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Dietary copper and cholesterol metabolism
NUTRITION RESEARCH, Vol. 5, pp. 1021-1034, 1985 0271-5317/85 $3.00 + .00 Printed in the USA. Copyright (c) 1985 Pergamon Press Ltd. All rights reserved.
DIETARY Samir
COPPER
AND C H O L E S T E R O L
METABOLISM
Samman BSc and David C.K. R o b e r t s Human N u t r i t i o n Unit, D e p a r t m e n t of B i o c h e m i s t r y , U n i v e r s i t y of Sydney, Sydney, N.S.W. 2006, Australia
PhD
ABSTRACT Since the time when an a s s o c i a t i o n was first shown b e t w e e n water h a r d n e s s and i n c i d e n c e of c a r d i o v a s c u l a r d i s e a s e (I) there has been a search for a c a u s a t i v e link. V a r i o u s trace e l e m e n t s have been i m p l i c a t e d including calcium, m a n g a n e s e (2) and m a g n e s i u m (3). One theory based on s t u d i e s in rats s u g g e s t s that a r e l a t i v e copper d e f i c i e n c y caused by an increase in the z i n c : c o p p e r ratio may alter c h o l e s t e r o l m e t a b o l i s m . This Zn/Cu h y p o t h e s i s was first p r o p o s e d by Klevay (4) and was last r e v i e w e d by Klevay in 1982 (5). It is the p u r p o s e of this r e v i e w to e v a l u a t e some of the r e s e a r c h that this h y p o t h e s i s has generated. KEYWORDS:
Copper, c h o l e s t e r o l , m a c r o n u t r i e n t s , b i o a v a i l a b i l i t y , n u t r i t i o n a l design
INTRODUCTION A l t h o u g h the i n f l u e n c e of the v a r i o u s m a c r o n u t r i e n t s o n lipid m e t a b o l i s m has been well d o c u m e n t e d , the role of m i c r o n u t r i e n t s is less u n d e r s t o o d . Of the m i c r o n u t r i e n t s , trace e l e m e n t s r e p r e s e n t a p a r t i c u l a r problem, b e c a u s e of t h e i r complex i n t e r a c t i o n s (6) and the effect that the m a c r o n u t r i e n t s have on their b i o a v a i l a b i l i t y (7-9). A brief r e v i e w of the e f f e c t s on b l o o d lipids of Ca, Mg, Zn, Cu, V, Cr, Fe and I has been p u b l i s h e d (10) and more r e c e n t l y a d e t a i l e d review of iron and lipid m e t a b o l i s m has a p p e a r e d (Ii). The effect of Cu on c h o l e s t e r o l m e t a b o l i s m and its interrelatic~nship with Zn in that effect was first p r o p o s e d by K l e v a y (4). This Zn:Cu h y p o t h e s i s states that "...the r e l a t i v e or a b s o l u t e d e f i c i e n c y of Cu c h a r a c t e r i s e d by a high ratio of zinc to copper is a major factor in the e t i o l o g y of c o r o n a r y heart, d i s e a s e " ( 1 2 ) . I t is the p u r p o s e of this paper to r e v i e w recent d e v e l o p m e n t s in this area, with p a r t i c u l a r r e f e r e n c e to the n u t r i t i o n a l d e s i g n of the studies. THE EFFECT
OF CU
IMBALANCE
Most of this work has been limited number of r e s e a r c h e r s . c o n s i d e r a b l e v a r i a t i o n in basal strain of rat used (Table I). d i s c r e p a n c i e s in results. The which the Cu or Zn is p r e s e n t e d
ON P L A S M A
LIPIDS
IN ANIMALS
u n d e r t a k e n in rats and only by a D e s p i t e this latter fact, there is diet. c o m p o s i t i o n , time on diet, and This may a c c o u n t for some of the o t h e r major v a r i a t i o n is the way in to the animals. In general, a 1021
1022
S. SAMMANand D.C.K. ROBERTS
d e f i c i e n t or marginal diet is fed with e i t h e r in the diet or in the water. Conflicting
supplementary
Cu and/or
Zn
reports
Several s t u d i e s ( 1 9 , 2 4 , 6 7 , 6 8 , 7 3 ) have failed to show a h y p e r c h o l e s t e r o l a e m i a at Cu intakes from 0.5 ~g/g to 6~g/g (in the diet) and with Zn/Cu ratios of 3 . 3 to 30:1. However, in one of these, the e x p e r i m e n t a l p e r i o d was only 3 weeks (24). At a similar Cu intake (1.6 ~g/g) to that study, Lin and Lei (27) also failed to show an increase in c h o l e s t e r o l after 8 weeks on diet but by 18 weeks there was a marginal but s i g n i f i c a n t h y p e r c h o l e s t e r o l a e m i a . Halving the Cu in the diet (28) p r o d u c e d a s i g n i f i c a n t increase in 7 weeks. It is t h e r e f o r e r e a s o n a b l e to suggest, that had the e x p e r i m e n t of Caster and D o s t e r (24) c o n t i n u e d for a longer time, there may have been a s u b s e q u e n t increase in plasma c h o l e s t e r o l . G e i g e r et al (67) using diets based on a p r e v i o u s study (4) s h o w e d a d e c r e a s e in h a e m a t o c r i t but ne effect on plasma c h o l e s t e r o l . However, the Cu and Zn were a d d e d to the water as Zn a c e t a t e (10 ~g/ml) and Cu s u l p h a t e (2~g/ml) r e s u l t i n g in only 3,5 ~g Zn and 0,5 ~g as c o m p a r e d to the levels used by K l e v a y (4), (10 and 2 ~g r e s p e c t i v e l y ) . Other s t u d i e s (20,21,28) using this level of Cu intake and for s i m i l a r p e r i o d s (40 days) did p r o d u c e raised p l a s m a lipid levels r e l a t i v e to their r e s p e c t i v e control diets. It is p o s s i b l e that the lack of effect in this study may have been due to the low Cu intake in both control and treated g r o u p s such that both were mildly h y p e r c h o l e s t e r o l a e m i c and no d i f f e r e n t i a l effect was seen. A longer e x p e r i m e n t a l period or a h i g h e r Cu intake in the control diet may have shown a response. S h e r m a n (68) fed copper d e f i c i e n t d i e t s (Cu = Iug/g) to rats for a p e r i o d of 18 weeks. From the r e s u l t a n t d e c r e a s e in c e r u l o p l a s m i n and liver copper c o n c e n t r a t i o n , the a u t h o r c o n c l u d e d that the rats were copper depleted. D e s p i t e this, there was not effect on p l a s m a cholesterol. Female r a t h e r than male rats were used for this study however. A n o t h e r n e g a t i v e study (19), using a diet c o n t a i n i n g a s i m i l a r a m o u n t of Cu (1.5 ~g/g) could not d e m o n s t r a t e a h y p e r c h o l e s t e r o l a e m i a after 15 weeks on diet. They used Wistar, r a t h e r than S p r a g u e Dawley rats and the d i e t s d i f f e r e d in fat content and type of p r o t e i n and c a r b o h y d r a t e s (Table i) from those used in the other s t u d i e s (27,28). Diet
interactions
These diet i n t e r a c t i o n s may well i n f l u e n c e the copper effect. It is well e s t a b l i s h e d that the source of p r o t e i n has a s i g n i f i c a n t effect on p l a s m a c h o l e s t e r o l in rats (32,33) and other animals (34,35). This could be due to the amino acid c o m p o s i t i o n of the p r o t e i n (36-38) or the n o n - p r o t e i n c o m p o n e n t s of the p r o t e i n source (39) in p a r t i c u l a r the mineral i n t e r a c t i o n s (40-42). Other d i e t a r y c o m p o n e n t s such as the type of c a r b o h y d r a t e have been shown to affect. the c h o l e s t e r o l r e s p o n s e (43) and g l u c o s e and insulin r e s p o n s e s p a r t i c u l a r l y in Cu d e f i c i e n t rats (44).
