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Nucleic and Free Amino Acid Response in Avian Tissues During Tuberculosis Infection
NITRATE AND NITRITE TOXICITY
O'Dell, B. L., Z. Erek, L. Flynn, G. B. Garner and M. E. Muhrer, 1960. Effects of nitrite containing rations in producing vitamin A and vitamin E deficiencies in rats. J. Animal Sci. 19: 1280. Riggs, C. W., 1945. Nitrite poisoning from ingestion of plants high in nitrate. Am. J. Vet. Res. 6: 194-197. Roberts, W. K., and J. L. Sell, 1963. Vitamin A destruction by nitrate in vitro and in vivo J. Animal Sci. 22: 1081-1085. Sell, J. L., and W. K. Roberts, 1963. Effects of dietary nitrate on the chicks growth, liver vitamin A stores and thyroid weight. J. Nutr. 79: 171-178. Smith, G. S., E. E. Hatfield, W. M. Durdle and A. L. Neumann, 1962. Vitamin A status of cattle and sheep as affected by nitrate added to rations of hay or silage and by supplementation with carotene on performed vitamin A. J. Animal Sci. 21: 1013-1014. Vogel, A. I., 1948. Textbook of Quantitative Inorganic Analysis. 1st Ed. Longmans, Green and Co., London. Wallace, J. D., R. J. Raleigh and P. H. Weswig, 1964. Performance and carotene conversion in Hereford heifers fed different levels of nitrate. J. Animal Sci. 24: 1042-1045. Weichenthal, B. A., L. B. Embry, R. J. Emerick and F. W. Whetzal, 1963. Influence of sodium nitrate, vitamin A and protein level on feed lot performance and vitamin A status of fattening cattle. J. Animal Sci. 22: 979-984.
Nucleic and Free Amino Acid Response in Avian Tissues During Tuberculosis Infection R. L. SQUIBB, H. SIEGEL AND M. SOLOTOROVSKY Bureau of Biological Research, Rutgers—The State University, New Brunswick, N. J. 08903 AND M. M.
LYONS
Department of Pathology, New York University School of Medicine, New York, N.Y. 10016 (Received for publication July 21, 1967)
INTRODUCTION
S
OLOTOROVSKY et al. (1949) pointed out that M. tuberculosis, avian strain, was an excellent disease model for the study of biochemical and nutritional interactions. Later studies by these workers
on the effect of dietary vitamin A and fat on avian tuberculosis demonstrated that chicks survived longer when fed diets with lower fat content or adequate vitamin A (Solotorovsky et al., 1961) and protein (Squibb et al., 1965). Deficient or surfeit
Downloaded from http://ps.oxfordjournals.org/ at Yale University on May 15, 2015
Garner, G. B., B. L. O'Dell, P. Radar and M. E. Muhrer, 1958. Further studies on the effects of nitrate upon reproduction and vitamin A storage with rats and swine. J. Animal Sci. 17: 1213. Goodrich, R. D., R. J. Emerick and L. B. Embry, 1964. Effect of sodium nitrate on the vitamin A nutrition of sheep. J. Animal Sci. 23: 100-104. Guberlet, J. E., 1922. Potassium nitrate poisoning in chickens with a note on its toxicity. J.A.V.M.A. 62: 362-365. Jones, D. I. H., and G. ap Griffith, 1965. Reduction of nitrate to nitrite in moist feeds. J. Sci. Fd. Agric. 16: 721-725. Kienholz, E. W., H. L. Enos and T. A. McPherron, 1965. The effects of some water sources and treatments upon turkey performance. Poultry Sci. 44: 1390. Krista, L. M., C. W. Carlson and 0 . E. Olson, 1961. Some effects of saline waters on chicks, laying hens, poults, and ducklings. Poultry Sci. 40: 938-944. Kurup, C. K., and C. S. Vaidyanathan, 1964. Mechanism of nitrate reduction by Agrobacterium tumefaciens. Indian J. Biochemistry, 1: 111-116. Mayo, N. S., 1895. Cattle poisoning by nitrate of potash. Kansas Agr. Expt. Sta. Bull. 49. Njaa, L. R., F. Utne, O. R. Braekkan, J. Minsaas, B. Laksesvela and G. Sand, 1955. An experiment with sodium nitrite and herring meal from herring preserved with sodium nitrite for chickens. Fiskeridirektoret. Skrifter Ser. Teknol. Undersf(k3:65-86.
