Britishjournul oflfuemutology, 1979,43,451-456.

Influence of Dietary Protein Concentration and Quality on Response to Erythropoietin in the Polycythaemic Rat R. M. ALIPPI, J. M. GIGLIO, A. C . BARCEL~), C . E. BOZZINI, R. FARINA* AND M. E. RIO* Cdtedra de Fisiologia, Facultad de Odontologia, and *Departamento de Bromatologia y Nutricidn Experimental, Facultad de Farmacia y Bioquimica, University of Buenos Aires, Argentina (Received 4 October 1978; accepted for publication 3 April 1979)

SUMMARY. The effects were examined of dietary protein concentration and quality on the response of polycythaemic hypertransfused rats to 6 units of human urinary erythropoietin. Rats were either starved or fed one of 14 different diets. Four protein sources were used, having a quality gradient from 100 to about 24. Two proteinscasein and wheat gluten-were used at five different levels of concentration (5-25%) in the diet. The response of rats maintained on the standard diet (Purina rat chow, 23.4% protein/g) was taken as the normal standard. The response to erythropoietin was 25% of normal in starved rats and 35% of normal in rats put on a protein-free diet. When 10% protein in the diet was obtained by using high biological value proteins (egg yolk or casein) the response to erythropoietin was normal. When the same concentration was achieved by using low biological value proteins (wheat gluten or corn protein) the response to erythropoietin was undistinguishable from that of rats put on the protein-free diet. When rats were maintained on diets with different concentrations of casein (5-25%) a normal response was observed whin protein concentration was 10% with no further changes at higher concentrations. When rats were fed diets with different wheat gluten concentrations (5-25%) the response to erythropoietin was subnormal. These data suggest that the ability of rats to respond normally to erythropoietin is dependent on a continuous dietary intake of proteins at levels which are dependent on their biological values.

Naets & Wittek (1974) found that starvation markedly reduces the response to erythropoietin (erythropoiesis-stimulating hormone, ESH) in normal and polycythaemic rats. Since the disappearance rate of ESH from the plasma compartment was normal and no inhibitor was detectable in the plasma of starved rats, the authors concluded that starvation decreases the ESH-sensitive stem cell pool. Studies performed in our laboratory have confirmed these findings and extended them to the water-deprived rat (Giglio et al, 1979). The depressed Correspondence: Dr Carlos E. Bozzini, Citedra de Fisiologia, Facultad de Odontologia, M. T. de Alvear 2142, 1122 Buenos Aires, Republica Argentina. 0007-1048/79/110~0451$02.00

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response to ESH seen in the starved rat may be the result of either absolute food deprivation or lack ofa specific constituent of the diet. Therefore, it would be important to know the effect of a diet limited in one of its constituents on the response to the hormone. Protein is perhaps the constituent of the diet which exerts the most important influence on erythropoiesis. Proteinenergy malnutrition is a well recognized cause of anaemia, which can be reproduced experimentally by feeding animals protein-deficient diets (Orten & Smith, 1937;’Bethardet al, 1958; Reissmann, 1964a). The object of the experiments described in this paper was to examine the influence of dietary protein concentration and quality on the response to ESH in the polycythaemic rat. MATERIALS AND METHODS Male Wistar rats weighing 160f10 g were used in groups of five, and all data refer to mean+ SEM of a group. Purina rat chow (23.4% protein, energy value 4.4 calories/g) was considered as standard diet. Rats were fed freely with one of 14 diets containing four protein sources having a quality gradient from 100 to about 24, evaluated as chemical score (CS) against human milk as reference protein (Energy and Protein Requirements: World Health Organisation Technical Reports Series no. 522, 1973). A protein free diet containing 5.0% oil, vitamins and minerals according to Harper (1959) and 89.6% dextran was included in every experiment as a control. Two proteins, casein and wheat gluten, were used at five different levels of concentration: 5, 10, 15, 20 and 25% in the diet. T w o other proteins, egg yolk and corn, were used at 10%. All the diets were isocaloric and protein was included in the protein-free diet by substituting an equivalent amount of dextran. The effect of starvation, biological quality and protein levels in the diet on the response to 6.0 IRP units of human urinary ESH* was investigated in polycythaemic rats. Rats were hypertransfused by being given packed red cells, 2.5 m1/100 g body weight (washed once with saline), intraperitoneally on two consecutive days. Three days after the second transfusion (day 1)rats were placed on either starvation or experimental diets. ESH was injected subcutaneously citrate) was injected in the femoral in divided doses on days 3 and 4. Radioiron (0.5 pCi SYFe vein on day 5. Incorporation into red cells was measured on Day 7, 48 h later. Blood was obtained by cardiac puncture and incorporation was calculated estimating the blood volume to be 7 % of the initial body weight (Bozzini & Kofoed, 1966). The statistical significance of differences was determined by Student’s t test. RESULTS 1. Response to Erytkropoietin in Polycytkaemic rats Placed on Starvation, Experimental or Standard Diets Groups of polycythaemic rats were placed on either starvation or experimental (protein-free or casein 20%) or standard (Purina lab chow) diets. Their average responses to 6 u ESH are shown in Fig 1. Rats fed an experimental diet which contained 20% casein (group 4) showed a Collected and concentrated by the Department of Physiology, University of the Northeast, Corrientes, Argentina, further processed and assayed by Hematological Research Laboratories, Children’s Hospital of LOS Angeles, under research grant HL 00880 (National Heart and Lung Institutes).

