- Email: [email protected]
Serum Protein and Amino Acid Levels of Escherichia coli-Infected Chicks on High and Low Levels of Dietary Protein
1S80
K. SATO AND B. GLICK
platelet histamine release. Am. J. Physiol. 205: 348-350. Tidball, M. E., and P. A. Shore, 1962. Release of rabbit platelet histamine by serum in vitro. Am. J. Physiol. 202: 265-267.
Weigle, W. O., C. G. Cochrane and F. J. Dixon, 1960. Anaphylactogenic properties of soluble antigen antibody complexes in the guinea pig and rabbit. J. Immunol. 85: 469-477.
JOHN R. COLE, JR. AND FRANK M. BOYD The Poultry Disease Research Center, University of Georgia, Athens (Received for publication June 16, 1965) INTRODUCTION
T
HE effect of different levels of dietary proteins on bacterial infections has been investigated. Boyd and Edwards (1963) reported that mortality from Escherichia coli infection was higher in protein-deficient chicks, while mortality from Newcastle disease and Salmonella gallinarum infections was greater in chicks on a high protein diet. Koerner et al. (1949) and Dubos and Schaedler (1958) found that rats and mice were more susceptible to tuberculosis infection when on a low protein diet. Twenty-one amino acids in the plasma or serum of chickens of different ages have been identified by paper chromatography using diversified color tests (Chubb, 1959). Owings and Balloun (1961), Hill et al. (1961), Hill and Olsen (1963), and Askelson and Balloun (1963) found that the plasma amino acid concentrations were dependent on the type of protein supplementation in the diet. Ross et al. (1955) and Woodward et al. (1954) reported that chicks and rats have reduced blood levels of some amino acids when inJournal Paper No. 422 of the College Experiment Station, University of Georgia College of Agriculture Experiment Stations.
fected with Salmonella pullorum and Pasteurella tularensis, respectively. Protein nutrition and its effect on serum proteins and antibody production have been studied. Leveille et al. (1961) reported that increasing the dietary protein level of chicks from 10 to 25% resulted in an increase in albumin, but the globulin level remained unchanged. Shelton and Olson (1960) found that chicks infected with synovitis virus and Mycoplasma showed an increase in total serum protein and serum globulin, and a decrease in serum albumin. Sturkie's (1954) compilation of data indicates that the albumin/globulin ratio (A /G) is usually less than 1 in chickens, and in pigeons more than 1; whereas Medway and Kare (1959), Leveille et al. (1961), Narain et al. (1961), and Wagner and Worstman (1961) obtained A/G values greater than 1 for chickens. Since it is possible that metabolic disturbances or alterations which occur in organs of functional importance are manifested in the blood of chicks, the following blood constituents were selected to be analyzed: albumin, amino acids and total globulin. Alterations of these components could be indicative of E. coli infections or the level of dietary protein on which they were maintained.
Downloaded from http://ps.oxfordjournals.org/ at West Virginia University, Health Science Libraryy on May 5, 2015
Serum Protein and Amino Acid Levels of Escherichia ro/z'-Infected Chicks on High and Low Levels of Dietary Protein
1581
SERUM PROTEIN AND E. COLI MATERIALS AND METHODS
Dietary pr e in (^ ) 15 15 25 25 a
Treatment Normal Infected Normal Infected
Number Albumin (A) Globulin (G) chicks gm./lOO ml. gm./100 ml. 10 10 10 10
2.68±.44 3 . 1 2 ± .60 3.22±.52 2 . 8 5 + .11
a
1.04+.10 1.17+.22 1.20+.11 1.17± .22
A /
^
2.58 2.67 2.68 2.43
Mean with the standard error of the mean.
A visual comparison of the color intensities and spot sizes of the amino acids was made among sera from the 4 groups of chicks. There was no difference detected in the serum amino acid concentration of normal chicks on 15 and 25% protein, but there was a general decrease in the concentration in infected chicks maintained on either diet. Albumin and globulin determinations were made on individual chick sera and the values averaged (Table 1). Although the albumin values varied and were inconsistent, infected chicks fed a 15% protein ration had a greater serum albumin concentration than the normal chick and the controls fed a 25% protein ration had a higher level than the infected chicks.
