Chitin and Chitosan: Influence on Element Absorption in Rats - ACS

12 Chitin and Chitosan: Influence on Element Absorption in Rats DENNIS T. GORDON

Downloaded by UNIV OF CALIFORNIA SAN DIEGO on September 4, 2015 | http://pubs.acs.org Publication Date: April 11, 1983 | doi: 10.1021/bk-1983-0214.ch012

University of Missouri-Columbia, Department of Food Science & Nutrition, Columbia, MO 65211 CYNTHIA BESCH WILLIFORD University of Missouri-Columbia, College of Veterinary Medicine, Research Animal Diagnostic and Investigative Laboratory, Columbia, MO 65211

Chitin and chitosan are reported to bind essential elements in v i t r o . To evaluate these unconventional sources of dietary fiber in vivo, the apparent absorption of six (6) elements ( i . e . , P, Ca, Mg, Fe, Zn and Cu) from diets containing chitin and chitosan were measured by balance t r i a l s in growing rats. At the 5% level, and at three particle sizes, neither chitin nor chitosan affected animal growth or food consumption over a 3 week period compared to a 5% cellulose control diet. Apparent absorption of the six elements were positive in all animals. Increasing both chitin and chitosan to 10% and 20% levels in the diet depressed Fe absorption except in animals consuming 10% chitosan. There was no difference in growth or food consumption between animals groups receiving higher fiber levels. Animals were in positive balance for P, Ca, Mg, Zn and Cu. Greater morphological changes were observed histologically in the small intestine and cecum of animals consuming chitosan and chitin compared to animals consuming cellulose. Increased numbers of neutrophils were seen in the lamina propria of the small intestine and cecum. More crypt e p i t h e l i a l cells of the small intestine were undergoing mitosis. The i n a b i l i t y of rats to u t i l i z e dietary Fe and lose body Fe is believed to be due to the inflammatory state of the small intestine, caused by chitosan, and to a lesser degree c h i t i n . Reasons for mortality in rats consuming high levels of chitosan, >10%, are also discussed. Dietary fiber (DF) has taken on a larger meaning than its original definition of plant c e l l wall material resistant to the digestive process (JL) in animals or man. 0097-6156/83/0214-0155$08.50/0 © 1983 American Chemical Society

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


156

UNCONVENTIONAL SOURCES OF DIETARY FIBER

Many non-metabolizable materials not obtained from plant sources that are consumed i n trace and p o s s i b l y bulk amounts both i n t e n t i o n a l l y and u n i n t e n t i o n a l l y are now recognized. Examples include mucopolysaccharides from animal t i s s u e (2) m i c r o b i a l c e l l w a l l s (3) exoskeletons of arthopods (4) and s y n t h e t i c b u l k i n g agents (5) . Consideration must also be given to i n t e s t i n a l t r a c t residues derived from the processing of foods which M a i l l a r d r e a c t i o n products (6) are an e x c e l l e n t example. E a r l y i n the l a s t decade, two, now almost c l a s s i c p u b l i c a t i o n s , (1, 7) caused a r e i n v e s t i g a t i o n of thought as to the importance of f i b e r i n man's d i e t . This was not a new r e v e l a t i o n ( 8 ) . What was new, was the r e c o g n i t i o n of the m u l t i p l i c i t y of h e a l t h - r e l a t e d d i s o r d e r s a t t r i b u t e d to the l a c k of DF in the d i e t (9)· Increased DF consumption i s advocated (10), but there may be adverse consequences with excessive i n t a k e . The biochemical and/or p h y s i o l o g i c a l a c t i o n of DF i n the gut have not been c l e a r l y d e l i n e a t e d . One of the b e n e f i t s a t t r i b u t e d to DF has been i t s a b i l i t y to sequester b i l e acids preventing t h e i r reabsorption (11). The o b j e c t i v e of decreasing plasma c h o l e s t e r o l by a l t e r i n g b i l e a c i d homeostasis i s s t r o n g l y championed. However, DF has been i m p l i c a t e d i n the impairment of e s s e n t i a l element absorption (12) . A p o s s i b l e n u t r i t i o n a l or h e a l t h status t r a d e - o f f e x i s t s for the host consuming DF. Two unconventional, but p o t e n t i a l sources of DF are c h i t i n and chitosan. C h i t i n , along with c e l l u l o s e and c o l l a g e n , are the p r i n c i p a l s k e l e t a l or support matrices i n a l l l i v i n g systems. Reviews of the c h i t i n system i n animals and p l a n t s (13) and the chemistry of c h i t i n (14) are a v a i l a b l e . Commercial c h i t i n i s mainly derived from waste products of C r u s t a c e a ( i . e . c r a b and shrimp) processed f o r human food. C h i t i n i s s i m i l a r to c e l l u l o s e , b u t i s a polymer of N-acetyl-Dglycosamine u n i t s , l i n k e d by β (1 -»· 4) g l y c o s i d i c bonds. I t may have p o t e n t i a l f o r use as DF (15), b u t i t does not have wide a p p l i c a t i o n and has not been approved f o r use i n foods. At p r e s e n t , i n g e s t i o n of c h i t i n by man i s only i n c i d e n t a l . Treatment of c h i t i n with strong base causes d e a c y l a t i o n , r e s u l t i n g i n the product chitosan (Figure 1). This compound reportedly has p o t e n t i a l f o r lowering blood c h o l e s t e r o l i n r a t s (4, lj5, 17)• Chitosan i s being compared to and suggested as a s u b s t i t u t e f o r cholestyramine; t h i s l a t t e r compound has proved to be a very e f f e c t i v e agent i n the treatment of p a t i e n t s w i t h elevated serum c h o l e s t e r o l (18). However, the mechanisms of a c t i o n may be d i f f e r e n t . The s a f e t y of chitosan or c h i t i n has not been thoroughly e s t a b l i s h e d . Both c h i t i n and chitosan have been reported to bind e s s e n t i a l elements from s o l u t i o n (19) and chitosan i s used to remove suspended organic m a t e r i a l s i n waste treatment (20). These observations again suggest a r e c i p r o c a l r e l a t i o n s h i p between c h o l e s t e r o l and e s s e n t i a l element status may occur with the i n g e s t i o n of c h i t o s a n .



