Absorption of Altered Amino Acids from the Intestine - ACS Publications

Absorption of Altered Amino Acids from the Intestine

11

DANIEL E. SCHWASS—Western Regional Research Center, U.S. Department of Agriculture, ARS, Berkeley, CA 94710 L. RAÚL TOVAR—Departamento de Alimentos, DEPg, Facultad de Química, Universidad de México, México 04510, D. F. 1

Downloaded by EMORY UNIV on February 21, 2016 | http://pubs.acs.org Publication Date: October 25, 1983 | doi: 10.1021/bk-1983-0234.ch011

JOHN W. FINLEY —Nutritional Biochemistry, Ralston Purina, Checkerboard Square, St. Louis, MO 63188 Heat or alkaline treatment of protein-containing materials may cause racemization of a portion of the amino acids as well as crosslinking between amino acids. Very little is understood about how these altered amino acids interact with transport systems for normal amino acids and peptides in the gut. This study was designed to examine the effects of racemization on in vitro digestibility of alkali-treated protein and on in vivo uptake of digested, treated protein, in the absence of crosslinking. We report that significant decreases in in vitro digestibility and in vivo availability can be attributed to racemization effects. Two major chemical m o d i f i c a t i o n s of p r o t e i n s that occur during a l k a l i n e treatment are c r o s s l i n k i n g and racemization. Lysine, o r n i t h i n e ( v i a a r g i n i n e ) , c y s t i n e and O-substituted serine can p a r t i c i p a t e i n base-catalyzed reactions forming the c r o s s l i n k e d amino acids l y s i n o a l a n i n e , o r n i t h i n o a l a n i n e and l a n t h i o n i n e ( 1^-4). Under the same c o n d i t i o n s , i n v e r s i o n can occur when the or hydrogen of an amino a c i d residue i s abstracted by the base, r e s u l t i n g i n a planar, o p t i c a l l y i n a c t i v e carbanion ( 7 ) , as i l l u s t r a t e d i n Figure 1. The carbanion may be reprotonated from e i t h e r face of the plane, which causes i n v e r s i o n when t h i s occurs from the opposite f a c e . The formation of l y s i n o a l a n i n e (LAL) i n treated p r o t e i n s has been the subject of s e v e r a l i n v e s t i g a t i o n s ( f o r reviews, see 6^7) since i t was discovered that a l k a l i - t r e a t e d soy p r o t e i n could cause a kidney l e s i o n i n r a t s and LAL appeared to be r e s p o n s i b l e ( 8 ) . Human t o x i c i t y has not been e s t a b l i s h e d and no l e s i o n s were observed i n rhesus monkeys, mice, hamsters, dogs and Japanese q u a i l f e d d i e t s i n c l u d i n g f r e e LAL or p r o t e i n containing LAL (8^ and see 7 ) . 1

Current address: Department of Pediatrics, University Hospitals and Clinics, University of Iowa, Iowa City, IA 52242

©

0097-6156/83/0234-0187$06.00/0 1983 A m e r i c a n C h e m i c a l S o c i e t y

In Xenobiotics in Foods and Feeds; Finley, John W., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1983.


188

X E N O B I O T I C S IN

FOODS A N D

FEEDS

In a d d i t i o n to LAL, D-serine has been shown to be nephrotoxic i n r a t s , with l e s i o n s s i m i l a r to those observed a f t e r LAL administ r a t i o n ( 9 ) . When included i n the d i e t , D-serine causes n e c r o s i s of the proximal tubule of the kidney which can be f a t a l (10). A l though r a t s are s e n s i t i v e to D-serine, s e v e r a l other species such as dogs, hamsters, r a b b i t s and mice do not develop l e s i o n s (8,11). T o x i c i t y has not been e s t a b l i s h e d f o r humans. Over and above the t o x i c e f f e c t s of D-serine, which are l i k e l y to be n e g l i g i b l e i n a r e a l i s t i c d i e t , s e v e r a l studies have provided evidence that racemization of amino a c y l residues i n a l k a l i - t r e a t e d and heat-treated proteins decreases d i g e s t i b i l i t y . Dakin, i n 1908, was the f i r s t to examine the e f f e c t s of a l k a l i treatment on in vitro d i g e s t i b i l i t y (12). He showed that treated c a s e i n was h i g h l y - r e s i s t a n t to pepsin, t r y p s i n and e r e p s i n hyd r o l y s i s . Dakin and Dudley followed up the in vitro studies with an experiment i n which dogs were fed a l k a l i - t r e a t e d c a s e i n , and found that the p r o t e i n was l a r g e l y unabsorbed and was recoverable i n the feces (13). In 1969, deGroot and Slump a l s o demonstrated decreases f o r in vitro d i g e s t i b i l i t y of a l k a l i - t r e a t e d soy p r o t e i n i s o l a t e and decreases i n absorption of some amino acids by everted i n t e s t i n a l sacs (14)· These workers a l s o observed decreases i n net p r o t e i n u t i l i z a t i o n f o r treated soybean meal, treated soy p r o t e i n i s o l a t e and treated c a s e i n . These decreases i n net p r o t e i n u t i l i z a t i o n were c o r r e l a t e d with increases i n LAL formation. In these e x p e r i ments, therefore, i t was p o s s i b l e that p r o t e i n u t i l i z a t i o n was hindered by the presence of LAL. Provansal and co-workers (15) treated sunflower p r o t e i n i s o l a t e with sodium hydroxide and observed formation of LAL, racemization of i s o l e u c i n e and l y s i n e (only these two were analyzed f o r racemization), and decreased in vitro d i g e s t i b i l i t y by pronase. While these workers suggested that the decreased pronase h y d r o l y s i s may have been due to c r o s s l i n k i n g r e a c t i o n s , they a l s o pointed out that i t was l i k e l y that other amino acids were a l s o racemized which could compromise the n u t r i t i o n a l q u a l i t y of the protein. More r e c e n t l y , Hayashi and Kameda (16) investigated the e f f e c t s of a l k a l i treatment on c a s e i n and soybean p r o t e i n by looking a t in vitro pepsin d i g e s t i b i l i t y , racemization and LAL f o r mation. These authors observed that pepsin released fewer amino a c i d s from treated p r o t e i n than c o n t r o l samples. They suggested that decreases i n in vitro d i g e s t i b i l i t y were due to racemization (which was measured i n d i r e c t l y by t r i t i u m exchange) and not c r o s s l i n k i n g because they f e l t LAL formation occurred too slowly. However, i t appears that LAL formation was high enough i n these experiments to question t h i s c o n c l u s i o n . In 1981, Friedman, Zahnley and Masters measured the in vitro d i g e s t i b i l i t y of a l k a l i - t r e a t e d c a s e i n by t r y p s i n and chymotrypsin as a f u n c t i o n of temperature, time and pH of the treatment (17). They a l s o measured LAL formation and the racemization of aspartate



