Dietary protein quality and zinc levels in growing rats

Nutrition Research. Vol. 19, No. 7, pp. 1089-1095,1999 Copyright 0 1999 Elsevier Science Inc. Printed in the USA. All rights reserved 0271.5317/99&see front matter

ELSEVIER

PIISO271-5317(99)00069-X

DIETARY PROTEIN QUALITY AND ZINC LEVELS IN GROWING RATS

Department

Anabel Pallarol, PhD, Nora. H. Slobodianik, PhD. of Nutrition. School of Pharmacy & Biochemistry. University of Buenos Aires, Buenos Aires, Argentina.

ABSTRACT Previous studies from our laboratory have shown that the intake of a low quality dietary protein during either a short or a long period, causes thymus atrophy of growing rats with a significant reduction in the number of cells and the mature T cell population. Moreover, thymus atrophy was associated with Zinc deficiency. The aim of this study was to determine if the intake of this diet induced a concomitant Zinc deficiency. Rats of the Wistar strain were fed from weaning and during 10 days a 6.5% maize protein diet (MlO) or a 6.5% casein diet (ClO). An age-matched control group was run simultaneously (C). The only dietary variable was the protein quality, being the Zinc content the same. Food intake (g/day) was similar: (MlO: 6521.4, ClO: 6821.1). Zn levels were determined by atomic absorption spectrophotometry in plasma (P), whole blood (WB), liver (L) and thymus (T) and expressed as ug/ml and ug/g wet tissue, respectively. No differences were found in WB (MlO: 38520.24, ClO: 3X3+1.00, C: 3.6720.34) nor L (MlO: 42.50+11.30, ClO: 36.3026.70, C: 39.1028.60). However, P levels decreased in Ml0 and Cl0 when compared to C (MlO: 1.843.63, ClO: 1.7920.41, C: 2.433.37, PcO.04) while a significant increase in thymus Zn was only found in Ml0 compared to Cl0 and C (MlO: 40.00~13.20, ClO: 26.5022.70, C: 27.1023.60, pcO.01). These results show that the intake of a low quality dietary protein might alter the distribution of plasma Zinc with a concomitant uptake of Zinc by the thymus. 8 1999 Elsevia Science Inc. Key words:

Dietary protein quality, Growing rats, Zinc levels.

INTRODUCTION Several reviews of immunological studies in malnutrition provide detailed descriptions about the impairment of host defense mechanisms. Delayed hypersensitivity to common microbial antigens, evident decrease in lymphocyte number and response to mitogens, diminished complement levels, phagocytic capacity of macrophages and intracellular bactericidal capacity of neutrophils and remarkable reduction in the proportion of helper T cells were described among many other topics investigated (1,2). Much has been written about the effects of dietary protein concentration. Moreover, ’ADDRESS FOR CORRESPONDENCE: Anabel Pallaro. Department of Nutrition. School of Pharmacy & Biochemistry. University of Buenos Aires. Junin 956.2nd floor. (1113) Buenos Aires, Argentina. Telephone: 1 - 964-8242 FAX: 00-54-I-964-8243 E-mail: apallaro@ffyb. uba. ar

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A. PALLARO and N.H. SLOBODIANIK

it has been shown that the type of dietary protein has a significant effect on humoral immunity related to T cell dependent and independent antigens in mice (3-S), and provokes atrophy of lymphoid organs (thymus, spleen) with altered histology, decrease in organ weight and cellularity (6,7) and lower rosette forming cells and splenic lymphocyte responses to mitogens in rats fed beans as the only source of protein (8). Previous studies from our laboratory have shown that the intake of a low quality cereal protein , during either a short or a long period , causes thymus atrophy of growing rats with a significant decrease in cellular proliferation and altered differentiation and maturation (9-l 1). Besides, it is known that Zinc (Zn) plays a critical role in the integrity of the immune system and its deficiency is associated with the loss of lymphoid tissue in general and of the thymus in particular and influences various T cell functions (12,13). It has been demonstrated that dietary protein level affects serum Zn, associated with low total serum proteins. On the other hand, the depression in plasma Zn may be related to the concomitant uptake of this oligoelement by the liver and other tissues (14). The aim of this study was to assess if the intake of the low quality cereal protein used in our experimental model induces a concomitant Zn deficiency in the thymus of growing rats. For this purpose, Zn levels were determined in whole blood, plasma, thymus and liver of rats fed a corn diet and compared to those of a high quality control group fed casein diet.

