Physiological Effects of Carrageenan - ACS Symposium Series (ACS

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Downloaded by NANYANG TECHNOLOGICAL UNIV on August 26, 2015 | http://pubs.acs.org Publication Date: June 1, 1975 | doi: 10.1021/bk-1975-0015.ch018

Physiological Effects of Carrageenan DIMITRI J. STANCIOFF and DONALD W. RENN Marine Colloids, Inc., Rockland, Maine 04841

Abstract

Breakdown absorption and toxicity of carrageenan in the gas trointestinal tract is reviewed. Carrageenan, a linear sulfated galactan (molecular weight 250,000) derived from red seaweeds, is used as a food stabilizer, particularly in dietetic and dairy products. Breakdown in the gut appears to be insignificant and there is no evidence that it is absorbed by test animals with the possible exceptions of guinea pigs and rabbits. In large doses it inhibits pepsin activity (in vitro), depresses gastric juice secretion and reduces food absorption in rats. There is no evi dence of such effects with the low levels used in food. Depolymerized carrageenan (molecular weight < 20,000), used in France for treatment of peptic ulcers, has shown no toxicity in man but caused mucosal erosions in the cecum of guinea pigs and at high doses also effected cell changes in the colon of rats and monkeys. Degraded carrageenan was demonstrated as being partly absorbed by the epithelial cells, deposited in the Kupffer cells of the liver and found in the urine. A broad margin of safety for food grade carrageenan in the diet is assured by low func tional use levels and a minimum molecular weight of 100,000. Introduction E x c e l l e n t reviews on the p h y s i o l o g i c a l e f f e c t s o f carrageenan have been p u b l i s h e d i n the l a s t s i x years: two of them by Anderson (1,2) and the most recent by DiRosa ( 3 ) . Although these r e views are very comprehensive, t h e i r emphasis i s on the pharmacolo g i c a l p r o p e r t i e s o f carrageenan administered by p a r e n t e r a l routes. For example, carrageenan introduced subcutaneously i n duces c o l l a g e n p r o l i f e r a t i o n w i t h the r e s u l t a n t formation o f a granuloma (4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14). When i n j e c t e d i n t o a rodent's paw, a r e p r o d u c i b l e inflammatory edematous condit i o n r e s u l t s which has been s u c c e s s f u l l y used i n screening compounds f o r anti-inflammatory a c t i v i t y (15, 16, 17, 18, 19, 20). 282 In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.


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A n t i - c o a g u l a n t (21, 22, 23 24) and hypotensive p r o p e r t i e s (25) have a l s o been observed. I n a d d i t i o n , v a r i o u s immunological r e sponses have been reported which i n c l u d e i n v i t r o and i n v i v o complement d e p l e t i o n and delayed h y p e r s e n s i t i v i t y (26, 27, 28, 29, 30, 31, 32, 33). Although these p h y s i o l o g i c a l m a n i f e s t a t i o n s o f carrageenan may seem somewhat foreboding, 99.9% o f the carrageenan consumed by humans i s i n food r a t h e r than i n drugs, and the emphasis of t h i s symposium i s on d i e t a r y r a t h e r than pharmacological aspects of carbohydrates. I n t h i s context the e f f e c t s o f p a r e n t e r a l l y administered carrageenan i s about as r e l e v a n t as the e f f e c t s o f an intravenous i n j e c t i o n o f beef stew. Though the l a t t e r i s a wholesome food, i t s p h y s i o l o g i c a l e f f e c t i n the bloodstream would, no doubt, be q u i t e d e v a s t a t i n g . We w i l l , t h e r e f o r e , r e s t r i c t the s u b j e c t o f t h i s review to carrageenan taken o r a l l y . The use o f p u r i f i e d carrageenan i n modern food technology dates back to the e a r l y t h i r t i e s , w h i l e the use o f crude preparat i o n s by O r i e n t a l and European c o a s t a l d w e l l e r s i s a t l e a s t seve r a l c e n t u r i e s o l d (34). In s p i t e of t h i s long h i s t o r y o f food use, or perhaps because of i t , f a r more has been p u b l i s h e d on i t s p h y s i o l o g i c a l e f f e c t as an experimental drug than as a food i n g r e d i e n t . I n f a c t p u b l i c a t i o n s a r e i n i n v e r s e r a t i o to use: 10,000,000 l b s / y e a r used i n food - 20 papers 10,000 l b s / y e a r a n t i - u l c e r drug - 70 papers 1 l b / y e a r p a r e n t e r a l use - 150 papers N e v e r t h e l e s s , we can glean enough from the l i t e r a t u r e to est a b l i s h that carrageenan i s not broken down o r absorbed and that at the low l e v e l s used i n food has no adverse p h y s i o l o g i c a l e f f e c t s i n humans. Source P r o p e r t i e s and Uses o f Carrageenan But f i r s t l e t ' s say a few words about the o r i g i n , chemistry, and p h y s i c a l p r o p e r t i e s o f carrageenan. Carrageenan can be obtained from about 250 species o f c l o s e l y r e l a t e d red algae o f the order G i g a r t i n a l e s but only about h a l f a dozen o f them are used commercially: These i n c l u d e species o f Chondrus, Eucheuma and G i g a r t i n a . I t i s the major i n t e r c e l l u l a r c o n s t i t u e n t o f these p l a n t s and represents about 607o o f t h e i r s a l t - f r e e dry weight. Carrageenan i s g e n e r a l l y prepared by hot aqueous e x t r a c t i o n of the seaweed, f o l l o w e d by f i l t r a t i o n to remove i n s o l u b l e matter, c o a g u l a t i o n i n a l c o h o l , vacuum d r y i n g , and g r i n d i n g . Chemically, carrageenan i s a s t r a i g h t c h a i n s u l f a t e d g a l a c t a n w i t h a backbone o f a l t e r n a t i n g 1-4 l i n k e d ^ - D - g a l a c t o s e and 1-3 l i n k e d ^ - D - g a l a c t o s e o r i t s 3,6 anhydride. Commercial e x t r a c t s u s u a l l y have a weight average molecular weight 100,000 to 500,000.

