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The Effect of Dietary Diammonium Citrate on Glutamine and Glutamic Acid Concentration in the Blood Plasma of Chicks1
1177
EGG QUALITY
showed slight stains after washing. Those either oiled or not oiled and held at room temperature for a period of one week were not adequately cleaned by washing. Additional study is needed to determine the saving in time, water and fuel from washing eggs once a week instead of washing egg each day. REFERENCES Bigbee, D. C , L. E. Dawson, W. L. Mallmann and G. A. Houghtby, 1961. Evaluation of an oil-inwater emulsion for cleaning shell eggs. Poultry
Sci. 40: 1380. Froning, G. W., and M. H. Swanson, 1961. Effect of time of oiling and holding temperature on cloudy whites and percent outer thin white of shell eggs. Poultry Sci. 40:140S. Schmidt, F. J., and W. J. Stadelman, 1957. Effects of antibiotics and heat treatment of shell eggs on quality after storage. Poultry Sci. 36: 1023-1026. Stewart, G. F., and S. Bose, 1948. Factors influencing the efficiency of solvent-oil mixtures in the preservation of shell eggs. Poultry Sci. 27: 270276. Davis, H. B., and C. C. Brunson, 1961. Relative effectiveness of oil and cellophane in protecting eggs. Poultry Sci. 40: 1393.
The Effect of Dietary Diammonium Citrate on Glutamine and Glutamic Acid Concentration in the Blood Plasma of Chicks1 ELLEN M. OLSEN, D. C. HILL AND H. D. BRANION Department of Nutrition, Ontario Agricultural College, Guelph, Ontario, Canada (Received for publication March 25, 1963)
G
LUTAMINE forms a major fraction of the non-protein nitrogen in the blood plasma of humans and other mammals (Hamilton, 1945; Prescott and Waelsch, 1947; Bessman et al., 1948), and it has been suggested that blood glutamine functions as a non-toxic carrier of ammonia (Meister, 1956), and provides the major source of ammonia excreted in the urine (Van Slyke et al., 1943). Recently it has been reported that the blood plasma of the chicken also contains considerable glutamine (Olsen et al., 1962) and it seems likely that in the avian species glutamine plays a particularly important part in ammonia metabolism since it is a major contributor of nitrogen during the synthesis of uric acid (Buchanan and Hartman, 1959). The experiments to be described were 1
Presented at the Sth annual meeting of the Canadian Fecderation of Biological Societies held at Laval University, Quebec, P.Q., June, 1962.
designed to provide evidence that the chick uses glutamine as an ammonia carrier in the blood. To this end the response of plasma glutamine to the ingestion of ammonia in the form of diammonium citrate both with and without supplemental glutamic acid was studied. Tigerman and Mac Vicar (1951) reported that when ammonia and glutamic acid were administered together to rats a considerable increase occurred in the concentration of glutamine in various body tissues. EXPERIMENTAL
Day-old White Plymouth Rock pullets, obtained from a commercial hatchery, were placed in starter batteries and fed a semi-purified diet containing either purified soybean protein or casein. The composition of the diets is given in Table 1. At 2 weeks of age the chicks were divided into lots of approximately equal average weight, 12-15 chicks per lot, and
1178
E. M. OLSEN, D. C. HILL AND H. D. BRANION
TABLE 1.—Composition of the basal diets Ingredient Dextrose Cellulose1 Corn oil Isolated soybean protein2 Vitamin-free casein DL-Methionine Glycine L-Arginine HC1 Choline chloride3 Vitamin premix4 Salts6 Chromium oxide Protein (NX 6.25) Glutamic acid
Basal 1
Basal 2
%
%
61.36 7.00 4.00 18.00
62.08 7.00 4.00
—
16.60 0.25 0.66 0.83 0.28 2.00 6.00 0.30 17.0 4.2
0.54 0.36
—
0.44 2.00 6.00 0.30 15.4 2.6
—
1 Alphacel, Nutritional Biochemicals Corporation, Cleveland. 2 C-l assay protein purchased from ArcherDaniels-Midland Company, Cincinnati. 3 An equal parts mixture of pure choline chloride and a 25% choline chloride concentrate. 4 Contained in gm. per kilogram: vitamin E concentrate (20,000 I.U./gm.), 40.8; vitamin A concentrate (250,000 I.U./gm.), 2.12; vitamin D 3 concentrate (16,500 I.C.U./gm.), 2.40; riboflavin premix (1 gm. riboflavin/oz.), 16.3; Ca pantothenate, 2.03; niacin, 5.29; biotin, 0.0176; folic acid, 0.11; menadione, 1.01; thiamine HC1, 0.40; pyridoxine HC1, 0.70; vitamin B12 (0.1% triturate), 1.01; p-aminobenzoic acid, 1.00; inositol, 55.1. 5 Briggs et al. (1943).
