Atherosclerosis, 82 (1990) 69-74 Elsevier Scientific Publishers Ireland,
ATHERO
69 Ltd.
04460
Influence of dietary minerals on apolipoprotein B metabolism in rabbits fed semipurified diets containing casein Samir Samman, Pramod Khosla * and Kenneth K. Carroll Department
of Biochemistry,
University of Western Ontario, London, Ontario, N6A 5CI (Canada) (Received 18 April, 1989) (Revised, received 7 November, 1989) (Accepted 20 January, 1990)
Summary
Rabbits were fed semipurified casein diets containing either 4% or 2.5% mineral mix for 8 weeks. Both groups maintained weight throughout the experimental period. The plasma total cholesterol concentration was significantly higher after 4 weeks on diet and slightly higher after 8 weeks in animals fed the lower level of minerals. Plasma IDL- and LDL-cholesterol concentrations after 4 weeks and HDL-cholesterol concentrations after 8 weeks were significantly higher in animals fed the 2.5% compared to those fed the 4% mineral mix. Kinetic experiments showed that in rabbits fed the lower level, the fractional catabolic and production rates of VLDL-apo B were lower and a greater proportion of IDL-apo B was derived from sources other than VLDL compared to the animals fed the higher level. LDL-apo B kinetics were not significantly different between the 2 groups. These data suggest that a reduction in dietary minerals enhances casein-induced hypercholesterolemia.
Key words: Minerals; Casein; Rabbits; Cholesterol;
Introduction
Micronutrients have been implicated in cholesterol metabolism in experimental animals and humans [l-6]. Supplementing humans with zinc [4] or calcium [5] results in a decrease in LDL- and total cholesterol, respectively. In contrast, copper
Correspondence to: S. Samman, Ph.D., Dept. of Clinical Biochemistry, Royal Prince Alfred Hospital, Missenden Rd. Camperdown, Sydney, N.S.W., 2050, Australia. * Current address: Foster Biomedical Research Laboratory, Brandeis University, Waltham, MA 02254-9110, U.S.A.
0021-9150/90/$03.50
0 1990 Elsevier Scientific
Publishers
Ireland,
Plasma lipoproteins;
Apolipoprotein
B metabolism
deficiency is associated with elevated levels of plasma cholesterol [6]. In rats, increasing the mineral content of the diet has been shown to decrease hepatic cholesterol and raise plasma HDL-cholesterol concentrations [7]. In rabbits fed casein, increases in dietary calcium [8] and zinc and copper [9] are associated with a lower concentration of plasma total cholesterol, while decreasing the mineral content of the diet resulted in a marked hypercholesterolemia [ 10,111. To investigate this effect further, rabbits were fed semipurified casein diets containing either 4% or 2.5% mineral mix and lipoprotein turnover studies were carried out. The results showed that a Ltd.
70
reduction in dietary minerals has significant effects on lipoprotein metabolism indicating that minerals influence the hypercholesterolemic response to dietary casein. Materials and methods
1.019) LDL (1.019 < d < 1.063) and HDL (1.063 < d < 1.21) were isolated by sequential ultracentrifugation in a Ti50 rotor at 40000 rpm at 15°C [14]. Total and lipoprotein-cholesterol was determined using enzymatic kits (CHOD-PAP, Boehringer Mannheim, Canada).
Animals and diets
Preparation of labelled lipoproteins
Young, male, New Zealand white rabbits (starting weight 1.8-2.0 kg) were obtained from Reimen’s Fur Ranches (Guelph, Ontario) and housed under conditions described previously [12]. Rabbits were fed low-fat, cholesterol-free, semipurified diets containing casein with either 4% mineral mix (standard minerals) or 2.5% mineral mix (reduced minerals). Both groups were fed the semipurified diets for 8 weeks following a l-week adaptation period. The composition of the diets is shown in Table 1.
After 6 weeks on diet, VLDL and LDL were isolated [14] and each lipoprotein was recentrifuged once at its optimal density. VLDL and LDL were radiolabelled with Na12’I and Na1311 (Amersham, Oakville, Ontario) respectively by a modification [15] of the McFarlane method [16]. The labelled lipoproteins were sterilized by passage through a 0.45 pm Millex-HA filter and gentamycin sulphate (1 pg/ml) was added. Within 36 h of iodination, labelled lipoproteins were reinjetted into recipient rabbits. The intramolecular distribution (IMD) of radioactivity in the tracer was determined as described previously [15,17]. The IMD for 12’I-VLDL and l3 I-LDL were similar in the 2 groups. For VLDL, the percent distribution in protein, apo B, lipid and as free iodine was 91, 73, 6 and 5, respectively. For LDL, the distribution was 97, 92, 3 and 2, respectively.