In Physiological Effects of Food Carbohydrates; Jeanes, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1975.


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This simple p i c t u r e i s somewhat complicated by the f a c t that the number and p o s i t i o n of the e s t e r s u l f a t e groups and the r a t i o of g a l a c t o s e to 3,6-anhydrogalactose may v a r y c o n s i d e r a b l y . Greek l e t t e r p r e f i x e s are used f o r c a t e g o r i z i n g the v a r i o u s com binations . Two major groups of carrageenan are recognized. In the f i r s t , the 1 , 3 - l i n k e d u n i t s are s u l f a t e d i n the 4 - p o s i t i o n w h i l e i n the second, the s u l f a t e i s i n the 2 - p o s i t i o n (Figure 1). The f i r s t group i s s u b d i v i d e d a c c o r d i n g to the nature of the 1- 4 l i n k e d u n i t s . These may be present as g a l a c t o s e 6 - s u l f a t e (>i-carrageenan) or g a l a c t o s e 2-6 d i s u l f a t e ( ^-carrageenan) or as the corresponding 3,6-anhydrides i n fC- and L -carrageenan. In nature the 3,6-anhydrides of /(- and £-carrageenan are formed by enzymatic e l i m i n a t i o n of the 6 - s u l f a t e from the μ- and p-forms, but the c o n v e r s i o n i s not always complete. In some seaweeds these carrageenan types can be i s o l a t e d i n almost pure form w h i l e i n others they e x i s t as copolymers. In the second group, the 1 , 4 - l i n k e d u n i t s are s u l f a t e d i n the 2- p o s i t i o n . In λ-carrageenan the 6 - p o s i t i o n i s a l s o s u l f a t e d w h i l e i n ξ-carrageenan i t i s not. Aqueous s o l u t i o n s of carrageenan are h i g h l y v i s c o u s . Kappa and C -carrageenan form heat r e v e r s i b l e gels i n the presence of potassium and c a l c i u m i o n s . Carrageenans a l s o r e a c t s t r o n g l y w i t h l a r g e p o s i t i v e l y charged i o n s , notably w i t h p r o t e i n s below their i s o e l e c t r i c point. The main uses of carrageenan are as t h i c k e n e r s , s t a b i l i z e r s , and g e l l i n g agents i n food. I t s strong i n t e r a c t i o n w i t h c a s e i n i s u t i l i z e d i n d a i r y products such as c h o c o l a t e m i l k , i c e cream and puddings. The g e l l i n g p r o p e r t i e s are u s e f u l f o r making j e l l i e s , r e l i s h e s , and p i e f i l l i n g s . The l i s t of a p p l i c a t i o n s a l s o i n c l u d e s whipped t o p p i n g , non-dairy c o f f e e w h i t e n e r s , and a host of convenience foods. The f u n c t i o n a l p r o p e r t i e s of carrageenan are h i g h l y dependent on molecular weight. When the m o l e c u l a r weight i s below 100,000 the s t a b i l i z i n g p r o p e r t i e s are almost completely l o s t . More de t a i l e d i n f o r m a t i o n on the sources, p r o p e r t i e s , and uses of c a r r a geenan has been p u b l i s h e d i n reviews by Rees ( 3 5 ) , by Glicksman (36), Sand and Glicksman ( 3 7 ) , and by Towle ( 3 8 ) . Degraded Carrageenan When speaking o f p h y s i o l o g i c a l p r o p e r t i e s of ingested c a r r a geenan we must d i s t i n g u i s h between food grade carrageenan, which has a m o l e c u l a r weight of 100,000 to 500,000 and s o - c a l l e d de graded carrageenan. Degraded carrageenan i n which the g l y c o s i d i c l i n k a g e s are h y d r o l i z e d to reduce the molecular weight to l e s s than 20,000 i s used i n France f o r p e p t i c u l c e r treatment. I t i s i n e f f e c t i v e as a food s t a b i l i z e r and a l s o has d i f f e r e n t p h y s i o l o g i c a l p r o p e r t i e s . We w i l l come back to i t l a t e r , but f i r s t l e t s t a l k about food grade carrageenan. 1



