Citrus Pectic PolysaccharidesâTheir in Vitro Interaction with Low

13 Citrus Pectic Polysaccharides—Their in Vitro Interaction with Low Density Serum Lipoproteins M . M A N S O O R B A I G and J A M E S J . C E R D A

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University of Florida College of Medicine, Department of Medicine, Division of Gastroenterology and Nutrition, Gainesville, FL 32610

Dietary fiber," the skeletal remains of plant cells that are resistant to digestion by enzymes of man" (1) include at least five different known components: l i g n i n , cellulose, hemicellulose, gums and pectins. Lignin is a non-carbohydrate component and appears to have no known role in human n u t r i tion. Cellulose and hemicellulose affect large bowel function and cause an increase in stool weight and decrease in transit time. Gums and pectins can also increase intestinal transit time, adsorb fecal water, influence glucose and l i p i d metabol ism, mineral absorption and, in addition, may possibly play a role in colon cancer. With the exception of cellulose, a polymer of glucose, the chemistry of hemicelluloses, gums and pectins is extremely complex (2). Pectin, a heterogeneous mixture of a number of complex pectic polysaccharides, appears early during plant c e l l wall biogenesis. A series of complex biochemical steps result in the formation of c e l l plate followed f i r s t by i t s growth in area (primary c e l l wall) then in thickness (secondary c e l l wall). Exclusive of randomly oriented cellulose f i b r i l s , p r i mary c e l l wall is composed mainly of pectic polysaccharides (3). These pectic polysaccharides are rich in D-galacturonic acid, D-galactose and L-arabinose residues. With growth in thickness, there occurs a replacement of pectic polysaccharide deposition with polysaccharides rich in D-glucuronic acid, D -xylose and D-glucose residues. These polysaccharides which add to the growth in thickness of the plant c e l l wall are classified as alkali-soluble hemicelluloses. Previous studies (4_) the chemistry of pectin suggested the presence of three major polysaccharides in pectin, namely 1) a glacturonan poly mer containing 1,4 linked α-D-galacturonic acid residues, 2) a galactan polymer containing chains of 1,4 linked a-D-galactopyranose residues and 3) and arabinan a branched polymer con taining 1,5 and 1,3 linked L-arabinofuranose residues. Com paratively recent studies (2-3,5-6), however, suggest that the chemistry of pectin is much more complex than previously 0097-6156/83/0214-0185$06.00/0 © 1983 American Chemical Society

Furda; Unconventional Sources of Dietary Fiber ACS Symposium Series; American Chemical Society: Washington, DC, 1983.

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b e l i e v e d . Presence of c o v a l a n t l y - l i n k e d n e u t r a l sugars e s p e c i a l l y g a l a c t o s e , rhamnose and arabinose to polymers of g a l a c t u r onic a c i d residues was demonstrated. A l s o , demonstrated was the presence of L-fucose, D-apiose and D-xylose c o v a l e n t l y l i n k e d to three major polysaccharides, i . e . , galacturonans, arabinans and galactans as mentioned e a r l i e r . The demonstrated c o v a l e n t l y - l i n k e d amino acids such as hydroxy p r o l i n e w i t h i n the s t r u c t u r a l framework of various p e c t i c polysaccharides added f u r t h e r complexity to the chemistry of p e c t i n . C h o l e s t e r o l Lowering Influence of P e c t i n ; Several i n v e s t i g a t o r s (7-9) have c l e a r l y suggested that p e c t i n when supplemented i n the d i e t of l a b o r a t o r y animals and/or human volunteers causes s i g n i f i c a n t reduction i n serum c h o l e s t e r o l l e v e l s . T h i s , indeed, i s an important observation, as i t has been shown that the e t i o l o g y of a t h e r o s c l e r o s i s and coronary a r t e r y disease i s a s s o c i a t e d with elevated serum c h o l e s t e r o l l e v e l s , and the observed reduction of serum c h o l e s t e r o l l e v e l by p e c t i n holds promise f o r the treatment and/or prevention of these d i s e a s e s . I t i s r e l e v a n t here to point out that d i e t a r y p e c t i n has shown to r e t a r d induced avian a t h e r o s c l e r o s i s (10). P e c t i n B i l e S a l t I n t e r a c t i o n . The biochemical b a s i s by which p e c t i n causes lowering of serum c h o l e s t e r o l l e v e l remains elusive. I t has been suggested that d i e t a r y f i b e r s i n general cause lowering of c h o l e s t e r o l l e v e l s by binding with b i l e s a l t s and consequently causing a break i n the enterohepatic c i r c u l a t i o n of b i l e a c i d s and i n c r e a s i n g c h o l e s t e r o l turnover (1113). This explanation i s s i m i l a r to one where cholestyramine, an i o n exchange r e s i n (Dowex 1x2 CI""), used as a drug (Cuemid, Questran, Quantalan) to a f f e c t the lowering of serum cholest e r o l l e v e l s when given to p a t i e n t s with hypercholesterolemia. C r i t i c a l e v a l u a t i o n of data suggesting the binding of b i l e s a l t s to d i e t a r y f i b e r reveals that the observed i n v i t r o a f f i n i t y of various d i e t a r y f i b e r s to b i l e acids i s much lower when compared with binding of b i l e acids with cholestyramine. The i n v i t r o a f f i n i t y of cholestyramine f o r acids i s 1.0 mg b i l e acid/mg of r e s i n estimated at pH 6.4 i n 0.9% s a l i n e (14). On the other hand, the i n v i t r o binding of b i l e acids to f i b e r s such as wheat straw, sugar cane pulp, bran, oat, e t c . , ranges from 0.007 mg to 0.024 mg of b i l e a c i d per mg of f i b e r i n normal s a l i n e . These values amount to 41-142 times l e s s than the a f f i n i t y of b i l e acids to cholestyramine. Although a l f a l f a , guar gum and p e c t i n , u n l i k e the various f i b e r s mentioned above, showed b e t t e r a f f i n i t y f o r b i l e a c i d s , t h i s a f f i n i t y i s about 5 times l e s s than the a f f i n i t y of b i l e acids to cholestyramine. For a physicochemical phenomenon to be of b i o l o g i c a l s i g n i f i cance, the reactants mush e x h i b i t a s t o i c h i o m e t r i c r e l a t i o n s h i p . In the studies mentioned above (12,13), although i n c r e a s i n g the amount of various f i b e r s i n the incubation