Dietary Proteins and Erythropoiesis 453 response to ESH which was similar to that of rats placed on the standard diet (group 5). Their responses were about 2.5 times greater than that of rats placed on a protein-free diet (group 3). The response to ESH of starved rats (group 2) was about 60% of that seen in rats fed a protein-free diet. 2. Response to Erythropoietin in Polycythaemic Rats as a Function of the Casein Content ofthe Diet Groups of rats were fed a diet that was either protein-free or contained 5%, lo%, 15%, 20% or 25% casein. Average responses to ESH are shown in Fig 2. Erythrocyte 59Feincorporation was 13.47+0.63% in rats maintained on a protein-free diet. There was a progressive increase in the response as the casein content ofithe diet increased from 0 to 10%. No further changes in the response were observed by increasing casein content up to 25%.

3. Response to Erythropoietin in Polycythaemic Rats Placed on Diets Containing 10% Proteins of Digerent Biological Values Groups of rats were fed a diet that was either protein-free or contained 10% egg yolk (BV=100), casein (BV=71.4), corn proteins (BV=36) or wheat gluten (BV=24.2). Average

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FIG 1 . Erythropoietic response of polycythaemic rats to 6.0 IRP units ESH measured by RBC "Fe incorporation. l=saline injected control rats, 2=starved rats, 3=rats put on a protein free diet, 4=rats put on a 20% casein diet, 5=rats put on standard diet.

FIG2. Erythropoietic response of polycythaemic rats to 6.0 IRP units ESH measured by RBC 5'Fe incorporation, as function of the casein concentration of the diet. Meanf SEM of five rats in each group.

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responses to ESH are shown in Fig 3. Rats fed diets that contained either egg yolk (group 3) or casein (group 4) showed responses to ESH which were about two times greater than those of rats maintained on a protein-free diet (group 2). Conversely, rats placed on diets that contained either corn proteins (group 5) or wheat gluten (group 6) showed responses which were not significantly different from those of rats put on a protein-free diet.

4. Response to Erythropoietin in Polycythaemic Rats as a Function of the Wheat Gluten Content ofthe Diet Groups of rats were fed a diet that was either protein-free or contained 5%, lo%, 15%, 20%” or 25% wheat gluten. Another group of rats was placed on a diet containing 10% casein for comparison. Their average responses to ESH are shown in Fig 4. Erythrocyte 5yFeuptake was 8.5 f 1.8% in rats fed a protein-free diet. The response to ESH was not modified by increasing wheat gluten content of the diet from 0 to 10%. A progressive increase in the response was observed as the wheat gluten concentration of the diet increased from 10% to 25%. However, the response to ESH seen in rats maintained on a diet containing 25% wheat gluten was about 25% below the value of rats fed a 10% casein diet (normal response).

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FIG3. Erythropoietic response of polycythaemic rats to 6.0 IRP units ESH measured by RBC 59Fe incorporation. 1=saline injected control rats, 2=rats put on a protein-free diet, 3=ratsput on a 10% egg yolk diet, 4=rats put on a 10% casein diet, 5=rats put on a 10% corn proteins diet, 6=rats put on a 10% wheat gluten diet. Mean* SEM of five rats in each group. FIG4. Erythropoietic response of polycythaemic rats to 6.0 IRP units ESH measured by RBC 5yFe incorporation, as function of the wheat gluten concentration in the diet. Meanf SEM of five rats in each group.