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Figure 1. Repeating units of carrageenans


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P h y s i o l o g i c a l E f f e c t s of Food Grade Carrageenan Breakdown i n the I n t e s t i n a l T r a c t . Breakdown of carrageenan i n the d i g e s t i v e system i s probably minimal, f o r i t seems that n e i t h e r man nor the experimental animals t e s t e d so f a r possess the necessary enzymes to h y d r o l y z e i t . In the stomach, where pH i s very low, a small amount o f a c i d h y d r o l y s i s undoubtedly does occur. However, i n v i t r o experiments w i t h simulated g a s t r i c j u i c e at pH 1.2 and 37°C showed that i n three hours (which i s about the maximum residence time i n the stomach) the breakdown of g l y c o s i d i c linkages was l e s s than 0.1% (39). Information about what happens i n the lower gut i s n o t o r i o u s l y l a c k i n g . I n c u b a t i o n of a carrageenan s o l u t i o n w i t h the c e c a l contents of r a t s f o r s e v e r a l hours at 37°C d i d not a l t e r i t s v i s c o s i t y , which i n d i c a t e s that the m i c r o b i a l f l o r a , of the r a t gut a t any r a t e , w i l l not break down carrageenan (40). There i s always the p o s s i b i l i t y t h a t , a f t e r s e v e r a l months of feeding large doses of carrageenan, the m i c r o b i a l f l o r a would be changed enough to cause some breakdown. However, there i s so f a r no suggestion that t h i s might occur. I n f a c t there seem to be very few b a c t e r i a , other than those of marine o r i g i n , that can decompose carrageenan. Our own waste d i s p o s a l d i f f i c u l t y w i t h d i g e s t i n g carrageenan w i t h a c t i v a t e d sewage sludge a t t e s t s to t h a t . A b s o r p t i o n . I f carrageenan i s not broken down, i s i t absorbed without breakdown? In three species of monkey (41, 42, 43), dog (44), p i g ( 4 5 ) , r a t (41, 46, 47, 48, 49), mouse ( 4 1 ) , f e r r e t ( 4 1 ) , hamster ( 4 1 ) , i t a p p a r e n t l y i s not. Houck (44) obtained 100% r e covery i n the feces of dogs. In a 2-year feeding study by N i l s o n and Wagner ( 4 8 ) , r a t s r e c e i v e d from 1 to 25% carrageenan mixed w i t h the dry d i e t . Growth r a t e s and i n t e r n a l organs of the a n i mals were normal a t dose l e v e l s up to 10%. At high doses growth was r e t a r d e d , and some animals fed 25% carrageenan showed l i v e r a b n o r m a l i t i e s . N i l s o n and Wagner a t t r i b u t e d the l a t t e r to a d i e t a r y d e f i c i e n c y caused by the large bulk of the ingested c a r r a geenan. Recovery i n the feces was 50% i r r e s p e c t i v e of the l e v e l fed. This r a t h e r s u r p r i s i n g r e s u l t was, however, obtained by an a n a l y t i c a l method which l e f t something to be d e s i r e d . Two l a t e r s t u d i e s , a l s o w i t h r a t s , r e s u l t e d i n 90-100% recovery (46, 47). In the case of guinea pigs and r a b b i t s , r e s u l t s have been cont r a d i c t o r y . Watt and Marcus (50) found that h i g h molecular weight carrageenan caused u l c e r a t i o n of the cecum and c o l o n of guinea p i g s . Grasso, S h a r r a t t , C a r p a n i n i and Gangoli (41) confirmed these r e s u l t s and a l s o showed that r a b b i t s were s i m i l a r l y a f f e c t ed. Although the bulk of the carrageenan was e x c r e t e d i n the f e c e s , a c e r t a i n amount was absorbed by the macrophages i n the s u b e p i t h e l i a l l a y e r s of the cecum and c o l o n . On the other hand, a study a t the Albany M e d i c a l College showed n e i t h e r a b s o r p t i o n nor l e s i o n s (49, 51). Anderson and Soman ( 5 2 ) , l i k e w i s e , showed