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mixture d i d cause an increase i n the amount of b i l e a c i d bound to the various f i b e r s s t u d i e d , t h i s observed increase was not stoichiometric. Furthermore, no binding of b i l e acids to p u r i f i e d d i e t a r y f i b e r s was observed by these i n v e s t i g a t o r s . I t i s of i n t e r e s t to mention the r e s u l t s of a recent study (15) which f a i l e d to demonstrate the i n v i t r o binding and/or i n t e r a c t i n of p e c t i n with b i l e acids when examined by u t i l i z i n g NMR spectroscopy. The observations made by these i n v e s t i g a t o r s are s i m i l a r to those where i n t e r a c t i o n of b i l e acids and d i e t a r y f i b e r s from a v a r i e t y of sources was studied i n v i t r o by u t i l i z i n g l a b e l l e d b i l e a c i d s , e q u i l i b r i u m d i a l y s i s and/or c e n t r i f u g a t i o n techniques (16). Increased e x c r e t i o n of b i l e s a l t s has a l s o been presented as an evidence f o r the binding of b i l e a c i d to d i e t a r y f i b e r s consequently r e s u l t i n g i n hypocholesterolemia (17,18). However, i n a recent study (19) where f e c a l b i l e a c i d e x c r e t i o n was studied i n response to intake of a v a r i e t y of f i b e r s , no s i g n i f i c a n t a l t e r a t i o n s i n the f e c a l b i l e a c i d content was observed. I t i s evident from the above that our current understanding of the biochemical basis by which d i e t a r y f i b e r causes lowering of c h o l e s t e o l l e v e l s i s f a r from comp l e t e , and therefore i t needs to be researched and r e evaluated. Pectin Lipoprotein Interaction. C h o l e s t e r o l i n serum i s not found f r e e but i s c a r r i e d by a number of l i p o p r o t e i n s found i n serum. Low density l i p o p r o t e i n (LDL) i s the major c a r r i e r of c i r c u l a t i n g c h o l e s t e r o l . Elevated l e v e l s of LDL i n the serum contribute s i g n i f i c a n t l y to the r i s k of coronary heart disease, and development of a t h e r o s c l e r o s i s . Atherosclerotic l e s i o n s are c h a r a c t e r i z e d by i n t i m a i p r o l i f e r a t i o n of smooth muscle c e l l s accompanied by accumulation of l a r g e amounts of connective t i s s u e components such as c o l l a g e n , e l a s t i n and glycosaminoglycans. In a t h e r o s c l e r o s i s , presence of l a r g e amounts of LDL and glycosaminoglycan complexes i n plaques have been demonstrated (20,21). Glycosaminoglycans, l i k e p e c t i n , are p o l y a n i o n i c complex carbohydrates found i n the connective t i s s u e of animals. In view of the f a c t that p e c t i n lowers serum c h o l e s t e r o l l e v e l s and c h o l e s t e r o l i s c a r r i e d by l i p o p r o t e i n s i n the serum, we i n v e s t i g a t e d the i n t e r a c t i o n of p e c t i n obtained from grapef r u i t albedo with various human serum l i p o p r o t e i n f r a c t i o n s i n v i t r o (22). I t was observed that p e c t i n i n t e r a c t e d s p e c i f i c a l l y with LDL, while no i n t e r a c t i o n was observed between p e c t i n and high density or very low density l i p o p r o t e i n s . The b i o l o g i c a l s i g n i f i c a n c e of t h i s observed i n v i t r o i n t e r a c t i o n of p e c t i n with LDL remain unclear, p r i m a r i l y because of l a c k of evidence suggesting entry of p e c t i n or some component of i t i n t o the blood streatm. Therefore, unless such entry and/or absorption of p e c t i n or one of i t s components i s demonstrated, the b i o l o g i c a l i m p l i c a t i o n of t h i s observed i n t e r a c t i o n



