Lipid malabsorption as a possible cause of anorexia in Zn-deficient juvenile common carp, Cyprinus carpio

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Aquaculture, 89 (1990) 327-335 Elsevier Science Publishers B-V., Amsterdam

Lipid malabsorption as a possible cause of anorexia in Zn-deficient juvenile common carp, Cyprinus carpio SK. Taneja, A. Lath and P. Arya Department of Zoology, Punjab University, Chandigarh 160 014 (India) (Accepted 20 December

1989 )

ABSTRACT Taneja, SK., Lath, A. and Arya, P., 1990. Lipid malabsorption as a possible cause of anorexia in Zndeficient juvenile common carp, Cyprinus carpio. Aquaculture, 89: 327-335. Zn-deficiency was induced in Cyprinus carpio juveniles by feeding them on purified Zn-deficient diet for 8 weeks. The gastrosomatic index (GSI), was, thereafter, calculated at 0 h, 3 h and 6 h of starvation after feeding the morning feed, and compared with those ofZn-supplemented and pair-fed groups. The data revealed that the GSI declined by 35.8% in Zn-deficient fish in contrast to 80% in controls at 6 h and this points towards slower stomach clearance rate leading to anorexia in the Zndeficient group. Cytochemical investigations of intestinal cells, after 3 h of starvation, revealed the presence of triglyceride-rich deposits in the form of clumps, throughout the cytoplasm of the absorptive epithelial cells in Zn-deficient fish, in contrast to granular deposits of different sizes in moderate concentrations lying mostly in a supranuclear position in the Zn-supplemented control. The concentrations of these deposits declined only slightly at 6 h in Zn-deficient fish, while they disappeared completely in the Zn-supplemented control group, reflecting poor lipid transportation from epithelial cells to lacteals in the Zn-deficient group compared with the control. Lipid malabsorption was envisaged as a possible cause of anorexia. This conclusion was based on the data obtained from the experiment extended for another 8 weeks after the induction of Zn-deficiency in the fish and feeding them on the same diets but free from corn oil. The gastrosomatic index, stomach clearance rate and weight gain improved and approached, in the Zn-deticient group, that of the control group after 8 weeks of dietary treatment.

INTRODUCTION

Anorexia is one of the earliest manifestations of clinical symptoms under Zn-deficiency conditions in mammals, birds and fishes that have been investigated so far. At present, the mechanism by which Zn-deficiency induces anorexia is unknown, although it has been associated with alteration in taste acuity (Henkin et al., 1969)) alterations in molar ratio of tryptophan (Ashley and Anderson, 1975 ) or tyrosine in blood plasma (Wallwork et al., 1979) to the sum of neutral amino acids, reduction of catecholamine levels in hypo-

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thalamus (Hollas et al., 1982) and membrane fluidity in receptors to neurotransmitters (Essatara et al., 1984; McClain et al., 1985). The ultrastructural (Koo and Turk, 1977) and cytochemical investigations (Taneja and Kaur, 1988) of the mucosal epithelium of the intestine have provided ample evidence of lipid malabsorption in rats and mice fed Zn-deficient diets, wherein triglycerides accumulate in mucosal epithelial absorptive cells. These reports led us to speculate that a possible cause of anorexia was the inhibition of gastric secretion and the stomach emptying process by the accumulated triglycerides. Accordingly, we tested this on the fish, Cyprinus carpio, by eliminating a part of the dietary lipid in the semisynthetic diet. The results thus obtained are reported in this paper. MATERIALS AND METHODS

About 200 juvenile common carp, Cyprinus carpio, were collected locally during August. They were divided into three groups and kept in plastic portable tanks. Water in the tanks was aerated continuously throughout the experiment which lasted for 16 weeks. The fish of the first group (ZD) were fed on pelletised Zn-deficient diet (Table 1) . The analysis of this diet by atomic TABLE 1 Composition of diet Test diet Casein (EDTA treated)” Gelatin Corn oil Cod liver oil Cellulose Sucrose vitamin mixture #Mineral mixture Water Total diet

