamin E and not excluding transient alterations for vitamin E distribution among lipoproteins and of lipoprotein vitamin E content. In vitro studies have shown that vitamin E distribution among lipoproteins depends on the lipoprotein mass ratio, lipoprotein half lives and PLTP activity [6,24]. In our study, HDL vitamin E content correlated positively with HDL cholesterol and no such relation was found with LDL-cholesterol. On the other hand, LDL and HDL vitamin E contents correlated negatively with TGRL levels, which is in accordance with the previously described role of these lipoproteins as a vitamin E acceptor. Therefore, the alteration in vitamin E distribution among lipoproteins observed during the postprandial period may be due to a net transfer of vitamin E
150
R. Couderc et al. / Clinica Chimica Acta 277 (1998) 141 – 152
into chylomicrons. It is noteworthy that the plasma vitamin E content in HDL was 10% lower in our study than in others [25,26]. Other than the opposite alterations of vitamin E distribution in TGRL and in cholesterol rich lipoproteins (LDL and HDL), vitamin E contents of all lipoprotein classes decreased to reach a lower level at t 4 on the average. These data suggest that chylomicrons originating from the intestine were rather vitamin E poor or that vitamin E left these lipoproteins at the very beginning of their catabolism leading to vitamin E poor chylomicron remnants which were still isolated during the first step of ultracentrifugation at d , 1.000. On the other hand, the decrease in LDL and HDL vitamin E contents may be due to a net transfer of vitamin E into chylomicrons, the mass of which rapidly increased during the post prandial period. Red blood cells (RBC) may also act as a vitamin E acceptor because in our study, their vitamin E content was found to be correlated to HDL and LDL vitamin E content, and in a previous study in children, to that of HDL [26]. Despite the fact that the total RBC vitamin E mass increase is not sufficient to explain the alterations observed in lipoproteins, this phenomenon may be enhanced by the presence of high levels of chylomicrons. In subjects on a diet without excess of lipids and rich enough in lipid-soluble antioxidants and exhibiting a fast postprandial clearance of chylomicrons, the postprandial depletion of LDL and HDL in vitamin E may be slight and brief. This is because the vitamin E transfer, controlled by the chylomicrons / HDL plus LDL mass ratio, may well be balanced by a vitamin E transfer into HDL during TG lipolysis. On the other hand, and in subjects on a high fat and low lipid-soluble antioxidant diet and slowly clearing chylomicrons, LDL and HDL vitamin E depletion may be high and prolonged. In our study, in the subject who presented the most prolonged chylomicron clearance, the LDL vitamin E content was decreased by 54%. Consequently, lipoproteins from this subject could be more susceptible to oxidation. This point needs further investigation better to understand the LDL susceptibility to oxidation in subjects selected on the basis of TG concentration which is still high 6 h after a meal. In conclusion our study links two pathophysiological hypotheses of atherogenesis: the Zilversmit’s hypothesis involving chylomicron hydrolysis alterations [1] and the modified lipoprotein hypothesis involving stress factors such as free radicals.
References [1] Zilversmit DB. Atherogenesis: a postprandial phenomenon. Circulation 1979;60:473–85. [2] Zilversmit DB. Atherogenic nature of triglycerides, postprandial lipemia, and triglyceriderich remnant lipoproteins. Clin Chem 1995;41:153–8.