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r e s u l t i n g i n the formation of pectin-LDL complexes and the metabolic f a t e of such complexes i f found remains h y p o t h e t i c a l i n nature. Our ongoing studies aimed to gain an i n s i g h t i n t o the b i o chemical b a s i s of t h i s observed i n t e r a c t i o n between LDL and p e c t i n have revealed that s e l e c t i v e m o d i f i c a t i o n of basic amino a c i d residues such as h i s t i d i n e , a r g i n i n e and l y s i n e , found i n LDL caused a s i g n i f i c a n t l o s s of i n t e r a c t i o n between p e c t i n and LDL. These r e s u l t s suggest that various b a s i c amino acids are involved i n the observed i n t e r a c t i o n between p e c t i n and LDL (23). Results from our recent experiments designed t o evaluate the comparative e f f e c t i v e n e s s of cholystyramine and p e c t i n i n lowering serum c h o l e s t e r o l l e v e l s i n r a t s (24), c l e a r l y suggest that p e c t i n i s comparatively as e f f e c t i v e as cholestyramine i n lowering serum c h o l e s t e r o l l e v e l s and t h i s observed lowering of serum c h o l e s t e r o l by p e c t i n i s mediated through an e x c l u s i v e lowering of c h o l e s t e r o l a s s o c i a t e d with LDL. S e l e c t i v e lowering of LDL by p e c t i n (25) and by other d i e t a r y f i b e r s such as oat bran (26) has a l s o been reported by other i n v e s t i g a t o r s . Based on these observations, i t could be concluded that the mediation of serum c h o l e s t e r o l lowering e f f e c t of p e c t i n i s accomplished by lowering of c h o l e s t e r o l associated with serum low density l i p o p r o t e i n s by mechanisms which remain e l u s i v e . Furthermore, experimental attempts to e l u c i d a t e the p o s s i b l e mechanism by which p e c t i n causes lowering of c h o l e s t e r o l l e v e l must take i n t o account the heterogeneous nature of p e c t i n , f o r a unique p e c t i c polysaccharide found i n p e c t i n may be s o l e l y responsible f o r the observed hypocholesterolemic a c t i v i t y of pectin. S e l e c t i v e I n t e r a c t i o n of various P e c t i c Polysaccharides with Low Density L i p o p r o t e i n s . As mentioned e a r l i e r , i n v i t r o i n t e r a c t i o n between unfractionated p e c t i n and LDL r e s u l t s i n the formation of i n s o l u b l e complexes (22). Further i n s i g h t i n t o t h i s observed i n t e r a c t i o n was gained by examining t h i s i n t e r a c t i o n between i s o l a t e d p e c t i c polysaccharides found i n p e c t i n and human LDL. Various p e c t i n polysaccharides found i n c i t r u s p e c t i c were resolved by employing DE-52 c e l l u l o s e i o n exchange column chromatography techniques (27). Four chemicall y d i s t i n c t p e c t i c polysaccharides were resolved f o l l o w i n g s e q u e n t i a l e l u t i o n of column bound p e c t i n with 0.025, 0.1, 0.25 and 0.5 M sodium phosphate b u f f e r , pH 6.0, and composed of 13% ( F r a c t i o n I ) , 5% ( F r a c t i o n I I ) , 62% ( F r a c t i o n I I I ) , and 20% ( F r a c t i o n IV), r e s p e c t i v e l y , of the t o t a l p e c t i n subjected to i o n exchange chromatography. The g a l a c t u r o n i c a c i d contents of these i n d i v i d u a l p e c t i c polysaccharide f r a c t i o n s were not s t r i k i n g l y d i f f e r e n t and ranged between 70-80% of the t o t a l c a r bohydrate contents. However, the n e u t r a l sugar composition of these p e c t i c polysaccharides was found to be d i f f e r e n t . A progressive decrease i n the degree of methylation of these p e c t i c