(g) 38 12 6 3 8 28 1 4 200 300

*Vitamin mixture

(mg)

*Mineral mixture

(g )

Ascorbic acid Biotin Calcium pantothenate Choline chloride Folic acid Inositol Menadione (K) Nicotinic acid Pyridoxine HCl Riboflavin Thiamine HCl cr-Tocopherol acetate (E ) Vit. Br2

100 0.5 50 500 1.5 200 4 75 5 20 5 4 0.01

CaHPO, coc12 CuCl* FeSo4*7Hz0 MnS04.S20 MgSOd.HrO KC1 Kl Na&O, NaF AU&

2.58 0.004 0.010 0.060 0.080 0.405 0.343 0.015 0.115 0.008 0.015

“Commercial vitamin-free casein was treated with 0.1% EDTA to chelate metallic ions so that it became free of Zn*+. After this treatment, the casein was washed twice with water to remove EDTA and was dehydrated with acetone followed by ether. After drying with blotting paper, it was kept in an oven at 60°C overnight. The hard granules of casein were then powdered.

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absorption spectrophotometry showed it to contain 0.5- 1 ppm Zn. The group II fish (ZS) were fed on Zn-supplemented diet containing 100 mg ZnS04*7H20/100 mg of diet and the group III fish (PF) on Zn-supplemented diet equal to the amount consumed by the Zn-deficient group. Food was provided to all fish twice a day at 8 a.m. and 6 p.m. After 8 weeks of dietary treatment, the Zn level in the intestine of six fishes of each group was estimated on an AAS Perkin Elmer (302). The 18 fishes were separated from the rest of the group and starved overnight. Next morning they were fed on their respective diets for 1 h. Thereafter, their gastrosomatic indices and stomach clearance rates were calculated at 0 h, 3 h and 6 h of starvation after their meals by the following methods: Gastrosomatic

Index, GSI =

Weight of the stomach x 1o. Weight of the body

Stomach Clearance Rate, SCR= ( GSIO - GSI,/GSIO x t ) x 100 where GSIo = initial gastrosomatic GSI, = gastrosomatic t

=

index at 0 h

index at time t

starvation period in hour.

Their midgut was accordingly fixed in form01 calcium and frozen sections were stained in Sudan Black B (SBB ) and Nile blue sulphate (NBS) following Pearse ( 1985 ) , for general lipids and triglycerides, respectively. Ten fishes in each group were kept for further use. They were fed on their respective diets but this time the diet lacked corn oil. After 8 weeks, they were sacriticed and gastrosomatic indices and stomach clearance rates were calculated in the same manner as described above. RESULTS

After 2 weeks, fish fed the ZD diet began to exhibit a gradual decrease in food intake in contrast to the fish fed the ZS diet (Table 2). In the beginning of the experiment, fish consumed 3 g of diet per 100 g of biomass per day. In the 3rd week, the ZD fish could consume only the morning ration, and the evening ration was left unconsumed. In the 4th and 5th weeks, their food intake decreased further and after the 6th week it stabilised to 1% of body weight while the ZS fish continued to utilize their usual ration. The zinc concentration in the intestine at this stage was found to be 178.92 it 16.29, 101.79 + 13.56 and 48.22 i- 8.83 pg/g of dry weight of the tissue in ZS, PF and ZD groups, respectively. The mean weights and food intake at weekly intervals and stomach clear-

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TABLE 2 Mean weights + s.d. and food intake in zinc-supplemented, Weeks

1 2 3 4 5 6 7 *8

Zinc supplemented

(ZS )

Mean weight ?s.d. (g)

Food intake/

3.166 + 1.748 3.160 !I 1.761 3.236 + 1.824 3.292 f 1.831 3.353 ?Y1.830 3.443 + 1.908 3.588 ?I 1.938 3.790 I!I2.06 1

0.9498

day (g)