RESULTS
Ten amino acids were separable into discrete spots on paper chromatograms by the methods employed. Two ninhydrin spots, each of which contained 2 coalesced amino acids, could not be precisely differentiated. Cystine, not included in the previous figures, was only evident after it had been oxidized to cysteic acid by treating the deproteinized serum with 30% hydrogen peroxide. The most abundant amino acids in the serum samples were alanine, arginine, glycine, lysine, serine, and threonine. In lesser concentrations, as evinced by more weakly reacting spots, were aspartic acid, cysteic acid, glutamic acid, histidine, leucine plus isoleucine, methionine plus valine, and tyrosine.
DISCUSSION Ross et al. (1955) and Chubb (1959) reported 13 and 21 amino acids, respectively, from chick serum on paper chromatograms. Eleven serum amino acids or, if the 2 groups with coalesced amino acids are considered, a maximum of 15 amino acids were identified in this study by paper chromatography. Ross' group did not find cysteic acid, histidine, leucine isomer, lysine, and serine, but they did detect cysteine and cystine (which would give cysteic acid). Phenylalanine and tryptophane, which were not demonstrated in the investigation, were found by Ross et al. (1955). Ross et al. (1955) have reported that chicks infected with S. pullorum have re-
Downloaded from http://ps.oxfordjournals.org/ at West Virginia University, Health Science Libraryy on May 5, 2015
Procedures for chick maintenance and inoculation are identical to those previously described by Cole and Boyd (1965). Protein-variable rations similar to those of Boyd and Edwards (1963), were used in these studies. For serum analyses, the blood was placed in plastic petri dishes and serum collected after coagulation. Four serum samples of 0.5 ml. each were pooled from each group of test chicks. Serum was deproteinized by the addition of 95% ethanol according to the procedure of Block, Durrum, and Zweig (1958). The methods of two-dimensional descending chromatography of Block et al. (1958) and Smith (1960) were used for amino acid analysis, employing ninhydrin as the developer. The albumin and globulin concentrations of serum were determined by the biuret procedure described in the Bausch and Lomb Clinical Methods Manual (Bausch and Lomb Optical Company, Rochester, N. Y.) and read at a wavelength of 540 my..
TABLE 1.—Serum albumin and globulin levels of chicks infected with Escherichia coli and fed 15 and 25% protein diets
1582
J. R. COLE, JR. AND F. M. BOYD
ACKNOWLEDGMENT
This investigation was supported in part by Public Health Service Research Grant, No. AM 07532 from the National Institutes of Health. REFERENCES Askelson, C. E., and S. L. Balloun, 1963. Influence of age and dietary protein on certain free amino acids in chick blood plasma. Poultry Sci. 42 : 140-146. Block, R. J., E. L. Durrum and G. Zweig, 1958. A Manual of Paper Chromatography and Paper Electrophoresis, 2nd. ed., revised. Academic Press, New York. Boyd, F. M., and H. M. Edwards, 1963. The effect of dietary protein on the course of various infections in the chick. J. Infect. Diseases, 112: 53-56. Chubb, L. G., 1959. Application of paper chromatography to avian pathology. The free amino acids in the blood, urine and body tissues of the normal fowl. Poultry Sci. 38: 668-676. Cole, J. R., Jr., and F. M. Boyd, 1965. Chemical analyses of the blood of chicks infected or endointoxicated with Escherichia coli. Poultry Sci. 44: 1551-1555. Dubos, R. J., and R. W. Schaedler, 1958. Effect of dietary proteins and amino acids on the susceptibility of mice to bacterial infections. J. Exptl. Med. 108: 69-81. Hill, D. C , E. M. Mclndoo and E. M. Olsen, 1961. Influence of dietary zein on the concentration of amino acids in the plasma of chicks. J. Nutrition, 74: 16-22.