519
520
R. L. SQUIBB, H. SIEGEL, M. SOLOTOROVSKY AND M. M. LYONS
METHODS AND RESULTS
Day-old White Leghorn cockerel chicks were maintained in isolated air-conditioned rooms with constant lighting and fed a standard diet ad libitum (Squibb, 1961) for 10 days. The growth rate of the chicks at this time compared favorably with a reference curve, indicating all conditions were normal. The chicks were then divided into two groups—one the noninfected controls, and the other to be inoculated with TB. Each chick was injected via heart puncture with an 0.5 ml. suspension of M. tuberculosis, avian strain Kirchberg. This was prepared by growing the organism in a Tween-albumin culture for a 10-day period (Solotorovsky et al., 1961). The same standard diet was continued ad libitum to both groups for the
duration of the experiment. At 0, 1, 2, 3 and 4 weeks post inoculation 10 birds from each treatment group were weighed and sacrificed between 900 and 1,000 hours. Samples of serum, pectoral muscle and liver were obtained and stored at — 20°C. and later analyzed individually for total protein, DNA, RNA and 7 free amino acids by methods previously described (Wannemacher et al., 1965; Squibb, 1963a). Liver sections were obtained for histopathologic examination. All values reported herein are in terms of mg./gm. fresh tissue for muscle and liver and mg./lOO ml. for serum. Statistical analyses were carried out according to Snedecor (1957). Table 1 shows that at 4 weeks post inoculation body weights were severely depressed and liver size increased by the infection. Numbers of tubercles in the liver in relation to liver and body weight (Table 2) were used to calculate an index of TB involvement (Squibb et al., 1965). This index showed the infection increased in severity with each week post inoculation. Further, it was apparent that a significant reaction had occurred in the liver during the incubation or early phase of the disease, defined as the interval prior to the first significant depression of body weight. Tubercles were positively identified 2 weeks post inoculation and had displaced T A B L E 1.—Body and liver weights of control and TB infected chicks Weeks post inoculation1 1 Body weight (gm.) Controls TB Percent change
2
3
4 603
210
322
450
206
328
406
462
0
0
—10
-23
Liver weight (gm.) Controls 6.2 9.5 10.6 14.2 TB 6.4 10.8 18.8 35.7 Percent change 0 +14 +80 +150 1 Each period, average of 10 chicks per treatment group.
Downloaded from http://ps.oxfordjournals.org/ at Yale University on May 15, 2015
quantities of these dietary essentials not only increased mortality but also the degree of involvement as determined from an index based on histopathologic studies (Squibb et al., 1965; Siegel et al., 1968). Squibb et al. (1965) later evaluated protein metabolism in the livers of chicks infected with the same avian TB strain by observing tissue changes in protein, DNA, RNA and 7 free amino acids. At 4 weeks post inoculation the infection caused an acceleration of protein metabolism which correlated with an increase in liver size and in the number and size of tubercles and displacement of normal tissue. A significant depression of diet intake with a concurrent wasting of pectoral musculature suggested that the source of the materia prima for the increased anabolism in the liver was the body's protein reserves. The experiment reported here was undertaken to study 1) tissue reactions during the very early developmental phases of avian tuberculosis; and 2) simultaneous relationships between liver, blood and muscle.