0.9480 0.9708 0.9876 1.1735 1.2050 1.2558 1.3265

Zinc-deficient Mean weight fs.d. (g) 3.208 f 1.041 3.208 f 1.040 3.154 & 1.035 3.124 + 1.036 3.049 f 1.039 2.942 + 1.072 2.878 -I 1.101 2.817 ? 1.128

zinc-deficient

(ZD ) Food intake/ day (g) 0.9624 0.9624 0.9462 0.9372 0.6098 0.5884 0.5776 0.5634

and pair-fed fish (n = 12) Pair-fed (PF ) Mean weight +s.d. (g)

Food intake/

2.119 + 1.020 2.105 f 1.022 2.123 f 1.029 2.166 f 1.077 2.149 + 1.030 2.155 f 1.020 2.144 + 1.017 **2.052 + 1.140

0.6357

day (g)

0.6315 0.6369 0.6498 0.4298 0.4310 0.4288 0.4104

*PC 0.00 1 between ZD and ZS groups. **P-c 0.05 between ZD and PF groups.

ante rate in each group determined after 8 weeks of dietary treatment, at 0 h, 3 h and 6 h of starvation, are summarized in Tables 2 and 4. The cytochemical localisation of lipids in mucosal epithelial cells of the midgut at 0 h, 3 h and 6 h of starvation of the three groups was analysed because of its relevance to gastric secretion and stomach emptying. The mucosal epithelial cells lining the lumen of the intestine appeared dark blue with SBB stain due to the presence of the lipid clumps distributed mostly at the supra- and infranuclear positions at 0 h in the three groups. The tips of the mucosal folds displayed more accumulation of SBB-positive material in the cytoplasm than in the cells situated at the bases of the folds. These lipid granules stained homogeneously pink with NBS due to their triglyceride nature. The concentration of these lipids varied in the three groups when examined at 3 h and 6 h of starvation. The SBB and NBS techniques did not exhibit any decrease in the ZD group at 3 h compared with the level observed at 0 h (Fig. 1) . In contrast, the intensity of these two stains decreased considerably at 3 h in the ZS and PF groups. The Sudanophilic material appeared as line granules instead of clumps at this stage. The overall picture depicted the presence of triglycerides in a much lesser amount than observed in the counterpart at 0 h (Fig. 3). At 6 h of starvation, the SBB and NBS reaction in the ZS and PF groups appeared almost negative while in ZD there was no significant difference over 3 h (Fig. 2). There was only a little reduction in the intensities of these reactions in a few cells situated on the side and basally of some of the

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Fig. l-3. T.S. midgut of (1) ZD fish after 3 h, (2) ZD fish after 6 h, (3) ZS fish after 3 h of starvation (form01 calcium/SBB) showing (arrows) deposition of lipids in absorptive epithelial cells.

mucosal folds. These reactions clearly indicate that the dietary lipid is completely absorbed within 6 h of feeding in the ZS and PF groups while in the ZD group it remains in the cells for a longer duration. These cytochemical alterations led us to speculate that dietary lipid malabsorption was a possible cause of low dietary intake in ZD fish. Accordingly, we examined the effect of depleting corn oil from the diets of the respective groups on their food intake, gain in body weight, gastrosomatic indices and stomach clearance rate. The results are summarized in Tables 3 and 4. The data revealed that the food intake in the ZD group started improving when

SK. TANEJA ET AL.

332 TABLE 3 Mean weights + s.d. and food intake in the zinc-supplemented oil-deficient and pair-fed fish (n = 6 ) Weeks

1 2 3 4 5 6 7 *s

Zinc-supplemented deficient

corn-oil-deficient,

com-

corn-oil-

Zinc-deficient deficient

Mean weight 2s.d. (g)

Food intake/ day (g)

Mean weight +_s.d. (g)

Food intake/ day (g)

Mean weight +s.d. (g)

Food intake/ day (g)

3.190 k2.061 3.817 k2.081 3.940 + 2.080 3.868 f2.081 3.893 + 2.075 3.914 + 2.074 3.924 + 2.075 3.986 k2.118

1.3266

2.817 + 1.128 2.808 ? 1.126 2.840 + 1.132 3.058 f. 1.241 3.438 * 1.542 3.819 + 1.708 4.152 + 1.943 4.909 + 2.250

0.5634

2.052 f1.140 2.139 + 1.008 2.371 f 1.048 2.476 k 1.056 2.895 + 1.081 3.176 f 1.095 3.632 k1.112 3.914 + 1.250

0.4104

1.3359 1.3440 1.3558 1.5572 1.5656 1.5708 1.5944

com-oil-

zinc-deficient

0.5616 0.5680 0.9174 1.3782 1.5276 1.6608 1.9636

Pair-fed

0.4278 0.4742 0.7428 1.1580 1.2704 1.4528 1.5656

*PC 0.05 between ZS, ZD and PF groups.