each lot was housed in an individual pen. The experimental diets were assigned to the pens at random, 2 pens per diet, and the feeding continued for an additional week. The experimental diets were formulated by replacing dextrose in the diets of Table 1 with various levels of reagent grade diammonium citrate (DAC) and L-glutamicacid (GA). Excreta samples were collected daily from each lot during the final 3 days of the experiments, the 3 collections pooled for each lot, dried in the frozen state and ground. Uric acid was determined in the dried excreta by the method of Shirley and Van Landingham (1939). Chromic oxide was used as a reference indicator to relate the uric acid found in the excreta to the feed consumption, and the ratio of chromic oxide per gm. of feed to chromic oxide per gm. of dried excreta was deter-
mined as described by Czarnocki, Sibbald and Evans (1961). At the conclusion of the experiments blood was removed from the carotid artery of each chick and equal quantities pooled for each lot. Heparin was used as an anticoagulant. The preparation of the protein-free plasma and the method for the determination of glutamic acid and glutamine have been described in previous papers (Gray et al., 1960; Olsen et ah, 1962). Statistical analyses of the data was by methods described by Snedecor (1956). RESULTS AND DISCUSSION
In all, 5 experiments were conducted, 3 with basal diet 1 and 2 with basal diet 2, in which various combinations of DAC and GA were used. Experiments with basal diet 1 {Table 2). Weight gains were influenced by the inclusion of DAC in the diet. A level of 4% of DAC was ineffective, a level of 8% produced a small but consistent reduction and a level of 12% (Experiment 3) resulted in the most marked reduction in weight gains. Elevated levels of glutamine in the blood plasma resulted from diets containing 8 or 12% of DAC and these levels were further increased when supplementary GA was added to the diets. The addition of GA alone was without effect on plasma glutamine levels. A statistical analysis of the combined data for the 3 experiments, encompassing the control diet and those with supplements of 8% of DAC, 4% of GA and the combination of these supplements, revealed a significant effect of DAC (P<0.01) and a non-significant effect of GA. Surprisingly, in view of the data, the interaction of DAC and GA proved nonsignificant (P<0.05) if the statistical test for this interaction was made on the basis of extending conclusions beyond the 3
D I E T A R Y DIAMMONIUM C I T R A T E AND B L O O D PLASMA G L U T A M I N E
TABLE 2.—Effect
1179
of the addition of diammonium citrate (DAC) and glutamic acid (GA) to basal J1 Av. gain, 2-3 wks., gm.
Plasma glutamine, mg./lOO ml.
Plasma glutamic acid, mg./lOO ml.
Excreted uric acid, gm./lOO gm. feed consumed
Experiment 1 None 2 % DAC 4 % DAC 8% DAC 4%GA 2 % D A C + 4 % GA 4 % D A C + 4 % GA 8% D A C + 4 % GA
110 116 110 88 HI 113 110 90
( 7) ( 2) ( 5) ( 2) ( 7) ( 7) (13) ( 2)
16.0(1.5) 19.2 (1.0) 19.2 (2.7) 21.5 (1.7) 15.1 (1.8) 16.4(2.5) 21.1 (0.3) 34.5 (3.4)
2.7 (0.1) 3.5(0.4) 2.8(0.2) 3.0(0.2) 3.5 (0.9) 3.0 (0.8) 3.6(0 ) 3.1 (0.2)
1.11 (0.02) 1.41 (0.02) 1.93 (0.02) 2.46(0.1) 1.74(0.11) 2.37 (0.13) 2.37 (0.27) 3.33 (0.25)
Experiment 2 None 8% DAC 4%GA 8% D A C + 4 % GA 8% D A C + 8 % GA
105 97 110 91 82
( ( ( ( (
0) 7) 6) 7) 2)
16.3 (0.4) 18.7 (1.0) 17.6(0.3) 22.3 (5.3) 26.5 (8.6)
4.0 (0.2) 2.8(0.3) 4.6(0.2) 3.3 (0.4) 4.4(0.3)
0.73 (0.02) 2.14(0.32) 1.43 (0.15) 2.77 (0.05) 3.37 (0.37)
Experiment 3 None 8% DAC 12% DAC 4%GA 8%GA 8% D A C + 4 % GA 8% D A C + 8 % GA 12% DAC+895, GA
116 (25) 95(6) 67 ( 6) 1242 116 ( 6) 85 ( 6) 80 (12) 60 (15)
14.7 (0.1) 24.2 (4.8) 31.7(7.4) 14.7 (0.6) 15.7 (2.3) 29.5 (1.8) 38.3 (1.8) 61.0(0 )
2.8(0.8) 1.2(0 ) 3.7 (0.9) 4.4 (0.9) 7.5(0 ) 3.0(0.1) 4.4 (0.2) 4.3(0.7)
0.94 2.41 3.02 1.67 2.32 3.22 3.95 4.50
Supplements
(0.19) (0.11) (0.43) (0.18) (0.15) (0.09) (0.28) (0.18)
1 Each value is the average of 2 lots of birds each containing 13 birds in experiment 1 and 2 and 15 birds in experiment 3. Figure in parentheses following each value is the difference between the duplicate lots. 2 Average for a single lot. Average gain for duplicate lot only 65 gm.
