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score of aortic atherosclerotic lesions (0-4 scale); soy isolate (L/A-0.9) gave ... atherosclerosis were performed by Ignatowski (1_) who fed rabbits ...
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13 Influence of Animal and Vegetable Protein on Serum Cholesterol, Lipoproteins, and Experimental Atherosclerosis David M . Klurfeld and David Kritchevsky The Wistar Institute of Anatomy and Biology, Philadelphia, PA 19104

One distinguishing characteristic between protein of animal and vegetable origin is the ratio of lysine to arginine (L/A). Feeding casein (L/A-1.9) to rabbits gave cholesterol levels of 268 mg/dl and 1.34 average score of aortic atherosclerotic lesions (0-4 scale); soy isolate (L/A-0.9) gave 145 mg/dl cholesterol and 0.55 lesions. Addition of lysine to soy to give L/A ratio of casein raised lipids and lesions significantly. Addition of arginine to casein had a slight lowering effect on lipids and atheroma. Feeding three animal proteins of varying L/A ratios (fish, 1.4; casein, 1.9; milk, 2.4) resulted in atherosclerosis significantly correlated (r = 0.99) with L/A ratio. Feeding beef or textured vegetable protein (TVP) yielded significantly higher serum cholesterol and atherosclerosis in the group fed beef; however, an equal mixture of beef:TVP gave results similar to TVP alone. Animal proteins are more atherogenic than those from vegetable sources and the L/A ratio may give an index of magnitude of effect. When animal protein is part of a mixed diet, the atherogenic effect is diminished or abrogated. The f i r s t experimental studies on n u t r i t i o n a l induction of atherosclerosis were performed by Ignatowski (1_) who fed rabbits milk, beef, and egg yolk. These experiments were interpreted as indicating that the animal foods consumed by the wealthy contributed to atherosclerotic heart disease which was observed only among the upper class at that time. However, Anitschkow and Chalatow (2) published studies shortly thereafter in which they fed p u r i f i e d cholesterol to rabbits and achieved the same results which led them to conclude that the sterol present in foods of animal o r i g i n , was the predominant factor in genesis of the a r t e r i a l lesions. This finding has had lasting and s i g n i f i c a n t influence on the d i r e c t i o n of research about atherogenesis. The e a r l i e s t comparison of animal and plant proteins in a model of experimental atherogenesis showed that feeding casein led to more atherosclerosis than feeding soy 0097-6156/86/0312-O150$06.00/0 © 1986 American Chemical Society

13.

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151

r o t e i n whether or not c h o l e s t e r o l was added to the d i e t s (_3) . i n c e the p u b l i c a t i o n of t h i s experiment over 45 y e a r s ago, t h e r e has been a r e c e n t r e s u r g e n c e of i n t e r e s t in the a r e a of p r o t e i n e f f e c t s on plasma l i p i d s and a t h e r o s c l e r o s i s . T h i s paper summarizes some of the d a t a g e n e r a t e d in our l a b o r a t o r y over the l a s t s e v e r a l y e a r s and d i s c u s s e s t h e i r s i g n i f i c a n c e in r e l a t i o n to h y p e r c h o l e s t e r o l e m i a and a t h e r o s c l e r o s i s in o t h e r s p e c i e s i n c l u d i n g humans. The s t a n d a r d d i e t used in our experiments is a s e m i p u r i f i e d , c h o l e s t e r o l - f r e e p r e p a r a t i o n that is composed o f 25% p r o t e i n , 40% s u c r o s e , 13% coconut o i l , 1% c o r n o i l , 15% c e l l u l o s e , 5% m i n e r a l mix, and 1% v i t a m i n mix. T h i s d i e t has been shown to induce an endogenous h y p e r c h o l e s t e r o l e m i a and l e a d to a t h e r o s c l e r o s i s in r a b b i t s and monkeys (4,_5). The s p e c i f i c q u e s t i o n a d d r e s s e d by our s e r i e s of i n v e s t i g a t i o n s is whether the type of d i e t a r y p r o t e i n , when a l l o t h e r d i e t a r y components are c o n s t a n t , can i n f l u e n c e the development of h y p e r l i p o p r o t e i n e m i a and a t h e r o s c l e r o s i s . More s p e c i f i c a l l y , we have examined the e f f e c t s of the i n d i v i d u a l amino a c i d s , l y s i n e and a r g i n i n e , and t h e i r r a t i o s in the d i e t on plasma and h e p a t i c l i p i d s as w e l l as the development of a r t e r i a l p l a q u e s . To study amino a c i d e f f e c t s , we have added s i n g l e amino a c i d s to the d i e t s to approximate the l y s i n e / a r g i n i n e (L/A) r a t i o of d i f f e r e n t proteins. For example, the L/A r a t i o of c a s e i n is about 2.0; t h e r e f o r e , enough l y s i n e was added to the d i e t c o n t a i n i n g soy p r o t e i n i s o l a t e to b r i n g i t s endogenous L/A r a t i o of O.9 up to t h a t of c a s e i n . S i m i l a r m a n i p u l a t i o n s were performed w i t h a d d i t i o n of a r g i n i n e to c a s e i n to g i v e the L/A r a t i o n o r m a l l y found in soy protein. Four e x p e r i m e n t a l d i e t s based upon t h a t d e s c r i b e d above were f e d to r a b b i t s f o r e i g h t to ten months in t h r e e s e p a r a t e experiments. The r e s u l t s are summarized in T a b l e I . Serum c h o l e s t e r o l c o n c e n t r a t i o n s in r a b b i t s f e d c a s e i n were s i g n i f i c a n t l y e l e v a t e d o v e r those observed w i t h soy p r o t e i n f e e d i n g . A d d i t i o n of a r g i n i n e to c a s e i n had i n c o n s i s t e n t e f f e c t s on serum c h o l e s t e r o l l e v e l s ; a d d i t i o n of l y s i n e to soy p r o t e i n u s u a l l y r a i s e d the c h o l e s t e r o l l e v e l s above those found by f e e d i n g soy a l o n e . Serum t r i g l y c e r i d e s were s i g n i f i c a n t l y lower in the animals fed soy than in the r a b b i t s fed c a s e i n . A d d i t i o n of the i n d i v i d u a l amino a c i d s to the p r o t e i n s had no c o n s i s t e n t s i g n i f i c a n t e f f e c t s on triglycerides. The d a t a f o r a o r t i c a t h e r o s c l e r o s i s are e x p r e s s e d on a 0 to 4 s c a l e w i t h 4 d e n o t i n g complete coverage of the i n t i m a i surface with l i p i d - c o n t a i n i n g l e s i o n s . T a b l e I shows t h a t f e e d i n g o f soy p r o t e i n l e a d s to l e s s than h a l f the a o r t i c a t h e r o s c l e r o s i s found when c a s e i n is the p r o t e i n . A d d i t i o n of a r g i n i n e to c a s e i n r e d u c e s the s e v e r i t y of a o r t i c a t h e r o s l e r o s i s by about 25% w h i l e a d d i t i o n of l y s i n e to soy p r o t e i n i n c r e a s e d the s e v e r i t y of a t h e r o s c l e r o s i s in the a o r t i c a r c h by 57% and t h a t in the t h o r a c i c a o r t a by 75%. Serum samples from some animals fed the four d i e t a r y regimens were p o o l e d and s u b j e c t e d to f r a c t i o n a t i o n of the l i p o p r o t e i n s by a g a r o s e column chromatography. The t o t a l amounts of l i p o p r o t e i n s found in the s e r a from the four groups (ug/ml) were: casein, 904; soy p r o t e i n , 807; c a s e i n / a r g i n i n e , 1,130; and, s o y / l y s i n e , 672. T a b l e I I g i v e s the d i s t r i b u t i o n of l i p o p r o t e i n s in the v a r i o u s d e n s i t y c l a s s e s from these a n i m a l s . S i n c e the t o t a l amounts of