TABLE 4 Gastrosomatic indices (GSI) and stomach clearance rate (SCR) (% of food clearance/h) in C. curpio fed on Zn-supplemented, Zn-deficient, pair-fed and Zn-deficient corn-oil-depleted diet, after 0 h, 3 h and 6 h of starvation (n=6) Time (h)

0 mean + s.d. s.e.m. *3 Mean + s.d. s.e.m. *6 Mean 5 s.d. s.e.m.

ZD

ZS

ZDCD

PF

GSI

SCR

GSI

SCR

GSI

SCR

GSI

SCR

2.410 f0.170 0.070 1.540 kO.120 0.050 0.500 * 0.080 0.030

11.93 -t 0.970 0.430 13.190 kO.350 0.150

3.44 5.0140 0.060 3.330 kO.090 0.040 2.80 f0.220 0.100

1.118 kO.669 0.299 5.630 20.720 0.320

1.580 kO.800 0.030 1.070 20.130 0.050 0.560 kO.050 0.020

10.820 k1.710 0.760 10.670 f0.348 0.155

2.385 kO.079 0.032 Y.695 kO.244 0.099 **0.670 kO.195 0.079

9.643 + 3.249 1.326 11.984 + 1.352 0.552

*PC 0.001 between ZS and ZD groups. **PC 0.05 between ZS and ZDCD, PF and ZDCD and ZS and PE groups.

LIPID MALABSORPTION AND ANOREXIA IN Zn-DERCIENT 5-l

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Data from ‘Zinc Def Study’

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Time (Weeks) Fig. 4. Mean body weight (g) of ZS, ZD and PF fish recorded at intervals of 1 week. The vertical line at 8 weeks represents the termination of the corn-oil-supplemented diet feeding and the start of the corn-oil-depleted diet feeding.

they were fed the modified diet. They consumed 2% of their body weight after 4 days and 4% after 8 days. Their weights increased beyond the level of the ZS control group (Fig. 4) and the GSI and stomach clearance rates were almost identical after the 8 weeks modified dietary treatment. DISCUSSION

The data revealed that the GSI of the ZD group was almost the same at 0 h and 3 h and declined only 30% at 6 h. In the ZS and PF groups, it declined by 35.8% to 40% at 3 h and by about 80% at 6 h. This indicates that the food stays in the stomach for longer in ZD than in PF and ZS fish. The ZS group clear their stomachs at the rate of 11.93% up to 3 h and 13.19% after that, and the PF at the rate of 10.82% up to 3 h and 10.67% after it. In contrast, the ZD group clear their stomachs at a much lower rate, i.e. 1.11%/h up to 3 h and 5.63%/h thereafter. This implies that the ZD group clear their stomachs in 15.8 h if 0.50 GSI is taken as an empty stomach. In other words, ZD tish would consume 2.63 times less food in 6 h, which approximately coincides with the actual daily food intake values in the groups. The stomach clearance rate increased from 1.118 5 0.669 to 9.643 +3.249/h at 3 h and from 5.63 + 0.720 to 11.984 2 1.352 at 6 h in the ZD group when corn oil was elim-