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that high molecular weight carrageenan was not absorbed through the gut of guinea pigs and could not be detected i n the blood or u r i n e unless a d m i n i s t e r e d i n t r a v e n o u s l y . Grasso et a l . hypothes i z e that a b s o r p t i o n by macrophages may be p e c u l i a r to guinea pigs and r a b b i t s , herbivorous rodents, both o f which possess an unu s u a l l y large cecum, and suggest that a b s o r p t i o n may be due to i n complete neonatal c l o t u r e of the i n t e s t i n a l b a r r i e r to macromolecules . Other E f f e c t s . Now, i f carrageenan i s not digested and not absorbed, j u s t what p h y s i o l o g i c a l e f f e c t s does i t have? So f a r , s e v e r a l e f f e c t s have been reported i n the l i t e r a t u r e : Reduction of p e p t i c a c t i v i t y Reduced flow of g a s t r i c s e c r e t i o n s i n the stomach Antilipemic a c t i v i t y Increase i n water content of the gut A l l these e f f e c t s can be a t t r i b u t e d to the h y d r o p h i l i c and p o l y a n i o n i c p r o p e r t i e s of the macromolecule, however, they a l l r e s u l t only from very h i g h dosages of carrageenan and cannot be detected at the low l e v e l s a t which carrageenan i s used i n food. A n t i p e p t i c a c t i v i t y . The f i r s t and the best documented of these e f f e c t s i s the a n t i p e p t i c a c t i v i t y . Carrageenan i n t e r f e r e s w i t h the p r o t e o l y t i c a c t i v i t y of p e p s i n , both ixi v i t r o and i n v i v o (44, 52, 53). Several researchers have concluded that the i n h i b i t i o n i s due to the i n t e r a c t i o n of carrageenan w i t h the subs t r a t e and not w i t h the p e p s i n . This view i s supported by the f a c t that the degree of i n h i b i t i o n depends on the r a t i o of c a r r a geenan to s u b s t r a t e and not on the amount of p e p s i n (54, 55, 56). Anderson ( 1 , 54) has pointed out that a t pH 1.3 most p r o t e i n s become p o s i t i v e l y charged and form i n s o l u b l e complexes w i t h c a r r a geenan, whereas pepsin w i t h an i s o e l e c t r i c p o i n t of 1.0 remains a n i o n i c under these c o n d i t i o n s and i s u n l i k e l y to r e a c t . The noni n t e r a c t i o n of carrageenan and p e p s i n has a l s o been shown e l e c t r o p h o r e t i c a l l y (57). You may ask then, i f carrageenan i n h i b i t s p e p t i c a c t i v i t y , w i l l i t not then i n t e r f e r e w i t h d i g e s t i o n and cause reduced prot e i n intake? This c e r t a i n l y would have been a p o s s i b i l i t y i f carrageenan i n t e r a c t e d w i t h the p e p s i n , because then, even small doses should i n t e r f e r e . However, since i t r e a c t s w i t h the subs t r a t e , the amount of p r o t e i n d i g e s t e d depends on how much of i t remains unreacted w i t h the carrageenan. Since the amount of proe i n ingested i n a normal d i e t i s c o n s i d e r a b l e , very l a r g e doses of carrageenan would be needed to i n a c t i v a t e a l l the s u b s t r a t e . Indeed, such seems to be the case. Hawkins and Yaphe (47) found that young r a t s gained weight more s l o w l y only i f t h e i r d i e t contained more than 107 carrageenan (10,000 mg/kg). Such an excess i s , however, completely u n r e a l i s t i c and the work of o