GORDON AND WILLIFORD

Chitin and Chitoson

Chitin 1. dilute HCI 2. dilute ~OH 3. cone hot "OH 4. wash

Jη Chitosan Figure 1.

The chemical structures of chitin and chitosan.



158

The primary o b j e c t i v e of t h i s i n v e s t i g a t i o n was to examine the e f f e c t of c h i t i n and c h i t o s a n on e s s e n t i a l element absorpt i o n i n r a t s . Since previous r e p o r t s (4, 21) and unpublished observations by one of the authors (D.T.G.) have i n d i c a t e d h i g h l e v e l s of c h i t o s a n impair growth and cause death i n r a t s , a second o b j e c t i v e was e s t a b l i s h e d to more thoroughly e x p l a i n these f i n d i n g s .


M a t e r i a l s and Methods C h i t i n and Chitosan. C h i t i n and chitosan were r e c e i v e d through the courtesy of Kypro Co., S e a t t l e , Washington* After s i f t i n g to remove f i n e s and extraneous n o n - s h e l l f l a k e s , the remaining m a t e r i a l was ground i n a Thomas Wiley l a b o r a t o r y m i l l (Model 4, A. H. Thomas Co., P h i l a d e l p h i a , PA) to pass a 2.00 mm screen. Using a Ro Tap T e s t i n g Sieve Shaker (Model B, W. S. T y l e r , Inc., Mentor, OH), three f r a c t i o n s of each m a t e r i a l were c o l l e c t e d :

0.50 - 0.25

5

193.5±11.4

20% c e l l u l o s e > 10% c h i t i n > 10% c e l l u l o s e > 10% c h i t i n . Another morphological change observed i n animals consuming 20% c h i t o s a n included



12.

Chitin and Chitoson

GORDON AND wiLLiFORD

0

1

2

3

4

5

169

6

7

8

9

60 HR PERIODS DAYS ON DIET ANIMAL AGE, DAYS

0

1

2

23 24

3

4

25 26

5

6

27 28

7

8

10

11

12

29

30 31 32

9

33

34 35 36 37

13

14

15 16

17

18 19 20 21

38 39 40 41

22 23

42 43 44 45

Figure 2. Apparent utilization (i.e., absorption) of Ρ by rats consuming three dietary fibers at two levels. Key O - O , 10% chitin; · - · , 20% chitin; • - · - • , 10% chitosan; • - · · 20% chitosan; Δ - · · · - Δ , 10% cellulose; and . · . 20% cellulose. Each point represents the mean absorption ± SEM for a 60 h period. Days on the diet and animal age (in days) are also indicated on the x-axis.

6 0 HR PERIODS

Figure 3.

Apparent utilization of Ca by rats consuming three dietary fibers at two levels. Key is the same as in Figure 2.