R. Couderc et al. / Clinica Chimica Acta 277 (1998) 141 – 152
151
[3] Groot PH, Van Stiphout WH, Krauss XH, et al. Postprandial lipoprotein metabolism in normolipidemic men with and without coronary artery disease. Arteriosclerosis Thromb 1991;11:653–62. [4] Karpe F, Steiner G, Uffelman K, Olivecrona T, Hamsten A. Postprandial lipoproteins and progression of coronary atherosclerosis. Atherosclerosis 1994;106:83–97. ¨ [5] Miesenbock G, Patsch JR. Postprandial hyperlipidemia: the search for the atherogenic lipoprotein. Cur Opin Lipidol 1992;3:196–201. [6] Kostner GM, Oettl K, Jauhiainen M, Ehnholm C, Esterbauer H, Dieplinger H. Human plasma phospholipid transfer protein accelerates exchange / transfer of a-tocopherol between lipoproteins and cells. Biochem J 1995;305:659–67. [7] Kayden HJ, Traber MG. Absorption, lipoprotein transport, and regulation of plasma concentrations of vitamin E in humans. J Lipid Res 1993;34:343–58. [8] Esterbauer H, Puhl H, Waeg G, Krebs A, Tatzber F, Rabol H. Vitamin E and atherosclerosisAn overview. In: Mino M, et al, editors. Vitamin E - Its usefulness in Health and in Curing diseases. Basel: Karger, 1993:233–41. [9] Riemersma RA, Wood DA, MacIntyre CCA, Elton RA, Gey KF, Oliver MF. Risk of angina pectoris and plasma concentrations of vitamin A, C, and E and carotene. Lancet 1991;337:1– 5. [10] Rimm EB, Stampfer MJ, Ascherio A, Giovannucci E, Colditz GA, Willett WC. Vitamin E consumption and the risk of coronary disease in men. New Engl J Med 1993;328:1450–6. [11] Stampfer MJ, Hennekens CH, Manson JE, Colditz GA, Rosner B, Willett WC. Vitamin E consumption and the risk of coronary disease in women. New Engl J Med 1993;328:1444–9. [12] Rifici V, Khachadurian A. Oxidation of high density lipoproteins: characterization and effects on cholesterol efflux from J774 macrophages. Biochem Biophys Acta 1996;99:87–94. [13] Traber MG, Lane JC, Lagmay NR, Kayden HJ. Studies on the transfer of tocopherol between lipoproteins. Lipids 1992;27:657–63. [14] Kayden HJ, Traber MG. Transport of vitamin E in human lipoproteins. In: Mino M, et al, editors. Vitamin E-Its usefulness in Health and in Curing diseases. Basel: Karger, 1993:85– 93. [15] Biery JG, Tolliver TJ, Gatignani GL. Simultaneous determination of alpha-tocopherol and retinol in plasma or red blood cells by high pressure-chromatography. Am J Clin Nutr 1979;32:2143–9. [16] Karpe F, Tornvall P, Olivecrona T, Steiner G, Carlson LA, Hamsten A. Composition of human low density lipoprotein: effects of postprandial triglyceride-rich lipoproteins, lipoprotein lipase, hepatic lipase and cholesteryl ester tranfer protein. Atherosclerosis 1993;98:33– 49. [17] Stensvold I, Tverdal A, Urdal P, Graff-Iversen S. Non-fasting serum triglyceride concentration and mortality from coronary heart disease and any cause in middle aged Norwegian women. Br Med J 1993;307:1318–22. ¨ G, Hopferwieser T, et al. Relation of triglyceride metabolism and [18] Patsch JR, Miesenbock coronary artery disease. Arteriosclerosis Thromb 1992;12:1336–45. [19] Cara L, Dubois C, Borel P, et al. Effects of cat bran, rice bran, wheat fiber, and wheat germ on postprandial lipemia in adults. Am J Clin Nutr 1992;55:81–8. [20] Mattes RD. Oral fat exposure alters postprandial lipid metabolism in humans. Am J Nutr 1996;63:911–7. [21] Cohn JS, McNamara JR, Schaefer EJ. Lipoprotein cholesterol concentrations in the plasma of human subjects as measured in the fed and fasted states. Clin Chem 1988;34:2456–9. [22] Rifai N, Merrill JR, Holly RG. Postprandial effect of a high fat meal on plasma lipid, lipoprotein cholesterol and apolipoprotein measurements. Ann Clin Biochem 1990;27:489– 93.
152
R. Couderc et al. / Clinica Chimica Acta 277 (1998) 141 – 152
[23] Dubois C, Armand M, Ferezou J, et al. Postprandial appearance of dietary deuterated cholesterol in the chylomicron fraction and whole plasma in healthy subjects. Am J Nutr 1996;64:47–52. [24] Granot E, Tamir I, Deckelbaum R. Neutral lipid transfer protein does not regulate atocopherol transfer between human plasma lipoproteins. Lipids 1988;23:17–21. [25] Carcelain G, David F, Lepage S, et al. Simple method for quantifying a-tocopherol in low-density 1 very-low-density lipoproteins and in high-density lipoproteins. Clin Chem 1992;38:1792–5. [26] Morita T, Kitagawa M, Mino M. Tocopherol distribution in serum lipoproteins with respect to red blood cell tocopherol levels in children. J Nutr Sci Vitaminol (Tokyo) 1989;35:243– 51.