11.

SCHWASS E T A L .

Absorption

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189

and phenylalanine and observed that the decrease i n d i g e s t i b i l i t y occurred as both c r o s s l i n k i n g and racemization increased. However, i t was not p o s s i b l e from t h e i r r e s u l t s to determine whether c r o s s l i n k i n g r e a c t i o n s or racemization had the greater e f f e c t on digestibility. Because c r o s s l i n k i n g and racemization both occur during a l k a l i n e treatment of p r o t e i n s and an e f f e c t of both of these phenomena i s to decrease n u t r i t i o n a l q u a l i t y (18), i t i s of i n t e r e s t to know i f e i t h e r process has a greater e f f e c t on p r o t e i n d i g e s t i b i l i t y and uptake. While there i s no way known to prevent racem i z a t i o n during a l k a l i n e treatment, i t i s p o s s i b l e to prevent LAL formation by the a d d i t i o n of t h i o l s during processing (19) or by a c y l a t i o n of the p r o t e i n p r i o r to processing (20). The work of Bunjapamai, Mahoney and Fagerson (21) was the f i r s t s u c c e s s f u l attempt to separate the e f f e c t s of racemization from c r o s s l i n k i n g as measured by in vitro d i g e s t i o n . Alkalitreatment of c i t r a c o n y l a t e d ( l y s i n e - b l o c k e d ) or non-blocked c a s e i n r e s u l t e d i n racemized only (blocked) or racemized and LAL c r o s s l i n k e d (non-blocked) c a s e i n . In vitro multienzyme d i g e s t i o n of these preparations as w e l l as untreated c a s e i n revealed s i m i l a r l y decreased d i g e s t i b i l i t i e s f o r the treated p r o t e i n s whether c r o s s l i n k e d or not, i n d i c a t i n g that the primary cause f o r reduct i o n of c a s e i n d i g e s t i b i l i t y was racemization. I t i s important to know the separate e f f e c t s of racemization and c r o s s l i n k i n g f o r s e v e r a l p r o t e i n s , e s p e c i a l l y those important i n food systems. Therefore, the purpose of t h i s study was to i s o l a t e racemization from c r o s s l i n k i n g , examine the e f f e c t s of racem i z a t i o n on in vitro d i g e s t i b i l i t y of a l k a l i - t r e a t e d z e i n and in vivo accumulation of a l k a l i - t r a t e d p r o t e i n by i s o l a t e d r a t jejunum. Zein, which i s the major p r o t e i n i n corn, was chosen because i t contains no l y s i n e . T h i s precluded the formation of LAL during a l k a l i treatment. Therefore, any changes i n d i g e s t i b i l i t y or uptake could be a t t r i b u t e d to racemization e f f e c t s alone. Addit i o n a l l y , we were i n t e r e s t e d i n comparing the e f f e c t s of sodium hydroxide treatment with the e f f e c t s of calcium hydroxide t r e a t ment because lime i s used i n the p r e p a r a t i o n of corn meal f o r use in t o r t i l l a s . I f the t r a d i t i o n a l lime treatment of corn meal i s u n n e c e s s a r i l y harsh, i t could have important n u t r i t i o n a l consequences because a l a r g e segment of the Mexican population obtains much of t h e i r d i e t a r y p r o t e i n i n the form of t o r t i l l a s (22).