152

PLANT PROTEINS

l i p o p r o t e i n s in the s e r a o f r a b b i t s f e d the d i e t s c o n t a i n i n g d i f f e r e n t p r o t e i n s was so v a r i e d , a comparison of the p e r c e n t a g e d i s t r i b u t i o n o f the l i p o p r o t e i n c l a s s e s is more a p p r o p r i a t e than simply comparing a b s o l u t e amounts. The p e r c e n t a g e s o f v e r y low d e n s i t y (VLDL) and i n t e r m e d i a t e d e n s i t y l i p o p r o t e i n s (IDL) were s i m i l a r in the animals f e d the c a s e i n and s o y / l y s i n e d i e t s and were double the p e r c e n t a g e of l i p o p r o t e i n in these two d e n s i t y c l a s s e s found in the s e r a of animals f e d soy p r o t e i n or c a s e i n / a r g i n i n e d i e t s . The p e r c e n t a g e s o f low d e n s i t y l i p o p r o t e i n s (LDL) were s i m i l a r in a l l groups and the p e r c e n t a g e o f h i g h d e n s i t y l i p o p r o t e i n (HDL) was h i g h e s t in the a n i m a l s f e d soy p r o t e i n .

T a b l e I . Serum L i p i d s and A o r t i c A t h e r o s c l e r o s i s in R a b b i t s f e d C a s e i n , Soy P r o t e i n , C a s e i n & A r g i n i n e , o r Soy & L y s i n e Serum L i p i d s

Atherosclerosis

(mg/dl)~

Number of Thoracic Experiment 1 Casein Soy p r o t e i n Cas/arginine Soy/lysine

7 7 6 6

174 77 129 106

+ + + +

30 21 12 29

133 98 186 101

+ + + +

17 17 20 14

2.2 1.1 1.4 1.6

+ + + +

O.5 O.4 O.4 O.4

1.5 O.7 O.8 1.1

+ + + +

O.4 O.3 O.3 O.2

Experiment 2 Casein 8 Soy p r o t e i n 11 Cas/arginine 7 Soy/lysine 11

283 234 343 242

+ + + +

28 20 65 22

81 53 117 70

+ 11 + 7 + 58 + 7

1.1 O.5 1.3 O.9

+ + + +

O.3 O.2 O.4 O.3

O.8 O.4 O.7 O.5

+ + + +

O.3 O.1 O.2 O.2

Experiment 3 Casein Soy p r o t e i n Cas/arginine Soy/lysine

377 117 271 242

+ + + +

59 10 74 43

104 43 75 60

+ 28 + 4 + 13 + 5

1.6 O.6 1.2 O.5

+ + + +

O.2 O.2 O.2 O.2

O.9 O.1 1.3 O.7

+ + + +

O.3 O.1 O.2 O.1

5 7 7 8

T a b l e I I . P e r c e n t a g e D i s t r i b u t i o n of L i p o p r o t e i n s fed Various P r o t e i n s

Treatment Casein Soy p r o t e i n Cas/arginine Soy/lysine

from R a b b i t s

VLDL

IDL

LDL

HDL

2.3 1.1 O.9 2.2

14.4 7.7 7.8 17.8

31.9 30.0 35.8 30.2

51.4 61.2 55.5 49.9

A n o t h e r experiment compared the e f f e c t s of f e e d i n g p a r t i a l l y d e f a t t e d beef w i t h t e x t u r e d v e g e t a b l e p r o t e i n (TVP) d e r i v e d from soy

13.