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Vaughan, F i l e r and C h u r e l l a (56) shows what occurs under condit i o n s c l o s e r to r e a l i t y . They examined p e p t i c i n h i b i t i o n of c a r rageenan w i t h s e v e r a l p r o t e i n s i n v i t r o and a l s o measured i n v i v o d i g e s t i b i l i t y of m i l k p r o t e i n and carrageenan mixtures i n r a t s . They found that i n h i b i t i o n depended on the r a t i o of carrageenan to p r o t e i n . In a l l cases there was no d e t e c t a b l e i n h i b i t i o n a t r a t i o s s m a l l e r than 0.1 and i n some cases r a t i o s were as h i g h as 0.3 before i n t e r f e r e n c e occurred. In the i n v i v o s t u d i e s the d i g e s t i b i l i t y was 100% a t r a t i o s below 0.1. Even a t the h i g h r a t i o of 0.3 p e p t i c a c t i v i t y was reduced only 10 to 20%. The g r e a t e s t use of carrageenan i s i n the d a i r y i n d u s t r y , however, the l e v e l s a t which i t i s used are extremely low. In puddings the carrageenan to p r o t e i n r a t i o i s about 0.03, i n chocolate m i l k i t i s 0.01, and i n i c e cream, evaporated m i l k and i n f a n t f o r mulas i t i s about 0.005. Products, such as r e l i s h e s or low c a l o r i e j e l l i e s which may c o n t a i n up to 0.77 carrageenan and i n which the carrageenan exceeds the amount of p r o t e i n , g e n e r a l l y form o n l y a minor p a r t of the d i e t and are u s u a l l y eaten a t the same time as other foods which have a h i g h p r o t e i n content. Therefore, unless someone dec i d e d to go on a pure j e l l y and r e l i s h binge the l i k e l i h o o d of p r o t e i n d e f i c i e n c y due to a s u r f e i t of carrageenan i s q u i t e n e g l i g i b l e . In f a c t the average per c a p i t a consumption o f carrageenan i n the U n i t e d States i s l e s s than 0.5 mg/kg of body weight per day. The h i g h e s t l e v e l s are consumed by i n f a n t s r e c e i v i n g s p e c i a l d i e t a r y formulas during the f i r s t 2-3 months a f t e r b i r t h . The d a i l y l e v e l s average 25 to 50 mg/kg of body weight, but the r a t i o of carrageenan to p r o t e i n i s o n l y about 0.005. o

G a s t r i c s e c r e t i o n . Carrageenan diminishes the volume and a c i d i t y of h i s t a m i n e - s t i m u l a t e d g a s t r i c s e c r e t i o n , but on the other hand, i t a l s o r e s t o r e s normal s e c r e t i o n i n cases where supramaximal histamine s t i m u l a t i o n has caused submaximal a c i d output. According to Anderson ( 5 8 ) , carrageenan complexes w i t h mucin on the stomach w a l l and i t s a b i l i t y to i n h i b i t g a s t r i c s e c r e t i o n (59, 60) could be r e l a t e d by t h i s i n t e r a c t i o n . I t s a b i l i t y to r e store normal flow i s , however, more d i f f i c u l t to e x p l a i n (61). These phenomena are u n l i k e l y to take p l a c e w i t h carrageenan that i s ingested w i t h food -- f i r s t because of the small amounts present and, second, because the carrageenan would be complexed w i t h the food p r o t e i n . Lipemia c l e a r i n g . The t h i r d e f f e c t of carrageenan i s i t s hypocholesterolemic a c t i v i t y . A g a i n , large dosages are necessary. Fahrenbach et a l (62) obtained s i g n i f i c a n t r e d u c t i o n i n blood c h o l e s t e r o l of white Leghorn c h i c k s which were fed 1 to 3% c a r r a geenan i n the d i e t . E r s h o f f and W i l l s (63) obtained a s i m i l a r e f f e c t w i t h 10% carrageenan i n the d i e t of r a t s fed 1% c h o l e s t e r o l . L i v e r c h o l e s t e r o l remained a t normal l e v e l and t o t a l l i v e r