Hill, D. C , and E. M. Olsen, 1963. Effect of starvation and a nonprotein diet on blood plasma amino acids, and observations on the detection of amino acids limiting growth of chicks fed purified diets. J. Nutrition, 79: 296-302. Koerner, T. A., H. R. Getz and E. R. Long, 1949. Experimental studies on nutrition in tuberculoss. The role of protein in resistance to tuberculosis. Proc. Soc. Exptl. Biol. Med. 71: 154158. Leveille, G. A., H. Fisher and A. S. Feigenbaum, 1961. Dietary protein and its effects on the serum proteins of the chicken. Ann. New York Acad. Sci. 94: 265-271. Medway, W., and M. R. Kare, 1959. Blood and plasma volume, hematocrit, blood specific gravity and serum protein electrophoresis of the chicken. Poultry Sci. 38: 624-631. Narain, R., C. M. Lyman, C. W. Deyoe and J. R. Couch, 1961. Paper electrophoresis and albumin/globulin ratios of the serum of normal chickens and chickens fed free gossypol in the diet. Poultry Sci. 40: 21-25. Olsen, E. M., D. C. Hill, J. A. Gray and H. D. Branion, 1959. Effect of dietary protein on the concentration of free amino acids in the blood plasma of chicks. Poultry Sci. 38: 1234. Owings, W. J., and S. L. Balloun, 1961. Effect of protein sources and amino acid supplementation on intestinal microflora and plasma amino acids of the chick. Poultry Sci. 40: 1718-1730. Ross, R. T., D. F. Holtman and R. F. Gilfillan, 1955. The effect of Salmonella pullorum infection on amino acids of the chick. J. Bacteriol. 70: 212-215. Shelton, D. C , and N. O. Olson, 1960. Serum proteins of chicks with infectious and Mycoplasma synovitis. Poultry Sci. 39: 112-117. Smith, I., 1960. Chromatographic and Electrophoretic Techniques. Vol. 1. Chromatography, 2nd ed. Interscience Publishers, Inc., New York. Sturkie, P. D., 1954. Avian Physiology. Comstock Publishing Associates, Ithaca, New York. Wagner, M., and B. S. Wostman, 1961. Serum protein fractions and antibody studies in gnotobiotic animals reared germ free or monocontaminated. Ann. New York Acad. Sci. 94: 210217. Woodward, J. M., A. J. Sbarra and D. F. Holtman, 1954. The host parasite relationship in tularemia. I. A study of the influence of Bacterium tularense on the amino acid metabolism of white rats. J. Bacteriol. 67: 58-61.
Downloaded from http://ps.oxfordjournals.org/ at West Virginia University, Health Science Libraryy on May 5, 2015
duced blood levels of only arginine, glycine, methionine, and tyrosine. Woodward et al. (1954) have found that the levels of 12 amino acids in rats infected with P. tularensis were reduced. Results presented in this study are comparable to those of Woodward's group. Contrary to Olsen et al. (1959), we did not find that certain serum amino acids increased as the dietary protein level increased. Paper chromatography showed reduced serum amino acid levels in infected chicks regardless of the protein level of the diet.
K. SATO AND B. GLICK
platelet histamine release. Am. J. Physiol. 205: 348-350. Tidball, M. E., and P. A. Shore, 1962. Release of rabbit platelet histamine by serum in vitro. Am. J. Physiol. 202: 265-267.
Weigle, W. O., C. G. Cochrane and F. J. Dixon, 1960. Anaphylactogenic properties of soluble antigen antibody complexes in the guinea pig and rabbit. J. Immunol. 85: 469-477.
JOHN R. COLE, JR. AND FRANK M. BOYD The Poultry Disease Research Center, University of Georgia, Athens (Received for publication June 16, 1965) INTRODUCTION
T
HE effect of different levels of dietary proteins on bacterial infections has been investigated. Boyd and Edwards (1963) reported that mortality from Escherichia coli infection was higher in protein-deficient chicks, while mortality from Newcastle disease and Salmonella gallinarum infections was greater in chicks on a high protein diet. Koerner et al. (1949) and Dubos and Schaedler (1958) found that rats and mice were more susceptible to tuberculosis infection when on a low protein diet. Twenty-one amino acids in the plasma or serum of chickens of different ages have been identified by paper chromatography using diversified color tests (Chubb, 1959). Owings and Balloun (1961), Hill et al. (1961), Hill and Olsen (1963), and Askelson and Balloun (1963) found that the plasma amino acid concentrations were dependent on the type of protein supplementation in the diet. Ross et al. (1955) and Woodward et al. (1954) reported that chicks and rats have reduced blood levels of some amino acids when inJournal Paper No. 422 of the College Experiment Station, University of Georgia College of Agriculture Experiment Stations.
fected with Salmonella pullorum and Pasteurella tularensis, respectively. Protein nutrition and its effect on serum proteins and antibody production have been studied. Leveille et al. (1961) reported that increasing the dietary protein level of chicks from 10 to 25% resulted in an increase in albumin, but the globulin level remained unchanged. Shelton and Olson (1960) found that chicks infected with synovitis virus and Mycoplasma showed an increase in total serum protein and serum globulin, and a decrease in serum albumin. Sturkie's (1954) compilation of data indicates that the albumin/globulin ratio (A /G) is usually less than 1 in chickens, and in pigeons more than 1; whereas Medway and Kare (1959), Leveille et al. (1961), Narain et al. (1961), and Wagner and Worstman (1961) obtained A/G values greater than 1 for chickens. Since it is possible that metabolic disturbances or alterations which occur in organs of functional importance are manifested in the blood of chicks, the following blood constituents were selected to be analyzed: albumin, amino acids and total globulin. Alterations of these components could be indicative of E. coli infections or the level of dietary protein on which they were maintained.