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153

beans. In t h i s experiment, the f a t was b e e f t a l l o w and i t was added so t h a t a l l groups had a t o t a l of 14% f a t in the d i e t . T h i s a l l o w e d compensation f o r the f a t c o n t e n t of the p a r t i a l l y d e f a t t e d beef so t h a t o n l y 9.3% t a l l o w was added to the b e e f d i e t . S i m i l a r l y , c a r b o h y d r a t e and crude f i b e r were a d j u s t e d in the d i e t s c o n t a i n i n g TVP so t h a t each group consumed 40% d i g e s t i b l e c a r b o h y d r a t e and 15% crude f i b e r . In a d d i t i o n to the groups fed beef and TVP, a t h i r d group was fed an e q u a l m i x t u r e of the two p r o t e i n s o u r c e s . The d i e t s were fed to groups of twelve r a b b i t s f o r e i g h t months. At the end o f the study, the animals were k i l l e d and serum and h e p a t i c l i p i d s as w e l l as a o r t i c a t h e r o s c l e r o s i s were q u a n t i t a t e d . Table I I I g i v e s some of the d a t a from t h i s experiment.

Table I I I .

Serum L i p i d s and A o r t i c A t h e r o s c l e r o s i s in Beef, TVP, o r an E q u a l Mix o f Beef & TVP Beef

Serum l i p i d s (mg/dl) Cholesterol % HDL-cholesterol Triglycerides

185 20 60

Aortic atherosclerosis Arch 1.3 Thoracic O.8

+ 24 + 2 + 8

+ O.2 Τ O.1

TVP

Rabbits

fed

Beef-TVP

37 39 59

+ + +

4 4 7

O.8 O.2

+ +

O.1 O.1

6 61 + 4 43 + 70 + 13

O.7 O.4

+ O.1 + O.1

S t a t i s t i c a l l y s i g n i f i c a n t d i f f e r e n c e s were observed in serum t o t a l c h o l e s t e r o l , H D L - c h o l e s t e r o l , and a o r t i c a t h e r o s c l e r o s i s . Serum t r i g l y c e r i d e s were u n a f f e c t e d by d i e t a r y p r o t e i n . Textured v e g e t a b l e p r o t e i n as the s o l e d i e t a r y p r o t e i n s o u r c e r e s u l t e d in o n e - f i f t h the amount of serum c h o l e s t e r o l and about h a l f the a t h e r o s c l e r o s i s in the animals fed b e e f . Most i m p o r t a n t l y , the a n i m a l s fed an e q u a l mix o f b e e f and TVP had serum c h o l e s t e r o l l e v e l s and a t h e r o s c l e r o s i s almost e q u a l to those seen w i t h pure vegetable protein. H D L - c h o l e s t e r o l c o n c e n t r a t i o n s were s i m i l a r in the a l l TVP and beef-TVP d i e t a r y groups which were double t h a t seen in the a l l b e e f group. The r e s u l t s o b t a i n e d f o r serum l i p i d s and a t h e r o s c l e r o s i s w i t h f e e d i n g beef were almost i d e n t i c a l to a group f e d c a s e i n c o n c o m i t a n t l y as the s o l e p r o t e i n s o u r c e (6). In t h i s p a r t i c u l a r experiment, a s m a l l amount of c h o l e s t e r o l was consumed w i t h the b e e f and t a l l o w . However, due to the d e s i g n of the d i e t s which made up f o r the endogenous f a t c o n t e n t of the b e e f , the c h o l e s t e r o l i n t a k e (10-12 mg/day) was s i m i l a r in a l l groups. F u r t h e r m o r e , a t t h i s l e v e l of c h o l e s t e r o l i n t a k e l i t t l e or no e f f e c t would be expected on plasma l i p i d s and the development of atherosclerosis (3). An i m p o r t a n t p o i n t r e l e v a n t to the above r e s u l t s is t h a t the diet was i d e n t i c a l f o r a l l groups w i t h i n a g i v e n experiment except for p r o t e i n source. T h i s is n e c e s s a r y to d i s t i n g u i s h s p e c i f i c n u t r i e n t e f f e c t s but does not r e f l e c t a c t u a l food consumption s i n c e i n t a k e of animal p r o t e i n is a l s o a s s o c i a t e d w i t h i n c r e a s e d ingestion of t o t a l f a t , s a t u r a t e d f a t , and c h o l e s t e r o l . T h e r e f o r e , i t is o n l y