experiments which were conducted. However, it seems apparent t h a t the non-significant interaction revealed b y the test can be attributed chiefly to the rather small responses to the supplements obtained in experiment 2, and it would be unreasonable on the basis of this test to conclude t h a t supplemental GA did not increase glutamine formation in the presence of ammonia. Certainly, in experiments 2 and 3 the addition of 8 % of GA to diets containing either 8 % or 1 2 % of DAC resulted in increased plasma glutamine. In the latter case the plasma glutamine rose from 31.2 to 61 mg. per 100 ml., a highly significant effect ( P < 0 . 0 1 ) . Also, results of experiments 4 and 5, to be discussed later, support a role for dietary GA in promoting glutamine synthesis. However, it is noteworthy t h a t
the depressed weight gains given b y diets containing 8 % or 1 2 % of DAC were not improved by the addition of GA to these diets. I t is evident, therefore, t h a t the decreased weight gains could not be attributed to a diversion of glutamic acid, needed for protein synthesis, to glutamine formation. Plasma GA concentrations also exhibited a definite relationship to the dietary supplements. Statistical analysis of the combined d a t a from the 3 experiments utilizing the 4 diets common to the 3 experiments, revealed a significant decrease in plasma GA resulting from DAC in the diet and a significant increase of plasma GA from supplementary GA ( P < 0 . 0 5 ) . There was no evidence for an interaction effect of the two supplements. The observed decrease of plasma GA is
1180
E. M. O L S E N , D . C. H I L L AND H. D .
probably a reflection of the use of GA for the formation of glutamine. Uric acid excretion was increased when either D A C or GA were added to the basal diet and the effects of the two fed in combination seemed to be additive. Statistical analysis of the combined d a t a from the 3 experiments supported these conclusions ( P < 0 . 0 1 ) . The proportion of the total nitrogen contributed to the diets by D A C and GA excreted as uric acid nitrogen was calculated, assuming a constant excretion of uric acid from the nitrogen contributed by the basal diet. The values so obtained were variable, 3 9 - 6 1 % for DAC and 5 4 8 2 % for GA. There was no evidence t h a t the amount of the supplement added to the diet influenced the percentage excretion of the nitrogen as uric acid. Experiments with basal diet 2 {Table 3). While results were in general similar to those obtained with basal diet 1 there were some quantitative differences. I n experiment 5 the addition of 1 2 % of D A C to the diet resulted in an average plasma glutamine level of 21.2 mg. per 100 ml., and the further addition of 8 % of GA increased this value only to an average of 36.8 mg. per 100 ml. This result contrasts with levels of 31.7 and 61.0 mg. per 100 TABLE 3.—Effect
Supplements
BRANION
ml. given by similar levels of supplements in experiment 3. While it is not clear why the two basal diets, differing by formula, only in the protein supplements, should yield a quantitatively different result, the two sets of experiments lead to the same conclusions. Statistical analysis of the d a t a of experiment 5 revealed significant effects (P <0.01) for DAC and GA and the interaction between them. Plasma GA levels and uric acid excretion followed patterns similar to those observed in experiments 1, 2 and 3. Utilizing the d a t a of experiment 5 it was found t h a t the effects of dietary DAC and GA were statistically significant ( P < 0 . 0 1 ) and their interaction non-significant. I n these experiments, and in the 3 preceding, possible influence of the dietary supplements on blood plasma volume was not studied. However, it seems unlikely t h a t blood plasma volume changes could explain the observed results. Very large alterations in plasma volume would be needed to account for the observed effects on glutamine and GA concentrations. Furthermore, the differential effects of the supplements on plasma glutamine and GA argue against the possibility t h a t plasma volume change could be a major factor influencing the results.
of the addition of diammonium citrate (DAC) and glutamic acid (GA) to basal 2l Av. gain, 2-3 wks., gm.
Plasma glutamine, mg./lOO ml.
Plasma glutamic acid, mg./lOO ml.