170



16

H

0

1

2

3

4 60

Figure 4.

6

7

8

9

PERIODS

Apparent utilization of Mg by rats consuming three dietary fibers at two levels. Key is the same as in Figure 2.

I

0

1

1

1

•

1

1

2

3 60

Figure 5.

5 HR

—

1

4

1

1

5

1

6

1

7

1

8

9

HR PERIODS

Apparent utilization of Fe by rats consuming three dietary fibers at two levels. Key is the same as in Figure 2.


Chitin and Chitoson



171

60 HR PERIODS

Figure 6.

160

Apparent utilization of Zn by rats consuming three dietary fibers at two levels. Key is the same as in Figure 2.

A

0

1

2

3

4

5

6

7

8

9

60 HR PERIODS

Figure 7.

Apparent utilization of Cu by rats consuming three dietary fibers at two levels. Key is the same as in Figure 2.



172


Table 5·

Mean apparent element absorption among animal groups

consuming c h i t i n , chitosan or c e l l u l o s e at l e v e l s of 10 and 20%. Dietary fiber

Number of animals

Level

Ρ

Ca 2

%

Chitin

Chitosan

Cellulose

mg/60 hr ,f '

10

5

174.1±3.6

a,b

96.0±4.8

a,e

20

5

166.3±3.4

a,b

122.2±4.5

b,C

10

4

130.0±3.8

b

110.7±5.1

b,C

20

3

101.8±5.0°

l4l.3±6.5

C,d

10

4

156.0±3.4

d

87.2±4.5 »

20

5

152.0±3.8

d

90.5±5.1 ' »

4 C h i t i n vs chitosan C h i t i n vs c e l l u l o s e ^

** 4

a

f

e

f

*

**

*

**

**

Chitosan vs c e l l u l o s e

a,e

Mean of three balance periods,

) 'Mean ± S.D.; values i n same column not sharing a common superscript are s i g n i f i c a n t l y d i f f e r e n t (P 0.05). ^Values i n parenthesis are negative. ^Pooled d i e t comparisons; *(P

**

**

**

N.S.

**

N.S.

**

**

**

N.S.

**


f


Figure 8. Photomicroscopy of duodenum sections from two rats, one consuming 20% cellulose (A) and the second, 20% chitosan (Β) 220χ. Cross-section of crypt and longitudinal section of villus illustrated in A, and longitudinal section of crypt area only illustrated in B. Arrows indicate mitotic figures.



12.


Chitin and Chitoson

175

Table 6. Inflammatory c e l l i n f i l t r a t e of i n t e s t i n a l t r a c t s from r a t s consuming c h i t i n , c h i t o s a n or c e l l u l o s e a t l e v e l s of 10 and 20%.

Dietary

Level

fiber

Duodenum

%

Chitin

Cellulose

Cecum

Ileum

Neutrophils/HPF

1 , 2 , 3

-

3.6±0.8

5.4±1.4

4.9±2.1

2.7±1.3

3.3±1.4

3.5±1.4

2.5±1.0

10

6.3±2.3

5.8±1.1

4.9±1.1

4.4±1.4E

20

3.3±1.1

4.6±1.6

6.1±1.6

8.0±2.6E

10

3.7±1.3

3.7±1.1

3.3±0.8

4.1+1.2

20

7.1±1.4

3.6±1.3

2.8±1.0

3.7±1.4

10

Chitosan

Jejunum

20

3.7±1.1

See M a t e r i a l and Methods f o r explanation of determining n e u t r o p h i l e s per high powered f i e l d (HPF).

number of

Mean ± S.D.; d i f f e r e n c e s between means were not s t a t i s t i c a l l y tested. l

E = edema.



176


Table 7. M i t o t i c a c t i v i t y i n crypts of i n t e s t i n a l t r a c t s from r a t s consuming c h i t i n , chitosan or c e l l u l o s e at l e v e l s of 10 and 20%.

Dietary

Level

fiber

%

Chitin

Chitosan

Cellulose

Duodenum

Jejunum

Mitotic

Cecum

Ileum

figures/HPF

1 , 2

'

3

10

3.0±1.0

3.2±1.0

3.4±1.0

1.1±0.6

20

4.1±1.4

3.7±1.3

3.4±1.3

0.710.6

10

6.5±1.2

6.7±1.1

3.9±0.9

1.3±0.9E

20

6.1±1.5

5.0±1.5

6.9±1.9

1.0±0.7E

10

2.8±1.0

3.5±0.9

3.2±1.2

0.7±0.7

20

4.7±1.4

4.5±1.3

3.3±0.9

1.0±0.7

"See M a t e r i a l and Methods f o r explanation of determining number of m i t o t i c f i g u r e s per h i g h powered f i e l d (HPF). Mean ± S.D.; d i f f e r e n c e s between means were not s t a t i s t i c a l l y t e s t e d . ^E = edema.