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through m a n i p u l a t i o n of d i e t s of e x p e r i m e n t a l animals t h a t the q u e s t i o n of p r o t e i n e f f e c t s on l i p i d s and a t h e r o s c l e r o s i s can be a d d r e s s e d in an adequate manner. I t is d i f f i c u l t to e x t r a p o l a t e f i n d i n g s in r a b b i t s to humans but o t h e r i n v e s t i g a t o r s have c o n f i r m e d our r e s u l t s in s e v e r a l animals s p e c i e s . S t u d i e s w i t h humans fed soy p r o t e i n have a l s o been r e p o r t e d but these have been l e s s c o n s i s t e n t than the animal s t u d i e s f o r reasons t h a t w i l l be d i s c u s s e d below. Kim e t a l (_7) f e d swine d i e t s c o n t a i n i n g e i t h e r c a s e i n or soy protein. These d i e t s resembled the t y p i c a l American d i e t in t h a t they d e r i v e d about 42% o f c a l o r i e s from f a t and 21% o f c a l o r i e s from p r o t e i n w i t h the remainder from c a r b o h y d r a t e . In these t e s t d i e t s , 90% o f the f a t was b u t t e r and the r e s t was c o r n o i l . In a d d i t i o n , c r y s t a l l i n e c h o l e s t e r o l was added to b r i n g t o t a l c h o l e s t e r o l in each d i e t to 1055 mg per day. These i n v e s t i g a t o r s a l s o fed one group of swine a d i e t w i t h e q u a l p a r t s of c a s e i n and soy. F e e d i n g these d i e t s f o r four or s i x weeks l e d to a d o u b l i n g of serum c h o l e s t e r o l c o n c e n t r a t i o n s when the p r o t e i n was c a s e i n but only a s m a l l n o n s i g n i f i c a n t r i s e when soy p r o t e i n or the m i x t u r e of soy and c a s e i n was f e d . In a d d i t i o n , these r e s e a r c h e r s found t h a t s u b s t i t u t i o n of soy p r o t e i n f o r c a s e i n c o m p l e t e l y i n h i b i t e d the i n c r e a s e in t o t a l body c h o l e s t e r o l t h a t they had demonstrated in swine fed a c a s e i n - b a s e d d i e t . F u r t h e r s t u d i e s by t h i s group of i n v e s t i g a t o r s r e v e a l e d t h a t the h y p o c h o l e s t e r o l e m i c e f f e c t of soy p r o t e i n was e v i d e n t in swine o n l y when they were fed a hypercholesterolemic d i e t (8). I n c l u s i o n of soy p r o t e i n in the basal hypercholesterolemic d i e t r e s u l t e d in s i g n i f i c a n t i n c r e a s e s in f e c a l e x c r e t i o n of n e u t r a l and a c i d i c s t e r o i d s that was not accompanied by i n c r e a s e s in c h o l e s t e r o l s y n t h e s i s . The d i f f e r e n c e between soy and c a s e i n was not observed when i n c l u d e d in a b a s a l mash d i e t , i n d i c a t i n g t h a t t h i s d i e t a r y e f f e c t in swine was i m p o r t a n t o n l y under c o n d i t i o n s f a v o r a b l e to net c h o l e s t e r o l retention. S e m i p u r i f i e d d i e t s whose b a s i c c o m p o s i t i o n was s i m i l a r to d i e t s consumed in Western c o u n t r i e s were fed to cynomolgus monkeys; the d i f f e r e n c e s between the groups were t h a t one d i e t c o n t a i n e d c a s e i n and the o t h e r c o n t a i n e d soy p r o t e i n i s o l a t e (9). In a c r o s s o v e r experiment, soy p r o t e i n was exchanged f o r c a s e i n and v i c e v e r s a f o r three-week p e r i o d s . A l t h o u g h t h e r e were no s i g n i f i c a n t d i f f e r e n c e s in t o t a l serum c h o l e s t e r o l c o n c e n t r a t i o n s , b e n e f i c i a l e f f e c t s on i n d i v i d u a l l i p o p r o t e i n c l a s s e s were demonstrated f o l l o w i n g f e e d i n g of the soy p r o t e i n d i e t . HDL c h o l e s t e r o l l e v e l s were s i g n i f i c a n t l y h i g h e r and VLDL c h o l e s t e r o l l e v e l s were s i g n i f i c a n t l y lower when soy p r o t e i n r e p l a c e d c a s e i n . S i n c e most s t u d i e s c o n c e r n i n g a l t e r a t i o n s in l i p i d m e t a b o l i s m among a n i m a l s fed d i f f e r e n t types of p r o t e i n have been performed u s i n g r o d e n t s , the remainder of t h i s paper w i l l c o n c e n t r a t e on those findings. C a r r o l l and h i s a s s o c i a t e s have i n v e s t i g a t e d t h i s a r e a extensively. H a m i l t o n and C a r r o l l (_10) surveyed a v a r i e t y of animal and p l a n t p r o t e i n s f o r t h e i r e f f e c t s on c h o l e s t e r o l l e v e l s in r a b b i t s fed a c h o l e s t e r o l - f r e e , s e m i p u r i f i e d d i e t . These p r o t e i n s were d e l i p i d i z e d by s o l v e n t e x t r a c t i o n and f e d to the animals f o r 28-day p e r i o d s . A l t h o u g h t h e r e was r e l a t i v e l y wide v a r i a t i o n in the r e s p o n s e of serum c h o l e s t e r o l to f e e d i n g of the p r o t e i n s , i n g e s t i o n of animal p r o t e i n s r e s u l t e d in s i g n i f i c a n t l y h i g h e r serum c h o l e s t e r o l c o n c e n t r a t i o n s than d i d consumption of p l a n t p r o t e i n s

13.

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KLURFELD AND KRITCHEVSKY

155

( T a b l e I V ) . H u f f e t a l (11) extended these o b s e r v a t i o n s to i n c l u d e enzymatic h y d r o l y s a t e s of the n a t i v e p r o t e i n s and amino a c i d m i x t u r e s e q u i v a l e n t to soy or c a s e i n . In t h i s experiment amino a c i d c o n t e n t s were i d e n t i c a l among the d i e t s f e d as the t h r e e forms o f each p r o t e i n but the s t r u c t u r e was d i f f e r e n t ( T a b l e V ) .

T a b l e IV.

Serum C h o l e s t e r o l L e v e l s in R a b b i t s Proteins

Protein Animal p r o t e i n Whole egg Skim m i l k Lactalbumin Beef Pork Egg w h i t e

Serum C h o l e s t e r o l 235 230 215 160 110 105

Plant protein Wheat g l u t e n Peanut p r o t e i n Soy p r o t e i n c o n c e n t r a t e Promine-R From: H a m i l t o n and C a r r o l l

80 80 25 15

fed D i f f e r e n t

(mg/dl) + + + + + +

89 40 69 60 17 28

+ 21 + 10 + 5 + 5

(10)

T a b l e V. Serum C h o l e s t e r o l L e v e l s in R a b b i t s f e d C a s e i n , Soy P r o t e i n , T h e i r H y d r o l y s a t e s and T h e i r Amino A c i d M i x t u r e s D i e t a r y treatment Intact casein Enzymatic h y d r o l y s a t e Amino a c i d m i x t u r e I n t a c t soy p r o t e i n Enzymatic h y d r o l y s a t e Amino a c i d m i x t u r e

Serum C h o l e s t e r o l (mg/dl) 213 + 53 178 + 30 213 +_ 42 69 + 12 41 + 8 124 + 30

From: H u f f e t a l (11)

Table VI.