521
TUBERCULOSIS INFECTION
Concurrently in the serum (Fig. 1) there was a significant ( P < 0 . 0 1 ) increase in total protein. Correlated with the rise in serum protein were increases of free valine and the leucines and a pronounced depression of lysine. As a result, the ratios of the individual free amino acids to one another in the serum (Table 3) were significantly altered; t h a t is, lysine was depressed linearly ( P < 0 . 0 1 ) b y the advancing infection, while valine and the leucines were increased ( P < 0 . 0 1 ) . Ratios of histidine, arginine, aspartic acid and alanine, on the other hand, were similar to the controls. I n the liver only the ratio of lysine was affected, being significantly ( P < 0 . 0 1 ) lower at 4 weeks post inoculation. DISCUSSION With avian tuberculosis the liver is the principal site of involvement; hence the increase in size of the infected livers is of interest. I n the normal chick, liver weight as percent of body weight remains fairly constant for this specific age span (Squibb, 1963b) b u t in the tuberculous
TABLE 2.—Histological changes in livers of TB infected chicks Weeks post inoculation1 1 2 2
Number of tubercles Tissue displaced—% Index3
3
4
— 9 26 52 — — 10-25 25-50 — 0.30 1.30 4.15
1
Each period, average of 10 chicks. Number of tubercles per low power field. No. tuberclesX liver weight (gm.) 3 _ , Body weight (gm.) 2
chick the liver as percent of body weight becomes significantly greater (Squibb et al., 1965). Estimates of the total a m o u n t of normal liver tissue, excluding the amount accounted for by the granulomatous tuberculous tissue, i.e., the remaining liver parenchyma, show a similar percentage ratio to body weight as in noninfected chicks, suggesting t h a t the rapid increase in liver weight is for the maintenance of the integrity of this vital organ. T h e pathways involved have not been defined, b u t similar liver enlargement, appetite loss and wasting of musculature have been observed in chicks injected with hydrocortisone, suggesting mediation through the adrenals (Squibb et al., 1965). I t is evident from the d a t a t h a t several biochemical changes of magnitude occurred in liver, serum and muscle of the infected birds during the incubation stage of the T B , defined as t h a t period prior to the first significant depression of body weight. While the changes in nucleic acid and protein levels in the liver and in the D N A and protein of muscle had no definite trend during the developing tuberculous state, muscle R N A was significantly depressed which would be expected in a tissue undergoing catabolism. T h e serum hyperproteinemia observed in these studies has been shown to consist of a decrease in albumin and increases in alpha-3, beta and g a m m a globulins. T h e
Downloaded from http://ps.oxfordjournals.org/ at Yale University on May 15, 2015
as much as 5 0 % of normal liver tissue b y the end of the 4th week or just prior to the first mortality (Table 2). Liver D N A , R N A and protein values were within control ranges (Fig. 1) even though the tuberculous tissue was displacing the normal a t a rapid rate. D N A and protein content in the muscle also were unaffected, b u t R N A synthesis showed a significant ( P < 0 . 0 1 ) decline. T h e average increase of all the liver free amino acids was significant ( P < 0 . 0 3 ) at the end of the 2nd week when the tubercles were first discernible. T h e tubercles at this early stage were essentially epithelioid with practically no necrosis. Liver and muscle free amino acids became depressed ( P < 0.01) at the same time the histopathologic examination showed tubercle number and size significantly increased ( P < 0 . 0 1 ) .
522
R. L. SQUIBB, H. SIEGEL, M. SOLOTOROVSKY AND M. M. LYONS
MUSCLE
Downloaded from http://ps.oxfordjournals.org/ at Yale University on May 15, 2015
4
WEEKS
POST
0
I
2
INOCULATION
FIG. 1. Effect of M. tuberculosis on total protein, nucleic and free amino acids in avian liver, muscle and serum.
523
TUBERCULOSIS INFECTION TABLE 3.—Ratios of free amino acids in the serum and liver of control and TB infected chicks 2 Weeks post inoculation
Amino acids1
2
1
4
3
TB
Control
TB
Control
TB
Control
TB
Serum Lysine Histidine Arginine Aspartic acid Alanine Valine Leucines3
22 8 15 11 26 9 9
21 9 16 12 23 9 10
19 12 18 9 19 11 12
17 11 18 10 23 9 12
20 8 18 11 22 10 11
17 6 21 11 18 13 14
20 9 17 10 25 9 10
9 7 16 11 22 18 17
Liver Lysine Histidine Arginine Aspartic acid Alanine Valine Leucines
12 8 8 19 27 12 14
10 7 8 18 30 12 15
11 7 9 21 26 11 15
10 7 9 22 24 11 17
10 7 9 24 27 10 13
7 7 10 26 24 12 14
11 8 9 21 27 10 14
5 9 7 24 29 12 14
1 2 3
In percent of the total of the 7. Each period, average of 10 chicks per treatment group. Leucine and isoleucine combined.