Effects of postprandial hyperlipemia on the vitamin E content of lipoproteins ´ ` Cambillaud c ,f , Remy Couderc a ,f , *, Jacqueline Peynet b ,f , Michele d ,f e ,f ` a ,f , Frank Tallet , Claudine Cosson , Guillaume Lefevre ´ Veronique Atger c ,f a b
ˆ Service de Biochimie, Hopital Tenon, 4 rue de la Chine, 75020, Paris, France ˆ ` , Assistance Publique, Paris, France Lariboisiere Service de Biochimie, Hopital c ˆ Broussais, Assistance Publique, Paris, France Service de Biochimie, Hopital d ˆ Service de Biochimie, Hopital Cochin, Assistance Publique, Paris, France e ˆ ˆ , Assistance Publique, Paris, France Bicetre Service de Biochimie, Hopital f 1 ´ , Paris, France GERBAP , Section Lipoproteines
Received 23 February 1998; received in revised form 13 July 1998; accepted 15 July 1998
Abstract Delayed postprandial clearance of triglyceride-rich lipoproteins (TGRL) could induce a decrease of low-density lipoprotein (LDL) and high-density lipoprotein (HDL) vitamin E content through its transfer into TGRL. We thus studied lipoprotein vitamin E content during postprandial hypertriglyceridemia induced by a high fat meal without vitamin E supplement. Venous blood was drawn from five healthy male volunteers following a 12 h fast at (t 0 ) then 2 h (t 2 ), 4 h (t 4 ), 6 h (t 6 ) and 8 h (t 8 ) after a 80 g fat meal. In plasma, only TG significantly varied during the postprandial period with a large interindividual variability. Mean composition of lipoproteins in terms of mass was not significantly modified. The amount of vitamin E significantly increased in TGRL and decreased in LDL plus HDL at t 4 and t 6 relative to t 0 . Vitamin E content of TGRL and LDL but not of HDL decreased significantly at t 4 . The mean decrease was 20% (range 5%–54%) for the LDL. LDL– and HDL vitamin E content correlated inversely with plasma TGRL levels. Our data suggest that LDL from subjects with delayed chylomicron clearance could be less protected against oxidation. 1998 Elsevier Science B.V. All rights reserved. *Corresponding author. Tel.: 1 33-1-56017989; Fax: 1 33-1-56017840; E-mail: [email protected] 1 ˆ GERBAP (Groupe d’Evaluation et de Recherche de l’Assistance Publique des Hopitaux de Paris) Section ´ Lipoproteines; Members (other than the authors): Bailleul Sophie, Bonneau Christine, Davit-Sprault Anne, ` Marie Bernadette, Myara Isaac, Turpin Elisabeth. Erlich Daniele, 0009-8981 / 98 / $ – see front matter 1998 Elsevier Science B.V. All rights reserved. PII: S0009-8981( 98 )00121-1
142
R. Couderc et al. / Clinica Chimica Acta 277 (1998) 141 – 152
Keywords: Vitamin E (alpha-tocopherol); Lipoproteins; Postprandial
1. Introduction Reduced postprandial clearance of triglyceride (TG)-rich lipoproteins (TGRL) may be involved in the development of premature coronary atherosclerosis [1,2]. Indeed, postprandial hypertriglyceridemia is delayed in subjects with coronary atherosclerosis in comparison to control subjects [3] and postprandial plasma levels of small chylomicron remnants were found to relate to the progression of coronary atherosclerosis [4]. Reduced postprandial clearance of TGRL generates potentially atherogenic lipoproteins through mechanisms involving exchanges and transfers of lipids and apolipoproteins between TGRL and low-density lipoproteins (LDL) and high-density lipoproteins (HDL) [5]. These transfers also involve lipid-soluble molecules carried by lipoproteins and especially vitamin E: this exchange between lipoproteins is greatly facilitated by a specific phospholipid transfer protein [6]. Vitamin E is incorporated into chylomicrons and secreted by intestinal cells and its delivery to tissues is closely linked to