Serum C h o l e s t e r o l

% C a s e i n / % Soy P r o t e i n 100 0 75 25 50 50 0 100 From: H u f f e t a l (11)

L e v e l s in R a b b i t s and C a s e i n

f e d M i x t u r e s o f Soy

Serum C h o l e s t e r o l 239 + 138 + 70 + 67 +

(mg/dl) 28 20 15 12

PLANT PROTEINS

156

While the enzymatic h y d r o l y s a t e o f c a s e i n and i t s e q u i v a l e n t amino a c i d m i x t u r e gave serum c h o l e s t e r o l l e v e l s s i m i l a r t o i n t a c t c a s e i n , such was not the case w i t h f e e d i n g soy p r o t e i n . The enzymatic soy h y d r o l y s a t e was almost e q u i v a l e n t to the i n t a c t p r o t e i n but f e e d i n g the amino a c i d m i x t u r e gave serum c h o l e s t e r o l l e v e l s double t h a t o f the n a t i v e p r o t e i n ( a l t h o u g h s t i l l s i g n i f i c a n t l y lower than f e e d i n g casein). T h i s i n d i c a t e s t h a t the t e r t i a r y s t r u c t u r e o f the p r o t e i n , or some minor n o n - p r o t e i n contaminant, has a s i g n i f i c a n t e f f e c t o f serum c h o l e s t e r o l l e v e l s . The experiment in which d i l u t i o n o f c a s e i n w i t h soy p r o t e i n was s t u d i e d ( T a b l e V I ) showed a r e d u c t i o n in serum c h o l e s t e r o l p r o p o r t i o n a l t o the amount o f soy p r o t e i n in the d i e t up t o 50% which was e q u a l to an a l l soy d i e t . If a c o n t a m i n a t i n g f a c t o r were p r e s e n t , i t would have t o be e x t r a o r d i n a r i l y p o t e n t to e x e r t such an e f f e c t . When the soy and c a s e i n d i e t s were f e d f o r t e n months s i g n i f i c a n t d i f f e r e n c e s in a o r t i c a t h e r o s c l e r o s i s were found ( Γ 2 ) . F e e d i n g c a s e i n r e s u l t e d in average atheroma s c o r e s o f 1.8 + O.3 (0-4 s c a l e ) w h i l e soy p r o t e i n gave a mean v a l u e o f O.2 + O.06, which is s i g n i f i c a n t a t ρ < O.01. T e r p s t r a and h i s a s s o c i a t e s have been a c t i v e in i n v e s t i g a t i n g the e f f e c t s of d i e t a r y p r o t e i n and s i n g l e amino a c i d s on serum c h o l e s t e r o l l e v e l s in s e v e r a l s p e c i e s ( r e v i e w e d in J^3). They c o n f i r m e d t h a t f e e d i n g c a s e i n r e s u l t s in s i g n i f i c a n t l y h i g h e r serum c h o l e s t e r o l c o n c e n t r a t i o n s than does soy p r o t e i n . In t h e i r s t u d i e s , a d d i t i o n o f O.8% a r g i n i n e t o a 20.8% c a s e i n d i e t r a i s e d serum c h o l e s t e r o l o f r a b b i t s , in c o n t r a s t to our f i n d i n g s . A d d i t i o n o f g l y c i n e r e s u l t e d in h a l f the serum c h o l e s t e r o l o b t a i n e d w i t h f e e d i n g casein alone. I n t e r e s t i n g l y , a d i e t c o n t a i n i n g a m i x t u r e o f three animal p r o t e i n s ( 8 % c a s e i n , 5.6% g e l a t i n , and 8% f i s h p r o t e i n ) gave serum c h o l e s t e r o l l e v e l s s l i g h t l y h i g h e r than those found w i t h f e e d i n g soy p r o t e i n and s i g n i f i c a n t l y lower than those observed when c a s e i n was f e d a l o n e . A c h a r a c t e r i s t i c of c a s e i n and o t h e r animal p r o t e i n s t h a t d i s t i n g u i s h e s them from p l a n t p r o t e i n s is the h i g h l y s i n e c o n t e n t . We have demonstrated t h a t a d d i t i o n of l y s i n e to soy p r o t e i n causes a s i g n i f i c a n t i n c r e a s e of serum c h o l e s t e r o l and development o f atherosclerosis. Another approach to t h i s q u e s t i o n was t o feed t h r e e p r o t e i n s of animal o r i g i n t h a t c o n t a i n e d almost i d e n t i c a l amounts o f l y s i n e b u t d i f f e r e n t amounts o f a r g i n i n e . We used f i s h p r o t e i n ( l y s i n e , 6.81; a r g i n i n e , 4.74; L/A r a t i o , 1.44), c a s e i n ( l y s i n e , 6.91; a r g i n i n e , 3.65; L/A r a t i o , 1.89), and m i l k p r o t e i n ( l y s i n e , 6.61; a r g i n i n e , 2.71; L/A r a t i o , 2.44) ( 1 4 ) . The p r o t e i n s Table VII. Serum L i p i d s and A t h e r o s c l e r o s i s in R a b b i t s f e d Three Animal P r o t e i n s w i t h D i f f e r e n t L y s i n e / A r g i n i n e R a t i o s

C h o l e s t e r o l (mg/dl) T r i g l y c e r i d e s (mg/dl) Atherosclerosis Arch Thoracic From: K r i t c h e v s k y

Fish protein 238 + 40 122 + 20

1.6 + O.2 1.0 + O.2 e t a l (14)

Casein 530 + 76 177 + 47

2.1 + O.3 1.1 + O.3

Milk protein 462 + 62 251 + 56

2.6 + O.2 1.6 + O.2

13.