Plasma cholesterol analyses
Blood was collected from animals in the fasted state at the start of the experimental period for the determination of plasma total cholesterol. Also, after 4 and 8 weeks on diet, blood was collected and VLDL (d < 1.006 g/ml), IDL (1.006 < d < TABLE
Lipoprotein kinetic studies
1
COMPOSITION RABBITS
Casein ’ Dextrose b Celluflour ’ Mineralmix ac Molasses b (50% v/v) Corn oil b Vitamins d
OF THE SEMIPURIFIED
DIETS FED TO
Standard minerals
Reduced minerals
(g/kg)
(g/kg)
270 598 50 40
270 613 50 25
30 10 2
30 10 2
a Obtained from ICN Life Science Group, Nutritional Biochemicals Division, Cleveland, OH. b Obtained from a local feed mill. ’ Mineral mix (Phillips and Hart). Composition (mg/kg diet when used at the 4% level): calcium, 5.097; chloride, 3.646; copper, 0.0027; iodine, 0.0219; iron, 0.164; magnesium, 0.361; manganese, 0.0031; phosphorous, 2.668; potassium, 5.195; sodium, 2.361; sulphur, 0.479; zinc, 0.0043. d The composition of the vitamin mix has been reported elsewhere [13].
Following an overnight fast, unanesthetised rabbits were injected simultaneously with 2-8 $i ‘251-VLDL-apo B and l-3 PCi 1311-LDL-apo B in the marginal ear vein. Twelve blood samples (4-5 ml each) were obtained from each animal at timed intervals up to 48 h after injection. The protocol used has been described in more detail elsewhere [18]. There were 6 turnover studies, 3 per dietary group. In each of the studies, equal volumes of plasma from 3 rabbits were pooled from each of the 12 time points. VLDL, IDL and LDL were isolated from 4-6 ml plasma by ultracentrifugation [14] and the lipoproteins were recentrifuged once at their optimal densities. The protein content of all the lipoproteins was determined by the Markwell modification [19] of the Lowry method
WI. To determine the dose injected, aliquots of the first blood sample, at 5 min post-injection, were taken and the radioactivity in apo B was determined [21]. The dose injected was then calcu-
71
lated by assuming that the plasma volume is 32.8 ml/kg body weight [22]. The assumption was made that there was similar metabolism of apo B within the first 5 min. The lipoprotein apo B concentrations were determined [23] and the apo B pellets were processed for the determination of specific activity (SA) as described previously [24]. The SA time curves of VLDL and LDL plotted semi-logarithmically were best described by a double-exponential curve and were resolved using a least squares method. Curve parameters were calculated according to the twopool model [25].
A
A-A VLDL .---. IDL .- - ??LDL
1000~
I
I
6
12
18
Results
30
3t
42
48
TIME (hr)
Feeding diets with a reduced content of minerals had no significant effect on the weight of A
24
B 1000r
A---A .---. .-
VLOL IOL
- 0 LDL
/
35aC
*
??
m
Standard
I
Reduced
Minerals Minerals
*t
0 plasma
VLDL
IDL
LDL
HDL
0
t 6
12
I8
24
310
3c
42
48
TIME (hr)
m Standard IReduced
plCWTM
VLDL
IDL
LDL
Minerals Minerals
HDL
Fig. 1. Plasma and lipoprotein cholesterol concentrations in rabbits fed diets containing standard or reduced minerals for (A) 4 weeks or (B) 8 weeks. Each value is the mean (*SE) of 6-8 observations. Statistical significance was determined by Student’s t test, ** P < 0.01, * P-e 0.05.
Fig. 2. Precursor-product relationships between VLDL-, IDLand LDL-apo B in the plasma of animals fed diets containing either (A) standard or (B) reduced minerals. Each graph represents data obtained from the pooled plasma of 3 animals.
the animals during the 8 week experimental period compared to those fed standard minerals (2.3 f 0.1 vs. 2.6 f 0.1 kg, mean f SE). However, the plasma cholesterol concentrations of the animals fed diets with reduced minerals was increased significantly (P < 0.01) after 4 weeks and slightly higher after 8 weeks on diet (Fig. 1). The rise in plasma total cholesterol after 4 weeks was primarily due to significant increases in the IDL- (P < 0.05) and LDL- (P < 0.01) cholesterol concentrations (Fig. 1A). After 8 weeks on diet, HDL-cholesterol concentrations were significantly higher (P -z 0.05)
72 TABLE 2 KINETIC PARAMETERS FOR VLDL- AND LDL-APO B IN RABBITS FED DIETS CONTAINING STANDARD OR REDUCED MINERALS Values are expressed as the mean of 3 experiments f SE. Values for each experiment were obtained from the pooled plasma of 3 rabbits. Standard minerals VLDL-apo B Pool (mg/kg) 8.49 zb1.99 FCR (per h) 0.172 kO.014 Production rate (mg/kg/h) 1.43 kO.27 IDL SA : VLDL SA a 0.95 f 0.10 LDL-apo B Pool (mg/kg) 29.90 k6.99 FCR (per hr) 0.035 f 0.003 Production rate (mg/kg/h) 1.04 +0.28
Reduced minerals 10.46 k1.29 0.137 f 0.020 0.95 *IO.10 0.55 +0.05 * 29.96 +7.00 0.027 f 0.001 0.81 i-O.19
’ Ratios of r2%apo B specific activity when IDL specific activity reaches its maximum. * Significantly lower than standard minerals counterpart using Student’s t test, P c: 0.01.