(45).
The effect of fat on c h o l e s t e r o l m e t a b o l i s m is well e s t a b l i s h e d D i e t a r y fat content, in p a r t i c u l a r the s a t u r a t e d fat, will
COPPER AND CHOLESTEROL METABOLISM
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S. SAMMANand D.C.K. ROBERTS
d e t e r m i n e how q u i c k l y h y p e r c h o l e s t e r o l a e m i a is e s t a b l i s h e d (46). As a result of d i f f e r e n c e s in d i e t a r y fat b e t w e e n e x p e r i m e n t s , the time taken to d e v e l o p h y p e r c h o l e s t e r o l a e m i a at v a r i o u s levels of copper will vary. This undoubtedly c o n t r i b u t e s to some of the r e p o r t e d tlme d i f f e r e n c e s in response. It is e v i d e n t that the source of the m a c r o n u t r i e n t s must also be c o n t r o l l e d in i n v e s t i g a t i o n s of this nature. Not only have the m a c r o n u t r i e n t s varied in these s t u d i e s but so have the salt mixes. These d i f f e r in a b s o l u t e amount as well as r a t i o s of minerals. S t u d i e s in a n i m a l s have shown that copper a b s o r p t i o n can be i n h i b i t e d by high c o n c e n t r a t i o n s of zinc (47,48) and other ions with s i m i l a r c h a r a c t e r i s t i c s (49,50) thus the v a r i a t i o n s in a m o u n t s and ratios add a further c o n f o u n d i n g factor to the p o s s i b i l i t y of u n i f i e d results. Cholesterol
response
at
lower Cu
intakes
D e s p i t e this, the r e m a i n i n g papers, even t h o u g h d i f f e r i n g in type and c o n t e n t of major n u t r i e n t s (Table I), r e p o r t i n c r e a s e s in plasma c h o l e s t e r o l with r e s t r i c t e d Cu intake. There is a trend for the g r e a t e r i n c r e a s e s to o c c u r at lower copper intakes and/or longer time on diet. In 6 r e p o r t s (one set of data p r e s e n t e d twice: 16,17) s i g n i f i c a n t i n c r e a s e s after 3 to 26 weeks were seen in a n i m a l s fed diets containing 0.5-0.6 ~g Cu/g ( 1 6 - 1 8 , 2 0 , 2 1 , 2 5 ) . At h i g h e r Cu intakes (2~g/g) p l a s m a c h o l e s t e r o l v a r i e d with time on diet and other m a c r o n u t r i e n t changes, In the a b s e n c e of fibre, there was only a marginal increase in p l a s m a c h o l e s t e r o l after 8 weeks (25) and i n c r e a s i n g the fibre c o n t e n t of the diet reduced the degree of h y p e r c h o l e s t e r o l a e m i a at 9 weeks (22). C h o l e s t e r o l feeding (2% w/w) a b o l i s h e d the d i f f e r e n t i a l effect of Copper (23), Thus at this level of Cu intake (2 #g/g) any increase in plasma c h o l e s t e r o l is much less m a r k e d and can be i n f l u e n c e d by other d i e t a r y variables. It is t h e r e f o r e p o s s i b l e that the Zn/Cu r a t i o may now become i m p o r t a n t (51). S i n t h u s e k et al (69) using d i e t s c o n t a i n i n g d i f f e r e n t fats, corr. oil or c o c o n u t oil, showed that p l a s m a c h o l e s t e r o l rose as the Zn/Cu ratio increased from 1.25 to 20. However, at equal ratios the diet with the least copper (1.5 .~g/g) was the most h y p e r c h o l e s t e r o l a e m i c when corn oil was the fat used. With coconut oil in the diet, a Zn/Cu ratio os 5 p r o d u c e d the h i g h e s t plasma cholesterol, but plasma c h o l e s t e r o l was not d i f f e r e n t at r a t i o s of 1.25 and 20. Thus in thi~ study, feeding corn oil to rats and m a n i p u l a t i n g the Zn/Cu ratio, p r o d u c e d a h i g h e r p l a s m a c h o l e s t e r o l than when c o c o n u t oil was used. K l e v a y (4), a n a l y s i n g his data in a s l i g h t l y d i f f e r e n t way (number of days on diet before the g r o u p s were first s i g n i f i c a n t l y d i f f e r e n t ) showed that i n c r e a s i n g the ratio of Zn/Cu in the water from 5:1 to 40:1 r e s u l t e d in an a d d i t i o n a l increase in plasma cholesterol. It is also c o n s i s t e n t with his data to say that the rats o f f e r e d the least copper (0.25~g/ml and 0.5 ~g/ml) p r o d u c e d a g r e a t e r h y p e r c h o l e s t e r o l a e m i a than their r e s p e c t i v e c o n t r o l s (2~g Cu/ml~. Two f u r t h e r s t u d i e s ( 1 3 , 1 5 ) have s h o w n that at v a r y i n g r a t i o s of Zn/Cu (0.16 to 160:1) in the water, the g r e a t e r i n c r e a s e s in plasma
COPPER AND CHOLESTEROL METABOLISM
1027
c h o l e s t e r o l were seen at the lower copper intakes. Thus the major h y p e r c h o l e s t e r o l a e m i c effect in rats seems to be caused by copper d e f i c i e n c y r a t h e r than by a l t e r e d Zn/Cu ratio. Studies
in other
species
There have been s t u d i e s in other species. In mice fed diets containing 0.410 ~g/ml Cu, p l a s m a c h o l e s t e r o l did not change (70). In c h i c k e n s (29) there was no effect on p l a s m a c h o l e s t e r o l of diets c o n t a i n i n g 6 to 60 ~g Cu/g and r a t i o s of Zn/Cu of I to 20:1. However, the lowest level of Cu o f f e r r e d was 3 times h i g h e r than the amount r e q u i r e d to p r o d u c e a marginal e f f e c t in rats. One study in plgs (30) at a constant Cu intake (i.% ~g/g) d e m o n s t r a t e d a fall in c h o l e s t e r o l level when the d i e t a r y Zn content was r e d u c e d to 7.2 ~g/g (i.e. the Zn/Cu ratio d e c r e a s e d from 67:1 to 4:1). In a second study in pigs (31) using Zn/Cu r a t i o s of 0.5:1 to 73:1 there was no c o n s i s t e n t effect on plasma c h o l e s t e r o l and none of the a n i m a l s became hypercholesterolaemic. However, again the lowest level of d i e t a r y Cu (8 ~g/g) was 4 times that r e q u i r e d to show a h y p e r c h o l e s t e r o l a e m i a iT. rats. STUDIES