KLURFELD AND KRITCHEVSKY

Influence of Protein on Serum Cholesterol

157

were fed a t 25% in our s t a n d a r d c h o l e s t e r o l - f r e e , s e m i p u r i f i e d d i e t f o r e i g h t months. R e s u l t s from t h i s study are g i v e n in T a b l e V I I . A n a l y s i s of l i n e a r r e g r e s s i o n between a o r t i c a t h e r o s c l e r o s i s and the l y s i n e / a r g i n i n e r a t i o of the three p r o t e i n s y i e l d e d r = O.99 (p < .05). These r e s u l t s i n d i c a t e t h a t not o n l y does the amount of l y s i n e a f f e c t serum c h o l e s t e r o l and a t h e r o s c l e r o s i s but in p r o t e i n s w i t h s i m i l a r l y s i n e c o n t e n t s the p r o p o r t i o n of l y s i n e to a r g i n i n e may be a s i g n i f i c a n t d e t e r m i n a n t of t h e i r e f f e c t s on these parameters. A r g i n i n e has been r e p o r t e d t o be a g l u c a g o n s e c r e t a g o g u e and may have g l u c a g o n - l i k e a c t i v i t y ( 1 5 ) . Sugano et a l (_15) r e p o r t e d t h a t a d d i t i o n of a r g i n i n e to c a s e i n r e s u l t e d in a s i g n i f i c a n t i n c r e a s e in plasma g l u c a g o n l e v e l s in r a t s . In t h i s study, c a s e i n and soy f e e d i n g y i e l d e d s i m i l a r l e v e l s of g l u c a g o n but s i g n i f i c a n t l y lower immunoreactive i n s u l i n c o n c e n t r a t i o n s were found in the r a t s fed soy protein. S i n c e p e p t i d e s formed d u r i n g p r o t e i n d i g e s t i o n can f u n c t i o n as g l u c a g o n s e c r e t a g o g u e s through some mechanism o t h e r than a l t e r a t i o n s of amino a c i d l e v e l s in the plasma ( 1 6 ) , i t is n e c e s s a r y to study d i f f e r e n c e s these p r o t e i n s may e x e r t in the i n t e s t i n e . To t h i s end, we have examined the i n f l u e n c e of c a s e i n and soy p r o t e i n on i n t e s t i n a l a b s o r p t i o n and l y m p h a t i c t r a n s p o r t of c h o l e s t e r o l and o l e i c a c i d (17). A l t h o u g h o v e r a l l r e c o v e r y of c h o l e s t e r o l and o l e i c a c i d was s i m i l a r among a n i m a l s fed d i f f e r e n t p r o t e i n s , a n a l y s i s of timed lymph c o l l e c t i o n s r e v e a l e d t h a t a b s o r p t i o n of l i p i d s was more r a p i d in r a t s fed c a s e i n than in those f e d soy p r o t e i n . F u r t h e r m o r e , a d d i t i o n of l y s i n e to soy p r o t e i n markedly i n c r e a s e d the r a t e of l i p i d a b s o r p t i o n and a d d i t i o n of a r g i n i n e to c a s e i n slowed l i p i d a b s o r p t i o n . The slowed a b s o r p t i o n of l i p i d s in animals fed soy p r o t e i n is s i m i l a r to that r e p o r t e d f o r s o l u b l e f i b e r s such as p e c t i n and guar gum t h a t a c t to lower serum c h o l e s t e r o l c o n c e n t r a t i o n s in a number of animal s p e c i e s , i n c l u d i n g humans. In a d d i t i o n to more r a p i d a b s o r p t i o n of l i p i d s in a n i m a l s fed c a s e i n , a n o t h e r mechanism t h a t may be o p e r a t i v e is d e c r e a s e d c l e a r a n c e of c i r c u l a t i n g l i p i d s . R a b b i t s fed a c a s e i n - b a s e d s e m i p u r i f i e d d i e t e x c r e t e d s i g n i f i c a n t l y l e s s c h o l e s t e r o l but more b i l e a c i d s in t h e i r f e c e s than a n i m a l s f e d a commercial d i e t ( 1 8 ) . The t o t a l s t e r o l e x c r e t i o n in f e c e s of the animals f e d the c a s e i n d i e t was h a l f t h a t of the r a b b i t s fed the s t o c k d i e t . H u f f and C a r r o l l (_19) found t h a t r a b b i t s fed soy p r o t e i n had a much f a s t e r t u r n o v e r r a t e of c h o l e s t e r o l and a s i g n i f i c a n t l y reduced r a p i d l y exchangeable c h o l e s t e r o l p o o l compared w i t h r a b b i t s fed c a s e i n . S i m i l a r s t u d i e s performed in our l a b o r a t o r y r e v e a l e d t h a t the mean t r a n s i t time f o r c h o l e s t e r o l was 18.4 days in r a b b i t s fed soy p r o t e i n , 36.8 days in r a b b i t s fed c a s e i n , 33.7 days in r a b b i t s fed soy p l u s l y s i n e , and 36.3 days in r a b b i t s fed c a s e i n p l u s a r g i n i n e . These d a t a suggest that a d d i t i o n of l y s i n e to soy p r o t e i n

T a b l e V I I I . D i e t a r y P r o t e i n E f f e c t s on C h o l e s t e r o l K i n e t i c s Rabbits

Soy p r o t e i n Soy & l y s i n e Commercial d i e t

Pool A 427 791 200

(mg)

Pool

Β (mg) 711 1260 690

in

Mean t r a n s i t time 18.4 33.7 11.8

(days)