decrease in albumin is believed to be related to liver damage while the increase in gamma globulin is considered to be associated with antibody production (Wogan et al., 1961) The significant changes in the free amino acid pools must be interpreted in terms of the diseased state and the function of the specific tissue As the infection progresses in the bird there is a concomitant lower food intake which apparently triggers a catabolism of body protein reserves (Squibb et al., 1965). The decrease in the free amino acid pool in muscle therefore can be attributed to the diversion of these essentials to the fueling of the enlarging tuberculous liver. The free amino acids of the liver increased during the early formation of the tubercles and then as the tubercles became larger and more numerous they significantly decreased, with lysine being especially affected. Some of the free amino acids were used in the synthesis of the serum proteins while others served to continue
the normal functions of the body and the needs of the infected liver. Serum or transport levels of these amino acids may be indicative of need. For example, valine and the leucines increased, indicating less need or less utilization. At the other extreme, lysine became significantly lower, which correlated with lower levels in muscle and liver. Increases or decreases in levels of free amino acids of body tissues occur under normal conditions. For example, the needs of rapid growth can deplete free amino acid pools (Squibb, 1964) but the ratios of the free amino acids to one another remain remarkably constant (Squibb, 1966). Since availability of materia prima is essential for the synthesis of any protein, the functioning state of a tissue should be reflected in the ratios of free amino acids. Therefore, during TB infection one can postulate that synthesis of protein for the tuberculous tissue requires not only a different combination of free amino acids than does the normal protein
Downloaded from http://ps.oxfordjournals.org/ at Yale University on May 15, 2015
Control
524
R. L. SQUIBB, H. SIEGEL, M. SOLOTOROVSKY AND M. M. LYONS
but also a different level of metabolism. Together, these phenomena would cause an alteration in the utilization of the normal free amino acid pool, resulting in a greater requirement for synthesis or catabolism of specific amino acids. Of the free amino acids observed in this study, availability of lysine appeared to be the most critical. SUMMARY
ACKNOWLEDGEMENTS The work was supported in part by the U. S. Army Medical Research and Development Command, Contract DA-49-193MD-2694. REFERENCES Siegel, H., R. L. Squibb, M. Solotorovsky and W. H. Ott, 1968. A quantitative pathologic study
SEPTEMBER 8-12. INTERNATIONAL POULTRY INDUSTRY MANUFACTURERS' COUNCIL, INTERNATIONAL AMPHITHEATRE, CHICAGO, ILLINOIS.
Downloaded from http://ps.oxfordjournals.org/ at Yale University on May 15, 2015
Protein, nucleic and free amino acids were determined in the liver, serum and pectoral muscle of chicks infected with avian tuberculosis. Weekly observations showed significant biochemical changes in the tissues prior to the first significant loss in body weight which occurred 28 days post inoculation. Of particular importance was the depression of RNA in muscle which correlated with a high rate of catabolism. Serum valine and the leucines increased while alanine, histidine and lysine decreased. During the latter stage of the incubation period all the free amino acids were depressed in the liver. Changes in ratios between the free amino acids of the liver indicated that lysine was utilized to the greatest extent, either for synthesis of the tuberculous tissue or an increased catabolism related to the diseased state.
of avian tuberculosis in the chick: Effect of protein and lysine dietary levels. Am. J. Path. 52: 349-367. Solotorovsky, M., H. Siegel and W. H. Ott, 1949. The use of avian tuberculosis in the chick for experimental studies. Ann. New York Acad. Sci. 52: 696-702. Solotorovsky, M., R. L. Squibb, G. N. Wogan, H. Siegel and R. Gala, 1961. The effect of dietary fat and vitamin A on avian tuberculosis in chicks. Amer. Rev. Resp. Dis. 84: 226-235. Snedecor, G. W., 1957. Statistical Methods, Iowa State University Press, Ames. Squibb, R. L., and H. Veros, 1961. Avian disease virus and nutrition relationships. I. Effect of vitamin A on growth, symptoms, mortality and vitamin A reserves of White Leghorn chicks infected with Newcastle disease virus. Poultry Sci. 40: 425-433. Squibb, R. L., 1963a. Thin-layer chromatographic separation and quantitative determination of several free amino acids of avian liver. Nature, 199: 1216. Squibb, R. L., 1963b. Nutrition and biochemistry of survival during Newcastle disease virus infection. I. Liver nucleic acid, protein and lipid patterns in chicks. J. Nutr. 81:48-54. Squibb, R. L., 1964. Nutrition and biochemistry of survival during Newcastle disease virus infection. II. Relation of clinical symptoms and starvation to nucleic and free amino acids of avian liver. J. Nutr. 82: 422-426. Squibb, R. L., 1966. Nature of the free amino-acid pool in avian tissues. Nature, 209: 710-711. Squibb, R. L., H. Siegel and M. Solotorovsky, 1965. Protein metabolism in livers of chicks fed deficient-to-excess quantities of protein and lysine and infected with tuberculosis. J. Nutr. 86: 133-142. Wannemacher, R. W., Jr., W. L. Banks, Jr. and W. H. Wunner, 1965. Use of a single tissue extract to determine cellular protein and nucleic acid concentrations and rate of amino acid incorporation. Anal. Biochem. 11: 320-326. Wogan, G. N., M. Solotorovsky, R. L. Squibb and H. Siegel, 1961. The serum protein and lipoprotein response to tuberculosis in chicks fed various levels of dietary fat. Amer. Rev. Resp. Dis. 84: 236-241.