OF
THE
MECHANISM
BY
WHICH
HYPERCHOLESTEROLAEMIA D e s p i t e the v a r i a t i o n s observed, to i n v e s t i g a t e the m e c h a n i s m by w h i c h induces hypercholesterolaemia.
CU
DEFICIENCY
PRODUCES
IN RATS
some r e s e a r c h e r s have a t t e m p t e d copper d e f i c i e n c y in rats
Several s t u d i e s have been c a r r i e d out to d e t e r m i n e the effect of copper d e f i c i e n c y on c h o l e s t e r o l turnover. It was found that newly s y n t h e s i z e d c h o l e s t e r o l (25,16) a c c u m u l a t e s in the plasma of copper d e f i c i e n t rats but that d e g r a d a t i o n is not a f f e c t e d (23). This data implies that copper d e f i c i e n c y has caused a shift of c h o l e s t e r e l from the liver to the serum pool and this is r e s p o n s i b l e for the hypercholesterolaemia. The e f f e c t s of copper on the major enzymes of l i p o p r o t e i n m e t a b o l i s m have also been studied. L e c i t h i n c h o l e s t e r o l acyl t r a n s f e r a s e , LCAT, (E.C. 2.3.1.43) is p r o d u c e d in the liver and was shown to be s t i m u l a t e d in v i t r o by copper (52). However, the nature of the b i o l o g i c a l l y a c t i v e cofactor is yet to be d e t e r m i n e d but it could be the i n t e r a c t i o n b e t w e e n c o p p e r - a l b u m i n and the enzyme (53). In rive s t u d i e s have shown that LCAT a c t i v i t y is r e d u c e d in copper d e f i c i e n c y (20,26). In one instance, (26), liver copper was r e d u c e d by 73% as c o m p a r e d to the control group. However, in both cases there was no a t t e m p t to c o r r e l a t e liver copper and LCAT activity. P l a s m a t r i a c y l g l y c e r o l c o n c e n t r a t i o n has been n e g a t i v e l y c o r r e l a t e d with both p l a s m a and liver c o n c e n t r a t i o n s of copper ~15). This may be due to a n o t h e r major enzyme involved in l i p o p r o t e i n m e t a b o l i s m , l i p o p r o t e i n lipase (LPL), (E.C. 3.1.1.34). LPL was a s s a y e d in vitro and a c t i v i t y was found to be r e d u c e d by 40-47% in copper d e f i c i e n c y (21). However, the a p p a r e n t d e c r e a s e in enzyme a c t i v i t y may result from the d i l u t i o n effect on both e n z y m e s and their
1028
S. SAMMANand D.C.K. ROBERTS
c o f a c t o r s caused by the r e d u c e d h a e m a t o c r i t and hence volume o b s e r v e d in copper d e f i c i e n t r a t s (28). EFFECT
OF COPPER
ON P L A S M A
LIPIDS
increased
plasma
IN HUMANS
S t u d i e s in h u m a n s have been few and g e n e r a l l y indirect involving r e l a t i o n s h i p s b e t w e e n p l a s m a Zn/Cu and c h o l e s t e r o l rather than d i e t a r y Zn/Cu. In a d i a b e t i c group, plasma Zn/Cu was shown to be p o s i t i v e l y c o r r e l a t e d with p l a s m a c h o l e s t e r o l c o n c e n t r a t i o n (54) but in a nond i a b e t i c h y p e r l i p i d a e m i c p o p u l a t i o n there was no such r e l a t i o n s h i p (55). In a g r o u p of u r a e m i c p a t i e n t s or. h a e m o d i a l y s i s , plasma Zn/Cu ratio was lower than in c o n t r o l s and p l a s m a HDL c h o l e s t e r o l was also lower (56). Thus the r e l a t i o n s h i p seen in rats b e t w e e n plasma Zn/Cu ratio and c h o l e s t e r o l c o n c e n t r a t i o n (57) is at best equivocal in man. With one exception, the effect of d i e t a r y change has been on zinc s u p p l e m e n t a t i o n r a t h e r than Cu d e f i c i e n c y . In h e a l t h y males an oral zinc s u p p l e m e n t of 29 mg/day had no e f f e c t on p l a s m a c h o l e s t e r o l (58). H o w e v e r 160 m g / d a y over 5 weeks r e d u c e d HDL c h o l e s t e r o l (59) and larger s u p p l e m e n t s (300 mg/day) r e d u c e d HDL after 4 weeks and i n c r e a s e d LDL a f t e r 6 weeks (71). In females t a k i n g 100 mg/day a nons i g n i f i c a n t h y p o c h o l e s t e r o l a e m i c trend was o b s e r v e d as the d i e t a r y ZD/Cu ratio i n c r e a s e d (72) and a t r a n s i e n t fall in HDL c h o l e s t e r o l was r e p o r t e d which was no longer d i f f e r e n t from control by 8 weeks (60). This a p p a r e n t sex d i f f e r e n c e in r e s p o n s e to d i e t a r y zinc (59,60) was also r e p o r t e d for a g r o u p of 75 h e a l t h y s u b ] e c t s c o n s u m i n g their normal d!et. In males, there was a n e g a t i v e c o r r e l a t i o n b e t w e e n dietary zinc Intake and plasma HDL chc,iester~i c o r ~ c e n t ~ i : r ~ but no r e l a t i o n s h i p in females. P l a s m a total c h o l e s t e r o l was not r e l a t e d t:} dietary zinc or Cu intake (6]). A single study (in a b s t r a c t form, 62) r e p o r t e d that p l a s m a total c h o l e s t e r o l increased after iS weeks in a h e a l t h y male when Cu intake was 1.3 mg/day. R e p l e t i o n with an extra 2 m g / d a y caused the p l a s m a c h o l e s t e r o l to fall. Thus, in humans, the r e s u l t s are less than convinclng, not w i t h s t a n d i n g the e p i d e m i o l o g i c a l a s s o c i a t i o n s w h i c h have been d e m o n s t r a t e d (4,63). On the evidence, any effect of dietary Zn/Cu would a p p e a r to be marginal unless very low intakes of copper or large s u p p l e m e n t s of zinc are given. The effect is also on males and not females. It is i n t e r e s t i n g to note that all the s t u d i e s in rats have been on males.