158

PLANT PROTEINS

s i g n i f i c a n t l y i n h i b i t e d c h o l e s t e r o l f l u x and t h i s was borne out by the f i n d i n g of expanded s i z e s of b o t h the r a p i d l y exchanging ( p o o l A) and the s l o w l y exchanging ( p o o l B) p o o l s of body c h o l e s t e r o l (Table V I I I ) . Recent work r e p o r t e d by Vahouny et a l (20) suggested t h a t in r a t s there was reduced c l e a r a n c e of b o t h c h o l e s t e r o l and t r i g l y c e r i d e s associated with chylomicron-like lipoprotein particles in a n i m a l s fed c a s e i n as compared w i t h those fed soy p r o t e i n . A d d i t i o n of l y s i n e to soy p r o t e i n had o n l y s l i g h t e f f e c t s but a d d i t i o n of a r g i n i n e to c a s e i n l e d to r e s u l t s s i m i l a r to those found w i t h f e e d i n g soy p r o t e i n . In t h i s study, serum i n s u l i n l e v e l s were c o r r e l a t e d i n v e r s e l y w i t h serum l i p i d c o n c e n t r a t i o n s . T h i s is a t odds w i t h the r e p o r t of Sugano et a l (JJ>) who r e p o r t e d a l t e r a t i o n s of g l u c a g o n , but not i n s u l i n , w i t h a d d i t i o n of a r g i n i n e to c a s e i n . Both of these r e p o r t s agree that f e e d i n g soy p r o t e i n r e s u l t e d in l e s s c h o l e s t e r o l e m i a than c a s e i n and t h a t t o t a l serum c h o l e s t e r o l was o n l y s l i g h t l y a f f e c t e d by a d d i t i o n of l y s i n e or a r g i n i n e . The d i f f e r e n c e s between the two i n v e s t i g a t i o n s may be a t t r i b u t a b l e to d i f f e r e n c e s in age or s t r a i n of r a t s , or d i f f e r e n t p r e p a r a t i o n s of soy and casein. Whatever the mechanism of h y p o c h o l e s t e r o l e m i a a s s o c i a t e d w i t h f e e d i n g v e g e t a b l e p r o t e i n s when compared w i t h most animal p r o t e i n s , s t u d i e s in humans i n d i c a t e a s i m i l a r e f f e c t o n l y when the i n i t i a l serum c h o l e s t e r o l c o n c e n t r a t i o n is r e l a t i v e l y h i g h . Individuals w i t h normal c h o l e s t e r o l l e v e l s do not respond w i t h a f u r t h e r d e c r e a s e in serum l i p i d s . S i r t o r i et a l (21) t r e a t e d type I I h y p e r l i p o p r o t e i n e m i c p a t i e n t s w i t h a d i e t t h a t was s i m i l a r in c o m p o s i t i o n to the low f a t , low c h o l e s t e r o l d i e t recommended by the American H e a r t A s s o c i a t i o n and compared t h i s w i t h a d i e t which was s i m i l a r in c o m p o s i t i o n except f o r s u b s t i t u t i o n of the m a j o r i t y of animal p r o t e i n w i t h t e x t u r e d v e g e t a b l e p r o t e i n in a c r o s s o v e r design. The soy p r o t e i n d i e t r e s u l t e d in a s i g n i f i c a n t r e d u c t i o n in l e v e l s of plasma t o t a l and LDL c h o l e s t e r o l , and t h i s d e c r e a s e was f a r g r e a t e r than w i t h the u s u a l t h e r a p e u t i c d i e t . When p a t i e n t s were g i v e n the soy d i e t and then s w i t c h e d to the u s u a l d i e t (based on meat as the p r o t e i n s o u r c e ) , t h e r e was an i n c r e a s e of t o t a l and LDL c h o l e s t e r o l . T h i s study had been c r i t i c i z e d because of s i g n i f i c a n t d i f f e r e n c e s in the p o l y u n s a t u r a t e d : s a t u r a t e d (P/S) f a t r a t i o between the two d i e t s . T h e r e f o r e , t h i s group of r e s e a r c h e r s performed another study in which the P/S r a t i o was O.1 or 2.7 in a c r o s s o v e r d e s i g n (_22). The P/S r a t i o d i d a f f e c t plasma c h o l e s t e r o l l e v e l s but the soy d i e t r e s u l t e d in r e d u c t i o n s of plasma c h o l e s t e r o l that always exceeded 15% r e g a r d l e s s of the P/S r a t i o . Not a l l s t u d i e s of soy p r o t e i n d i e t s in humans have found s i g n i f i c a n t r e d u c t i o n of plasma c h o l e s t e r o l . In a d d i t i o n to d i f f e r e n c e s in d i e t f o r m u l a t i o n s and soy p r o t e i n p r e p a r a t i o n s , one r e a s o n t h a t has been proposed f o r these d i s p a r i t i e s is t h a t there is a s t a t i s t i c a l l y s i g n i f i c a n t n e g a t i v e c o r r e l a t i o n between the i n i t i a l c h o l e s t e r o l l e v e l and the d e c r e a s e of c h o l e s t e r o l a c h i e v e d w i t h the soy p r o t e i n d i e t ( r = -O.48, ρ < O.01) ( 2 3 ) . In o t h e r words, those w i t h normal or s l i g h t l y e l e v a t e d c h o l e s t e r o l l e v e l s would be e x p e c t e d to show l e s s change in c h o l e s t e r o l as a r e s u l t of the soy d i e t than i n d i v i d u a l s w i t h markedly e l e v a t e d l i p i d s . S i r t o r i and a s s o c i a t e s (23^ have f o l l o w e d a number of type I I h y p e r l i p o p r o t e i n e m i c p a t i e n t s who have been m a i n t a i n e d on the soy

13.