and VLDL-, IDL- and LDL-cholesterol still tended to be higher (Fig. 1B). The kinetically defined VLDL-apo B pool was slightly higher in animals fed the reduced minerals (Table 2). In addition, the fractional catabolic rate (FCR) and the production rate of VLDL-apo B were lower in the animals fed the lower level TABLE 3 PLASMA LIPOPROTEIN PROTEIN CONCENTRATIONS AND PERCENT APO B IN RABBITS FED DIETS CONTAINING STANDARD OR REDUCED MINERALS Values are expressed as means* SE. Values were obtained from the pooled plasma of 3 rabbits taken at 12 time points during the kinetic study.
VLDL Protein (mg/lOO ml) % apo B IDL Protein (mg/lOO ml) ‘K,apo B LDL Protein (mg/lOO ml) % apo B
Standard minerals
Reduced minerals
17.7* 39.7*
2.6 1.4
30.4* 32.6*
4.5 3.0
11.7rfr 2.3 67.0* 5.6
19.8* 72.1*
5.7 3.5
81.7 f 12.8 88.7f 1.4
72.5 f 13.3 83.7* 0.6
(Table 2). LDL-apo B kinetics were similar between the two groups (Table 2). The SA curves (Fig. 2) were examined for precursor-product relationships. As defined by Zilversmit [26], the fraction of a product derived from its precursor is calculated by examining the ratio of SA of the product to that of the precursor when the SA of the product is at its maximum. Estimates of the ratios of the maximum apo B SA in IDL to VLDL are shown in Table 2. These show that only 55% of IDL-apo B is derived from VLDL in animals fed reduced minerals compared to 95% in animals fed standard minerals. Thus, in animals fed reduced minerals there is greater input of IDL-apo B independently of the VLDL-apo B pathway compared to animals fed the higher level of minerals. The plasma lipoprotein protein concentrations and the percent of protein which was apo B showed little change over the 48 h of the kinetic study (Table 3). The coefficient of variation of the percent apo B was 8.3% which suggests the animals were in a steady state. Discussion Our results show that dietary minerals have an effect on plasma apo B metabolism and HDL cholesterol levels in rabbits fed semipurified diets containing casein. After 4 weeks on diet, the lower level of dietary minerals was associated with increased plasma total IDL- and LDL-cholesterol concentrations. These differences were no longer evident after 8 weeks on diet. Thus, the reduction in dietary minerals accelerates the rate with which the hypercholesterolemia is produced in rabbits fed casein. The VLDL kinetic studies showed that the efficiency of removal of VLDL-apo B was reduced in animals fed the lower level of minerals. In addition, the SA values obtained were consistent with the interpretation that more IDL-apo B was derived from sources other than VLDL-apo B in animals fed the lower level of minerals. LDL-apo B metabolism was not different between the 2 groups. However, in both cases, as in other lipoprotein studies in rabbits [18,27,28] and other species [24,29,30], production of LDL from sources other than VLDL was observed. Independent pro-
73
duction of LDL, or IDL, are subject to much debate. It is thought that these may be particles synthesized and secreted directly from the liver, or they may originate from VLDL that is converted rapidly to IDL and LDL [31]. The enhancement of the hypercholesterolemia in animals fed a casein/low mineral diet may be mediated through 2 processes. Firstly, a more rapid down-regulation of LDL receptors may be taking place. It has been shown that LDL receptors are down-regulated in casein-fed rabbits compared to those fed soy protein [32] and the reduction in mineral mix may accelerate the process. This would result in a more rapid increase in plasma LDLcholesterol, as seen after 4 weeks (Fig. 1A). A second possibility is the reduction in fecal sterol excretion. Casein-fed animals excrete less fecal sterols than their soy protein counterparts [33] possibly due to the lower calcium content of the diet [8]. A reduction in the mineral mix, of which calcium is a major component (Table l), may decrease further the excretion of sterols [34]. It has been proposed [35] that the enhanced reabsorption of sterols (or the reduced excretion) results in a larger hepatic pool of cholesterol [33] and in turn stimulates the secretion of cholesterol-rich lipoproteins such as LDL [18] and to a lesser extent, IDL. A reduction in dietary minerals may be interacting at the receptor level and/or by interfering with the enterohepatic circulation to elicit these effects. However, from these experiments, it is unclear which mineral is of importance in the observed changes in metabolism. Given the paucity of information available on the effect of micronutrients on cholesterol metabolism, further research is warranted to elucidate the role of individual elements in the cholesterolemic effect of dietary minerals.