CONCLUSION Before a s s o c i a t i n g d i e t a r y copper intake with lipid metabolism: two i m p o r t a n t factors must be considered. Firstly the b i o a v a i l a b i l i t y of copper from the small i n t e s t i n e must be taken into account. Thls will involve a g r e e m e n t upon a s t a n d a r d e • diet which will s t a n d a r d i s e the response. Secondly, more s y s t e m a t i c studies have to be carried out to d e t e r m i n e w h e t h e r a l t e r a t i o n s to the enzymes of lipid m e t a b o l i s m are not just part of a general effect of copper d e f i c i e n c y on the many copper c o n t a i n i n g e n z y m e s (64).
COPPER AND CHOLESTEROL METABOLISM
1029
In animals the balance of evidence points to an effect of copper d e f i c i e n c y and only in male rats, although further study in female rats may revise this conclusion. In humans, zinc s u p p l e m e n t a t i o n at pharmacological levels rather than copper d e f i c i e n c y appears to reduce plasma HDL cholesterol but again only in males. Copper d e f i c i e n c y in humans is rarely found (65). It is a c o m p l i c a t i o n of other disease states (6) and occurs s i m u l t a n e o u s l z with iron d e f i c i e n c y (66). I n view of this and of the lack of systematic studies in humans, the role of copper in cholesterol m e t a b o l i s m r e m a i n s c o n t r o v e r s i a l and the Z n / C u h y p o t h e s i s remains to be validated.
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Nut.
1030 15.
S. SAMMANand D.C.K. ROBERTS
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~i
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SHAO, H.J.S. and LEI, K.Y. Conversion of [2-14C] mevalonate into cholesterol, lanosterol and squalene in copper-deficient rats. J. Nutr., 1980; 110: 859-867.
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HARVEY, P.W. and ALLEN, K.G.D. Decreased plasma lecithin: cholesterol acyltransferase activity in copper-deficient rats. J. Nutr., 1981 111: 1855-1858.
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LIN, I.M. and LEI, K.Y. Cholesterol kinetic analyse~: deficient rats. J. Nutr., 1981" 111: 450-457.
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in copper-
COPPER AND CHOLESTEROLMETABOLISM
1031
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BURCH, R.E., WILLIAMS, R.V., HAHN, H.K.J., JETTON, M.H. and SULLIVAN, J.F. Serum and tissue enzyme activity and traceClin. element content in response to zinc deficiency in the pig. Chem., 1975 21: 568-577.
31.
EISEMANN, J.H., POND, W.G. and THONNEY, M.L. Effect of dietary zinc and copper on performance and tissue mineral and cholesterol concentratins in swine. J. Anim. Sci., 1979: 48: 1123-1128.
32.
COHN, J.S., KIMPTON, W.G. and NESTEL, P.J. The effect of dietary casein and soy protein on cholesterol and very low density lipoprotein metabolism in the rat. Atherosclerosis 1984: 52: 219-231.
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SUGANO, M., ISHIWAKI, N., NAGATA, Y., and IMAIZUMI, K. Effects of arginine and lysine addition to casein and soya-bean protein on serum lipids, apolipoproteins, insulin and glucagon in rats. Br. J. Nutr. 1982: 48: 211-221.
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KRITCHEVSKY, D. Vegetable Protein and Atherosclerosis. Oil Chem. Soc. 1979: 56: 135-140.
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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. (in press) 1985.
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LEE, Y.S,, NOGUCHI, T., and NAITO, H. Intestinal absorption of calcium in rats given diets containing casein or amino acid mixture: the role of casein phosphopeptides. Br. J. Nutr, 1983: 49: 67-76.
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VAN DER MEER, R. Is the hypercholesterolemic effect of dietary casein related to its phosphorylation state? Atherosclerosis, 1983: 49: 339-341.
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SAMMAN, S. and ROBERTS. D.C.K. The p h o s p h o r y l a t i o n state of casein and its h y p e r c h o l e s t e r o l a e m i c effect - the role of divalent cations. Atherosclerosis, 1984: 52: 347-348.
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CARROLL, K.K. and HAMILTON. R.M.G. Effects of dietary p r o t e l n and c a r b o h y d r a t e on plasma cholesterol levels in relatlon %0 atherosclerosis. J. Food. Sci., 1975: 40: 18-23.
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FIELDS, M., FERRETTI, R.J., SMITH, J.C. and REISER, S. Impairment of glucose t o l e r a n c e in c o p p e r - d e f l c l e n t rats: dependency on the type of carbohydrate. J. Nut r. 1984" 114: 397. llpids-
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Am.
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45.
Truswell, A.S. Diet and plasma Clin. Nutr., 1978: 31: 977-989.
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48.
FISCHER, P.W., GIROUX, A. and L'ABBE, M.R. mucosal copper binding and on the kinetics J. Nutr., 1983: 113: 462-469.