M a t e r i a l s and Methods The b a s i c o u t l i n e of our experiment (Figure 2) was to t r e a t z e i n with sodium hydroxide or calcium hydroxide causing racemizat i o n . The treated or untreated z e i n was then enzymatically hydrolyzed and values f o r d i g e s t i b i l i t y determined. Samples of the


190

XENOBIOTICS

lOH-J

IN FOODS A N DF E E D S

RH

Ji-C-N-PROT L-ISOMER

o=c

IOT' PROT' Η

•

R /N-PROT 0"C PLANAR =c CARBANION v

0


PROT'

4^

H R ^ P R O T - N - Ç - H . ^ D-ISOMER C=0

PROT' Figure

1. Mechanism

of base-catalyzed inversion protein.

of an amino

acyl residue in a

ZEIN

NaOH ^Heat Na-ZEIN Pronase Hydrolysis

Step

ZEIN HYDROLYSATES

2.

Intestinal Segment

a. Everted Sac or

b. Perfusion

Amino Acid Analysis Determines Absorption Figure 2. Outline of in vitro digestibility and in vitro (everted sac) or in vivo (intestinal perfusion) uptake experiments. Amino acid analysis of everted sac contents or of original and final intestinal perfusate allows uptake determination.


11.

SCHWASS E T A L .

Absorption

of Altered

Amino

Acids

191

enzymatic hydrolysates were then presented to segments of r a t i n t e s t i n e to determine uptake of the pre-digested treated or un treated material*


A l k a l i Treatment. Zein (ICN N u t r i t i o n a l Biochemicals, Cleveland, OH) was suspended i n 0.1N sodium hydroxide or 0.1N calcium hydrox i d e , h e l d at 85°C f o r 4 hours, n e u t r a l i z e d with 2 Ν h y d r o c h l o r i c a c i d , cooled to 4°C (3 hours) and d i a l y z e d against 25 volumes of 0.1N acetate b u f f e r , pH 4.5 f o r 24 hours with one change a t e i g h t hours. Untreated z e i n was suspended i n deionized water and taken through the d i a l y s i s procedure. A f t e r d i a l y s i s , the zeins were l y o p h i l i z e d . At t h i s p o i n t , samples were taken f o r amino a c i d a n a l y s i s (Durrum D-500, Dionex Corp., Sunnyvale, CA; or Beckman 121 MB, Palo A l t o , CA) and isomer a n a l y s i s . H y d r o l y s i s by Pronase. In vitro pronase ( Calbiochem-Behring Corp L a J o l l a , CA) hydrolyses were performed according to the method of Rayner and Fox (23). Mixtures of z e i n , pronase and 40 mM borate b u f f e r (pH 8.0) i n the r a t i o 1 gm z e i n to 150 mg pronase to 100 ml b u f f e r were placed i n screw-cap f l a s k s and the pH readjusted to 8.0 i f necessary. A sample without z e i n was a l s o prepared as a c o n t r o l . The suspensions were incubated at 40°C f o r 48 hours with shaking. A f t e r the i n c u b a t i o n , a 3 ml a l i q u o t was taken f o r amino a c i d a n a l y s i s and the remainder was f r o z e n at -20°C. The 3 ml of hydrolysate was mixed with 6 ml 1% aqueous p i c r i c a c i d and c e n t r i fuged f o r 30 minutes a t 3000 rpm at 4°C to remove r e s i d u a l p r o t e i n and peptides. F i v e ml of the supernatant was placed on a 1.2 cm by 9 cm AG-50W-X8(H+), 100-200 mesh c a t i o n exchange column ( B i o Rad L a b o r a t o r i e s , Richmond, CA). P i c r i c a c i d was washed from the column with deionized water and the bound hydrolysate was e l u t e d with 3M ammonium hydroxide. The eluates were r o t a r y evaporated a t 60°C and washed twice with deionized water, before being d i s solved i n 0.2N c i t r a t e b u f f e r (pH 2.2) f o r amino a c i d a n a l y s i s . Everted Sac Experiments. Hydrolysate s o l u t i o n s f o r use i n the everted sac experiments were prepared by mixing 15 ml of the thawed pronase hydrolysates with 5 ml of a s o l u t i o n c o n t a i n i n g 4.50 g/1 N a d , 0.74 g/1 KCL and 2.10 g/1 NaH003. The s o l u t i o n s were e q u i l i b r a t e d with 95%:5% oxygen:carbon dioxide at 37°C (pH 8.0) and o s m o l a l i t y (Wescor, Inc., Logan, UT) was observed to be w i t h i n 10% of 302 mOsm. Everted i n t e s t i n a l sacs were prepared f o l l o w i n g the method developed by Wilson and Wiseman (25). Rats (mean weight of 210g, Simonsen L a b o r a t o r i e s , G i l r o y , CA) which were fed a chow d i e t (Ralston Purina, Inc., S t . L o u i s , MO), were k i l l e d by a blow to the head a f t e r a 13 hour f a s t . The small i n t e s t i n e was sectioned a t a distance of 10% of the t o t a l length of the i n t e s t i n e from pylorus to the i l e o - c a e c a l j u n c t i o n . Any r e s i d u a l , undigested food was washed out with c h i l l e d 0.9% NaCl before the i n t e s t i n e