References
9
10
11
12
13
14
Acknowledgements
Supported by the Heart and Stroke Foundation of Ontario. Samir Samman is a Postdoctoral Fellow of the Canadian Heart Foundation and a recipient of a travel grant from the National Heart Foundation of Australia. Kenneth K. Carroll is a Career Investigator of the Medical Research Council of Canada.
15
16 17
Mertz, W., Trace elementsand atherosclerosis,Fed. Proc., 41 (1982) 2807. Medeiros, D.M., The copper: zinc hypothesis and cardiovascular disease, B&hem. Arch., 1 (1985) 67. Samman, S. and Roberts, D.C.K., Zinc and cholesterol metabolism, Nutr. Res., 8 (1988) 559. Samman, S. and Roberts, D.C.K., The effect of zinc supplements on lipoproteins and copper status, Atherosclerosis, 70 (1988) 247. Bierenbaum, M.L., Fleischman, A.1. and Raichelson, R.I., Long-term human studies on lipid effects of oral calcium, Lipids, 7 (1972) 202. Klevay, L.M., Inman, L., Johnson, L.K., Lawler, M.R., Mahalko, J.R., Milne, D.B., Lukaskt, “iii?., Bolonchuk, W. and Sandstead, H.H., Increased cholesterol in plasma in a young man during experimental copper depletion, Metabolism, 33 (1984) 1112. Murthy, L., Finelli, V.N., Garside, P.S. and Petering, H.G., Relationship of sufficient and abundant level of minerals to lipid and lipoprotein metabolism in male rats, Nutr. Res.. 2 (1982) 699. Van der Meer, R., De Vries, H., West, C.E. and De Waard, H., Casein-induced hypercholesterolaemia in rabbits is calcium-dependent, Atherosclerosis, 56 (1985) 139. Samman, S. and Roberts, D.C.K., The importance of the non-protein components of the diet in the plasma cholesterol response of rabbits to casein. Zinc and copper, Br. J. Nutr., 57 (1987) 27. Samman, S. and Roberts, D.C.K.. The phosphorylation state of casein and its hypercholesterolaemic effect - the role of divalent cations, Atherosclerosis, 52 (1984) 347. Allotta, E.C., Samman, S. and Roberts, D.C.K., The importance of the non-protein components of the diet in the plasma cholesterol ,response of rabbits to casein, Br. J. Nutr., 54 (1985) 87. Hrabek-Smith, J.M. and Carroll, K.K.. A comparative study of serum lipoproteins in rabbits fed a natural ingredient diet or low-fat, cholesterol-free, semipurified diets containing casein or isolated soy protein, Biochem. Cell Biol., 65 (1987) 610. Roberts, D.C.K., Stalmach, M.E., Khalil, M.W., Hutchinson, J.C. and Carroll, K.K., Effects of dietary protein on composition and turnover of apoproteins in plasma lipoproteins of rabbits, Can. J. B&hem., 59 (1981) 642. Havel, R.J., Eder, H.A. and Bragdon, J.H., The distribution and chemical composition of ultracentrifugally separated lipoproteins in human serum, J. Clin. Invest., 34 (1955) 1345. Fidge, N.H. and Poulis, P., Studies on the radioiodination of very low density lipoprotein obtained from different mammalian species, Clin. Chim. Acta, 52 (1974) 15. McFarlane, AS., Efficient trace-labelling of proteins with iodine, Nature, 182 (1958) 53. Samman, S., Khosla, P. and Carroll, K.K., Effects of dietary casein and soy protein on metabolism of radiolabelled
74 low density apolipoprotein B in rabbits, Lipids, 24 (1989) 169. 18 Khosla, P., Samman, S., Carroll, K.K. and Huff, M.W., Turnover of i2’I-VLDL and I31I-LDL apohpoprotein B in rabbits fed semipurified diets containing casein or soy protein, Biochirn. Biophys. Acta, 1002 (1989) 157. 19 Markwell, M.A.K., Haas, S.M., Bieber, L.L. and Tolbert, N.E., A modification of the Lowry procedure to simplify protein determination in membrane and lipoprotein samplea, Anal B&hem., 87 (1978) 206. 20 Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J., Protein measurement with the Folin phenol reagent, J. Biol. Chem., 193 (1951) 267. 21 Yamada, Y. and Havel, R.J., Measurement of apolipoprotein B radioactivity in whole blood plasma by precipitation with isopropanol, J. Lipid Res., 27 (1986) 910. 22 Roth, RI., Gaubatz, J.W., Gotto, A.M., Jr. and Patsch, J.R., Effect of cholesterol feeding on the distribution of plasma lipoproteins and on the metabolism of apolipoprotein E in the rabbit, J. Lipid Res., 24 (1983) 1. 