49
HILL, C.H. and MATRONE, G. Chemical p a r a m e t e r s in vivo and in vitro interactions of t r a n s i t i o n Proc., 1970; 29: 1474-1481.
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DAVIES, N.T. and CAMPBELL, J.K. The effect of cadmium on intestinal copper a b s o r p t i o n and binding in the rat. Life sciences, 1977~ 20: 955-960.
51
KATYA-KATYA, M., ENSMINGER, A., MEJEAN, L. and DEBPY, C. The effect of zinc s u p p l e m e n t a t i o n on plasma cholesterol levels. Nutr. Res., 1984; 4: 633-638.
52
SUZUE, G., VEZINA, C. and MARCEL, Y.L. P u r i f i c a t i o n of human plasma l e c i t h i n : c h o l e s t e r o l a c y l t r a n s f e r a s e and its a c t i v a t i o n metal ions. Can. J. Biochem., 1980: 58: 539-541.
K.K. Plasma llpoproteln fed s e m l p u r l f i e d diets.
copper
Effects of zlnc on of copper absorption.
on the study of elements. Fed.
by
53
LAU, S-J., and SARKAR, B. Ternary c o o r d i n a t i o n complex between human serum albumin, copper (II), and L-histidine. J. Biol. Chem., 1971: 246: 5938-5943.
54
MATEO, M.C.M., BUSTAMANTE, J,B. and CANTALAPIEDRA, M.A.G. Serum zinc, copper and insulin in d i a b e t e s mellitus. Biomedicine, 1978: 29: 56-58.
55
MEISSNER, D., LEONHARDT, W., SCHULZE J. and JULIUS, U. C o r r e l a t i o n s between p a r a m e t e r s of lipoprotein m e t a b o l i s m and magnesium, copper and zinc. In: M . P a l k o v i c H. ed. Advances in P h y s i o l o g i c a l Sciences. vol. 35. p. 91-96, 1981.
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OKUYAMA, S., MISHINA, H,, HASEGAWA, K., NAKANO, N. and ISE, K. Probable atherogenic role of zinc and copper as studied in chronic hemodialysis patients. Tohoku J. Exp. Med., 1982; 138: 227-229.
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FREELAND-GRAVES, J.H., FRIEDMAN, B.J., HAN, W.-H., SHOREY, R.L. and YOUNG, R. Effect of zinc supplementation on plasma highdensity lipoprotein cholesterol and zinc. Am. J. Clin. Mutt., 1982: 35: 98S-992.
61.
MEDEIROS, D., PELLUM, L. and BROWN, B. Serum lipids and glucose as associated with hemoglobin levels and copper and zinc intake in young adults. Life Science 1983: 32: 1897-1904.
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KLEVAY, L.M., INMAN, L., JOHNSON, L.K., LAWLER, M.R., MAHALKO, J.R., MILNE, D.B. and SANDSTEAD, H.H. Effects of a diet low in copper on a healthy man. Clin. Res., 1980: 28: 758A.
63.
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64.
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GEIGER, J.D., SETH P.K., KLEVAY, L.M. and PARMAR, S.S. binding changes in copper deficient rats. Pharmacology 196-202.
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Coronary heart disease. Am. J. Clin. Nutr. 1975:
G.E. JAMA
Zinc 1980;
The zinc/copper 28: 774-764.
Receptor1984: 28:
1034
S. SAMMANand D.C.K. ROBERTS
70.
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71.
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73.
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Accepted for publication July 11, 1985.
of zinc
impairs
immune responses.
DIETARY Samir
COPPER
AND C H O L E S T E R O L
METABOLISM
Samman BSc and David C.K. R o b e r t s Human N u t r i t i o n Unit, D e p a r t m e n t of B i o c h e m i s t r y , U n i v e r s i t y of Sydney, Sydney, N.S.W. 2006, Australia
PhD
ABSTRACT Since the time when an a s s o c i a t i o n was first shown b e t w e e n water h a r d n e s s and i n c i d e n c e of c a r d i o v a s c u l a r d i s e a s e (I) there has been a search for a c a u s a t i v e link. V a r i o u s trace e l e m e n t s have been i m p l i c a t e d including calcium, m a n g a n e s e (2) and m a g n e s i u m (3). One theory based on s t u d i e s in rats s u g g e s t s that a r e l a t i v e copper d e f i c i e n c y caused by an increase in the z i n c : c o p p e r ratio may alter c h o l e s t e r o l m e t a b o l i s m . This Zn/Cu h y p o t h e s i s was first p r o p o s e d by Klevay (4) and was last r e v i e w e d by Klevay in 1982 (5). It is the p u r p o s e of this r e v i e w to e v a l u a t e some of the r e s e a r c h that this h y p o t h e s i s has generated. KEYWORDS:
Copper, c h o l e s t e r o l , m a c r o n u t r i e n t s , b i o a v a i l a b i l i t y , n u t r i t i o n a l design