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l i p i d were g r e a t l y reduced. The mechanism o f lipemia c l e a r i n g i s not known. Perhaps, carrageenan, l i k e other n o n - d i g e s t i b l e f i b e r may i n t e r f e r e w i t h c h o l e s t e r o l a b s o r p t i o n . I t i s unfortunate t h a t , i n order to take advantage o f the a n t i - l i p e m i c p r o p e r t i e s of carrageenan, the o r a l dosage has to be so h i g h . Water content o f the bowel. There i s nothing unusual about the f o u r t h e f f e c t o f carrageenan which increases the water content o f the bowel and causes s o f t e n i n g o f the s t o o l . This e f f e c t i s common to a l l h y d r o p h i l i c polymers s e v e r a l o f which, notably agar, p s y l l i u m gum, and p e c t i n , are used as m i l d l a x a t i v e s because o f t h e i r w a t e r - h o l d i n g p r o p e r t i e s . P h y s i o l o g i c a l E f f e c t s o f Degraded

Carrageenan

Now a few words about degraded o r depolymerized carrageenan. This product, which has a molecular weight of 10-20,000 was developed as a more convenient, l e s s v i s c o u s , dosage form f o r the treatment o f p e p t i c and duodenal u l c e r s i n humans. I t i s produced by p a r t i a l h y d r o l y s i s o f the g l y c o s i d i c l i n k a g e s w i t h a c i d . Carrageenan, both i n the degraded and undegraded form, prevents o r diminishes histamine-induced experimental p e p t i c u l c e r a t i o n (44, 60) and a l l e v i a t e s p e p t i c and duodenal u l c e r s i n humans (64). Anderson (58) a t t r i b u t e s t h i s e f f e c t to the a b i l i t y o f carrageenan t o complex w i t h g a s t r i c mucin, thus p r o t e c t i n g the stomach w a l l a g a i n s t a t t a c k by the g a s t r i c j u i c e s . The degraded product i s as e f f e c t i v e i n p r o t e c t i n g the stomach w a l l as undegraded carrageenan and i s much e a s i e r f o r a pat i e n t t o take because o f i t s very low v i s c o s i t y . In c o n t r a s t to food grade carrageenan, the degraded product i s p a r t l y absorbed through the gut. I t has been detected i n the u r i n e o f baboons (42) and guinea pigs ( 5 2 ) . The amount absorbed was l e s s than 1%. I n Rhesus monkeys i t accumulated i n the l y s o somes o f the r e t i c u l o e n d o t h e l i a l c e l l s o f the l i v e r , s p l e e n , and lymph nodes, and could s t i l l be detected s i x months a f t e r t r e a t ment (43). Degraded carrageenan a l s o causes u l c e r a t i o n of the cecum and c o l o n o f s e v e r a l t e s t s p e c i e s . G e r b i l s and mice were not a f f e c t ed (51). Guinea pigs and r a b b i t s were p a r t i c u l a r l y s u s c e p t i b l e (40, 41, 50, 51, 65). Lesions were a l s o produced i n Rhesus monkeys (43) a t high dose l e v e l s (3,000 mg/kg/day), but not i n s q u i r r e l monkeys (41). C l i n i c a l t e s t s by B o n f i l s (64) on 200 p a t i e n t s r e c e i v i n g 5 grams per day (100/mg/kg/day) o f degraded carrageenan i n p e p t i c u l c e r treatment showed no adverse e f f e c t s on the c o l o n a f t e r s i x months to two years of treatment. The a b s o r p t i o n and u l c e r a t i o n a p p a r e n t l y are h i g h l y dependent on dose l e v e l and molecular weight. Above a molecular weight o f 50,000 no a b s o r p t i o n can be detected (51, 52).