23 Egusa, G., Brady, D.W., Grundy, SM. and Howard, B.V., Isopropanol precipitation method for the determination of apolipoprotein B specific activity and plasma concentration during metabolic studies of very low density lipoprotein and low density lipoprotein apohpoprotein B, J. Lipid Res., 24 (1983) 1261. 24 Huff, M.W., Telford, D.E., Woodcroft, K. and Strong, W.L.P., Mevinolin and cholestyramine inhibit the direct synthesis of low density lipoprotein apolipoprotein B in miniature pigs, J. Lipid Res., 26 (1985) 1175. 25 Gurpide, E., Mann, J. and Sandberg, E., Determination of kinetic parameters in a two-pool system by administration of one or more tracers, Biochemistry, 3 (1964) 1250. 26 Zilversmit, D.B., The design and analysis of isotope experiments, Am. J. Med., 29 (1960) 832. 27 Ghiselli, G., Evidence that two synthetic pathways contribute to the apolipoprotein B pool of the low density lipopro-
28
29
30
31
32
33
34
35
tein fraction of rabbit plasma, Biochirn. Biophys. Acta, 711 (1982) 311. La Ville, A., Turner, P.R., Pittilo, M., Martini, S., Marenah, C.B., Rowles, P.M., Morris, G., Thomson, G.A., Woolf, N. and Lewis, B., Hereditary hyperlipidemia in the rabbit due to overproduction of lipoproteins l.Biochemical studies, Arteriosclerosis, .7 (1987) 105. Goldberg, I.J., Le., N.A., Ginsberg, H.N., Patemiti, J.R. and Brown, W.V., Metabolism of apohpoprotein B in cynomologous monkey: evidence for independent production of low-density apoprotein B, Am. J. Physiol., 244 (1983) E196. Janus, E.D., Nicoll, A., Wootton, R., Turner, P.R., Magill, P.J. and Lewis, B., Quantitative studies of very low density lipoprotein: conversion to low density lipoprotein in normal controls and primary hyperlipidaernic states and the role of direct secretion of low density lipoprotein in heterozygous familial hypercholesterolaemia, Eur. J. Clin. Invest., 10 (1980) 149. Steiner, G., Poapst, M.E., Shumak, S.L. and Foster, D.M., Metabolism of the apolipoprotein B-containing lipoproteins. In: AIbers, J.J. and Segrest, J.P. (Eds.), Methods In Enzymology, vol. 129, Orlando, 1986, 395. Khosla, P., Samman, S. and Carroll, K.K., Receptor-mediated catabolism of LDL in rabbits fed casein or soy protein, FASEB J., 3 (1989) A4245. Huff, M.W. and Carroll, K.K., Effects of dietary protein on turnover, oxidation, and absorption of cholesterol, and on steroid excretion in rabbits, J. Lipid Res., 21 (1980) 546. Van der Meer, R., Is the hypercholesterolemic effect of dietary casein related to its phosphorylation state?, Atherosclerosis, 49 (1983) 339. Beynen, AC., Van der Meer, R. and West C.E., Mechanism of casein-induced hypercholesterolemia: primary and se0 ondary features, Atherosclerosis, 60 (1986) 291.
ATHERO
69 Ltd.
04460
Influence of dietary minerals on apolipoprotein B metabolism in rabbits fed semipurified diets containing casein Samir Samman, Pramod Khosla * and Kenneth K. Carroll Department
of Biochemistry,
University of Western Ontario, London, Ontario, N6A 5CI (Canada) (Received 18 April, 1989) (Revised, received 7 November, 1989) (Accepted 20 January, 1990)
Summary
Rabbits were fed semipurified casein diets containing either 4% or 2.5% mineral mix for 8 weeks. Both groups maintained weight throughout the experimental period. The plasma total cholesterol concentration was significantly higher after 4 weeks on diet and slightly higher after 8 weeks in animals fed the lower level of minerals. Plasma IDL- and LDL-cholesterol concentrations after 4 weeks and HDL-cholesterol concentrations after 8 weeks were significantly higher in animals fed the 2.5% compared to those fed the 4% mineral mix. Kinetic experiments showed that in rabbits fed the lower level, the fractional catabolic and production rates of VLDL-apo B were lower and a greater proportion of IDL-apo B was derived from sources other than VLDL compared to the animals fed the higher level. LDL-apo B kinetics were not significantly different between the 2 groups. These data suggest that a reduction in dietary minerals enhances casein-induced hypercholesterolemia.