the lipoprotein metabolism [7]. Vitamin E, mainly a-tocopherol, by its antioxidant activity, protects lipoprotein polyunsaturated fatty acids against oxidation [8] and may reduce the development of arteriosclerosis [9–11]. Moreover, in vitro studies have shown that oxidatively modified LDL are taken up, via the scavenger receptor, by monocyte / macrophage cells. Oxidized HDL have a reduced efficiency in mediating the cholesterol reverse transport and are less effective in protecting LDL against oxidation [12]. Lipoprotein oxidation depends on the balance between the free radical generation rate and protection systems which include vitamin E. Thus it is of interest to explore situations in which LDL and HDL could be depleted in vitamin E. In vitro, LDL and HDL tocopherol readily exchange with other lipoproteins and only the tocopherols in TGRL do not readily exchange [13]. In humans, the peak in tocopherol secretion in chylomicrons occurs between 6 and 12 h following the oral administration of deuterated tocopherols [14]. The rapid hydrolysis of TGRL, with the production of excess surface, allows the transfer of tocopherol from TGRL to HDL and then to other lipoproteins. There is no study, to our knowledge, on the variations of LDL and HDL vitamin E content after a high fat meal without tocopherol supplement. Our study was based on the following physiopathological hypothesis that delayed postprandial clearance of TGRL could induce a decrease of LDL and HDL vitamin E content through its transfer into TGRL. We therefore studied
R. Couderc et al. / Clinica Chimica Acta 277 (1998) 141 – 152
143
lipoprotein vitamin E content during postprandial hypertriglyceridemia induced by a high fat meal in healthy volunteers.
2. Methods Venous blood was drawn from five healthy male volunteers following a 12 h fast (t 0 ) then 2 h (t 2 ), 4 h (t 4 ), 6 h (t 6 ) and 8 h after (t 8 ) an 80 g fat meal including 20 g butter, 60 g cheese, two eggs, 40 g mayonnaise, 100 g white bread and a glass of orange juice. This meal contained about 10 mg of vitamin E; that is, the mean daily intake. A second protocol performed on four of these five men was designed to estimate the reproducibility of the postprandial variation of LDL vitamin E content. Lipoproteins were isolated from plasma EDTA by sequential ultracentrifugation. The chylomicron fraction was isolated from 3 ml plasma layered under 9 ml 0.9% NaCl for 0.5 h at 20 000 RPM in a Beckman SW41 rotor. VLDL, LDL and HDL were ultracentrifugated in the Beckman vertical NVT rotor at the densities of 1.006, 1.063 and 1.210 g / ml respectively. Lipids and proteins were assayed by using classical techniques and vitamin E by HPLC adapted from Biery [15]. For red blood cell (RBC) vitamin E assay, blood collected onto EDTA was centrifuged at 2000 3 g for 10 min at 48C to separate plasma and RBC. The RBC were washed three times with a 0.9% NaCl solution containing 0.5% of pyrogallol (Merck, Darmstadt, Germany) as antioxidant agent. The final RBC suspension solution was made up to about 50% with addition of distilled water containing 0.1% of BHT (butylated hydroxytoluene, Sigma, St Louis, MO, USA). RBC samples for vitamin E determination were stored frozen at 2 808C until time analysis. We checked that no loss of erythrocyte a-tocopherol occurred at 2 808C during at least six months. Statistical analyses were performed by using STATVIEW 4 software (Abacus Concept, Berkeley, CA). Unless otherwise stated, data are presented as mean6standard deviation. Statistical significance of the variation between t 0 and the other times after the meal was assessed by using the Student’s t-test for paired values.