12.


Chitin and Chitoson

177

l a c t e a l d i l a t i o n or edema of the lamina p r o p r i a of the cecum (Figure 9B)· T h i s l e s i o n was not observed i n ceca of the a n i mals i n the 20% c h i t i n or c e l l u l o s e groups (Figure 9A). For a l l animals, the e n t i r e small i n t e s t i n e showed the greatest changes with respect to d i e t a r y f i b e r . Two b a c t e r i a i s o l a t e d from the ceca of a l l t e s t animals were E s c h e r i c h i a c o l i and Enterobacter sp. No pathogenic bact e r i a (e.g. Salmonella typhimurium) were i d e n t i f i e d . The absence of t h i s l a t t e r organism helped r u l e out enterotoxemia as a cause of death i n one animal and impaired growth i n a second animal consuming 20% c h i t o s a n i n t h e i r d i e t s . Chitosan and c h i t i n d i d not appear to change the i n t e s t i n a l f l o r a popu l a t i o n e i t h e r 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 . Screening of anaerobic b a c t e r i a was not performed. Gross and h i s t o p a t h o l o g i c a l examination of lungs, kidneys, l i v e r s and s a l i v a r y glands from a l l animal groups revealed no evidence of n a t u r a l l y o c c u r r i n g i n f e c t i o u s d i s e a s e s . Discussion Chitosan i s being considered as a p o s s i b l e blood c h o l e s t e r o l lowering agent. Only at l e v e l s above 5% i n the d i e t of r a t s does c h i t o s a n and i t s parent compound c h i t i n appear to cause harmful e f f e c t s . As commercially a v a i l a b l e , n e i t h e r compound can be considered homogenous. Extensive c l e a n i n g and s t a n d a r d i z a t i o n of both m a t e r i a l s , and s p e c i f i c a l l y c h i t o s a n , may be necessary f o r f u t u r e e v a l u a t i o n . R e s u l t s observed i n t h i s study can p o s s i b l y be a t t r i b u t e d to some unknown c o n s t i tuent (s) i n c h i t i n or c h i t o s a n , as may be the case when using n a t u r a l products. Animals i n g e s t i n g c h i t i n and c h i t o s a n at l e v e l s of 5, 10 and 20% d i d not show any s i g n i f i c a n t decrease i n growth a f t e r 23 days compared to animals f e d c e l l u l o s e at equivalent l e v e l s . E a r l i e r r e p o r t s have i n d i c a t e d impaired growth i n r a t s when l e v e l s of c h i t o s a n i n the d i e t exceeded 10% 04, 21). At l e v e l s of 5%, d i f f e r e n t p a r t i c l e s i z e s of c h i t i n or chitosan d i d not a l t e r element a b s o r p t i o n to any s i g n i f i c a n t degree. Animals were i n p o s i t i v e balance f o r each of the s i x elements examined. Body element composition v a r i e d , but, again, d i f f e r e n c e s were s m a l l . At the 5% l e v e l i n the d i e t and below, f u r t h e r long term s t u d i e s are needed to e s t a b l i s h i f c h i t i n or c h i t o s a n have any adverse e f f e c t s on animal h e a l t h . O r i g i n a l l y t h i s experiment was designed to determine the degree of p o s i t i v e or n e g a t i v e a b s o r p t i o n of elements as i n f l u e n c e d by c h i t i n and c h i t o s a n . D i e t a r y element l e v e l s could not be standardized among d i e t s because of the v a r i a t i o n i n amounts provided by the t e s t DF sources. Although d i e t s were adequate i n a l l elements, d i f f e r e n c e s observed i n p o s i t i v e balance v a l u e s may only r e f l e c t d i f f e r e n c e s i n amounts consumed. An example of the d i f f e r e n c e between d i e t a r y l e v e l s and



Figure 9. Photomicroscopy of longitudinal sections of cecum from two rats, one consuming 20% cellulose (A) and the second, 20% chitosan (Β) 220χ. Arrows with Ν indicate neutrophils, short arrows indicate mitotic figures.



12.