Key words: Minerals; Casein; Rabbits; Cholesterol;
Introduction
Micronutrients have been implicated in cholesterol metabolism in experimental animals and humans [l-6]. Supplementing humans with zinc [4] or calcium [5] results in a decrease in LDL- and total cholesterol, respectively. In contrast, copper
Correspondence to: S. Samman, Ph.D., Dept. of Clinical Biochemistry, Royal Prince Alfred Hospital, Missenden Rd. Camperdown, Sydney, N.S.W., 2050, Australia. * Current address: Foster Biomedical Research Laboratory, Brandeis University, Waltham, MA 02254-9110, U.S.A.
0021-9150/90/$03.50
0 1990 Elsevier Scientific
Publishers
Ireland,
Plasma lipoproteins;
Apolipoprotein
B metabolism
deficiency is associated with elevated levels of plasma cholesterol [6]. In rats, increasing the mineral content of the diet has been shown to decrease hepatic cholesterol and raise plasma HDL-cholesterol concentrations [7]. In rabbits fed casein, increases in dietary calcium [8] and zinc and copper [9] are associated with a lower concentration of plasma total cholesterol, while decreasing the mineral content of the diet resulted in a marked hypercholesterolemia [ 10,111. To investigate this effect further, rabbits were fed semipurified casein diets containing either 4% or 2.5% mineral mix and lipoprotein turnover studies were carried out. The results showed that a Ltd.
70
reduction in dietary minerals has significant effects on lipoprotein metabolism indicating that minerals influence the hypercholesterolemic response to dietary casein. Materials and methods
1.019) LDL (1.019 < d < 1.063) and HDL (1.063 < d < 1.21) were isolated by sequential ultracentrifugation in a Ti50 rotor at 40000 rpm at 15°C [14]. Total and lipoprotein-cholesterol was determined using enzymatic kits (CHOD-PAP, Boehringer Mannheim, Canada).
Animals and diets
Preparation of labelled lipoproteins
Young, male, New Zealand white rabbits (starting weight 1.8-2.0 kg) were obtained from Reimen’s Fur Ranches (Guelph, Ontario) and housed under conditions described previously [12]. Rabbits were fed low-fat, cholesterol-free, semipurified diets containing casein with either 4% mineral mix (standard minerals) or 2.5% mineral mix (reduced minerals). Both groups were fed the semipurified diets for 8 weeks following a l-week adaptation period. The composition of the diets is shown in Table 1.
After 6 weeks on diet, VLDL and LDL were isolated [14] and each lipoprotein was recentrifuged once at its optimal density. VLDL and LDL were radiolabelled with Na12’I and Na1311 (Amersham, Oakville, Ontario) respectively by a modification [15] of the McFarlane method [16]. The labelled lipoproteins were sterilized by passage through a 0.45 pm Millex-HA filter and gentamycin sulphate (1 pg/ml) was added. Within 36 h of iodination, labelled lipoproteins were reinjetted into recipient rabbits. The intramolecular distribution (IMD) of radioactivity in the tracer was determined as described previously [15,17]. The IMD for 12’I-VLDL and l3 I-LDL were similar in the 2 groups. For VLDL, the percent distribution in protein, apo B, lipid and as free iodine was 91, 73, 6 and 5, respectively. For LDL, the distribution was 97, 92, 3 and 2, respectively.
Plasma cholesterol analyses
Blood was collected from animals in the fasted state at the start of the experimental period for the determination of plasma total cholesterol. Also, after 4 and 8 weeks on diet, blood was collected and VLDL (d < 1.006 g/ml), IDL (1.006 < d < TABLE
Lipoprotein kinetic studies
1
COMPOSITION RABBITS
Casein ’ Dextrose b Celluflour ’ Mineralmix ac Molasses b (50% v/v) Corn oil b Vitamins d
OF THE SEMIPURIFIED
DIETS FED TO
Standard minerals
Reduced minerals
(g/kg)
(g/kg)
270 598 50 40
270 613 50 25
30 10 2
30 10 2
a Obtained from ICN Life Science Group, Nutritional Biochemicals Division, Cleveland, OH. b Obtained from a local feed mill. ’ Mineral mix (Phillips and Hart). Composition (mg/kg diet when used at the 4% level): calcium, 5.097; chloride, 3.646; copper, 0.0027; iodine, 0.0219; iron, 0.164; magnesium, 0.361; manganese, 0.0031; phosphorous, 2.668; potassium, 5.195; sodium, 2.361; sulphur, 0.479; zinc, 0.0043. d The composition of the vitamin mix has been reported elsewhere [13].