O'Dell, B. L., Z. Erek, L. Flynn, G. B. Garner and M. E. Muhrer, 1960. Effects of nitrite containing rations in producing vitamin A and vitamin E deficiencies in rats. J. Animal Sci. 19: 1280. Riggs, C. W., 1945. Nitrite poisoning from ingestion of plants high in nitrate. Am. J. Vet. Res. 6: 194-197. Roberts, W. K., and J. L. Sell, 1963. Vitamin A destruction by nitrate in vitro and in vivo J. Animal Sci. 22: 1081-1085. Sell, J. L., and W. K. Roberts, 1963. Effects of dietary nitrate on the chicks growth, liver vitamin A stores and thyroid weight. J. Nutr. 79: 171-178. Smith, G. S., E. E. Hatfield, W. M. Durdle and A. L. Neumann, 1962. Vitamin A status of cattle and sheep as affected by nitrate added to rations of hay or silage and by supplementation with carotene on performed vitamin A. J. Animal Sci. 21: 1013-1014. Vogel, A. I., 1948. Textbook of Quantitative Inorganic Analysis. 1st Ed. Longmans, Green and Co., London. Wallace, J. D., R. J. Raleigh and P. H. Weswig, 1964. Performance and carotene conversion in Hereford heifers fed different levels of nitrate. J. Animal Sci. 24: 1042-1045. Weichenthal, B. A., L. B. Embry, R. J. Emerick and F. W. Whetzal, 1963. Influence of sodium nitrate, vitamin A and protein level on feed lot performance and vitamin A status of fattening cattle. J. Animal Sci. 22: 979-984.
Nucleic and Free Amino Acid Response in Avian Tissues During Tuberculosis Infection R. L. SQUIBB, H. SIEGEL AND M. SOLOTOROVSKY Bureau of Biological Research, Rutgers—The State University, New Brunswick, N. J. 08903 AND M. M.
LYONS
Department of Pathology, New York University School of Medicine, New York, N.Y. 10016 (Received for publication July 21, 1967)
INTRODUCTION
S
OLOTOROVSKY et al. (1949) pointed out that M. tuberculosis, avian strain, was an excellent disease model for the study of biochemical and nutritional interactions. Later studies by these workers
on the effect of dietary vitamin A and fat on avian tuberculosis demonstrated that chicks survived longer when fed diets with lower fat content or adequate vitamin A (Solotorovsky et al., 1961) and protein (Squibb et al., 1965). Deficient or surfeit
Downloaded from http://ps.oxfordjournals.org/ at Yale University on May 15, 2015
Garner, G. B., B. L. O'Dell, P. Radar and M. E. Muhrer, 1958. Further studies on the effects of nitrate upon reproduction and vitamin A storage with rats and swine. J. Animal Sci. 17: 1213. Goodrich, R. D., R. J. Emerick and L. B. Embry, 1964. Effect of sodium nitrate on the vitamin A nutrition of sheep. J. Animal Sci. 23: 100-104. Guberlet, J. E., 1922. Potassium nitrate poisoning in chickens with a note on its toxicity. J.A.V.M.A. 62: 362-365. Jones, D. I. H., and G. ap Griffith, 1965. Reduction of nitrate to nitrite in moist feeds. J. Sci. Fd. Agric. 16: 721-725. Kienholz, E. W., H. L. Enos and T. A. McPherron, 1965. The effects of some water sources and treatments upon turkey performance. Poultry Sci. 44: 1390. Krista, L. M., C. W. Carlson and 0 . E. Olson, 1961. Some effects of saline waters on chicks, laying hens, poults, and ducklings. Poultry Sci. 40: 938-944. Kurup, C. K., and C. S. Vaidyanathan, 1964. Mechanism of nitrate reduction by Agrobacterium tumefaciens. Indian J. Biochemistry, 1: 111-116. Mayo, N. S., 1895. Cattle poisoning by nitrate of potash. Kansas Agr. Expt. Sta. Bull. 49. Njaa, L. R., F. Utne, O. R. Braekkan, J. Minsaas, B. Laksesvela and G. Sand, 1955. An experiment with sodium nitrite and herring meal from herring preserved with sodium nitrite for chickens. Fiskeridirektoret. Skrifter Ser. Teknol. Undersf(k3:65-86.