Excreted uric acid, gm./lOO gm. feed consumed
Experiment 4 None 8% DAC 4 % GA 8% D A C + 4 % GA
89 68 92 68
(14) ( 0) ( 2) (18)
16.8(1.7) 26.5 (0.4) 18.0 (0.7) 29.0 (1.7)
3.3 2.7 3.5 3.5
(0) (0.1) (0.7) (0.8)
1.29 2.85 1.93 3.17
(0) (0.47) (0.07) (0.14)
Experiment 5 None 8% DAC 12% DAC 8% GA 8% D A C + 8 % GA 12% D A C + 8 % GA
120 105 91 141 118 86
(12) (18) ( 1) ( 2) ( 3) ( 6)
14.4(0.3) 19.4(1.4) 21.2 (2.0) 14.3 (0 ) 30.2 (4.1) 36.8 (21.1)
3.1 (0.4) 2.8(0.3) 2.4(0.5) 5.5 (0.1) 5.8 (0.3) 4.0 (0.6)
1.30 2.58 3.23 2.72 4.27 4.34
(0.25) (0.37) (0.6) (0.11) (0.04) (0.24)
1 Each value is the average of 2 lots of birds each containing 14 birds in experiment 4 and 12 birds in experiment 5. Figure in parentheses following each value is the difference between the duplicate lots.
DIETARY DIAMMONIUM CITRATE AND BLOOD PLASMA GLUTAMINE
Taken together the 5 experiments provide indirect evidence that the chick, like other species, utilizes glutamine as a carrier of ammonia in the blood, synthesizing the glutamine from ammonia and GA.The rise in plasma glutamine, observed following ammonia ingestion, appears to be variable and under certain conditions, not elucidated here, may be rather small (experiment 2). Probably the rate at which glutamine is utilized for uric acid synthesis or is broken down to give urinary ammonia are major factors affecting its level in the plasma. SUMMARY
Chicks were fed semipurined diets containing either 18% of isolated soybean protein or 16.6% of casein. The glutamine content of the deproteinized blood plasma ranged from 14.4 to 16.8 mg. per 100 ml. and the glutamic acid content from 2.7 to 4.0 mg. per 100 ml. The incorporation of up to 12% of diammonium citrate (DAC) into these diets, replacing equivalent quantities of dextrose, resulted in marked increases in the concentration of glutamine in the plasma, and these increases were greater when supplements of 4 to 8% of glutamic acid (GA) were included in addition to DAC. Supplemental GA when added without DAC did not affect the concentration of glutamine in the plasma but did increase the concentration of GA. Uric acid excretion increased with increasing levels of DAC and/or GA in the diet and the effects of the two supplements when fed in combination were essentially additive. Results support the thesis that the formation of glutamine is a major mechanism for the removal of excess ammonia from the blood of chicks. ACKNOWLEDGMENTS
This work was supported in part by a
1181
grant in aid of research by the National Research Council of Canada. The authors are indebted to Merck and Co. Ltd. and Pfizer Canada both of Montreal, Quebec, and Distillation Products Industries, Rochester, N.Y. for several of the vitamins used in the experimental diets. REFERENCES Bessman, S. P., J. Magnes, P. Schwerin and H. Waelsch, 1948. The absorption of glutamic acid and glutamine. J. Biol. Chem. 175:817-823. Briggs, G. M., Jr., T. D. Luckey, C. A. Elvehjem and E. B. Hart, 1943. Studies on two chemically unidentified water-soluble vitamins necessary for the chick. J. Biol. Chem. 148: 163-172. Buchanan, J. M., and S. C. Hartman, 1959. Enzymatic reactions in the synthesis of the purines. Adv. Enzymology, 21: 199-261. Czarnocki, J., I. R. Sibbald and E. V. Evans, 1961. The determination of chromic oxide in samples of feed and excreta by acid digestion and spectrophotometry. Can. J. An. Sci. 41:167-179. Gray, J. A., E. M. Olsen, D. C. Hill and H. D. Branion, 1960. Effect of a dietary lysine deficiency on the concentration of amino acids in the deproteinized blood plasma of chicks. Can. J. Biochem. Physiol. 38: 435-441. Hamilton, P. B., 1945. Glutamine: A major constituent of free a amino acids in animal tissues and blood plasma. J. Biol. Chem. 158:397-409. Meister, A., 1956. Metabolism of glutamine. Physiol. Rev. 36: 103-127. Olsen, E. M., D. C. Hill and H. D. Branion, 1962. Determination of glutamine and glutamic acid in the blood plasma of chicks by a combined chromatographic and microbiological method. Can. J. Biochem. Physiol. 40: 381-390. Prescott, B. A., and H. Waelsch, 1947. Free and combined glutamic acid in human blood plasma and serum. J. Biol. Chem. 167: 855-860. Shirley, R. L., and R. H. Van Landingham, 1939. Determination of uric acid in the mixed excreta of birds. Ind. Eng. Chem. (Anal. Ed.) 11:381-383. Snedecor, G. W. 1956. Statistical Methods Applied to Experiments in Agriculture and Biology, 5th ed., Iowa State College Press, Ames, Iowa. Tigerman, H., and R. MacVicar, 1951. Glutamine, glutamic acid, ammonia administration and tissue glutamine. J. Biol. Chem. 189: 793-799. Van Slyke, D. D., R. A. Phillips, P. B. Hamilton, R. M. Archibald, P. H. Futcher and A. Hiller, 1943. Glutamine as a source material of urinary ammonia. J. Biol. Chem. 150: 481-482.