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polysaccharides was a l s o observed i n that p e c t i c polysaccharide e l u t e d with 0.1 M s a l t ( F r a c t i o n I I ) was highest i n i t s methoxy content (14.85%), whereas p e c t i c polysaccharide e l u t e d with 0.5 M s a l t ( F r a c t i o n IV) was lowest i n i t s methoxy content (4.42%). The methoxy content of F r a c t i o n I, the f r a c t i o n e l u t e d with 0.025 M s a l t , and not r e t a i n e d by i o n exchange r e s i n was found to be s i m i l a r to u n f r a c t i o n a t e d p e c t i n (8.9%). Examination of n e u t r a l sugar composition of various p e c t i c polysaccharides revealed that F r a c t i o n I and F r a c t i o n IV were r i c h i n rhamnose content, when compared with other p e c t i c p o l y saccharides ( 2 8 ) . Examination of i n t e r a c t i o n between these p e c t i c p o l y s a c charide f r a c t i o n s and LDL i n v i t r o by employing experimental techniques described e a r l i e r (22), revealed the s e l e c t i v e nature of t h i s i n t e r a c t i o n . P e c t i c polysaccharides I and IV, i . e . , polysaccharides r i c h i n rhamnose content were the only polysaccharides which i n t e r a c t e d s p e c i f i c a l l y with human LDL, causing t h e i r p r e c i p i t a t i o n i n v i t r o . No or l i t t l e i n t e r a c t i o n between LDL and p e c t i c polysaccharide F r a c t i o n s I I and I I I was observed. These observations suggest that p e c t i c polysacchar i d e s r i c h i n rhamnose sugar r e s i d u e s , s p e c i f i c a l l y i n t e r a c t e x c l u s i v e l y with human LDL, and t h i s i n t e r a c t i o n appears to be independent of the methoxy content of the p o l y s a c c h a r i d e s . In summary, s t u d i e s demonstrating i n v i t r o i n t e r a c t i o n between a v a r i e t y of d i e t a r y f i b e r and b i l e a c i d s (12,13) may be of importance i n part i n e x p l a i n i n g the biochemical b a s i s by which i n g e s t i n of d i e t a r y f i b e r causes lowering of serum chol e s t e r o l l e v e l s . However, i n view of r e s u l t s obtained from comparatively recent i n v i t r o (15,16) and/or i n v i t r o (19) studies i n d i c a t i n g l a c k of i n t e r a c t i o n between d i e t a r y f i b e r and b i l e s a l t , the existance of a much more complex mechanism by which d i e t a r y f i b e r causes lowering of serum c h o l e s t e r o l l e v e l could not be o v e r r u l e d . The f i n d i n g of s p e c i f i c i n v i t r o e l e c t r o s t a t i c i n t e r a c t i o n between p e c t i n and human low d e n s i t y l i p o p r o t e i n (22) adds f u r t h e r complexity to the f i b e r and serum c h o l e s t e r o l connection. Further complexity i s added to t h i s connection by observations suggesting heterogeneous nature of d i e t a r y p e c t i n , and s e l e c t i v e i n t e r a c t i o n of rhamnose r i c h p e c t i c polysaccharides with human low d e n s i t y l i p o p r o t e i n . I t i s , t h e r e f o r e , safe to conclude t h a t , only through s t u d i e s aimed towards d e f i n i n g the psysicochemical nature, metabolism and c h o l e s t e r o l - l o w e r i n g c a p a b i l i t y of w e l l defined i n d i v i d u a l polysaccharides found i n d i e t a r y f i b e r , can an i n s i g h t i n t o the e l u s i v e biochemical b a s i s by which d i e t a r y f i b e r causes lowering of serum c h o l e s t e r o l be gained.

Literature Cited 1. Trowell, H. C. Lancet, 1974, 1, 503. 2. Aspinall, G. O. "Carbohydrates, Chemistry and Biochemistry"; Pigmann, W. W. and Horton, D., Eds., Academic: New York, 1970, Vol. IIB, p. 515.



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Citrus Pectic PolysaccharidesâTheir in Vitro Interaction with Low

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