3. Results Only plasma TG significantly varied during the postprandial period (Table 1). There was a large interindividual variability, with particularly wide differences in TG levels which occurred at the late postprandial hours, i.e., between 6 and 8 h after ingestion of the test meal (Fig. 1). Among the five subjects, only three had TG , 1.5 mmol / l after t 8 . This high interindividual variability was
144
R. Couderc et al. / Clinica Chimica Acta 277 (1998) 141 – 152
Table 1 Lipid and vitamin E levels in plasma from five healthy men following a 12 h fast (t 0 ) then after an 80 g fat meal Time after meal (h)
TC mmol / l
TG mmol / l
PL mmol / l
Vit E mg / l
t0 t2 t4 t6 t8
5.8260.66 5.9060.54 5.6460.56 5.6260.63 5.8960.73
1.0560.18 1.646O.24** 1.8860.65* 1.6960.46* 1.6760.86
3.0260.38 3.1660.34 3.0860.37 3.1260.27 3.2760.29
14.362.1 13.761.9* 13.762.1 14.362.1 15.461.8*
Significantly different from t 0 : *P # 0.05; **P # 0.01. Mean61 standard deviation (n 5 5). TC: total cholesterol; TG: triglycerides; PL: phospholipids; Vit E: vitamin E.
confirmed by the second protocol. The mean composition of lipoproteins in terms of mass was not significantly modified. However, the cholesterol to TG molar ratio in HDL was significantly decreased at t 4 relative to t 0 (9.8962.95 vs
Fig. 1. Plasma triglyceride levels in five healthy men following a 12 h fast then after a high fat meal. Subject No. 1: s; No. 2: h; No. 3: n; No. 4: 앳; No. 5: 1 .
R. Couderc et al. / Clinica Chimica Acta 277 (1998) 141 – 152
145
7.7462.05; P # 0.05). The value of this ratio correlated negatively with the TG concentration in the plasma (r 5 2 0.616; P # 0.001). This relationship was particularly strong in the patient No. 5 (r 5 2 0.935). After the high-fat meal intake, the distribution of vitamin E between the lipoprotein classes was modified (Table 2). The amount of vitamin E significantly increased in triglyceride rich lipoproteins (chylomicrons plus very-low density lipoproteins) and decreased in LDL plus HDL at t 4 and t 6 relative to t 0 . The vitamin E content of all lipoprotein classes decreased 4 h after the meal (Fig. 2). However, significance was not reached for HDL. The mean decrease was 20% (range: 5–54%) for the LDL. The most significant decrease in LDL vitamin E content was found in the subject whose TG were still rising at the end of t 8 . The second protocol was designed to estimate the reproducibility of the variation of LDL vitamin E content. Indeed, in experimental conditions identical to those of the first protocol, we again found a significant (P # 0.05) decrease of vitamin E in LDL at t 2 (1.2260.09 mg / g) and t 4 (1.2060.07 mg / g) relative to t 0 (1.3960.09 mg / g). Vitamin E content of red blood cells (RBC), which provides a good model for the study of vitamin E exchanges between lipoproteins and cells, slightly increased at t 2 and t 8 relative to t 0 (Fig. 3). LDL vitamin E content correlated highly with the HDL vitamin E content (Fig. 4), and the latter (x) correlated highly with the esterified cholesterol HDL content ( y 5 10.2x 1 5.5, r 5 0.842, P # 0.0002), and with esterified cholesterol-to-TG molar ratio ( y 5 5.35x 1 1.33, r 5 0.592, P # 0.002) in these lipoproteins. LDL-vitamin E content correlated significantly with plasma TGRL levels (Fig. 5). There was also a weak correlation between LDL–vitamin E content and TGRL–vitamin E content (data not shown). Table 2 Distribution of vitamin E (percent) between the lipoprotein classes from five healthy men following a 12 h fast (TO) then after an 80 g fat meal Time after the meal (h)
TGRL (%)
LDL (%)
HDL (%)
t0 t2 t4 t6 t8
1967 2565 2669** 25611* 20612
4667 4366 4264** a 4267* a 4665
3564 3263 3265** a 3366* a 3468
Significantly different from t 0 : *P # 0.05; **P # 0.01; a Significantly different from t 0 when considering the sum of LDL- and HDL vitamin E rates. Mean61 standard deviation (n 5 5). TGRL: triglyceride rich lipoproteins; the amount of vitamin E in TGRL is the sum of the amount of vitamin E in very low-density lipoproteins and in chylomicrons.
146
R. Couderc et al. / Clinica Chimica Acta 277 (1998) 141 – 152
Fig. 2. Vitamin E content of plasma lipoproteins in five healthy men following a 12 h fast then after a high fat meal. Mean6standard deviation; *significantly different from t 0 (P # 0.05; Student’s t-test for paired values). TGRL (triglyceride-rich lipoprotein) vitamin E content is the sum of chylomicron- and very low-density lipoprotein vitamin E contents.