EGG QUALITY
showed slight stains after washing. Those either oiled or not oiled and held at room temperature for a period of one week were not adequately cleaned by washing. Additional study is needed to determine the saving in time, water and fuel from washing eggs once a week instead of washing egg each day. REFERENCES Bigbee, D. C , L. E. Dawson, W. L. Mallmann and G. A. Houghtby, 1961. Evaluation of an oil-inwater emulsion for cleaning shell eggs. Poultry
Sci. 40: 1380. Froning, G. W., and M. H. Swanson, 1961. Effect of time of oiling and holding temperature on cloudy whites and percent outer thin white of shell eggs. Poultry Sci. 40:140S. Schmidt, F. J., and W. J. Stadelman, 1957. Effects of antibiotics and heat treatment of shell eggs on quality after storage. Poultry Sci. 36: 1023-1026. Stewart, G. F., and S. Bose, 1948. Factors influencing the efficiency of solvent-oil mixtures in the preservation of shell eggs. Poultry Sci. 27: 270276. Davis, H. B., and C. C. Brunson, 1961. Relative effectiveness of oil and cellophane in protecting eggs. Poultry Sci. 40: 1393.
The Effect of Dietary Diammonium Citrate on Glutamine and Glutamic Acid Concentration in the Blood Plasma of Chicks1 ELLEN M. OLSEN, D. C. HILL AND H. D. BRANION Department of Nutrition, Ontario Agricultural College, Guelph, Ontario, Canada (Received for publication March 25, 1963)
G
LUTAMINE forms a major fraction of the non-protein nitrogen in the blood plasma of humans and other mammals (Hamilton, 1945; Prescott and Waelsch, 1947; Bessman et al., 1948), and it has been suggested that blood glutamine functions as a non-toxic carrier of ammonia (Meister, 1956), and provides the major source of ammonia excreted in the urine (Van Slyke et al., 1943). Recently it has been reported that the blood plasma of the chicken also contains considerable glutamine (Olsen et al., 1962) and it seems likely that in the avian species glutamine plays a particularly important part in ammonia metabolism since it is a major contributor of nitrogen during the synthesis of uric acid (Buchanan and Hartman, 1959). The experiments to be described were 1
Presented at the Sth annual meeting of the Canadian Fecderation of Biological Societies held at Laval University, Quebec, P.Q., June, 1962.
designed to provide evidence that the chick uses glutamine as an ammonia carrier in the blood. To this end the response of plasma glutamine to the ingestion of ammonia in the form of diammonium citrate both with and without supplemental glutamic acid was studied. Tigerman and Mac Vicar (1951) reported that when ammonia and glutamic acid were administered together to rats a considerable increase occurred in the concentration of glutamine in various body tissues. EXPERIMENTAL
Day-old White Plymouth Rock pullets, obtained from a commercial hatchery, were placed in starter batteries and fed a semi-purified diet containing either purified soybean protein or casein. The composition of the diets is given in Table 1. At 2 weeks of age the chicks were divided into lots of approximately equal average weight, 12-15 chicks per lot, and
1178
E. M. OLSEN, D. C. HILL AND H. D. BRANION
TABLE 1.—Composition of the basal diets Ingredient Dextrose Cellulose1 Corn oil Isolated soybean protein2 Vitamin-free casein DL-Methionine Glycine L-Arginine HC1 Choline chloride3 Vitamin premix4 Salts6 Chromium oxide Protein (NX 6.25) Glutamic acid
Basal 1
Basal 2
%
%
61.36 7.00 4.00 18.00
62.08 7.00 4.00
—
16.60 0.25 0.66 0.83 0.28 2.00 6.00 0.30 17.0 4.2
0.54 0.36
—
0.44 2.00 6.00 0.30 15.4 2.6
—
1 Alphacel, Nutritional Biochemicals Corporation, Cleveland. 2 C-l assay protein purchased from ArcherDaniels-Midland Company, Cincinnati. 3 An equal parts mixture of pure choline chloride and a 25% choline chloride concentrate. 4 Contained in gm. per kilogram: vitamin E concentrate (20,000 I.U./gm.), 40.8; vitamin A concentrate (250,000 I.U./gm.), 2.12; vitamin D 3 concentrate (16,500 I.C.U./gm.), 2.40; riboflavin premix (1 gm. riboflavin/oz.), 16.3; Ca pantothenate, 2.03; niacin, 5.29; biotin, 0.0176; folic acid, 0.11; menadione, 1.01; thiamine HC1, 0.40; pyridoxine HC1, 0.70; vitamin B12 (0.1% triturate), 1.01; p-aminobenzoic acid, 1.00; inositol, 55.1. 5 Briggs et al. (1943).