Downloaded from http://ps.oxfordjournals.org/ at West Virginia University, Health Science Libraryy on May 5, 2015
Serum Protein and Amino Acid Levels of Escherichia ro/z'-Infected Chicks on High and Low Levels of Dietary Protein
1581
SERUM PROTEIN AND E. COLI MATERIALS AND METHODS
Dietary pr e in (^ ) 15 15 25 25 a
Treatment Normal Infected Normal Infected
Number Albumin (A) Globulin (G) chicks gm./lOO ml. gm./100 ml. 10 10 10 10
2.68±.44 3 . 1 2 ± .60 3.22±.52 2 . 8 5 + .11
a
1.04+.10 1.17+.22 1.20+.11 1.17± .22
A /
^
2.58 2.67 2.68 2.43
Mean with the standard error of the mean.
A visual comparison of the color intensities and spot sizes of the amino acids was made among sera from the 4 groups of chicks. There was no difference detected in the serum amino acid concentration of normal chicks on 15 and 25% protein, but there was a general decrease in the concentration in infected chicks maintained on either diet. Albumin and globulin determinations were made on individual chick sera and the values averaged (Table 1). Although the albumin values varied and were inconsistent, infected chicks fed a 15% protein ration had a greater serum albumin concentration than the normal chick and the controls fed a 25% protein ration had a higher level than the infected chicks.
RESULTS
Ten amino acids were separable into discrete spots on paper chromatograms by the methods employed. Two ninhydrin spots, each of which contained 2 coalesced amino acids, could not be precisely differentiated. Cystine, not included in the previous figures, was only evident after it had been oxidized to cysteic acid by treating the deproteinized serum with 30% hydrogen peroxide. The most abundant amino acids in the serum samples were alanine, arginine, glycine, lysine, serine, and threonine. In lesser concentrations, as evinced by more weakly reacting spots, were aspartic acid, cysteic acid, glutamic acid, histidine, leucine plus isoleucine, methionine plus valine, and tyrosine.
DISCUSSION Ross et al. (1955) and Chubb (1959) reported 13 and 21 amino acids, respectively, from chick serum on paper chromatograms. Eleven serum amino acids or, if the 2 groups with coalesced amino acids are considered, a maximum of 15 amino acids were identified in this study by paper chromatography. Ross' group did not find cysteic acid, histidine, leucine isomer, lysine, and serine, but they did detect cysteine and cystine (which would give cysteic acid). Phenylalanine and tryptophane, which were not demonstrated in the investigation, were found by Ross et al. (1955). Ross et al. (1955) have reported that chicks infected with S. pullorum have re-
Downloaded from http://ps.oxfordjournals.org/ at West Virginia University, Health Science Libraryy on May 5, 2015
Procedures for chick maintenance and inoculation are identical to those previously described by Cole and Boyd (1965). Protein-variable rations similar to those of Boyd and Edwards (1963), were used in these studies. For serum analyses, the blood was placed in plastic petri dishes and serum collected after coagulation. Four serum samples of 0.5 ml. each were pooled from each group of test chicks. Serum was deproteinized by the addition of 95% ethanol according to the procedure of Block, Durrum, and Zweig (1958). The methods of two-dimensional descending chromatography of Block et al. (1958) and Smith (1960) were used for amino acid analysis, employing ninhydrin as the developer. The albumin and globulin concentrations of serum were determined by the biuret procedure described in the Bausch and Lomb Clinical Methods Manual (Bausch and Lomb Optical Company, Rochester, N. Y.) and read at a wavelength of 540 my..