R. Couderc et al. / Clinica Chimica Acta 277 (1998) 141 – 152
147
Fig. 3. Vitamin E content of red blood cells in five healthy men following a 12 h fast then after a high fat meal. Mean6standard deviation; *significantly different from t 0 (P # 0.05; Student’s t-test for paired values). Hb: haemoglobin.
4. Discussion This study shows that LDL– and HDL–vitamin E contents decrease after ingestion of a high fat test meal and reach a minimum at 4 h after the meal; especially in the alteration in LDL vitamin E correlated to the plasma triglyceride-rich lipoprotein level. TGRL vitamin E content was also decreased 4 h after the meal but these lipoproteins carried a larger fraction of total plasma vitamin E than in the fasting state. Composition and metabolism of LDL and HDL depend on chylomicron clearance by lipases [16]. Moreover, many epidemiologic studies have highlighted the significance of triglycerides as a coronary atherosclerosis risk factor, and several of them have been realized in non-fasting subjects [17,18]. Nevertheless, protocol for testing fat tolerance on a small number of subjects must be standardized in terms of quality and quantity of the meal ingested. We chose to give a test meal consisting of commercially available food and providing 1 g fat per kg body weight and almost 3 g of fiber because preparations of liquid fat emulsions do not provide fiber which can attenuate the
148
R. Couderc et al. / Clinica Chimica Acta 277 (1998) 141 – 152
Fig. 4. Scattergram illustrating the relationship between the HDL-( y) and LDL (x) vitamin E content in five healthy men following a 12 h fast then after a high fat meal. Subject No. 1: s; No. 2: h; No. 3: n; No. 4: 앳; No. 5: 1 . The r-value for all subjects and all times (n 5 25) obtained by linear regression was 0.79 (P # 0.001); the equation of the linear regression was: y 5 0.768x 2 0.019.
postprandial triglyceride response [19] and may not induce the same oronasal stimulation as a solid meal [20]. The meals were ingested over 20–30 min and no subjects suffered from gastrointestinal pain. The alterations of lipoprotein composition in terms of cholesterol, TG, and phospholipids were in accord with previous studies, particularly given the presence of two peaks of TG in the same subjects [21]. However, in our study there was no significant alteration in postprandial plasma LDL-cholesterol concentration (data not shown). These results conflict with those from previous studies [21–23], in which LDL-cholesterol concentration, as estimated by Friedewald formula, decreased after the fat-rich meal. That could be due to the difference in meal composition, and to the fact that we have measured LDLcholesterol in LDL isolated by ultracentrifugation. The large inter-individual variations explain the fact that no significant alteration of the mass / mass lipoprotein composition was found.
R. Couderc et al. / Clinica Chimica Acta 277 (1998) 141 – 152
149
Fig. 5. Scattergram illustrating the relationship between the LDL-vitamin E contents and TGRL (triglyceride-rich lipoprotein) plasma levels in five healthy men following a 12 h fast then after a high fat meal. The r-value for all subjects and all times (n 5 25) obtained by linear regression was 0.53 (P # 0.01); the equation of the linear regression was: y 5 2 0.757x 1 1.935. TGRL levels were calculated by adding chylomicron and very low-density lipoprotein plasma levels expressed in g / l.