INTRODUCTION A l t h o u g h the i n f l u e n c e of the v a r i o u s m a c r o n u t r i e n t s o n lipid m e t a b o l i s m has been well d o c u m e n t e d , the role of m i c r o n u t r i e n t s is less u n d e r s t o o d . Of the m i c r o n u t r i e n t s , trace e l e m e n t s r e p r e s e n t a p a r t i c u l a r problem, b e c a u s e of t h e i r complex i n t e r a c t i o n s (6) and the effect that the m a c r o n u t r i e n t s have on their b i o a v a i l a b i l i t y (7-9). A brief r e v i e w of the e f f e c t s on b l o o d lipids of Ca, Mg, Zn, Cu, V, Cr, Fe and I has been p u b l i s h e d (10) and more r e c e n t l y a d e t a i l e d review of iron and lipid m e t a b o l i s m has a p p e a r e d (Ii). The effect of Cu on c h o l e s t e r o l m e t a b o l i s m and its interrelatic~nship with Zn in that effect was first p r o p o s e d by K l e v a y (4). This Zn:Cu h y p o t h e s i s states that "...the r e l a t i v e or a b s o l u t e d e f i c i e n c y of Cu c h a r a c t e r i s e d by a high ratio of zinc to copper is a major factor in the e t i o l o g y of c o r o n a r y heart, d i s e a s e " ( 1 2 ) . I t is the p u r p o s e of this paper to r e v i e w recent d e v e l o p m e n t s in this area, with p a r t i c u l a r r e f e r e n c e to the n u t r i t i o n a l d e s i g n of the studies. THE EFFECT
OF CU
IMBALANCE
Most of this work has been limited number of r e s e a r c h e r s . c o n s i d e r a b l e v a r i a t i o n in basal strain of rat used (Table I). d i s c r e p a n c i e s in results. The which the Cu or Zn is p r e s e n t e d
ON P L A S M A
LIPIDS
IN ANIMALS
u n d e r t a k e n in rats and only by a D e s p i t e this latter fact, there is diet. c o m p o s i t i o n , time on diet, and This may a c c o u n t for some of the o t h e r major v a r i a t i o n is the way in to the animals. In general, a 1021
1022
S. SAMMANand D.C.K. ROBERTS
d e f i c i e n t or marginal diet is fed with e i t h e r in the diet or in the water. Conflicting
supplementary
Cu and/or
Zn
reports
Several s t u d i e s ( 1 9 , 2 4 , 6 7 , 6 8 , 7 3 ) have failed to show a h y p e r c h o l e s t e r o l a e m i a at Cu intakes from 0.5 ~g/g to 6~g/g (in the diet) and with Zn/Cu ratios of 3 . 3 to 30:1. However, in one of these, the e x p e r i m e n t a l p e r i o d was only 3 weeks (24). At a similar Cu intake (1.6 ~g/g) to that study, Lin and Lei (27) also failed to show an increase in c h o l e s t e r o l after 8 weeks on diet but by 18 weeks there was a marginal but s i g n i f i c a n t h y p e r c h o l e s t e r o l a e m i a . Halving the Cu in the diet (28) p r o d u c e d a s i g n i f i c a n t increase in 7 weeks. It is t h e r e f o r e r e a s o n a b l e to suggest, that had the e x p e r i m e n t of Caster and D o s t e r (24) c o n t i n u e d for a longer time, there may have been a s u b s e q u e n t increase in plasma c h o l e s t e r o l . G e i g e r et al (67) using diets based on a p r e v i o u s study (4) s h o w e d a d e c r e a s e in h a e m a t o c r i t but ne effect on plasma c h o l e s t e r o l . However, the Cu and Zn were a d d e d to the water as Zn a c e t a t e (10 ~g/ml) and Cu s u l p h a t e (2~g/ml) r e s u l t i n g in only 3,5 ~g Zn and 0,5 ~g as c o m p a r e d to the levels used by K l e v a y (4), (10 and 2 ~g r e s p e c t i v e l y ) . Other s t u d i e s (20,21,28) using this level of Cu intake and for s i m i l a r p e r i o d s (40 days) did p r o d u c e raised p l a s m a lipid levels r e l a t i v e to their r e s p e c t i v e control diets. It is p o s s i b l e that the lack of effect in this study may have been due to the low Cu intake in both control and treated g r o u p s such that both were mildly h y p e r c h o l e s t e r o l a e m i c and no d i f f e r e n t i a l effect was seen. A longer e x p e r i m e n t a l period or a h i g h e r Cu intake in the control diet may have shown a response. S h e r m a n (68) fed copper d e f i c i e n t d i e t s (Cu = Iug/g) to rats for a p e r i o d of 18 weeks. From the r e s u l t a n t d e c r e a s e in c e r u l o p l a s m i n and liver copper c o n c e n t r a t i o n , the a u t h o r c o n c l u d e d that the rats were copper depleted. D e s p i t e this, there was not effect on p l a s m a cholesterol. Female r a t h e r than male rats were used for this study however. A n o t h e r n e g a t i v e study (19), using a diet c o n t a i n i n g a s i m i l a r a m o u n t of Cu (1.5 ~g/g) could not d e m o n s t r a t e a h y p e r c h o l e s t e r o l a e m i a after 15 weeks on diet. They used Wistar, r a t h e r than S p r a g u e Dawley rats and the d i e t s d i f f e r e d in fat content and type of p r o t e i n and c a r b o h y d r a t e s (Table i) from those used in the other s t u d i e s (27,28). Diet
interactions
These diet i n t e r a c t i o n s may well i n f l u e n c e the copper effect. It is well e s t a b l i s h e d that the source of p r o t e i n has a s i g n i f i c a n t effect on p l a s m a c h o l e s t e r o l in rats (32,33) and other animals (34,35). This could be due to the amino acid c o m p o s i t i o n of the p r o t e i n (36-38) or the n o n - p r o t e i n c o m p o n e n t s of the p r o t e i n source (39) in p a r t i c u l a r the mineral i n t e r a c t i o n s (40-42). Other d i e t a r y c o m p o n e n t s such as the type of c a r b o h y d r a t e have been shown to affect. the c h o l e s t e r o l r e s p o n s e (43) and g l u c o s e and insulin r e s p o n s e s p a r t i c u l a r l y in Cu d e f i c i e n t rats (44).
(45).