TABLE 1.—Serum albumin and globulin levels of chicks infected with Escherichia coli and fed 15 and 25% protein diets
1582
J. R. COLE, JR. AND F. M. BOYD
ACKNOWLEDGMENT
This investigation was supported in part by Public Health Service Research Grant, No. AM 07532 from the National Institutes of Health. REFERENCES Askelson, C. E., and S. L. Balloun, 1963. Influence of age and dietary protein on certain free amino acids in chick blood plasma. Poultry Sci. 42 : 140-146. Block, R. J., E. L. Durrum and G. Zweig, 1958. A Manual of Paper Chromatography and Paper Electrophoresis, 2nd. ed., revised. Academic Press, New York. Boyd, F. M., and H. M. Edwards, 1963. The effect of dietary protein on the course of various infections in the chick. J. Infect. Diseases, 112: 53-56. Chubb, L. G., 1959. Application of paper chromatography to avian pathology. The free amino acids in the blood, urine and body tissues of the normal fowl. Poultry Sci. 38: 668-676. Cole, J. R., Jr., and F. M. Boyd, 1965. Chemical analyses of the blood of chicks infected or endointoxicated with Escherichia coli. Poultry Sci. 44: 1551-1555. Dubos, R. J., and R. W. Schaedler, 1958. Effect of dietary proteins and amino acids on the susceptibility of mice to bacterial infections. J. Exptl. Med. 108: 69-81. Hill, D. C , E. M. Mclndoo and E. M. Olsen, 1961. Influence of dietary zein on the concentration of amino acids in the plasma of chicks. J. Nutrition, 74: 16-22.
Hill, D. C , and E. M. Olsen, 1963. Effect of starvation and a nonprotein diet on blood plasma amino acids, and observations on the detection of amino acids limiting growth of chicks fed purified diets. J. Nutrition, 79: 296-302. Koerner, T. A., H. R. Getz and E. R. Long, 1949. Experimental studies on nutrition in tuberculoss. The role of protein in resistance to tuberculosis. Proc. Soc. Exptl. Biol. Med. 71: 154158. Leveille, G. A., H. Fisher and A. S. Feigenbaum, 1961. Dietary protein and its effects on the serum proteins of the chicken. Ann. New York Acad. Sci. 94: 265-271. Medway, W., and M. R. Kare, 1959. Blood and plasma volume, hematocrit, blood specific gravity and serum protein electrophoresis of the chicken. Poultry Sci. 38: 624-631. Narain, R., C. M. Lyman, C. W. Deyoe and J. R. Couch, 1961. Paper electrophoresis and albumin/globulin ratios of the serum of normal chickens and chickens fed free gossypol in the diet. Poultry Sci. 40: 21-25. Olsen, E. M., D. C. Hill, J. A. Gray and H. D. Branion, 1959. Effect of dietary protein on the concentration of free amino acids in the blood plasma of chicks. Poultry Sci. 38: 1234. Owings, W. J., and S. L. Balloun, 1961. Effect of protein sources and amino acid supplementation on intestinal microflora and plasma amino acids of the chick. Poultry Sci. 40: 1718-1730. Ross, R. T., D. F. Holtman and R. F. Gilfillan, 1955. The effect of Salmonella pullorum infection on amino acids of the chick. J. Bacteriol. 70: 212-215. Shelton, D. C , and N. O. Olson, 1960. Serum proteins of chicks with infectious and Mycoplasma synovitis. Poultry Sci. 39: 112-117. Smith, I., 1960. Chromatographic and Electrophoretic Techniques. Vol. 1. Chromatography, 2nd ed. Interscience Publishers, Inc., New York. Sturkie, P. D., 1954. Avian Physiology. Comstock Publishing Associates, Ithaca, New York. Wagner, M., and B. S. Wostman, 1961. Serum protein fractions and antibody studies in gnotobiotic animals reared germ free or monocontaminated. Ann. New York Acad. Sci. 94: 210217. Woodward, J. M., A. J. Sbarra and D. F. Holtman, 1954. The host parasite relationship in tularemia. I. A study of the influence of Bacterium tularense on the amino acid metabolism of white rats. J. Bacteriol. 67: 58-61.
Downloaded from http://ps.oxfordjournals.org/ at West Virginia University, Health Science Libraryy on May 5, 2015
duced blood levels of only arginine, glycine, methionine, and tyrosine. Woodward et al. (1954) have found that the levels of 12 amino acids in rats infected with P. tularensis were reduced. Results presented in this study are comparable to those of Woodward's group. Contrary to Olsen et al. (1959), we did not find that certain serum amino acids increased as the dietary protein level increased. Paper chromatography showed reduced serum amino acid levels in infected chicks regardless of the protein level of the diet.