Following an overnight fast, unanesthetised rabbits were injected simultaneously with 2-8 $i ‘251-VLDL-apo B and l-3 PCi 1311-LDL-apo B in the marginal ear vein. Twelve blood samples (4-5 ml each) were obtained from each animal at timed intervals up to 48 h after injection. The protocol used has been described in more detail elsewhere [18]. There were 6 turnover studies, 3 per dietary group. In each of the studies, equal volumes of plasma from 3 rabbits were pooled from each of the 12 time points. VLDL, IDL and LDL were isolated from 4-6 ml plasma by ultracentrifugation [14] and the lipoproteins were recentrifuged once at their optimal densities. The protein content of all the lipoproteins was determined by the Markwell modification [19] of the Lowry method
WI. To determine the dose injected, aliquots of the first blood sample, at 5 min post-injection, were taken and the radioactivity in apo B was determined [21]. The dose injected was then calcu-
71
lated by assuming that the plasma volume is 32.8 ml/kg body weight [22]. The assumption was made that there was similar metabolism of apo B within the first 5 min. The lipoprotein apo B concentrations were determined [23] and the apo B pellets were processed for the determination of specific activity (SA) as described previously [24]. The SA time curves of VLDL and LDL plotted semi-logarithmically were best described by a double-exponential curve and were resolved using a least squares method. Curve parameters were calculated according to the twopool model [25].
A
A-A VLDL .---. IDL .- - ??LDL
1000~
I
I
6
12
18
Results
30
3t
42
48
TIME (hr)
Feeding diets with a reduced content of minerals had no significant effect on the weight of A
24
B 1000r
A---A .---. .-
VLOL IOL
- 0 LDL
/
35aC
*
??
m
Standard
I
Reduced
Minerals Minerals
*t
0 plasma
VLDL
IDL
LDL
HDL
0
t 6
12
I8
24
310
3c
42
48
TIME (hr)
m Standard IReduced
plCWTM
VLDL
IDL
LDL
Minerals Minerals
HDL
Fig. 1. Plasma and lipoprotein cholesterol concentrations in rabbits fed diets containing standard or reduced minerals for (A) 4 weeks or (B) 8 weeks. Each value is the mean (*SE) of 6-8 observations. Statistical significance was determined by Student’s t test, ** P < 0.01, * P-e 0.05.
Fig. 2. Precursor-product relationships between VLDL-, IDLand LDL-apo B in the plasma of animals fed diets containing either (A) standard or (B) reduced minerals. Each graph represents data obtained from the pooled plasma of 3 animals.
the animals during the 8 week experimental period compared to those fed standard minerals (2.3 f 0.1 vs. 2.6 f 0.1 kg, mean f SE). However, the plasma cholesterol concentrations of the animals fed diets with reduced minerals was increased significantly (P < 0.01) after 4 weeks and slightly higher after 8 weeks on diet (Fig. 1). The rise in plasma total cholesterol after 4 weeks was primarily due to significant increases in the IDL- (P < 0.05) and LDL- (P < 0.01) cholesterol concentrations (Fig. 1A). After 8 weeks on diet, HDL-cholesterol concentrations were significantly higher (P -z 0.05)
72 TABLE 2 KINETIC PARAMETERS FOR VLDL- AND LDL-APO B IN RABBITS FED DIETS CONTAINING STANDARD OR REDUCED MINERALS Values are expressed as the mean of 3 experiments f SE. Values for each experiment were obtained from the pooled plasma of 3 rabbits. Standard minerals VLDL-apo B Pool (mg/kg) 8.49 zb1.99 FCR (per h) 0.172 kO.014 Production rate (mg/kg/h) 1.43 kO.27 IDL SA : VLDL SA a 0.95 f 0.10 LDL-apo B Pool (mg/kg) 29.90 k6.99 FCR (per hr) 0.035 f 0.003 Production rate (mg/kg/h) 1.04 +0.28
Reduced minerals 10.46 k1.29 0.137 f 0.020 0.95 *IO.10 0.55 +0.05 * 29.96 +7.00 0.027 f 0.001 0.81 i-O.19
’ Ratios of r2%apo B specific activity when IDL specific activity reaches its maximum. * Significantly lower than standard minerals counterpart using Student’s t test, P c: 0.01.
and VLDL-, IDL- and LDL-cholesterol still tended to be higher (Fig. 1B). The kinetically defined VLDL-apo B pool was slightly higher in animals fed the reduced minerals (Table 2). In addition, the fractional catabolic rate (FCR) and the production rate of VLDL-apo B were lower in the animals fed the lower level TABLE 3 PLASMA LIPOPROTEIN PROTEIN CONCENTRATIONS AND PERCENT APO B IN RABBITS FED DIETS CONTAINING STANDARD OR REDUCED MINERALS Values are expressed as means* SE. Values were obtained from the pooled plasma of 3 rabbits taken at 12 time points during the kinetic study.