EFFECTS

OF

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CARBOHYDRATES


PHYSIOLOGICAL



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AND

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Despite the apparent l a c k of t o x i c i t y to humans we must, n e v e r t h e l e s s , be c a u t i o u s i n the event that degraded carrageenan might have adverse e f f e c t s a f t e r prolonged i n g e s t i o n . For t h i s reason the Food and Drug A d m i n i s t r a t i o n r e q u i r e s a minimum molecu l a r weight of 100,000 f o r carrageenan i n food. This i s no problem because the s t a b i l i z i n g p r o p e r t i e s of carrageenan depend on high molecular weight. However, carrageenan i s p o l y d i s p e r s e and there i s always the p o s s i b i l i t y that low molecular weight m a t e r i a l may be present. At Marine C o l l o i d s we have developed a r a p i d method f o r determining molecular weight and molecular weight d i s t r i b u t i o n of c a r rageenan. The method i s based on the r e l a t i v e e l e c t r o p h o r e t i c m o b i l i t y of d i f f e r e n t s i z e carrageenan molecules i n a t h i n f i l m agarose g e l (66). S e p a r a t i o n i s p r i m a r i l y according to molecular weight, w i t h charge d e n s i t y e x e r t i n g o n l y a minor e f f e c t . The separated species can be q u a n t i t a t e d by t r a n s m i s s i o n densitometry. By comparing a s e r i e s of p r o g r e s s i v e l y depolymerized carrageenans whose molecular weights had been determined by u l t r a c e n t r i f u g a t i o n , a l i n e a r r e l a t i o n s h i p was found between e l e c t r o p h o r e t i c m o b i l i t y and the cube root of the molecular weight. The method has the advantage that o n l y about 50 micrograms of sample i s r e q u i r e d , s e v e r a l samples can be t e s t e d at the same time, r e s u l t s are obtained i n a day, and s e p a r a t i o n of high and low molecular weight carrageenan i s very good. The densitometer scan of a pherogram of a mixture of high and low molecular weight carrageenan i s shown i n F i g u r e 2. Our work has shown that o n l y minute amounts of low molecular weight m a t e r i a l i s present i n food grade carrageenan. C u r r e n t l y we are t e s t i n g carrageenan processed under c o n d i t i o n s used i n food p r o c e s s i n g . Our r e s u l t s to date show l i t t l e or no change i n mol e c u l a r weight d i s t r i b u t i o n under normal p r o c e s s i n g c o n d i t i o n s . Conclusion In c o n c l u s i o n , the s a f e t y o f carrageenan as a food a d d i t i v e has been the t a r g e t of many in-depth s t u d i e s supported by government agencies here and abroad as w e l l as by the v a r i o u s c a r r a geenan producers. From the r e s u l t s of these s t u d i e s , we can conclude that food grade carrageenan -- an i s o l a t e d component of n a t u r a l food products used e x t e n s i v e l y f o r over two c e n t u r i e s -has no adverse p h y s i o l o g i c a l e f f e c t s and that i t s s a f e t y i n foods i s assured. Addendum Development since September, 1974. Recently d u r i n g the course of a current t h r e e - g e n e r a t i o n feeding study, researchers at the Food and Drug A d m i n i s t r a t i o n noted changes i n the s u r f a c e appearance of the l i v e r s of animals being fed 5% and 2.5% carrageenan i n the dry d i e t (67). L i v e r s u r f a c e s appeared to be lumpy and i r r e g -



292

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O F FOOD

CARBOHYDRATES

u l a r . A t the 5% carrageenan l e v e l the frequency o f t h i s occur rence was 93% while a t the 2.5% l e v e l i t was 40%. Male animals fed the highest l e v e l gained l e s s weight than normal. In other respects the r a t s were q u i t e h e a l t h y and r e p r o d u c t i o n was normal. The l i v e r changes are d i f f i c u l t to e x p l a i n i n the l i g h t o f so many previous s t u d i e s , p a r t i c u l a r l y since the changes were very obvious and could hardly have been missed i f they had occurred before. On the other hand, a very thorough six-month feeding study at Wyeth Labs., Inc. (68), with 4% carrageenan i n the d i e t o f r a t s , revealed no a b n o r m a l i t i e s whatsoever. In t h i s case the carrageenan had been mixed i n t o skim m i l k a t a c o n c e n t r a t i o n equal to that o f the p r o t e i n a f t e r which the mixture was spraydried or lyophilized. The carrageenan had no i n f l u e n c e on growth r a t e , d i e t energy e f f i c i e n c y , a b s o r p t i o n o f p r o t e i n , f a t , or c a l cium, u t i l i z a t i o n o f p r o t e i n f o r growth o r the u t i l i z a t i o n o f i r o n . Gross examination o f the animals' organs showed no abnor malities. F u r t h e r t e s t s are now i n progress to c l a r i f y the d i s crepancies between the v a r i o u s s t u d i e s . Literature Cited

1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16.