192

XENOBIOTICS IN FOODS

A N D FEEDS


was turned i n s i d e out ( e v e r t e d ) . Two sacs from the duodenum end, of approximately 6 cm each, were made from each r a t . The sacs were t i e d o f f from the i l e a l end and f i l l e d with 2 ml KrebsH e n s e l e i t Ringer i s o t o n i c b u f f e r , pH 7.4. The sacs were then t r a n s f e r r e d i n t o Erlenmeyer f l a s k s containing 20 ml of the sam p l e s o l u t i o n . Incubation was c a r r i e d out a t 37° f o r 1 hour with constant s t i r r i n g . A t the end of the incubation p e r i o d , the volume of the s e r o s a l f l u i d was measured and an a l i q u o t taken f o r amino a c i d a n a l y s i s . I n t e s t i n a l P e r f u s i o n Experiments. Hydrolysate s o l u t i o n s f o r use i n the perfused i n t e s t i n e experiments were prepared by concentrat ing the pronase hydrolysates approximately two-fold i n a r o t a r y evaporator a t 60°C ( t o i n a c t i v a t e pronase, 24) and mixing the e q u i v a l e n t of 157.5 mg (untreated z e i n ) , 289.2 mg (Ca(OH)2~treated z e i n ) or 150.0 mg (NaOH-treated zein) with 0.05M HEPES ( C a l b i o chem-Behring Corp., L a J o l l a , CA) b u f f e r (pH 8.0). The f i n a l s o l u tions had a pH of 8.10 ± 0.05. I n t e s t i n a l p e r f u s i o n experiments were performed using an adaptation of the technique o f Smithson and Gray (26). Rats (mean weight of 220 gm f a s t e d 13 hr.) were anesthetized with sodium pen t o b a r b i t a l (Abbot L a b o r a t o r i e s , N. Chicago, IL) and a m i d l i n e i n c i s i o n made i n the abdomen which allowed access to the small i n t e s t i n e . A length of jejunum adjacent to the duodenum was i s o l a t e d by i n s e r t i n g a tube catheter a t both ends of the segment and the sample was perfused f o r 40 minutes. Even though the i n t e s t i n a l loop was outside the animal, i t continued to r e c e i v e blood from the i n t a c t c i r c u l a t o r y system and remained q u i t e v i a b l e over the course of the experiment. F i f t e e n ml of the pronase hydrolysate s o l u t i o n s were c i r c u l a t e d through the i n t e s t i n a l segments over a 40 minute i n t e r v a l a t a r a t e of 0.39 ml/min a t 37°C, using a p e r i s t a l t i c pump (Buchler " P o l y s t a l t i c " , F o r t Lee, NJ). A t the end of the p e r f u s i o n i n t e r v a l , the perfusates were f l u s h e d from the system using 0.9% s a l i n e a t 3 7 ° C a n d s t o r e d a t -20°C f o r l a t e r a n a l y s i s . The perfused seg ments of jejumum were e x c i s e d and weighed. The f r o z e n perfusates were thawed, adjusted to pH 6.5 with 0.1N h y d r o c h l o r i c a c i d , heated 10 minutes i n a b o i l i n g water bath ( t o p r e c i p i t a t e any p r o t e i n sloughed from the mucosal c e l l s ) , c e n t r i f u g e d 10 minutes and the supernatant c o l l e c t e d . The deproteinized supernatants were made up to 25 ml with deionized water and an a l i q u o t taken f o r h y d r o l y s i s i n 6 Ν h y d r o c h l o r i c a c i d . A l i q u o t s of the pronase hydrolysate s o l u t i o n s (which were not perfused) were a l s o adjusted to pH 6.5, b o i l e d , c e n t r i f u g e d and hydrolyzed as above. Amino a c i d analyses of the hydrolyzed samples were then performed. Isomer A n a l y s i s . Isomer analyses were performed using an o p t i c a l l y a c t i v e C h i r a s i l - V a l 25m column (Applied Science, State College, PA) i n a Hewlett-Packard 5840 gas chromatograph (Avon-


11.

SCHWASS E T A L .

Absorption

of Altered

Amino

Acids

193

dale, PA) f i t t e d with an i n l e t stream s p l i t t e r and flame i o n i z a t i o n d e t e c t o r . Hydrolysates were d e r i v a t i z e d (27) using 3N hydroc h l o r i c a c i d i n dry i s o p r o p y l a l c o h o l f o r es t e r i f i c a t i o n and pent a f l u o r o p r o p i o n i c anhydride ( P i e r c e Chemical Co., Rockford, IL) for acylation.