MATERIAL AND METHODS Weanling rats (n= 24) of the Wistar strain (closed colony from the breeding unit kept at the Department of Nutrition) of either sex and the same age, with an average weight of 35.721.4 g, were housed individually in screen-bottomed cages and exposed to 12-hours light-darkness cycle (7:00 AM to 7:00 PM). Room temperature was kept at 2 1.021 .O”C. Water and diets were offered ad lib&urn for a IO-days feeding period. Experimental group (n=8, MlO) received from weaning pre-cooked maize flour as the only source of protein. Quality control group (n=8, C 10) receiving a casein diet at the same concentration was run simultaneously. Experimental isocaloric diets, providing 6.5% protein and all the essential nutrients, were prepared according to the recommendations of the American Institute of Nutrition (Table 1). Dietary Zn concentration was the same in both diets. An age -matched wellnourished control group (n=8) receiving stock diet {Cargill, % of protein: 22.824.9, Biological value: 70 (15)) was included. Food intake was recorded and expressed as g/day and daily total and complete protein and energy intake were calculated and expressed on the basis of metabolic mass [bw(g)O.75 , (being bw = bw initial + bw final I 2)] (mg/ bwO.75 /day and kcal/bw 0 75 /day, respectively). Complete protein intake was calculated as total protein intake x Biological Value/loo. At the end of the feeding period, the diets were withdrawn and after 4-hr fasting, the animals were weighted, anesthesized and bled by venous puncture. Whole blood (WB) was collected and plasma was obtained from heparinized WB. Thymuses and livers were removed. These organs were weighed, dried and approximately 100 mg were ashed by acid digestion in a microwave oven with 1.5 ml IINO3 using microwave acid digestion bombs (Parr Instrument Company). The processed tissues, plasma and whole blood were adequately diluted with deionized water. The standard curve was prepared from a 1 mg/ml Zn solution (Efluequant). Zn content was determined by atomic absorption spectrophotometry (Varian Spectra A-20) and expressed either as pg/ml or ug/g wet tissue.

DIETARY PROTEIN AND ZINC LEVELS

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TABLE 1 Composition Component g/lOOg Precooked maize flour a

of Experimental

l-

Diets

Diets

t Maize Flour 84.42

Casein 7.75

calcium caseinate b Minerals c

5.00

5.00

Vitamin mixture c

0.25

0.25

Choline d

0.15

0.15

Oil e Dextrin

5.00

5.00

5.18

81.85

6.5

6.5

49

81

Total protein Biological value *

E

a containing 1.26 % of nitroger l( 7.70% protein b containing 13.42% of nitrogen (83.9% proteii c (17,18) d Choline chloride e containing liposoluble vitamins. fRNPRmethod(15, 16).

The statistical analysis was performed using a computer program.

by one way analysis of variance and Tukey post hoc,

RESULTS

No differences in food intake (g/day: MlO: 6.5+1.4 vs ClO: 6.821 .l) nor in the energy and total protein intakes,both expressed on the basis of metabolic mass were observed between Ml0 and CIO (kcal/bwO.75/day: MlO: 1.7+0.2 vs C10:1.6+0.2 and mg/bw 0.75/day: M10:29.2+3.8 vs ClO: 27.9+5.2, respectively); however, the complete protein intake was significantly lower in the group fed the maize flour (mg/bw 0.75/day: M10:14.3+1.9 vs ClO: 22.7+4.2). The food intake of control group was higher than both experimental groups. (17.52 1.1 g/day). Thymuses’ weight (mg) of Ml0 were significantly lower than Cl0 and the data of both were significantly lower than C ( MlO: 46.3 23.3; C10:117.9f 26.8 ; C: 223.12 29.3, pcO.01) (11). Livers’ weigth (g) of Ml0 and Cl0 were significantly lower than C ( MlO: 1.52 0.3; C10:2.lfO.3; C: 3.220.3). Data on whole blood (WB) and plasma (P) Zn levels are shown in Table 2.