Anderson, W., Can. J. Pharm. Sci., (1967), 2, 81-90. Anderson, W., Proc. Int. Seaweed Symp., (1969), 6, 627-635. DiRosa, Μ., J. Pharm. Pharmacol., (1972), 24, 89-102. Benitz, K.F. and Hall, L.M., Proc. Soc. Exp. Biol. Med. (1959), 108, 442-445. DiRosa, Μ., Giroud,J.P.and Willoughby, D.A., J. Pathol., (1971), 104, 15-29. DiRosa, M. and Sorrentino, L., Eur. J. Pharmacol., (1968), 4, 340-342. Garg, Bhagwan D. and McCandless, Esther L., Anat. Rec., (1968), 162, (1), 33-40. Hurley,J.V.and Willoughby, D.A., Pathol. (1973), 5, 9-21. Jackson, D.S., Biochem. J., (1957), 65, 277-284. Jackson, D.S., Biochem. J., (1957), 65, 459-464. McCandless, E.L., Ann. N.Y. Acad. Sci., (1965), 118, (22), 867-882. Monis, Β., Weinberg, T. and Spector, G.J., Brit.J.Exp. Pathol., (1968), 49, 302-310. Robertson, W., Hiwett, J. and Herman, L.C., J. Biol. Chem., (1959), 234, 105-108. Salvaggio, J. and Kundur, V., Proc. Soc. Exp. Biol. Med., (1970), 134, 1116-1119. Winter, C.A., Risley, E.A. and Nuss, G.W., Proc. Soc. Exp. Biol. Med., (1962), 111, 544-547. Van Arman, C.G., Begany, A.J., Miller, L.M., and Pless, H.H., J. Pharmacol. Exp. Ther., (1965), 150, 328-334.


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41. Grasso, P., Sharratt, Μ., Carpanini, F.M.B., and Gangolli, S.D., Food Cosmet. Toxicol., (1973), 2, 555-564. 42. Beattie, I.Α., Blakemore, W.R., Dewar, E.T. and Warwick, M.H., Food Cosmet. Toxicol., (1970), 8, 257-266. 43. Abraham, R., Golberg, L. and Coulston, F., Exp. Mol. Pathol., (1972), 17, 77-93. 44. Houck, J.C., Bhayana, J. and Lee, T., Gastroenterology, (1960), 39, 196-200. 45. Poulsen, Ε., Food Cosmet. Toxicol., (1973), 2, 219-227. 46. Dewar, E.T. and Maddy, M.L., J. Pharm. Pharmacol., (1970), 22, 791-793. 47. Hawkins, W.W. and Yaphe, W., Can. J. Biochem., (1965), 43, 479-484. 48. Nilson, H.W. and Wagner, J.A., Food Res., (1959), 24, 235-239. 49. Abraham, R., Golberg, L., and Coulston, F., Paper No. 192, presented at the Annual Meeting of the Society of Toxicology, March 10-14, 1974. 50. Watt, J. and Marcus, R., J. Pharm. Pharmacol., (1969), 21, Suppl. 187S. 51. "Safety Evaluation of Carrageenan," Albany Medical College, Institute Experimental Pathology and Toxicology, Albany, New York, (Unpublished report 1971). 52. Anderson, W., and Soman, P.D., J. Pharm. Pharmacol., (1966), 18, 825-827. 53. Anderson, W. and Harthill, J.E., J. Pharm. Pharmacol., (1967), 17, 647-654. 54. Anderson, W., and Baillie, A.J., J. Pharm. Pharmacol., (1967), 19, 720-728. 55. Anderson, W., Baillie, A.J. and Harthill, J.E., J. Pharm. Pharmacol., (1968), 20, 715-722. 56. Vaughan, O.W., Filer, L.J. Jr., and Churella, Μ., J. Agr. Food Chem., (1962), 10, 517-519. 57. Martin, F., Vagne, A.B., and Lambert, R., C.R. Soc. Biol., (1965), 159, 1582-1585. 58. Anderson, W. and Watt, J., J. Pharm. Pharmacol., (1959), 11:318. 59. Watt, J., Eagleton, J.B. and Marcus R., Nature, (1966), 211, 989. 60. Anderson, W., Marcus, R. and Watt, J., J. Pharm. Pharmacol., (1962), 14, 119-121. 61. Anderson, W. and Soman, P.D., Nature, (1967), 214, 823-824. 62. Fahrenbach, M.J., Riccardo, B.A. and Grant, W.C., Proc. Soc. Exp. Biol. Med., (1966), 123, 321. 63. Ershoff, B.H. and Wills, A.F., Proc. Soc. Exp. Biol. Med., (1962), 110, 580-582. 64. Bonfils, S., Lancet, (1970), 2, 414. 65. Watt, J. and Marcus, R., J. Pharm. Pharmacol., (1970), 22, 130-131.


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66. Stanley, N.F. and Renn, D.W., Proc. Int. Seaweed Symp., 8th, (1974), Bangor, Wales (In press). 67. Food Chemical News, (1974), 16, (29), 3-5. 68. Tomarelli, R.M., Tucker, W.D. Jr., Bauman, L.M., Savini, S. and Weaber, J.R., J. Agr. Food Chem., 22, 819-824.


Physiological Effects of Carrageenan - ACS Symposium Series (ACS

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