GORDON AND WILLIFORD

Chitin and Chitoson

179

absorption i s discussed f o r Ca. Animals consuming 20% c h i t o s a n had the lowest mean a b s o r p t i o n of Ca (102 mg/60 hr) while d i e t a r y l e v e l s were the highest (11.24 mg/g). D i e t a r y Ca l e v e l s i n the 20% c e l l u l o s e c o n t r o l d i e t s were lowest (4.95 mg/ g) , but a b s o r p t i o n was s i g n i f i c a n t l y higher (152 mg/60 hr) than i n the 20% c h i t o s a n f e d animals. Since both d i e t s contained the same amount of AIN s a l t mixture, i t appears that c h i t o s a n may impair Ca a b s o r p t i o n . However, c a u t i o n must be exercised i n making t h i s assumption. Growth was lower i n animals fed chitosan (20%) and t h i s may account f o r the lower needs as measured by balance i n t h i s study. Further work i s needed t o evaluate the accuracy of balance techniques i n measuring element a b s o r p t i o n a t low, adequate and excessive element intakes as employed i n t h i s study. For the elements P, Ca, Mg, Zn and Cu, v a r i a t i o n i n apparent a b s o r p t i o n l e v e l s e x i s t e d among animal groups i n g e s t i n g c h i t i n , c h i t o s a n or c e l l u l o s e a t v a r i o u s l e v e l s . A c l e a r trend of one DF source e x h i b i t i n g an i n h i b i t o r y or enhancing e f f e c t on the absorption of any one of these 5 elements was not c l e a r l y d e l i n e a t e d . D i f f e r e n c e s e x i s t e d i n a b s o r p t i o n of these 5 elements, but none of these changes r e s u l t e d i n animals being i n marginal balance or even near negative balance. The most s t r i k i n g r e s u l t of t h i s study was the c o n s i s t e n t l y negative Fe balance over time observed i n three of the four animal groups f e d c h i t i n or c h i t o s a n . The p o s i t i v e Fe balance i n which animals consuming 10% c h i t o s a n maintained themselves c o n t r a d i c t s the observations found with the other three d i e t s . This cannot be explained c o n s i d e r i n g other i n f o r m a t i o n obtained from the same group. Since a l l t e s t animal groups had lower hemoglobin l e v e l s than c e l l u l o s e c o n t r o l s (Table 8)» the 10% chitosan f e d animals would a l s o have been expected to be i n negative Fe balance. C o n f l i c t i n g r e s u l t s were a l s o observed i n animals consuming 20% c h i t i n . T h e i r hemoglobin value (13.7±1.1 g Hb/dl) would have been expected t o be lower c o n s i d e r i n g the manner i n which animals voided Fe v i a f e c a l e x c r e t i o n over time (Figure 5 ) . At the r a t e of Fe l o s s observed, t o t a l d e p l e t i o n of Fe was probable a f t e r 23 days on the d i e t or s h o r t l y t h e r e a f t e r . During the course of Expt. 2, one animal on a c h i t o s a n d i e t a t the 20% l e v e l died on day 6. H i s t o l o g i c a l observations of the i n t e s t i n a l t r a c t were i n c o n c l u s i v e because of the time delay between death and examination (ca. 12 h r ) . M i c r o b i o l o g i c a l survey of i n t e r n a l organs i n d i c a t e d the presence of Proteus sp i n the lung and extensive septicemia. This l a t t e r c o n d i t i o n was diagnosed as the cause of death. A second animal i n t h i s group began t o show signs of decreased food i n t a k e and growth. The animal was maintained on the d i e t u n t i l euthanized and immedia t e l y necropsied on day 9. Gross pathology i n d i c a t e d the i n t e s t i n e t o be g r e a t l y distended w i t h very f i r m luminal contents appearing to produce c o n s t i p a t i o n . M i l d e n t e r i t i s and




180

Table 8· F i n a l hemoglobin l e v e l i n animals consuming c h i t i n , chitosan or c e l l u l o s e at l e v e l s of 10 and 20% f o r 23 days.

Dietary f i b e r

No. of animals

Hemoglobin g/dl

10

5

12.7±0.6

a

20

5

13.7±l.l

a

10

4

13.5±0.9

a

20

3

13.5±0.9

a

10

4

14.5±0.7

b

20

5

14.4±l.l

b

Level

% Chitin

Chitosan

Cellulose

1

"Mean ± S.D.; values not sharing a common s u p e r s c r i p t are s i g n i f i c a n t l y d i f f e r e n t (P

Chitin and Chitosan: Influence on Element Absorption in Rats - ACS

Recommend Documents