519
520
R. L. SQUIBB, H. SIEGEL, M. SOLOTOROVSKY AND M. M. LYONS
METHODS AND RESULTS
Day-old White Leghorn cockerel chicks were maintained in isolated air-conditioned rooms with constant lighting and fed a standard diet ad libitum (Squibb, 1961) for 10 days. The growth rate of the chicks at this time compared favorably with a reference curve, indicating all conditions were normal. The chicks were then divided into two groups—one the noninfected controls, and the other to be inoculated with TB. Each chick was injected via heart puncture with an 0.5 ml. suspension of M. tuberculosis, avian strain Kirchberg. This was prepared by growing the organism in a Tween-albumin culture for a 10-day period (Solotorovsky et al., 1961). The same standard diet was continued ad libitum to both groups for the
duration of the experiment. At 0, 1, 2, 3 and 4 weeks post inoculation 10 birds from each treatment group were weighed and sacrificed between 900 and 1,000 hours. Samples of serum, pectoral muscle and liver were obtained and stored at — 20°C. and later analyzed individually for total protein, DNA, RNA and 7 free amino acids by methods previously described (Wannemacher et al., 1965; Squibb, 1963a). Liver sections were obtained for histopathologic examination. All values reported herein are in terms of mg./gm. fresh tissue for muscle and liver and mg./lOO ml. for serum. Statistical analyses were carried out according to Snedecor (1957). Table 1 shows that at 4 weeks post inoculation body weights were severely depressed and liver size increased by the infection. Numbers of tubercles in the liver in relation to liver and body weight (Table 2) were used to calculate an index of TB involvement (Squibb et al., 1965). This index showed the infection increased in severity with each week post inoculation. Further, it was apparent that a significant reaction had occurred in the liver during the incubation or early phase of the disease, defined as the interval prior to the first significant depression of body weight. Tubercles were positively identified 2 weeks post inoculation and had displaced T A B L E 1.—Body and liver weights of control and TB infected chicks Weeks post inoculation1 1 Body weight (gm.) Controls TB Percent change
2
3
4 603
210
322
450
206
328
406
462
0
0
—10
-23
Liver weight (gm.) Controls 6.2 9.5 10.6 14.2 TB 6.4 10.8 18.8 35.7 Percent change 0 +14 +80 +150 1 Each period, average of 10 chicks per treatment group.
Downloaded from http://ps.oxfordjournals.org/ at Yale University on May 15, 2015
quantities of these dietary essentials not only increased mortality but also the degree of involvement as determined from an index based on histopathologic studies (Squibb et al., 1965; Siegel et al., 1968). Squibb et al. (1965) later evaluated protein metabolism in the livers of chicks infected with the same avian TB strain by observing tissue changes in protein, DNA, RNA and 7 free amino acids. At 4 weeks post inoculation the infection caused an acceleration of protein metabolism which correlated with an increase in liver size and in the number and size of tubercles and displacement of normal tissue. A significant depression of diet intake with a concurrent wasting of pectoral musculature suggested that the source of the materia prima for the increased anabolism in the liver was the body's protein reserves. The experiment reported here was undertaken to study 1) tissue reactions during the very early developmental phases of avian tuberculosis; and 2) simultaneous relationships between liver, blood and muscle.