Plasma vitamin E levels in the subjects we studied were in the normal range before the meal (Table 1). In fasting sera, vitamin E distributed between VLDL, LDL and HDL with ratios of 1.0:2.4:1.8 in our five volunteers. These ratios are nearly the same as those published by Kostner et al who found ratios of 1.0:1.9:1.4 [6]. They slightly decreased at t 2 and slightly increased at t 8 . The fact that plasma vitamin E levels were not altered to a large extent may be the consequence of the high clearance of chylomicron vitamin E and not excluding transient alterations for vitamin E distribution among lipoproteins and of lipoprotein vitamin E content. In vitro studies have shown that vitamin E distribution among lipoproteins depends on the lipoprotein mass ratio, lipoprotein half lives and PLTP activity [6,24]. In our study, HDL vitamin E content correlated positively with HDL cholesterol and no such relation was found with LDL-cholesterol. On the other hand, LDL and HDL vitamin E contents correlated negatively with TGRL levels, which is in accordance with the previously described role of these lipoproteins as a vitamin E acceptor. Therefore, the alteration in vitamin E distribution among lipoproteins observed during the postprandial period may be due to a net transfer of vitamin E
150
R. Couderc et al. / Clinica Chimica Acta 277 (1998) 141 – 152
into chylomicrons. It is noteworthy that the plasma vitamin E content in HDL was 10% lower in our study than in others [25,26]. Other than the opposite alterations of vitamin E distribution in TGRL and in cholesterol rich lipoproteins (LDL and HDL), vitamin E contents of all lipoprotein classes decreased to reach a lower level at t 4 on the average. These data suggest that chylomicrons originating from the intestine were rather vitamin E poor or that vitamin E left these lipoproteins at the very beginning of their catabolism leading to vitamin E poor chylomicron remnants which were still isolated during the first step of ultracentrifugation at d , 1.000. On the other hand, the decrease in LDL and HDL vitamin E contents may be due to a net transfer of vitamin E into chylomicrons, the mass of which rapidly increased during the post prandial period. Red blood cells (RBC) may also act as a vitamin E acceptor because in our study, their vitamin E content was found to be correlated to HDL and LDL vitamin E content, and in a previous study in children, to that of HDL [26]. Despite the fact that the total RBC vitamin E mass increase is not sufficient to explain the alterations observed in lipoproteins, this phenomenon may be enhanced by the presence of high levels of chylomicrons. In subjects on a diet without excess of lipids and rich enough in lipid-soluble antioxidants and exhibiting a fast postprandial clearance of chylomicrons, the postprandial depletion of LDL and HDL in vitamin E may be slight and brief. This is because the vitamin E transfer, controlled by the chylomicrons / HDL plus LDL mass ratio, may well be balanced by a vitamin E transfer into HDL during TG lipolysis. On the other hand, and in subjects on a high fat and low lipid-soluble antioxidant diet and slowly clearing chylomicrons, LDL and HDL vitamin E depletion may be high and prolonged. In our study, in the subject who presented the most prolonged chylomicron clearance, the LDL vitamin E content was decreased by 54%. Consequently, lipoproteins from this subject could be more susceptible to oxidation. This point needs further investigation better to understand the LDL susceptibility to oxidation in subjects selected on the basis of TG concentration which is still high 6 h after a meal. In conclusion our study links two pathophysiological hypotheses of atherogenesis: the Zilversmit’s hypothesis involving chylomicron hydrolysis alterations [1] and the modified lipoprotein hypothesis involving stress factors such as free radicals.
References [1] Zilversmit DB. Atherogenesis: a postprandial phenomenon. Circulation 1979;60:473–85. [2] Zilversmit DB. Atherogenic nature of triglycerides, postprandial lipemia, and triglyceriderich remnant lipoproteins. Clin Chem 1995;41:153–8.