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DISCUSSION The experiments reported here examined the influence of dietary protein concentration and quality on the erythroid response of hypertransfused plethoric rats to ESH. Since variations in the response to the hormone were found, it was necessary to define the normal response. The normal standard was taken to be the response of rats maintained on the standard diet, which contained 23.4% protein and had an energy value of 4.4 calories/g. When compared to the normal value, starvation markedly reduced the response to ESH. In the experiment shown in Fig 1 the response in the starved group was approximately 25% of that of the group maintained on the standard diet. These findings confirm previous reports from Naets & Wittek (1974) and Giglio et al(1979). Changes in radioiron uptake in starved rats after ESH injection may represent real changes in erythropoiesis since the reticulocyte index changed in the same direction in the experiments reported by Naets & Wittek (1974). Rats maintained on a protein-free diet with an energy value similar to the standard diet showed a greater response to ESH than starved rats, although still subnormal. In the four experiments presented here it averaged 35% of the normal response. The quality and concentration ofprotein in the diet conditioned the response to ESH. When 10% protein in the diet was obtained by using high biological value proteins (egg yolk or casein) the response was normal. When the same protein concentration was achieved by using low biological value proteins (wheat gluten or corn proteins) the response to ESH was undistinguishable from that of protein-free diet. When rats were maintained on diets with normal redifferent concentrations (5-25%) of a high biological value protein-casein-a sponse was obtained when protein concentration was 10% with no further changes at higher concentrations. If, however, diets with less than 10% casein concentration were used, the response to ESH diminished. When rats were fed diets with different concentrations (5-25%) of a low biological value protein (wheat gluten) their responses to ESH, although increased in the 15-25% range of concentrations, were subnormal. These data indicate that the ability of rats to respond normally to ESH is dependent on a continuous dietary intake of proteins at levels which are dependent on their biological values. The results of the experiments reported here do not agree with those previously reported by Reissmann (1964b) who found that the erythroid responsiveness to ESH was normal in protein-deprived rats. The difference in the time of induction of polycythaemia relative to the initiation of protein starvation could perhaps explain the discrepancies between the two laboratories. O n the other hand, Fondu et al(1978), studying the regulation oferythropoiesis in protein-energy malnutrition in human beings, concluded that there must be some disturbances in the responsiveness of bone marrow to ESH in this condition. A satisfactory explanation as to why the erythroid response to ESH is dependent on the quality and concentration of dietary proteins is not apparent. Bell et al (1976) reported that mice maintained on a diet limited only in proteins (4% by weight) have lower numbers of colony forming units (CFU-S) and in vitro granulocytic colony forming cells (CFU-C) than mice maintained on a standard diet (18-20% protein). Some of these findings have been recently confirmed and extended by Fried et al(1978), who also found that PCV, lymphocyte counts and platelet counts of mice on a protein-free diet fall rapidly and also regenerate less rapidly and less completely after irradiation than in mice on normal diets. The effect may be

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due, in part, to a decrease in the number of CFU-S and consequently in the size of the precursor cell pool available for differentiation. If this explanation is valid it could be possible that quality and concentration of dietary proteins influence the ESH-responsive stem cell pool, thus conditioning the response to the hormone. ACKNOWLEDGMENTS

This work was partially supported by the Consejo Nacional de Investigacioncs Cientificas y Tkcnicas and by the Instituto Nacional de Farmacologia y Bromatologia de la Republica Argentina. R.M.A., C.E.B. and M.E.R. are Career Investigators, Consejo Nacional de Investigaciones Cientificas y Tkcnicas de la Repfiblica Argentina. REFERENCES BELL,R.G., HAZELL, L.A. & SHERIDAN, J.W. (1976) The influence of dietary protein deficiency on hematopoietin cells in the mouse. Cell and Tissue Kinetics, 9,305-31 1. BETHARD, W.F., WISSLER, R.W., THOMPSON, J.S., SCHROEDER, M.A. & ROBSON,M.T. (1958) The effect of acute protein deprivation upon erythropoiesis in rats. Blood, 13, 216-225. BOZZINI,C.E. 81 KOFOED, J.A. (1966) Erythroid responsiveness to erythropoietin in hypophysectomized rats. Acta Physiologica Latino-Americana, 16, 13-16. FONDU,P., HAGA,P. & HALVORSEN, S. (1978) The regulation of erythropoiesis in protein-energy malnutrition. Britishjournal of Haematology, 38,29-36. FRIED,W., SHAPIRO, S., BARONE, J. & ANAGNOSTOU, A. (1978) Effect ofprotein deprivation on hematopoietic stem cells and on peripheral blood counts. Journal $ Laboratory and Clinical Medicine, 92, 303-310. GIGLIO. J.M., ALIPPI,R.M., B A R C E A.C. L ~ , & Boz-

C.E. (1979) Mechanism of the decreased erythropoiesis in the water deprived rat. Britidi Journal of’Haematology, 42,93-100. HARPER, A.E. (1959) Amino acid balance and inbalance. I. Dietary level of proteins and amino acids inbalance. Journal ofNutrition, 68, 605418. NAETS, J.P. & WITTEK, M. (1974) Effect of starvation on the response to erythropoietin in the rat. Arta Haematologica, 52, 141-150. ORTEN, A.V. & SMTH, A.H. (1937) The effect of dietary protein on endocrine function and on the blood picture of female rats. American Journal of Physiology, 119,381-382. REISSMANN, K.R. (1964a) Protein metabolism and erythropoiesis. I. The anemia of protein deprivation. Blood, 23, 137-145. REISSMANN, K.R. (1964b) Protein metabolism and erythropoiesis. 11. Erythropoietin formation and erythroid responsiveness in protein-deprived rats. Blood, 23,146-153. ZINI,