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inated from the diet. These ZDCD fish (Table 4) empty 78% of their stomach contents against 35.5% recorded in corn-oil supplemented ZD fish in 6 h - almost a two-fold increase. This suggests that dietary lipid plays some role in stomach clearance rate. The cytochemical investigations of ZS and ZD groups clearly show that the dietary lipid is completely absorbed within 6 h of feeding by the ZS group while it stays in the cells of the ZD group, and that the transportation from mucosal epithelial cells to lacteals takes a longer time in the ZD than in the PF and ZS groups. Identical defects in lipid absorption were also obtained by Koo and Turk ( 1977) and Taneja and Kaur ( 1988) in rats and mice, respectively. Based upon electron microscopic and chromatographic studies, Koo and Turk ( 1977) concluded that the movement of the lipid droplets out of the mucosal cells was blocked due to failure of mucosal synthesis of protein required for the formation of chylomicrons in ZD animals. Very similar results were also obtained following treatment of rats with inhibitors of protein synthesis (Isselbacher and Budz, 1963; Sabesin and Isselbacher, 1965 ). The essentiality of Zn for protein synthesis is well established (Williams and Chesters, 1970; Im et al., 1975). The presence of lipids in mucosal epithelial cells of the midgut has been reported to impose an inhibitory effect on gastric secretion and stomach emptying through a feedback mechanism (Konturek and Grosoman, 1965; Long and Brooks, 1965 ) . A similar mechanism appears to operate under Zn-deficiency condition. The depression of stomach emptying caused by the persistence of inordinate lipid deposits in mucosal epithelial cells in the ZD group is evident from the recorded data on stomach clearance rate which increased and approached very close to that of the ZS group when corn oil was depleted from the ZD diet. Therefore, from the data we conclude that the loss of appetite in the ZD group basically stems from the defects in lipid absorption which drastically reduce the movement of food from the stomach to the next part of the alimentary canal and the animals remain in a state of satiety for a longer duration. A reduction of lipids in the diet promotes both food intake and body weight gain.

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Henkin, R.I., Graziadei, P.P.G. and Bradley, D.F., 1969. The molecular basis of taste and its disorders. Ann. Intern. Med., 71: 791-919. Hollas, E.S., Wallwork, J.C. and Sandstead, H.H., 1982. Mild Zn-deficiency and undernutrition during the prenatal and postnatal periods in rats: effects on weight, food consumption and brain catecholamines concentrations. J. Nutr., 112: 542-55 1. Im, M.J.C., Hsu, J.M. and Hoopes, J.E., 1975. Enzyme activities in the epidermis of zinc-deficient rats. J. Nutr., 105: 1391-1394. Isselbacher, K.J. and Budz, 1963. Synthesis of lipoproteins by rat intestinal mucosa. Nature, 200: 363-365. Konturek, S. and Grosoman, M.I., 1965. Effect of perfusion of intestinal loops with acid, fat or dextrose on gastric secretion. Gastroenterology, 49: 48 l-489. Koo, S.I. and Turk, D.E., 1977. Effect of Zn-deficiency on intestinal transport of triglycerides in the rat. J. Nutr., 107: 909-919. Long, J.F. and Brooks, F.P., 1965. Relation between inhibition of gastric secretion and absorption of fatty acids. Am. J. Physiol., 209: 447-45 1. McClain, C.J., Kasarkis, E.J. and Allen, J.J., 1985. Functional consequences of Zn-deficiency. Prog. Food Nutr. Sci., 9: 185-226. Pearse, A.G.E., 1985. In: Histochemistry - Theoretical and Applied, Vol. II. Churchill Livingstone, London, pp. 830-838. Sabesin, S.M. and Isselbacher, K.J., 1965. Protein synthesis and inhibition. Mechanism for the production of impaired fat absorption. Science, 147: 1149- 115 1. Taneja, S.K. and Kaur, B., 1988. Lipids in mucosal epithelium of the intestine of mice fed on Zn-deficient diet. Curr. Sci., 57(9): 493-494. Wallwork, J.C., Fosmire, C.J. and Sandstead, H.H., 1979: Cyclic feeding patterns and plasma amino acid concentration in Zn-deficient rats. Fed. Proc., 38: 606. Williams, R.B. and Chesters, J.K., 1970. The effects of early Zn-deficiency on DNA and protein synthesis in rat. Br. J. Nutr., 24: 1053-1059.