KLURFELD AND KRITCHEVSKY

Influence of Protein on Serum Cholesterol

159

p r o t e i n d i e t f o r over two y e a r s . A f t e r at l e a s t two months of t o t a l s u b s t i t u t i o n of the d i e t a r y p r o t e i n , the p a t i e n t s were i n s t r u c t e d to consume at l e a s t s i x meals per week based on the t e x t u r e d soy protein. A d e c r e a s e in t o t a l c h o l e s t e r o l a v e r a g i n g 30% was m a i n t a i n e d and t h e r e was a s m a l l but s i g n i f i c a n t i n c r e a s e in HDL cholesterol levels. S t u d i e s on the h y p o c h o l e s t e r o l e m i c mechanism(s) a s s o c i a t e d w i t h s u b s t i t u t i o n of soy p r o t e i n f o r animal p r o t e i n have p r o v i d e d seemingly c o n f l i c t i n g r e s u l t s . F u m a g a l l i et a l (24) have r e p o r t e d t h a t type I I h y p e r l i p o p r o t e i n e m i c p a t i e n t s t r e a t e d w i t h the soy d i e t had no change in f e c a l s t e r o i d e x c r e t i o n nor any d i f f e r e n c e in h a l f - l i f e of plasma c h o l e s t e r o l . However, N e s t e l et a l (25) found t h a t v e g e t a r i a n s e x c r e t e d l e s s n e u t r a l s t e r o l s and b i l e a c i d s and had reduced s y n t h e s i s of LDL than omnivores. Taken t o g e t h e r , these s t u d i e s i n d i c a t e t h a t the h y p o c h o l e s t e r o l e m i c e f f e c t of p l a n t p r o t e i n s is not due to d i e t a r y f i b e r or some o t h e r n o n a b s o r b a b l e component of the d i e t . F u r t h e r m o r e , the mode of a c t i o n of soy p r o t e i n appears to be d i s s i m i l a r from p h a r m a c o l o g i c agents t h a t a f f e c t s t e r o i d e x c r e t i o n i n t o the i n t e s t i n a l lumen or b i n d b i l e a c i d s to p r e v e n t r e a b s o r p t i o n . A number of p o t e n t i a l mechanisms are under study by s e v e r a l groups. The i n s u l i n / g l u c a g o n r a t i o s may c o n t r o l l i p i d b i o s y n t h e s i s but d a t a on t h i s p o i n t in r e l a t i o n to f e e d i n g soy p r o t e i n have a l s o been c o n f l i c t i n g . D i f f e r e n t m o l e c u l a r weight forms of the hormones may be s e c r e t e d in response to f e e d i n g the d i f f e r e n t p r o t e i n s . D i g e s t i o n of p l a n t and a n i m a l p r o t e i n s may y i e l d a b s o r p t i o n of d i f f e r e n t p e p t i d e s or amino a c i d s t h a t have b i o l o g i c e f f e c t s . To t h i s end, S i r t o r i (23) found t h a t plasma c o n t e n t of f r e e a r g i n i n e i n c r e a s e d 20% when p a t i e n t s were fed a soy-based d i e t . Partial d i g e s t i o n p r o d u c t s of the p r o t e i n may b i n d l i p i d s in the i n t e s t i n e but d a t a on t h i s p o i n t are c o n f l i c t i n g . Rates of d i g e s t i o n of the p r o t e i n s may be d i f f e r e n t , e l i c i t i n g a l t e r e d hormonal or o t h e r b i o l o g i c responses. P r o d u c t s of p r o t e i n d i g e s t i o n may influence a p o l i p o p r o t e i n s e c r e t i o n in e i t h e r the i n t e s t i n e or l i v e r . Of r e l e v a n c e to t h i s p o i n t is the h y p o t h e s i s t h a t the l y s i n e / a r g i n i n e r a t i o of a p r o t e i n d e t e r m i n e s i t s a t h e r o g e n i c i t y . S i n c e l y s i n e and a r g i n i n e are the amino a c i d s r e s p o n s i b l e f o r net b i n d i n g by the a p o p r o t e i n m o l e c u l e , i f v e g e t a b l e p r o t e i n can a l t e r the a p o p r o t e i n moitiés q u a n t i t a t i v e l y or q u a l i t a t i v e l y t h i s is a n o t h e r p o t e n t i a l pathway f o r h y p o c h o l e s t e r o l e m i c activity. We have a l s o suggested t h a t the h i g h L/A r a t i o of animal p r o t e i n might i n h i b i t h e p a t i c a r g i n a s e a c t i v i t y , thus r e d u c i n g f o r m a t i o n of a r g i n i n e - r i c h a p o p r o t e i n s which are c h a r a c t e r i s t i c of e x p e r i m e n t a l l y induced hyperlipidemia (14). I t is p o s s i b l e t h a t more than one mechanism may be found f o r the h y p o c h o l e s t e r o l e m i c e f f e c t of v e g e t a b l e protein. S u b s t a n t i a l work remains to be done in i d e n t i f i c a t i o n of the h y p o c h o l e s t e r o l e m i c mechanism of f e e d i n g soy p r o t e i n . Whether t h e r e are g e n e r a l or s p e c i f i c d i f f e r e n c e s in e f f e c t s on l i p i d m e t a b o l i s m between a n i m a l and p l a n t p r o t e i n s a l s o remains to be determined.

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Acknowledgments Supported, in p a r t , by g r a n t HL-03229 and R e s e a r c h C a r e e r Award HL-00734 from t h e N a t i o n a l I n s t i t u t e s o f H e a l t h and by funds from t h e Commonwealth o f P e n n s y l v a n i a . The authors thank t h e f o l l o w i n g i n d i v i d u a l s who have c o n t r i b u t e d t o the work d i s c u s s e d in t h i s review: D r s . J o n A. S t o r y , Susanne K. C z a r n e c k i , and Ms. S h i r l e y A. Tepper.

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RECEIVED December 13, 1985