each lot was housed in an individual pen. The experimental diets were assigned to the pens at random, 2 pens per diet, and the feeding continued for an additional week. The experimental diets were formulated by replacing dextrose in the diets of Table 1 with various levels of reagent grade diammonium citrate (DAC) and L-glutamicacid (GA). Excreta samples were collected daily from each lot during the final 3 days of the experiments, the 3 collections pooled for each lot, dried in the frozen state and ground. Uric acid was determined in the dried excreta by the method of Shirley and Van Landingham (1939). Chromic oxide was used as a reference indicator to relate the uric acid found in the excreta to the feed consumption, and the ratio of chromic oxide per gm. of feed to chromic oxide per gm. of dried excreta was deter-
mined as described by Czarnocki, Sibbald and Evans (1961). At the conclusion of the experiments blood was removed from the carotid artery of each chick and equal quantities pooled for each lot. Heparin was used as an anticoagulant. The preparation of the protein-free plasma and the method for the determination of glutamic acid and glutamine have been described in previous papers (Gray et al., 1960; Olsen et ah, 1962). Statistical analyses of the data was by methods described by Snedecor (1956). RESULTS AND DISCUSSION
In all, 5 experiments were conducted, 3 with basal diet 1 and 2 with basal diet 2, in which various combinations of DAC and GA were used. Experiments with basal diet 1 {Table 2). Weight gains were influenced by the inclusion of DAC in the diet. A level of 4% of DAC was ineffective, a level of 8% produced a small but consistent reduction and a level of 12% (Experiment 3) resulted in the most marked reduction in weight gains. Elevated levels of glutamine in the blood plasma resulted from diets containing 8 or 12% of DAC and these levels were further increased when supplementary GA was added to the diets. The addition of GA alone was without effect on plasma glutamine levels. A statistical analysis of the combined data for the 3 experiments, encompassing the control diet and those with supplements of 8% of DAC, 4% of GA and the combination of these supplements, revealed a significant effect of DAC (P<0.01) and a non-significant effect of GA. Surprisingly, in view of the data, the interaction of DAC and GA proved nonsignificant (P<0.05) if the statistical test for this interaction was made on the basis of extending conclusions beyond the 3
D I E T A R Y DIAMMONIUM C I T R A T E AND B L O O D PLASMA G L U T A M I N E
TABLE 2.—Effect
1179
of the addition of diammonium citrate (DAC) and glutamic acid (GA) to basal J1 Av. gain, 2-3 wks., gm.
Plasma glutamine, mg./lOO ml.
Plasma glutamic acid, mg./lOO ml.
Excreted uric acid, gm./lOO gm. feed consumed
Experiment 1 None 2 % DAC 4 % DAC 8% DAC 4%GA 2 % D A C + 4 % GA 4 % D A C + 4 % GA 8% D A C + 4 % GA
110 116 110 88 HI 113 110 90
( 7) ( 2) ( 5) ( 2) ( 7) ( 7) (13) ( 2)
16.0(1.5) 19.2 (1.0) 19.2 (2.7) 21.5 (1.7) 15.1 (1.8) 16.4(2.5) 21.1 (0.3) 34.5 (3.4)
2.7 (0.1) 3.5(0.4) 2.8(0.2) 3.0(0.2) 3.5 (0.9) 3.0 (0.8) 3.6(0 ) 3.1 (0.2)
1.11 (0.02) 1.41 (0.02) 1.93 (0.02) 2.46(0.1) 1.74(0.11) 2.37 (0.13) 2.37 (0.27) 3.33 (0.25)
Experiment 2 None 8% DAC 4%GA 8% D A C + 4 % GA 8% D A C + 8 % GA
105 97 110 91 82
( ( ( ( (
0) 7) 6) 7) 2)
16.3 (0.4) 18.7 (1.0) 17.6(0.3) 22.3 (5.3) 26.5 (8.6)
4.0 (0.2) 2.8(0.3) 4.6(0.2) 3.3 (0.4) 4.4(0.3)
0.73 (0.02) 2.14(0.32) 1.43 (0.15) 2.77 (0.05) 3.37 (0.37)
Experiment 3 None 8% DAC 12% DAC 4%GA 8%GA 8% D A C + 4 % GA 8% D A C + 8 % GA 12% DAC+895, GA
116 (25) 95(6) 67 ( 6) 1242 116 ( 6) 85 ( 6) 80 (12) 60 (15)
14.7 (0.1) 24.2 (4.8) 31.7(7.4) 14.7 (0.6) 15.7 (2.3) 29.5 (1.8) 38.3 (1.8) 61.0(0 )
2.8(0.8) 1.2(0 ) 3.7 (0.9) 4.4 (0.9) 7.5(0 ) 3.0(0.1) 4.4 (0.2) 4.3(0.7)
0.94 2.41 3.02 1.67 2.32 3.22 3.95 4.50
Supplements
(0.19) (0.11) (0.43) (0.18) (0.15) (0.09) (0.28) (0.18)
1 Each value is the average of 2 lots of birds each containing 13 birds in experiment 1 and 2 and 15 birds in experiment 3. Figure in parentheses following each value is the difference between the duplicate lots. 2 Average for a single lot. Average gain for duplicate lot only 65 gm.