521
TUBERCULOSIS INFECTION
Concurrently in the serum (Fig. 1) there was a significant ( P < 0 . 0 1 ) increase in total protein. Correlated with the rise in serum protein were increases of free valine and the leucines and a pronounced depression of lysine. As a result, the ratios of the individual free amino acids to one another in the serum (Table 3) were significantly altered; t h a t is, lysine was depressed linearly ( P < 0 . 0 1 ) b y the advancing infection, while valine and the leucines were increased ( P < 0 . 0 1 ) . Ratios of histidine, arginine, aspartic acid and alanine, on the other hand, were similar to the controls. I n the liver only the ratio of lysine was affected, being significantly ( P < 0 . 0 1 ) lower at 4 weeks post inoculation. DISCUSSION With avian tuberculosis the liver is the principal site of involvement; hence the increase in size of the infected livers is of interest. I n the normal chick, liver weight as percent of body weight remains fairly constant for this specific age span (Squibb, 1963b) b u t in the tuberculous
TABLE 2.—Histological changes in livers of TB infected chicks Weeks post inoculation1 1 2 2
Number of tubercles Tissue displaced—% Index3
3
4
— 9 26 52 — — 10-25 25-50 — 0.30 1.30 4.15
1
Each period, average of 10 chicks. Number of tubercles per low power field. No. tuberclesX liver weight (gm.) 3 _ , Body weight (gm.) 2
chick the liver as percent of body weight becomes significantly greater (Squibb et al., 1965). Estimates of the total a m o u n t of normal liver tissue, excluding the amount accounted for by the granulomatous tuberculous tissue, i.e., the remaining liver parenchyma, show a similar percentage ratio to body weight as in noninfected chicks, suggesting t h a t the rapid increase in liver weight is for the maintenance of the integrity of this vital organ. T h e pathways involved have not been defined, b u t similar liver enlargement, appetite loss and wasting of musculature have been observed in chicks injected with hydrocortisone, suggesting mediation through the adrenals (Squibb et al., 1965). I t is evident from the d a t a t h a t several biochemical changes of magnitude occurred in liver, serum and muscle of the infected birds during the incubation stage of the T B , defined as t h a t period prior to the first significant depression of body weight. While the changes in nucleic acid and protein levels in the liver and in the D N A and protein of muscle had no definite trend during the developing tuberculous state, muscle R N A was significantly depressed which would be expected in a tissue undergoing catabolism. T h e serum hyperproteinemia observed in these studies has been shown to consist of a decrease in albumin and increases in alpha-3, beta and g a m m a globulins. T h e
Downloaded from http://ps.oxfordjournals.org/ at Yale University on May 15, 2015
as much as 5 0 % of normal liver tissue b y the end of the 4th week or just prior to the first mortality (Table 2). Liver D N A , R N A and protein values were within control ranges (Fig. 1) even though the tuberculous tissue was displacing the normal a t a rapid rate. D N A and protein content in the muscle also were unaffected, b u t R N A synthesis showed a significant ( P < 0 . 0 1 ) decline. T h e average increase of all the liver free amino acids was significant ( P < 0 . 0 3 ) at the end of the 2nd week when the tubercles were first discernible. T h e tubercles at this early stage were essentially epithelioid with practically no necrosis. Liver and muscle free amino acids became depressed ( P < 0.01) at the same time the histopathologic examination showed tubercle number and size significantly increased ( P < 0 . 0 1 ) .
522
R. L. SQUIBB, H. SIEGEL, M. SOLOTOROVSKY AND M. M. LYONS
MUSCLE
Downloaded from http://ps.oxfordjournals.org/ at Yale University on May 15, 2015
4
WEEKS
POST
0
I
2
INOCULATION
FIG. 1. Effect of M. tuberculosis on total protein, nucleic and free amino acids in avian liver, muscle and serum.
523
TUBERCULOSIS INFECTION TABLE 3.—Ratios of free amino acids in the serum and liver of control and TB infected chicks 2 Weeks post inoculation
Amino acids1
2
1
4
3
TB
Control
TB
Control
TB
Control
TB
Serum Lysine Histidine Arginine Aspartic acid Alanine Valine Leucines3
22 8 15 11 26 9 9
21 9 16 12 23 9 10
19 12 18 9 19 11 12
17 11 18 10 23 9 12
20 8 18 11 22 10 11
17 6 21 11 18 13 14
20 9 17 10 25 9 10
9 7 16 11 22 18 17
Liver Lysine Histidine Arginine Aspartic acid Alanine Valine Leucines
12 8 8 19 27 12 14
10 7 8 18 30 12 15
11 7 9 21 26 11 15
10 7 9 22 24 11 17
10 7 9 24 27 10 13
7 7 10 26 24 12 14
11 8 9 21 27 10 14
5 9 7 24 29 12 14
1 2 3
In percent of the total of the 7. Each period, average of 10 chicks per treatment group. Leucine and isoleucine combined.