The effect of fat on c h o l e s t e r o l m e t a b o l i s m is well e s t a b l i s h e d D i e t a r y fat content, in p a r t i c u l a r the s a t u r a t e d fat, will
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COPPER AND CHOLESTEROL METABOLISM
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1026
S. SAMMANand D.C.K. ROBERTS
d e t e r m i n e how q u i c k l y h y p e r c h o l e s t e r o l a e m i a is e s t a b l i s h e d (46). As a result of d i f f e r e n c e s in d i e t a r y fat b e t w e e n e x p e r i m e n t s , the time taken to d e v e l o p h y p e r c h o l e s t e r o l a e m i a at v a r i o u s levels of copper will vary. This undoubtedly c o n t r i b u t e s to some of the r e p o r t e d tlme d i f f e r e n c e s in response. It is e v i d e n t that the source of the m a c r o n u t r i e n t s must also be c o n t r o l l e d in i n v e s t i g a t i o n s of this nature. Not only have the m a c r o n u t r i e n t s varied in these s t u d i e s but so have the salt mixes. These d i f f e r in a b s o l u t e amount as well as r a t i o s of minerals. S t u d i e s in a n i m a l s have shown that copper a b s o r p t i o n can be i n h i b i t e d by high c o n c e n t r a t i o n s of zinc (47,48) and other ions with s i m i l a r c h a r a c t e r i s t i c s (49,50) thus the v a r i a t i o n s in a m o u n t s and ratios add a further c o n f o u n d i n g factor to the p o s s i b i l i t y of u n i f i e d results. Cholesterol
response
at
lower Cu
intakes
D e s p i t e this, the r e m a i n i n g papers, even t h o u g h d i f f e r i n g in type and c o n t e n t of major n u t r i e n t s (Table I), r e p o r t i n c r e a s e s in plasma c h o l e s t e r o l with r e s t r i c t e d Cu intake. There is a trend for the g r e a t e r i n c r e a s e s to o c c u r at lower copper intakes and/or longer time on diet. In 6 r e p o r t s (one set of data p r e s e n t e d twice: 16,17) s i g n i f i c a n t i n c r e a s e s after 3 to 26 weeks were seen in a n i m a l s fed diets containing 0.5-0.6 ~g Cu/g ( 1 6 - 1 8 , 2 0 , 2 1 , 2 5 ) . At h i g h e r Cu intakes (2~g/g) p l a s m a c h o l e s t e r o l v a r i e d with time on diet and other m a c r o n u t r i e n t changes, In the a b s e n c e of fibre, there was only a marginal increase in p l a s m a c h o l e s t e r o l after 8 weeks (25) and i n c r e a s i n g the fibre c o n t e n t of the diet reduced the degree of h y p e r c h o l e s t e r o l a e m i a at 9 weeks (22). C h o l e s t e r o l feeding (2% w/w) a b o l i s h e d the d i f f e r e n t i a l effect of Copper (23), Thus at this level of Cu intake (2 #g/g) any increase in plasma c h o l e s t e r o l is much less m a r k e d and can be i n f l u e n c e d by other d i e t a r y variables. It is t h e r e f o r e p o s s i b l e that the Zn/Cu r a t i o may now become i m p o r t a n t (51). S i n t h u s e k et al (69) using d i e t s c o n t a i n i n g d i f f e r e n t fats, corr. oil or c o c o n u t oil, showed that p l a s m a c h o l e s t e r o l rose as the Zn/Cu ratio increased from 1.25 to 20. However, at equal ratios the diet with the least copper (1.5 .~g/g) was the most h y p e r c h o l e s t e r o l a e m i c when corn oil was the fat used. With coconut oil in the diet, a Zn/Cu ratio os 5 p r o d u c e d the h i g h e s t plasma cholesterol, but plasma c h o l e s t e r o l was not d i f f e r e n t at r a t i o s of 1.25 and 20. Thus in thi~ study, feeding corn oil to rats and m a n i p u l a t i n g the Zn/Cu ratio, p r o d u c e d a h i g h e r p l a s m a c h o l e s t e r o l than when c o c o n u t oil was used. K l e v a y (4), a n a l y s i n g his data in a s l i g h t l y d i f f e r e n t way (number of days on diet before the g r o u p s were first s i g n i f i c a n t l y d i f f e r e n t ) showed that i n c r e a s i n g the ratio of Zn/Cu in the water from 5:1 to 40:1 r e s u l t e d in an a d d i t i o n a l increase in plasma cholesterol. It is also c o n s i s t e n t with his data to say that the rats o f f e r e d the least copper (0.25~g/ml and 0.5 ~g/ml) p r o d u c e d a g r e a t e r h y p e r c h o l e s t e r o l a e m i a than their r e s p e c t i v e c o n t r o l s (2~g Cu/ml~. Two f u r t h e r s t u d i e s ( 1 3 , 1 5 ) have s h o w n that at v a r y i n g r a t i o s of Zn/Cu (0.16 to 160:1) in the water, the g r e a t e r i n c r e a s e s in plasma
COPPER AND CHOLESTEROL METABOLISM
1027
c h o l e s t e r o l were seen at the lower copper intakes. Thus the major h y p e r c h o l e s t e r o l a e m i c effect in rats seems to be caused by copper d e f i c i e n c y r a t h e r than by a l t e r e d Zn/Cu ratio. Studies
in other
species
There have been s t u d i e s in other species. In mice fed diets containing 0.410 ~g/ml Cu, p l a s m a c h o l e s t e r o l did not change (70). In c h i c k e n s (29) there was no effect on p l a s m a c h o l e s t e r o l of diets c o n t a i n i n g 6 to 60 ~g Cu/g and r a t i o s of Zn/Cu of I to 20:1. However, the lowest level of Cu o f f e r r e d was 3 times h i g h e r than the amount r e q u i r e d to p r o d u c e a marginal e f f e c t in rats. One study in plgs (30) at a constant Cu intake (i.% ~g/g) d e m o n s t r a t e d a fall in c h o l e s t e r o l level when the d i e t a r y Zn content was r e d u c e d to 7.2 ~g/g (i.e. the Zn/Cu ratio d e c r e a s e d from 67:1 to 4:1). In a second study in pigs (31) using Zn/Cu r a t i o s of 0.5:1 to 73:1 there was no c o n s i s t e n t effect on plasma c h o l e s t e r o l and none of the a n i m a l s became hypercholesterolaemic. However, again the lowest level of d i e t a r y Cu (8 ~g/g) was 4 times that r e q u i r e d to show a h y p e r c h o l e s t e r o l a e m i a iT. rats. STUDIES
OF
THE
MECHANISM
BY
WHICH
HYPERCHOLESTEROLAEMIA D e s p i t e the v a r i a t i o n s observed, to i n v e s t i g a t e the m e c h a n i s m by w h i c h induces hypercholesterolaemia.