experiments which were conducted. However, it seems apparent t h a t the non-significant interaction revealed b y the test can be attributed chiefly to the rather small responses to the supplements obtained in experiment 2, and it would be unreasonable on the basis of this test to conclude t h a t supplemental GA did not increase glutamine formation in the presence of ammonia. Certainly, in experiments 2 and 3 the addition of 8 % of GA to diets containing either 8 % or 1 2 % of DAC resulted in increased plasma glutamine. In the latter case the plasma glutamine rose from 31.2 to 61 mg. per 100 ml., a highly significant effect ( P < 0 . 0 1 ) . Also, results of experiments 4 and 5, to be discussed later, support a role for dietary GA in promoting glutamine synthesis. However, it is noteworthy t h a t
the depressed weight gains given b y diets containing 8 % or 1 2 % of DAC were not improved by the addition of GA to these diets. I t is evident, therefore, t h a t the decreased weight gains could not be attributed to a diversion of glutamic acid, needed for protein synthesis, to glutamine formation. Plasma GA concentrations also exhibited a definite relationship to the dietary supplements. Statistical analysis of the combined d a t a from the 3 experiments utilizing the 4 diets common to the 3 experiments, revealed a significant decrease in plasma GA resulting from DAC in the diet and a significant increase of plasma GA from supplementary GA ( P < 0 . 0 5 ) . There was no evidence for an interaction effect of the two supplements. The observed decrease of plasma GA is
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E. M. O L S E N , D . C. H I L L AND H. D .
probably a reflection of the use of GA for the formation of glutamine. Uric acid excretion was increased when either D A C or GA were added to the basal diet and the effects of the two fed in combination seemed to be additive. Statistical analysis of the combined d a t a from the 3 experiments supported these conclusions ( P < 0 . 0 1 ) . The proportion of the total nitrogen contributed to the diets by D A C and GA excreted as uric acid nitrogen was calculated, assuming a constant excretion of uric acid from the nitrogen contributed by the basal diet. The values so obtained were variable, 3 9 - 6 1 % for DAC and 5 4 8 2 % for GA. There was no evidence t h a t the amount of the supplement added to the diet influenced the percentage excretion of the nitrogen as uric acid. Experiments with basal diet 2 {Table 3). While results were in general similar to those obtained with basal diet 1 there were some quantitative differences. I n experiment 5 the addition of 1 2 % of D A C to the diet resulted in an average plasma glutamine level of 21.2 mg. per 100 ml., and the further addition of 8 % of GA increased this value only to an average of 36.8 mg. per 100 ml. This result contrasts with levels of 31.7 and 61.0 mg. per 100 TABLE 3.—Effect
Supplements
BRANION
ml. given by similar levels of supplements in experiment 3. While it is not clear why the two basal diets, differing by formula, only in the protein supplements, should yield a quantitatively different result, the two sets of experiments lead to the same conclusions. Statistical analysis of the d a t a of experiment 5 revealed significant effects (P <0.01) for DAC and GA and the interaction between them. Plasma GA levels and uric acid excretion followed patterns similar to those observed in experiments 1, 2 and 3. Utilizing the d a t a of experiment 5 it was found t h a t the effects of dietary DAC and GA were statistically significant ( P < 0 . 0 1 ) and their interaction non-significant. I n these experiments, and in the 3 preceding, possible influence of the dietary supplements on blood plasma volume was not studied. However, it seems unlikely t h a t blood plasma volume changes could explain the observed results. Very large alterations in plasma volume would be needed to account for the observed effects on glutamine and GA concentrations. Furthermore, the differential effects of the supplements on plasma glutamine and GA argue against the possibility t h a t plasma volume change could be a major factor influencing the results.
of the addition of diammonium citrate (DAC) and glutamic acid (GA) to basal 2l Av. gain, 2-3 wks., gm.
Plasma glutamine, mg./lOO ml.
Plasma glutamic acid, mg./lOO ml.
Excreted uric acid, gm./lOO gm. feed consumed
Experiment 4 None 8% DAC 4 % GA 8% D A C + 4 % GA
89 68 92 68
(14) ( 0) ( 2) (18)
16.8(1.7) 26.5 (0.4) 18.0 (0.7) 29.0 (1.7)
3.3 2.7 3.5 3.5
(0) (0.1) (0.7) (0.8)
1.29 2.85 1.93 3.17
(0) (0.47) (0.07) (0.14)
Experiment 5 None 8% DAC 12% DAC 8% GA 8% D A C + 8 % GA 12% D A C + 8 % GA
120 105 91 141 118 86
(12) (18) ( 1) ( 2) ( 3) ( 6)
14.4(0.3) 19.4(1.4) 21.2 (2.0) 14.3 (0 ) 30.2 (4.1) 36.8 (21.1)
3.1 (0.4) 2.8(0.3) 2.4(0.5) 5.5 (0.1) 5.8 (0.3) 4.0 (0.6)
1.30 2.58 3.23 2.72 4.27 4.34
(0.25) (0.37) (0.6) (0.11) (0.04) (0.24)
1 Each value is the average of 2 lots of birds each containing 14 birds in experiment 4 and 12 birds in experiment 5. Figure in parentheses following each value is the difference between the duplicate lots.