R e s u l t s and D i s c u s s i o n The amino a c i d compositions of the untreated and treated zeins are given i n Table I, and are i n reasonable agreement with those of Boundy, et a l . (28). As expected, no LAL was observed i n these m a t e r i a l s . Table I I l i s t s the amino a c i d compositions of .the pronase hydrolysates a f t e r p i c r i c a c i d p r e c i p i t a t i o n ( i . e . amino a c i d s r e l e a s e d by pronase). For each amino a c i d but g l y c i n e , which was present only i n small q u a n t i t i e s , decreases i n released amino a c i d s were observed f o r the l i m e - and c a u s t i c soda-treated z e i n s . These r e s u l t s are shown i n Figure 3 where r e l e a s e i s expressed as a percent of that seen f o r untreated z e i n . The 32% decrease f o r the Ca(OH)2~treated z e i n and 41% decrease f o r NaOH-treated z e i n i n d i c a t e s that the in vitro d i g e s t i b i l i t y has been s i g n i f i c a n t l y reduced by both of the a l k a l i treatments. Hayashi and Kameda have reported 40% to 70% decreases i n peps i n - c a t a l y z e d h y d r o l y s i s of lysozyme, soybean p r o t e i n , c a s e i n and ribonuclease A due to a l k a l i - t r e a t m e n t under s l i g h t l y milder cond i t i o n s than ours (16, 29). Friedman, Zahnley and Masters r e p o r t ed an 80% decrease i n d i g e s t i b i l i t y of sodium hydroxide-treated c a s e i n measured as h y d r o l y s i s by t r y p s i n (17). However, t r y p s i n i s s p e c i f i c f o r l y s y l residues and l y s i n e l e v e l s decreased to about h a l f c o n t r o l values during the a l k a l i - t r e a t m e n t , with a concomitant increase i n LAL formation. The somewhat lower d i g e s t i b i l i t i e s reported by these l a b o r a t o r i e s compared to our observat i o n s may be due to LAL formation i n the p r o t e i n s other than z e i n .

100 80

Figure 3. In vitro pronase digestibilities of untreated zein (open bar), Ca (OH) treated zein (center, shaded bar) and NaOH-treated zein (right, shaded bar). Results expressed as percent free amino acids released relative to control. Standard error bars are shown. 2


194

X E N O B I O T I C S IN F O O D S A N D F E E D S

TABLE I .

Amino a c i d composition of the non-treated and a l k a l i - t r e a t e d z e i n obtained by a c i d h y d r o l y s i s , compared with published data* g/16 g n i t r o g e n

Amino Acid

2

non-treated

NaOH-•treatec

Ca(0H)2"treated

%


% Çysteic a c i d

nd

[1.5]

nd

[1.3]

nd

Methionine

nd

[2.0]

nd

[1.4]

nd

Aspartic acid

6.6

[5.6]

6.6

[6.1]

(100)

6.2

(94)

Threonine

3.5

[3.2]

2.6

[3.2]

(74)

2.5

(71)

Serine

6.8

[6.9]

5.1

[6.3]

(75)

4.8

(90)

27.7

[25.9]

25.5

[26.7]

(92)

24.8

(90)

Proline

12.6

[11.0]

11.6

[9.6]

(92)

10.8

(86)

Glycine

1.5

[1.4]

1.5

[1.2]

(100)

1.4

(93)

Alanine

12.2

[10.6]

12.0

[11.2]

(98)

11.4

(93)

Valine

4.4

[3.2]

4.4

[4.0]

(100)

3.9

(89)

Isoleucine

5.2

[3.1]

4.8

[4.4]

(92)

4.5

(87)

25.8

[20.4]

27.6

[22.4]

(107)

24.9

(97)

Glutamic

acid

Leucine Tyrosine

6.8

nd

6.2

nd

(91)

5.7

(84)

Phenylalanine

9.4

[7.4]

8.9

[7.8]

(95)

8.2

(87)

Histidine

1.7

nd

1.6

nd

(94)

1.5

(88)

Arginine

2.1

[1.6]

1.4

[1.5]

(67)

1.8

(86)

Totals

126.3 [103.8]

119.8 [107.1]

112.4

^-Values i n brackets are data gathered by Boundy e t a l . (28). T h e i r Ca(0H)2 treatment was c a r r i e d out a t 75° f o r 15 minutes ( c o n c e n t r a t i o n of the a l k a l i not i n d i c a t e d ) . Values i n parentheses are percentages of amino a c i d observed r e l a t i v e to the non-treated z e i n a c i d hydrolyzate; nd =* not determined. 2

L y s i n o a l a n i n e was not detected i n any of the samples.


11.

SCHWASS E T A L .

Table I I .

Absorption

of Altered

Amino

Acids

Amino a c i d s r e l e a s e d by the in vitro pronase h y d r o l y s i s i n non-treated and a l k a l i - t r e a t e d z e i n * g amino acid/16 g of o r i g i n a l n i t r o g e n Zein

Amino Acid

non-treated Ca(0H)2 0.42

Aspartic acid Threonine-*


Serine

3

Glutamic

acid

3

NaOH

0.52

-

0.0

-

[0]

-

Proline

0.0

[0]

0.0

[0.0]

0.0

Glycine

0.2

[13]

0.5

[33.3]

0.0

Alanine

16.94

11.7

[98]

8.8

[77]

2.6

[59]

2.3

[59]

Valine

3.4

Methionine

0.9

Isoleucine

4.0

Leucine

27.7

[77]

0.4 [77]

2.7

0.5 [56]

2.4

[53]

[107]

22.9

[83]