decrease in albumin is believed to be related to liver damage while the increase in gamma globulin is considered to be associated with antibody production (Wogan et al., 1961) The significant changes in the free amino acid pools must be interpreted in terms of the diseased state and the function of the specific tissue As the infection progresses in the bird there is a concomitant lower food intake which apparently triggers a catabolism of body protein reserves (Squibb et al., 1965). The decrease in the free amino acid pool in muscle therefore can be attributed to the diversion of these essentials to the fueling of the enlarging tuberculous liver. The free amino acids of the liver increased during the early formation of the tubercles and then as the tubercles became larger and more numerous they significantly decreased, with lysine being especially affected. Some of the free amino acids were used in the synthesis of the serum proteins while others served to continue
the normal functions of the body and the needs of the infected liver. Serum or transport levels of these amino acids may be indicative of need. For example, valine and the leucines increased, indicating less need or less utilization. At the other extreme, lysine became significantly lower, which correlated with lower levels in muscle and liver. Increases or decreases in levels of free amino acids of body tissues occur under normal conditions. For example, the needs of rapid growth can deplete free amino acid pools (Squibb, 1964) but the ratios of the free amino acids to one another remain remarkably constant (Squibb, 1966). Since availability of materia prima is essential for the synthesis of any protein, the functioning state of a tissue should be reflected in the ratios of free amino acids. Therefore, during TB infection one can postulate that synthesis of protein for the tuberculous tissue requires not only a different combination of free amino acids than does the normal protein
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but also a different level of metabolism. Together, these phenomena would cause an alteration in the utilization of the normal free amino acid pool, resulting in a greater requirement for synthesis or catabolism of specific amino acids. Of the free amino acids observed in this study, availability of lysine appeared to be the most critical. SUMMARY
ACKNOWLEDGEMENTS The work was supported in part by the U. S. Army Medical Research and Development Command, Contract DA-49-193MD-2694. REFERENCES Siegel, H., R. L. Squibb, M. Solotorovsky and W. H. Ott, 1968. A quantitative pathologic study
SEPTEMBER 8-12. INTERNATIONAL POULTRY INDUSTRY MANUFACTURERS' COUNCIL, INTERNATIONAL AMPHITHEATRE, CHICAGO, ILLINOIS.
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Protein, nucleic and free amino acids were determined in the liver, serum and pectoral muscle of chicks infected with avian tuberculosis. Weekly observations showed significant biochemical changes in the tissues prior to the first significant loss in body weight which occurred 28 days post inoculation. Of particular importance was the depression of RNA in muscle which correlated with a high rate of catabolism. Serum valine and the leucines increased while alanine, histidine and lysine decreased. During the latter stage of the incubation period all the free amino acids were depressed in the liver. Changes in ratios between the free amino acids of the liver indicated that lysine was utilized to the greatest extent, either for synthesis of the tuberculous tissue or an increased catabolism related to the diseased state.
of avian tuberculosis in the chick: Effect of protein and lysine dietary levels. Am. J. Path. 52: 349-367. Solotorovsky, M., H. Siegel and W. H. Ott, 1949. The use of avian tuberculosis in the chick for experimental studies. Ann. New York Acad. Sci. 52: 696-702. Solotorovsky, M., R. L. Squibb, G. N. Wogan, H. Siegel and R. Gala, 1961. The effect of dietary fat and vitamin A on avian tuberculosis in chicks. Amer. Rev. Resp. Dis. 84: 226-235. Snedecor, G. W., 1957. Statistical Methods, Iowa State University Press, Ames. Squibb, R. L., and H. Veros, 1961. Avian disease virus and nutrition relationships. I. Effect of vitamin A on growth, symptoms, mortality and vitamin A reserves of White Leghorn chicks infected with Newcastle disease virus. Poultry Sci. 40: 425-433. Squibb, R. L., 1963a. Thin-layer chromatographic separation and quantitative determination of several free amino acids of avian liver. Nature, 199: 1216. Squibb, R. L., 1963b. Nutrition and biochemistry of survival during Newcastle disease virus infection. I. Liver nucleic acid, protein and lipid patterns in chicks. J. Nutr. 81:48-54. Squibb, R. L., 1964. Nutrition and biochemistry of survival during Newcastle disease virus infection. II. Relation of clinical symptoms and starvation to nucleic and free amino acids of avian liver. J. Nutr. 82: 422-426. Squibb, R. L., 1966. Nature of the free amino-acid pool in avian tissues. Nature, 209: 710-711. Squibb, R. L., H. Siegel and M. Solotorovsky, 1965. Protein metabolism in livers of chicks fed deficient-to-excess quantities of protein and lysine and infected with tuberculosis. J. Nutr. 86: 133-142. Wannemacher, R. W., Jr., W. L. Banks, Jr. and W. H. Wunner, 1965. Use of a single tissue extract to determine cellular protein and nucleic acid concentrations and rate of amino acid incorporation. Anal. Biochem. 11: 320-326. Wogan, G. N., M. Solotorovsky, R. L. Squibb and H. Siegel, 1961. The serum protein and lipoprotein response to tuberculosis in chicks fed various levels of dietary fat. Amer. Rev. Resp. Dis. 84: 236-241.