R. Couderc et al. / Clinica Chimica Acta 277 (1998) 141 – 152
151
[3] Groot PH, Van Stiphout WH, Krauss XH, et al. Postprandial lipoprotein metabolism in normolipidemic men with and without coronary artery disease. Arteriosclerosis Thromb 1991;11:653–62. [4] Karpe F, Steiner G, Uffelman K, Olivecrona T, Hamsten A. Postprandial lipoproteins and progression of coronary atherosclerosis. Atherosclerosis 1994;106:83–97. ¨ [5] Miesenbock G, Patsch JR. Postprandial hyperlipidemia: the search for the atherogenic lipoprotein. Cur Opin Lipidol 1992;3:196–201. [6] Kostner GM, Oettl K, Jauhiainen M, Ehnholm C, Esterbauer H, Dieplinger H. Human plasma phospholipid transfer protein accelerates exchange / transfer of a-tocopherol between lipoproteins and cells. Biochem J 1995;305:659–67. [7] Kayden HJ, Traber MG. Absorption, lipoprotein transport, and regulation of plasma concentrations of vitamin E in humans. J Lipid Res 1993;34:343–58. [8] Esterbauer H, Puhl H, Waeg G, Krebs A, Tatzber F, Rabol H. Vitamin E and atherosclerosisAn overview. In: Mino M, et al, editors. Vitamin E - Its usefulness in Health and in Curing diseases. Basel: Karger, 1993:233–41. [9] Riemersma RA, Wood DA, MacIntyre CCA, Elton RA, Gey KF, Oliver MF. Risk of angina pectoris and plasma concentrations of vitamin A, C, and E and carotene. Lancet 1991;337:1– 5. [10] Rimm EB, Stampfer MJ, Ascherio A, Giovannucci E, Colditz GA, Willett WC. Vitamin E consumption and the risk of coronary disease in men. New Engl J Med 1993;328:1450–6. [11] Stampfer MJ, Hennekens CH, Manson JE, Colditz GA, Rosner B, Willett WC. Vitamin E consumption and the risk of coronary disease in women. New Engl J Med 1993;328:1444–9. [12] Rifici V, Khachadurian A. Oxidation of high density lipoproteins: characterization and effects on cholesterol efflux from J774 macrophages. Biochem Biophys Acta 1996;99:87–94. [13] Traber MG, Lane JC, Lagmay NR, Kayden HJ. Studies on the transfer of tocopherol between lipoproteins. Lipids 1992;27:657–63. [14] Kayden HJ, Traber MG. Transport of vitamin E in human lipoproteins. In: Mino M, et al, editors. Vitamin E-Its usefulness in Health and in Curing diseases. Basel: Karger, 1993:85– 93. [15] Biery JG, Tolliver TJ, Gatignani GL. Simultaneous determination of alpha-tocopherol and retinol in plasma or red blood cells by high pressure-chromatography. Am J Clin Nutr 1979;32:2143–9. [16] Karpe F, Tornvall P, Olivecrona T, Steiner G, Carlson LA, Hamsten A. Composition of human low density lipoprotein: effects of postprandial triglyceride-rich lipoproteins, lipoprotein lipase, hepatic lipase and cholesteryl ester tranfer protein. Atherosclerosis 1993;98:33– 49. [17] Stensvold I, Tverdal A, Urdal P, Graff-Iversen S. Non-fasting serum triglyceride concentration and mortality from coronary heart disease and any cause in middle aged Norwegian women. Br Med J 1993;307:1318–22. ¨ G, Hopferwieser T, et al. Relation of triglyceride metabolism and [18] Patsch JR, Miesenbock coronary artery disease. Arteriosclerosis Thromb 1992;12:1336–45. [19] Cara L, Dubois C, Borel P, et al. Effects of cat bran, rice bran, wheat fiber, and wheat germ on postprandial lipemia in adults. Am J Clin Nutr 1992;55:81–8. [20] Mattes RD. Oral fat exposure alters postprandial lipid metabolism in humans. Am J Nutr 1996;63:911–7. [21] Cohn JS, McNamara JR, Schaefer EJ. Lipoprotein cholesterol concentrations in the plasma of human subjects as measured in the fed and fasted states. Clin Chem 1988;34:2456–9. [22] Rifai N, Merrill JR, Holly RG. Postprandial effect of a high fat meal on plasma lipid, lipoprotein cholesterol and apolipoprotein measurements. Ann Clin Biochem 1990;27:489– 93.
152
R. Couderc et al. / Clinica Chimica Acta 277 (1998) 141 – 152
[23] Dubois C, Armand M, Ferezou J, et al. Postprandial appearance of dietary deuterated cholesterol in the chylomicron fraction and whole plasma in healthy subjects. Am J Nutr 1996;64:47–52. [24] Granot E, Tamir I, Deckelbaum R. Neutral lipid transfer protein does not regulate atocopherol transfer between human plasma lipoproteins. Lipids 1988;23:17–21. [25] Carcelain G, David F, Lepage S, et al. Simple method for quantifying a-tocopherol in low-density 1 very-low-density lipoproteins and in high-density lipoproteins. Clin Chem 1992;38:1792–5. [26] Morita T, Kitagawa M, Mino M. Tocopherol distribution in serum lipoproteins with respect to red blood cell tocopherol levels in children. J Nutr Sci Vitaminol (Tokyo) 1989;35:243– 51.