VLDL Protein (mg/lOO ml) % apo B IDL Protein (mg/lOO ml) ‘K,apo B LDL Protein (mg/lOO ml) % apo B
Standard minerals
Reduced minerals
17.7* 39.7*
2.6 1.4
30.4* 32.6*
4.5 3.0
11.7rfr 2.3 67.0* 5.6
19.8* 72.1*
5.7 3.5
81.7 f 12.8 88.7f 1.4
72.5 f 13.3 83.7* 0.6
(Table 2). LDL-apo B kinetics were similar between the two groups (Table 2). The SA curves (Fig. 2) were examined for precursor-product relationships. As defined by Zilversmit [26], the fraction of a product derived from its precursor is calculated by examining the ratio of SA of the product to that of the precursor when the SA of the product is at its maximum. Estimates of the ratios of the maximum apo B SA in IDL to VLDL are shown in Table 2. These show that only 55% of IDL-apo B is derived from VLDL in animals fed reduced minerals compared to 95% in animals fed standard minerals. Thus, in animals fed reduced minerals there is greater input of IDL-apo B independently of the VLDL-apo B pathway compared to animals fed the higher level of minerals. The plasma lipoprotein protein concentrations and the percent of protein which was apo B showed little change over the 48 h of the kinetic study (Table 3). The coefficient of variation of the percent apo B was 8.3% which suggests the animals were in a steady state. Discussion Our results show that dietary minerals have an effect on plasma apo B metabolism and HDL cholesterol levels in rabbits fed semipurified diets containing casein. After 4 weeks on diet, the lower level of dietary minerals was associated with increased plasma total IDL- and LDL-cholesterol concentrations. These differences were no longer evident after 8 weeks on diet. Thus, the reduction in dietary minerals accelerates the rate with which the hypercholesterolemia is produced in rabbits fed casein. The VLDL kinetic studies showed that the efficiency of removal of VLDL-apo B was reduced in animals fed the lower level of minerals. In addition, the SA values obtained were consistent with the interpretation that more IDL-apo B was derived from sources other than VLDL-apo B in animals fed the lower level of minerals. LDL-apo B metabolism was not different between the 2 groups. However, in both cases, as in other lipoprotein studies in rabbits [18,27,28] and other species [24,29,30], production of LDL from sources other than VLDL was observed. Independent pro-
73
duction of LDL, or IDL, are subject to much debate. It is thought that these may be particles synthesized and secreted directly from the liver, or they may originate from VLDL that is converted rapidly to IDL and LDL [31]. The enhancement of the hypercholesterolemia in animals fed a casein/low mineral diet may be mediated through 2 processes. Firstly, a more rapid down-regulation of LDL receptors may be taking place. It has been shown that LDL receptors are down-regulated in casein-fed rabbits compared to those fed soy protein [32] and the reduction in mineral mix may accelerate the process. This would result in a more rapid increase in plasma LDLcholesterol, as seen after 4 weeks (Fig. 1A). A second possibility is the reduction in fecal sterol excretion. Casein-fed animals excrete less fecal sterols than their soy protein counterparts [33] possibly due to the lower calcium content of the diet [8]. A reduction in the mineral mix, of which calcium is a major component (Table l), may decrease further the excretion of sterols [34]. It has been proposed [35] that the enhanced reabsorption of sterols (or the reduced excretion) results in a larger hepatic pool of cholesterol [33] and in turn stimulates the secretion of cholesterol-rich lipoproteins such as LDL [18] and to a lesser extent, IDL. A reduction in dietary minerals may be interacting at the receptor level and/or by interfering with the enterohepatic circulation to elicit these effects. However, from these experiments, it is unclear which mineral is of importance in the observed changes in metabolism. Given the paucity of information available on the effect of micronutrients on cholesterol metabolism, further research is warranted to elucidate the role of individual elements in the cholesterolemic effect of dietary minerals.
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Acknowledgements
Supported by the Heart and Stroke Foundation of Ontario. Samir Samman is a Postdoctoral Fellow of the Canadian Heart Foundation and a recipient of a travel grant from the National Heart Foundation of Australia. Kenneth K. Carroll is a Career Investigator of the Medical Research Council of Canada.
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