A. PALLARO and N.H. SLOBODIANIK

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TABLE 2 Plasma and Whole Blood Zn Levels in Experimental

and Control Groups.

GROUP

WHOLE BLOOD Zn*

PLASMA Zn*

Ml0

@g/ml) 3.85 + 0.24

(&ml) 1.84 2 0.63 1

Cl0

3.88 + 1.00

1.79+0.411

C

3.67 + 0.34

2.43 + 0.37 2

* Values are means + SD of 6 - 8 rats per group. 122 Means within columns not sharing a common superscript differ (pcO.04) No statistical differences were observed in WB Zn among MIO, Cl0 and C. When Ml0 and Cl0 were compared to C, P Zn levels tended to be lower (pcO.04).

Table 3 shows the thymus (T) and Liver (L) Zn contents.

TABLE 3 Thymus and Liver Zn Levels in Experimental

and Control Groups.

Ml0

LIVER Zn* (ug/ g tissue) 42.5 -+ 11.3

THYMUS Zn* (ug/ g tissue) 40.0 2 13.2 2

Cl0

36.3 + 6.7

26.5 + 2.7 1

GROUP

C 39.1 + 8.6 27.1 + 3.6 1 * Values are means 2 SD of 6 - 8 rats per group. 1~2Means within columns not sharing a common superscript differ (pcO.01) No statistical differences were observed in L Zn concentration between Ml0 and Cl0 nor differences were found when these groups were compared to C. However, the thymus Zn concentration of Ml0 was significantly higher than those of C 10 and C (~~0.0 1). Briefly, plasma Zn levels tended to decrease after feeding a low protein diet but a significant increase in thymic Zn was only found when rats were fed the cereal protein (a low quantity - low quality protein diet). DISCUSSION The results suggest that the decrease in plasma Zn circulating levels is probably dependent on the dietary concentration of protein while the thymus Zn content is dependent on the associated effects

DIETARY PROTEIN AND ZINC LEVELS

of the low quality - low quantity protein diet. It seems that the intake of natural proteins imbalanced in its amino acid pattern provokes movements of Zn within the body to certain tissues. This intake might, in some way, alter the distribution of plasma Zn with a concomitant uptake by the thymus, perhaps to avoid the deleterious effects that this diet provokes on proliferation and maturation. Probably, it may be a compensatory way to activate the low presence and/or activity of certain thymic hormones like thymulin (FTS-Zn) described in malnutrition (19-21), as Zn could act as an immune stimulating factor via its biological activation, which is known to stimulate the maturation and proliferation of thymocytes (22). It has been reported in murine AIDS, where the thymus suffered a similar atrophy to that observed in protein - calorie malnutrition , that a similar increase in thymic Zn occurs when compared with the thymus of non-infected animals (23). Our findings would suggest that the reason of the increase, observed in both models, may be due to the malnutrition condition , when the organ seems to need higher quantity of this mineral. The results obtained by Chevalier et al., who demonstrated that children suffering from severe PEM and receiving Zn supplements, presented faster recovery (assessed by the left lobe area of the thymus) while the level of immature lymphocytes decreased significantly (24), strengthen our hypothesis. The thymus atrophy described in our previous papers in rats fed preecooked maize flour, as the only source of protein (9-l 1,15), is then a consequence of the intake of a low quantity - low quality cereal protein; besides this diet would produce a disturbance in Zn metabolism, concomitant to or as a consequence of the state induced by the administration of this type of protein.

ACKNOWLEDGMENT

This work was partially supported by grants of the University of Buenos Aires (FA- 021 and FA - 077). We thank the skilfull assistance of Mrs Lia Culotta de Calafat in animal care, breeding and diet preparations, the technical assistance of Prof. Maria Luz Portela in Zn determinations and the revision of the manuscript by Prof. Patricia Ronayne de Ferrer.

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Accepted

for

publication

November

17,

1998.