CU
DEFICIENCY
PRODUCES
IN RATS
some r e s e a r c h e r s have a t t e m p t e d copper d e f i c i e n c y in rats
Several s t u d i e s have been c a r r i e d out to d e t e r m i n e the effect of copper d e f i c i e n c y on c h o l e s t e r o l turnover. It was found that newly s y n t h e s i z e d c h o l e s t e r o l (25,16) a c c u m u l a t e s in the plasma of copper d e f i c i e n t rats but that d e g r a d a t i o n is not a f f e c t e d (23). This data implies that copper d e f i c i e n c y has caused a shift of c h o l e s t e r e l from the liver to the serum pool and this is r e s p o n s i b l e for the hypercholesterolaemia. The e f f e c t s of copper on the major enzymes of l i p o p r o t e i n m e t a b o l i s m have also been studied. L e c i t h i n c h o l e s t e r o l acyl t r a n s f e r a s e , LCAT, (E.C. 2.3.1.43) is p r o d u c e d in the liver and was shown to be s t i m u l a t e d in v i t r o by copper (52). However, the nature of the b i o l o g i c a l l y a c t i v e cofactor is yet to be d e t e r m i n e d but it could be the i n t e r a c t i o n b e t w e e n c o p p e r - a l b u m i n and the enzyme (53). In rive s t u d i e s have shown that LCAT a c t i v i t y is r e d u c e d in copper d e f i c i e n c y (20,26). In one instance, (26), liver copper was r e d u c e d by 73% as c o m p a r e d to the control group. However, in both cases there was no a t t e m p t to c o r r e l a t e liver copper and LCAT activity. P l a s m a t r i a c y l g l y c e r o l c o n c e n t r a t i o n has been n e g a t i v e l y c o r r e l a t e d with both p l a s m a and liver c o n c e n t r a t i o n s of copper ~15). This may be due to a n o t h e r major enzyme involved in l i p o p r o t e i n m e t a b o l i s m , l i p o p r o t e i n lipase (LPL), (E.C. 3.1.1.34). LPL was a s s a y e d in vitro and a c t i v i t y was found to be r e d u c e d by 40-47% in copper d e f i c i e n c y (21). However, the a p p a r e n t d e c r e a s e in enzyme a c t i v i t y may result from the d i l u t i o n effect on both e n z y m e s and their
1028
S. SAMMANand D.C.K. ROBERTS
c o f a c t o r s caused by the r e d u c e d h a e m a t o c r i t and hence volume o b s e r v e d in copper d e f i c i e n t r a t s (28). EFFECT
OF COPPER
ON P L A S M A
LIPIDS
increased
plasma
IN HUMANS
S t u d i e s in h u m a n s have been few and g e n e r a l l y indirect involving r e l a t i o n s h i p s b e t w e e n p l a s m a Zn/Cu and c h o l e s t e r o l rather than d i e t a r y Zn/Cu. In a d i a b e t i c group, plasma Zn/Cu was shown to be p o s i t i v e l y c o r r e l a t e d with p l a s m a c h o l e s t e r o l c o n c e n t r a t i o n (54) but in a nond i a b e t i c h y p e r l i p i d a e m i c p o p u l a t i o n there was no such r e l a t i o n s h i p (55). In a g r o u p of u r a e m i c p a t i e n t s or. h a e m o d i a l y s i s , plasma Zn/Cu ratio was lower than in c o n t r o l s and p l a s m a HDL c h o l e s t e r o l was also lower (56). Thus the r e l a t i o n s h i p seen in rats b e t w e e n plasma Zn/Cu ratio and c h o l e s t e r o l c o n c e n t r a t i o n (57) is at best equivocal in man. With one exception, the effect of d i e t a r y change has been on zinc s u p p l e m e n t a t i o n r a t h e r than Cu d e f i c i e n c y . In h e a l t h y males an oral zinc s u p p l e m e n t of 29 mg/day had no e f f e c t on p l a s m a c h o l e s t e r o l (58). H o w e v e r 160 m g / d a y over 5 weeks r e d u c e d HDL c h o l e s t e r o l (59) and larger s u p p l e m e n t s (300 mg/day) r e d u c e d HDL after 4 weeks and i n c r e a s e d LDL a f t e r 6 weeks (71). In females t a k i n g 100 mg/day a nons i g n i f i c a n t h y p o c h o l e s t e r o l a e m i c trend was o b s e r v e d as the d i e t a r y ZD/Cu ratio i n c r e a s e d (72) and a t r a n s i e n t fall in HDL c h o l e s t e r o l was r e p o r t e d which was no longer d i f f e r e n t from control by 8 weeks (60). This a p p a r e n t sex d i f f e r e n c e in r e s p o n s e to d i e t a r y zinc (59,60) was also r e p o r t e d for a g r o u p of 75 h e a l t h y s u b ] e c t s c o n s u m i n g their normal d!et. In males, there was a n e g a t i v e c o r r e l a t i o n b e t w e e n dietary zinc Intake and plasma HDL chc,iester~i c o r ~ c e n t ~ i : r ~ but no r e l a t i o n s h i p in females. P l a s m a total c h o l e s t e r o l was not r e l a t e d t:} dietary zinc or Cu intake (6]). A single study (in a b s t r a c t form, 62) r e p o r t e d that p l a s m a total c h o l e s t e r o l increased after iS weeks in a h e a l t h y male when Cu intake was 1.3 mg/day. R e p l e t i o n with an extra 2 m g / d a y caused the p l a s m a c h o l e s t e r o l to fall. Thus, in humans, the r e s u l t s are less than convinclng, not w i t h s t a n d i n g the e p i d e m i o l o g i c a l a s s o c i a t i o n s w h i c h have been d e m o n s t r a t e d (4,63). On the evidence, any effect of dietary Zn/Cu would a p p e a r to be marginal unless very low intakes of copper or large s u p p l e m e n t s of zinc are given. The effect is also on males and not females. It is i n t e r e s t i n g to note that all the s t u d i e s in rats have been on males.
CONCLUSION Before a s s o c i a t i n g d i e t a r y copper intake with lipid metabolism: two i m p o r t a n t factors must be considered. Firstly the b i o a v a i l a b i l i t y of copper from the small i n t e s t i n e must be taken into account. Thls will involve a g r e e m e n t upon a s t a n d a r d e • diet which will s t a n d a r d i s e the response. Secondly, more s y s t e m a t i c studies have to be carried out to d e t e r m i n e w h e t h e r a l t e r a t i o n s to the enzymes of lipid m e t a b o l i s m are not just part of a general effect of copper d e f i c i e n c y on the many copper c o n t a i n i n g e n z y m e s (64).
COPPER AND CHOLESTEROL METABOLISM
1029
In animals the balance of evidence points to an effect of copper d e f i c i e n c y and only in male rats, although further study in female rats may revise this conclusion. In humans, zinc s u p p l e m e n t a t i o n at pharmacological levels rather than copper d e f i c i e n c y appears to reduce plasma HDL cholesterol but again only in males. Copper d e f i c i e n c y in humans is rarely found (65). It is a c o m p l i c a t i o n of other disease states (6) and occurs s i m u l t a n e o u s l z with iron d e f i c i e n c y (66). I n view of this and of the lack of systematic studies in humans, the role of copper in cholesterol m e t a b o l i s m r e m a i n s c o n t r o v e r s i a l and the Z n / C u h y p o t h e s i s remains to be validated.
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Accepted for publication July 11, 1985.
of zinc
impairs
immune responses.