19.4

[78]

Tyrosine

3.0

[44]

2.5

[40]

2.1

[37]

Phenylalanine

7.5

[80]

5.0

[56]

5.1

[62]

Histidine

0.5

[29]

0.4

[25]

0.3

[20]

Lysine

0

0

0.21

0.16

0.4

1.5

Cysteine

0.0

0.0

0.0

64Λ

48.9

41.5

TOTAL

5

[71]

0.6

[38]

Arginine

[22]

1 Corrections have been made f o r the reagent blanks i n each t e s t m a t e r i a l s . Values i n brackets r e f e r to the percentage of the amino a c i d that has been r e l e a s e d by pronase r e l a t i v e to i t s content i n the z e i n a c i d h y d r o l y z a t e s . These values represent one determination. ^ , . -, . Continued on next page


196

XENOBIOTICS

IN F O O D S A N D

FEEDS

Table I I . — C o n t i n u e d ^No percentage can be given r e l a t i v e to the values i n the a c i d hydrolyzates because of a l a c k of data of a c t u a l content of asparagine i n non-treated and treated z e i n . 3

S i n c e asparagine and glutamine peaks overlapped with the threonine, f o r s e r i n e and glutamic a c i d peaks i n the z e i n h y d r o l y z a t e s , values f o r these amino a c i d s are not i n c l u d e d .

4

A n i n t e r f e r i n g peak overlapped with the a l a n i n e peak: i s higher than that obtained i n a c i d h y d r o l y z a t e s .

t h i s value


5Neither ammonia nor tryptophan i s i n c l u d e d .

Accumulation of the measured amino acids from the pronase d i g e s t s i n t o everted sacs i s shown i n Figure 4. The r e s u l t s are expressed as percent accumulation r e l a t i v e to c o n t r o l which was d i r e c t l y measured i n the contents of the everted sac by amino acid analysis. Although accumulation i n the calcium hydroxidetreated cases i s not g r e a t l y decreased compared to the c o n t r o l , the sodium hydroxide-treated z e i n hydrolysate shows markedly de creased accumulation. However, s e r i n e shows s i g n i f i c a n t l y r e duced uptake i n both the l i m e - and soda-treated z e i n s . The r e a son f o r t h i s observation i s not c l e a r . The r e s u l t s from the everted sac experiments were that both calcium hydroxide and sodium hydroxide treatment s i g n i f i c a n t l y decrease in vitro d i g e s t i b i l i t y , but that only sodium hydroxide treatment a f f e c t s uptake of the z e i n h y d r o l y s a t e s . Because of the minimal e f f e c t s of calcium hydroxide treatment on accumula t i o n , we decided to reexamine the uptake phenomenon using an

120

z

ο

5 ZD Σ ZD (_) ^

100 80

1 60

_J

1

α

1

40 -

Ο

(_) ~

20

1

0

thr1

H ser

pro

1 ala

Figure 4. Accumulation of free amino acids by pronase digests of Ca(OH) -treated zein; and NaOH-treated zein. Results are expressed as for pronase digests of untreated (control) zein nations. 2

1

1

ile

I leu

tyr

phe

everted gut sacs. Key: open bars, shaded bars, pronase digests of percent relative to accumulation and represent duplicate determi



11.

SCHWASS E T A L .

Absorption

of Altered

Amino

Acids

197

in vivo technique that more c l o s e l y approximated the p h y s i o l o g i c a l gut, as the everted sac technique has been c r i t i c i z e d f o r l a c k of proper oxygenation of the t i s s u e (26). Figure 5 shows the r e s u l t s f o r the uptake of s e v e r a l amino a c i d s from the pronase hydrolysate of untreated z e i n . One d i s a d vantage of using t h i s technique i s that i t i s necessary to measure both the o r i g i n a l concentration of substrate as w e l l as the conc e n t r a t i o n of substrate remaining a f t e r the uptake i n t e r v a l . The accumulation must be determined by d i f f e r e n c e and has been expressed here as the percent of amino a c i d i n the o r i g i n a l h y d r o l y sate taken up. These r e s u l t s show that the gut i s able to remove 10 to 15% of the amino acids presented to i t over a 40 minute interval. Figure 6 shows the r e s u l t s f o r the calcium hydroxide-treated z e i n d i g e s t i n the hatched bars, while the open bars are the cont r o l values, f o r ease of comparison. A g e n e r a l l y s i m i l a r p a t t e r n of uptake was observed except most values were reduced about 5%, ( t o about 60% c o n t r o l ) , i n d i c a t i n g a decrease i n amino a c i d uptake. This i s i n c o n t r a s t to the r e s u l t s obtained using the everted sac technique, where l i t t l e d i f f e r e n c e was observed between c o n t r o l and calcium hydroxide-treated z e i n . Numbers below the bars i n d i c a t e the degree of racemization f o r the treated z e i n , which w i l l be discussed below. The r e s u l t s f o r the sodium hydroxide treated z e i n are shown i n F i g u r e 7. Again, the c o n t r o l values are given as open bars f o r comparison. As i n the calcium hydroxide-treated case, uptake i s reduced to the 5 to 10% l e v e l (about 50% c o n t r o l ) with a g e n e r a l l y s i m i l a r p a t t e r n of uptake over the amino a c i d s . As i n Figure 6, numbers below the bars i n d i c a t e the percent D-isomer present i n the treated z e i n . I t i s apparent that there i s not a l i n e a r c o r r e l a t i o n between the degree of racemization and the degree of uptake i n h i b i t i o n i n e i t h e r the soda or l i m e - t r e a t e d cases since the accumulation of phenylalanine, f o r example, i n the sodatreated case i s s i m i l a r to the accumulation of alanine i n the soda-treated sample even though there i s only one h a l f as much racemization of a l a n i n e . T h i s phenomenon may be explained i f one considers that a great deal of the amino a c i d s are taken up i n the form of pept i d e s rather than by a process of e s s e n t i a l l y complete hydrolys i s to f r e e amino acids i n the gut lumen. Evidence from many l a b o r a t o r i e s i n c l u d i n g those of Matthews (30) and A d i b i (31) has shown that perhaps more than 50% of amino a c i d uptake can be accounted f o r by transport of i n t a c t peptides. There appears to be s p e c i f i c i t y f o r some p o r t i o n of the peptide, but at t h i s time i t i s not c l e a r how l a r g e the r e c o g n i t i o n sequence must be (32) . Although there has been no evidence excluding a l l peptides c o n t a i n i n g more than three residues, s e v e r a l tetrapeptides have been tested i n mammalian systems and do not appear to be t r a n s ported i n t a c t , while t r i p e p t i d e s are good substrates f o r uptake (33) .