DIETARY DIAMMONIUM CITRATE AND BLOOD PLASMA GLUTAMINE
Taken together the 5 experiments provide indirect evidence that the chick, like other species, utilizes glutamine as a carrier of ammonia in the blood, synthesizing the glutamine from ammonia and GA.The rise in plasma glutamine, observed following ammonia ingestion, appears to be variable and under certain conditions, not elucidated here, may be rather small (experiment 2). Probably the rate at which glutamine is utilized for uric acid synthesis or is broken down to give urinary ammonia are major factors affecting its level in the plasma. SUMMARY
Chicks were fed semipurined diets containing either 18% of isolated soybean protein or 16.6% of casein. The glutamine content of the deproteinized blood plasma ranged from 14.4 to 16.8 mg. per 100 ml. and the glutamic acid content from 2.7 to 4.0 mg. per 100 ml. The incorporation of up to 12% of diammonium citrate (DAC) into these diets, replacing equivalent quantities of dextrose, resulted in marked increases in the concentration of glutamine in the plasma, and these increases were greater when supplements of 4 to 8% of glutamic acid (GA) were included in addition to DAC. Supplemental GA when added without DAC did not affect the concentration of glutamine in the plasma but did increase the concentration of GA. Uric acid excretion increased with increasing levels of DAC and/or GA in the diet and the effects of the two supplements when fed in combination were essentially additive. Results support the thesis that the formation of glutamine is a major mechanism for the removal of excess ammonia from the blood of chicks. ACKNOWLEDGMENTS
This work was supported in part by a
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grant in aid of research by the National Research Council of Canada. The authors are indebted to Merck and Co. Ltd. and Pfizer Canada both of Montreal, Quebec, and Distillation Products Industries, Rochester, N.Y. for several of the vitamins used in the experimental diets. REFERENCES Bessman, S. P., J. Magnes, P. Schwerin and H. Waelsch, 1948. The absorption of glutamic acid and glutamine. J. Biol. Chem. 175:817-823. Briggs, G. M., Jr., T. D. Luckey, C. A. Elvehjem and E. B. Hart, 1943. Studies on two chemically unidentified water-soluble vitamins necessary for the chick. J. Biol. Chem. 148: 163-172. Buchanan, J. M., and S. C. Hartman, 1959. Enzymatic reactions in the synthesis of the purines. Adv. Enzymology, 21: 199-261. Czarnocki, J., I. R. Sibbald and E. V. Evans, 1961. The determination of chromic oxide in samples of feed and excreta by acid digestion and spectrophotometry. Can. J. An. Sci. 41:167-179. Gray, J. A., E. M. Olsen, D. C. Hill and H. D. Branion, 1960. Effect of a dietary lysine deficiency on the concentration of amino acids in the deproteinized blood plasma of chicks. Can. J. Biochem. Physiol. 38: 435-441. Hamilton, P. B., 1945. Glutamine: A major constituent of free a amino acids in animal tissues and blood plasma. J. Biol. Chem. 158:397-409. Meister, A., 1956. Metabolism of glutamine. Physiol. Rev. 36: 103-127. Olsen, E. M., D. C. Hill and H. D. Branion, 1962. Determination of glutamine and glutamic acid in the blood plasma of chicks by a combined chromatographic and microbiological method. Can. J. Biochem. Physiol. 40: 381-390. Prescott, B. A., and H. Waelsch, 1947. Free and combined glutamic acid in human blood plasma and serum. J. Biol. Chem. 167: 855-860. Shirley, R. L., and R. H. Van Landingham, 1939. Determination of uric acid in the mixed excreta of birds. Ind. Eng. Chem. (Anal. Ed.) 11:381-383. Snedecor, G. W. 1956. Statistical Methods Applied to Experiments in Agriculture and Biology, 5th ed., Iowa State College Press, Ames, Iowa. Tigerman, H., and R. MacVicar, 1951. Glutamine, glutamic acid, ammonia administration and tissue glutamine. J. Biol. Chem. 189: 793-799. Van Slyke, D. D., R. A. Phillips, P. B. Hamilton, R. M. Archibald, P. H. Futcher and A. Hiller, 1943. Glutamine as a source material of urinary ammonia. J. Biol. Chem. 150: 481-482.