198

XENOBIOTICS

asp thr scr

glu pro gly

ala val

He

leu tyr

IN F O O D S A N D

FEEDS

phe

Figure 5. Uptake of amino acids from pronase hydrolysate of untreated zein by perfused jejunal segments. Results represent the portion of total amino acids taken up and are expressed as the percent of the difference between the original and final amino acid concentrations in acid hydrolysates of the perfusate (AA - AAf) divided by the original concentration (AA ):[(AA - AAf)/(AA )] x 100. Duplicate determinations were run. Q

0

0

0

20 15

asp thr ser 20

0

19

glu pro 15

O

gly -

ala val 8

Ο

He leu tyr Ο

β

14

phe 13

Figure 6. Uptake of amino acids from pronase hydrolysate of Ca(OH) -treated zein by perfused jejunal segments. Shaded bars show uptake for Ca(OH) -treated zein and open bars are control (see Figure 5) for ease of comparison. Results expressed as in Figure 5. Numbers below the bars indicate percent D-isomer in the treated hydrolysates. 2

2


11.

SCHWASS E T A L .

Absorption

of Altered

Amino

Acids

20


LU

asp thr ter 23

O

23

glu pro gly 25

O

-

ala val 13

0

Ile

leu

10

7

tyr 12

phe 23

Figure 7. Uptake of amino acids from pronase hydrolysate ofNaOH-treated zein by perfused jejunal segments. Shaded areas show uptake for NaOH-treated zein and open bars are control (see Figure 5) for ease of comparison. Results are expressed as in Figure 5. Numbers below the bars indicate percent D-isomer in the treated hydrolysates.

15

Figure 8. Mean uptake of amino acids from pronase hydrolysates of untreated zein (open bar), Ca(OH) -treated zein (center, shaded bar) and NaOH-treated zein (right, shaded bar). Averages were taken over the 12 amino acids measured in Figures 5-7. Uptake is expressed as in Figure 5. Standard error bars are shown. 2


199


200

X E N O B I O T I C S IN FOODS A N D F E E D S

We are l e d by our r e s u l t s to speculate that i f an amino a c i d residue i s present i n the peptide as the D-isomer, i t may render that peptide u n a v a i l a b l e f o r transport regardless of whether the i n v e r t e d residue i s an a l a n i n e , phenylalanine or any other amino a c i d . And each of the other L-residues i n the peptide w i l l be denied entry regardless of t h e i r i d e n t i t y or degree of racemiza t i o n . So phenylalanine uptake might be decreased as much as a l a n i n e uptake because both of these amino a c i d s f i n d s themselves i n D-isomer-containing peptides a t about the same frequency. A summary of the r e s u l t s from the perfused i n t e s t i n e e x p e r i ment i s shown i n Figure 8. The r e s u l t s are expressed as the mean uptake of a l l the measured amino a c i d s f o r the three z e i n h y d r o l y s a t e s . The r e s u l t s from t h i s s e r i e s c l e a r l y show that uptake has been reduced by both soda- and lime-treatments of z e i n , which i s a l s o c o n s i s t e n t with the r e s u l t s of the pronase study which show that both calcium hydroxide and sodium hydroxide treatments de crease d i g e s t i b i l i t y . However, i t i s not c l e a r why the r e s u l t s f o r Ca(OH)2-treated z e i n are d i f f e r e n t f o r the everted sac and p e r f u s i o n experiments, but t h i s may be r e l a t e d to the b e t t e r v i a b i l i t y of the in vivo p e r f u s i o n technique. In c o n c l u s i o n , racemization alone reduces in vitro d i g e s t i b i l i t y as w e l l as in vivo uptake of enzymatically digested pro t e i n . T h i s supports i n p a r t the r e s u l t s of Bunjapamai, e t a l . , (21) who concluded that racemization plays a greater r o l e than c r o s s l i n k i n g i n reducing d i g e s t i b i l i t y of treated p r o t e i n . These r e s u l t s suggest that racemization of c e r t a i n non-essen t i a l residues such as aspartate may have i n d i r e c t , negative e f f e c t s on the a v a i l a b i l i t y of other, r e l a t i v e l y non-racemized but e s s e n t i a l r e s i d u e s . Therefore, c o n d i t i o n s which cause racemiza t i o n should be minimized during processing.

Acknowledgments The authors wish to thank Dr. Gary M. Gray and h i s l a b o r a t o r y f o r demonstrating the i n t e s t i n a l p e r f u s i o n techniques, Amy Noma f o r many amino a c i d analyses and Β. E. Powell and L i l l i e Davis f o r typing the manuscript.

Literature Cited 1.

Whitaker, J.R., Feeney, R.E. in "Protein Crosslinking Nutritional and Medical Consequences"; Friedman, M., Ed.; Plenum Press: New York, 1977, p. 155. 2. Ziegler, K., J . Biol. Chem., 1964, 239, 2713. 3. Ziegler, K., Melchert, I., Lurken, C. Nature, 1967, 214, 404. 4. Asquith, R.S., Garcia-Dominguez, J . J . , J. Soc. Dyers Colour, 1968, 84, 155. 5. Neuberger, A. Adv. Prot. Chem. 1948, 4, 297.


11.

6. 7. 8. 9. 10.


11. 12. 13. 14. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33.

SCHWASS E TAL.

Absorption

of Altered

Amino

Acids

201

Gould, D.H., MacGregor, J.T. in "Protein Crosslinking-Nutritional and Medical Consequences"; Friedman, M., Ed.; Plenum Press: New York, 1977, p.29. Finley, J.W. This publication. deGroot, A.P., Slump, P., Feron, V.J., Van Beek, L . , J. Nutr. 1976, 106. 1527. Artom, C., Fishman, W.H., Morehead, R.P. Proc. Soc. Exptl. Biol. Med., 1945, 60, 284. Morehead, R.P., Fishman, W.H., Artom, C. Am. J . Path. 1945, 21, 803. Kaltenbach, J.P., Ganote, C.E., Carone, F.A. Exp. Mol. Pathol., 1979, 30, 209. Dakin, H.D., J. Biol. Chem., 1908, 4, 437. Dakin, H.D., Dudley, H.W., J . Biol, Chem., 1913, 15, 271. deGroot, A.P., Slump, P., J . Nutr. 1969, 98, 45. Hayashi, R., Kameda, I., J . Food Sci., 1980, 45, 1430. Friedman, M., Zahnley, J.C., Masters, P.M., J. Food Sci. 1981, 46, 127. deGroot, A.P., Slump, P., van Beek, L . , Feron, V.J. in "Evaluation of Protein for Humans"; Bodwell, C.E., Ed.; AVI: Westport, Connecticut, 1977, p. 270. Finley, J.W., Snow, J.T., Johnston, P.H., Friedman, M. in "Protein Crosslinking-Nutritional and Medical Consequences"; Friedman, M., Ed.; Plenum Press: New York, 1977, p. 85. Friedman, M. in "Nutritional Improvement of Food and Feed Proteins"; Friedman, M., Ed.; Plenum Press: New York, 1978, p. 613. Bunjapamai, S., Mahoney, R.R., Fagerson, I.S. J. Food Sci. 1982, 47, 1229. Del Valle, F., Perez, J . , J. Food Sci., 1974, 39, 244. Rayner, C.J., Fox, M., J . Food Sci. Agric., 1976, 27, 643. Ouchi, T. Agric. Biol. Chem., 1962, 26, 734. Wilson, T.H., Wiseman, G. J . Physiol., 1954, 123, 116. Smithson, K.W., Gray, G.M., J . Clin. Invest., 1977, 60, 665. Schwass, D.E., Finley, J.W. Manuscript in preparation. Boundy, J.Α., Turner, J.E., Wall, J.S., Dimler, R.J. Cereal Chem., 1967, 44, 281. Hayashi, R., Kameda, I., Agric. Biol. Chem., 1980, 44, 891. Matthews, D.M., Craft, I.L., Geddes, D.M., Wise, I.J., Hyde, C.W., Clin. Sci., 1968, 35, 415. Adibi, S.A., Phillips, E . , Clin. Res., 1968, 16, 446. Addison, J.M., Burston, D., Dalrymple, J.Α., Matthews, D.M., Payne, J.W., Sleisenger, M.H., Wilkinson, S., Clin. Sci. Mol. Med., 1975, 49, 313. Adibi, S.A., Kim, Y.S. in "Physiology of the Gastrointestinal Tract"; Johnson, L.R., Ed.; Raven Press: New York, 1981, p. 1073.

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August 4, 1983


Absorption of Altered Amino Acids from the Intestine - ACS Publications

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