$TUDIE$ ON SERUM TRANSAMENASE 2N CALVES EVALUATED BY THE USE OF LIVER BIOPSY Thais for fin Degree a-f M. S. MiCHiGAN STATE UNIVERSITY Edward J. Hinsman EQQO LIBRARY Michigan Stan University STUDIES ON SERUM TRANSAMINASE IN CALVES EVALUATED BY THE USE OF LIVER BIOPSY by Edward J. Kinsman AN ABSTRACT Submitted to the College of Veterinary Medicine of Michigan State University of Agriculture and Applied Science in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Surgery and Medicine 1960 '/ pi . ,«i/ '1 , x" / Approved /1/ x/ (w, _,,/ EDWARD J. HINSMAN ' ABSTRACT The enzymatic liver function tests are presently being evaluated in human medicine. The value of such tests as applied to veterinary medicine is yet to be established. This paper describes the evaluation of the serum transaminase level as a test of liver destruction in the bovine. Carbon tetrachloride was administered in varying dosages to two groups of calves. This is a known way of producing liver destruction. The first group of calves was two months of age and the second group was six months of age. Liver biopsy specimens for histopathological study were taken at intervals to evaluate the amount of damage occurring in the liver. The serum glutamic pyruvic and serum glutamic oxalacetic transaminase tests were run at the same time in an attempt to correlate the amount of liver damage and transaminase levels. - There appeared to be some correlation between the transaminase levels and the damage observed on liver biopsy specimens in the two-month old calves. The serum glutamic oxalacetic transaminase was more sensitive as an indicator of liver destruction than the serum glutamic pyruvic transaminase. EDWARD J. HINSMAN ABSTRACT There appeared to be no correlation between the amount of damage seen on histopathological examination of liver biopsy Specimens in the six-month old calves and the transaminase levels. Normal values for two- and six-month old calves were established and included in this paper. STUDIES ON SERUM TRANSAMINASE IN CALVES EVALUATED BY THE USE OF LIVER BIOPSY by Edward J. Kinsman A THESIS Submitted to the College of Veterinary Medicine of_ Michigan State University of Agriculture and Applied Science in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Surgery and Medicine 1960 A C KN OW LED G MICE-I TS The writer wishes to express his sincere appre- ciation to all those whose cooperation, assistance and encouragement made this paper possible. He is indebted to the entire Department of Surgery and Medicine. He especially wishes to express gratitude to Dr. Gabel H. Conner for his guidance and patience; to Dr. George R. Moore, Director of the Large Animal Clinic, for his constructive suggestions; to Dr. Wade 0. Brinker, Head of the Department, for his encouragement; to Dr. William V. Lumb for his valuable criticism; and to Dr. Albert R. Drury for the use of his laboratory and equipment. Drs. Clifford C. Beck and David J. Ellis, Ambula- tory Clinic, supplied encouragement and much needed friendly advice. A sincere thank you to Dr. Ralph D. Earner, Depart- ment of Pathology, for his assistance in evaluating the histopathological sections. TABLE OF CONTENTS PA GE INTRODUCTION . . . . . . . . . . . . . 1 REVIEW OF LITERATURE . . . . . . . . . . 2 MATERIALS AND METHODS . . . . . . . . . . In RESULTS. . . . . . . . . . . . . . . 22 DISCUSSION. . . . . . . . . . . . . . 50 Two-month old calves . . . . . . . . . 53 Six-month old calves . . . . . . . . . 56 SUMMARY. . . . . . . . . . . . . . . 60 CONCLUSIONS . . . . . . . . . . . . . 61 BIBLIOQAPHY O O O O 0 O C O O O O O O 62 LIST OF FIGURES FIGURE PAGE I Scale drawing of biopsy needle . . . . . 1) II Biopsy site (overlay) . . . . . . . . 20 III Right side of bovine with liver exposed . . 21 LIST OF GRAPRS GRAPH PAGE I SGO-T values, calf 163 . . . . . . . 30 II SOP-T values, calf 163 . . . . . . . 31 III SGO-T values, calf 164 . . . . . . . 32 IV SGP-T values, calf 164 . . . . . . . 33 V SGO-T values, calf 43 . . . . . . . 3h VI SGO-T values, calf 44 . . . . . . . 35 VII SGO-T values, calf #5 . . . . . . . 36 VIII SGO—T values, calf A6 . . . . . . . 37 IX SGO-T values, calf 47 . . . . . . . 38 X SGP-T values, average . . . . . . . 39 CHART II III IV d VIII IX XI LIST OF CHARTS Urine examination results . . . Urine examination results . . . Prothrombin clotting times. . . Normal transaminase values. Normal transaminase values. . . Blood examination results . . . Blood examination results . . . Serum protein and electrophoresis results . . . . . . . . Serum protein and electrophoresis results . . . . . . . . Serum protein and electrophoresis results . . . . . . . . Serum protein and electrophoresis results . . . . . . . . PAGE 41 42 43 45 46 #8 INTRODUCTION The diagnostic problem of hepatic malfunction has long been one of the enigmas of medicine. The functions of the liver are numerous. There are almost as many liver function tests as there are functions. No one function test can be expected to completely reflect the biological state of the liver. A battery of function tests are usually run in anticipation of obtaining a more accurate picture of the liver. The more recent tests being evaluated in hepatic conditions are enzymatic. The problem arises as to the value of such tests as applied to veterinary medicine. Liver biopsy is one method of following the course of hepatic disease with live animals. The major problem remaining in evaluation of function tests is the produc- tion of known liver damage. This paper describes the experimental procedure for causing known liver damage in calves, evaluating the amount of liver damage by liver biopsy, and evaluating two enzymatic liver function tests in the bovine. REVIEW OF LITERATURE The use of enzymatic tests in clinical medicine is rapidly becoming widespread. Two of the more recent enzymatic tests being evaluated in human medicine are the serum glutamic oxalacetic transaminase*, and the serum glutamic pyruvic transaminase**. Transamination is the chemical reaction in which there is an exchange of the alpha-amino group of one amino acid for the keto groups of an alpha-keto acid, with the resulting synthesis of a second alpha-amino acid and a new alpha-keto acid (21). Glutamic acid / Oxalacetic acid £323.. Alpha-keto glutaric acid / Aspartic acid Glutamic acid / Pyruvic acid.+§9§Zg>-Alpha-keto glutaric acid / Alanine This type of chemical reaction was first described in 1937, and thought to be a reaction which occurred with any amino acid and alpha-keto glutarate or oxalacetate using pigeon breast muscle as a source of transaminase (3). In 1940 pig heart was shown to transaminate other sub- strate systems, but was still more limited in scope than originally postulated (ll). By the use of a coenzyme, pyridoxal phosphate, muscle homogenates were capable of * Hereafter reTEred to in this paper as SGO-T ** Hereafter refered to in this paper as SOP-T catalyzing the transfer of alpha-amino groups of 25 different amino acids (6). This enzyme was present in pig heart, liver and kidney tissue (6). By 1952 it was reported that transaminase activity was not limited to pigeon breast, pig heart, liver and kidney, but present as well in varying activities in eight organs of the rat. Glutamic oxalacetic transaminase* was present in heart homogenates to the greatest extent. Smaller amounts were demonstrated in skeletal muscle, lung, brain, liver, spleen, prostate and testes in decreasing order (1). Subsequent observations indicated that the organ distribution of GC-T was species specific, and in man was present in heart, liver, skeletal muscle, and kidney in decreasing order (42). The transaminases have been measured in a multitude of ways. SGO-T has been measured chromatographically (29), Spectrophotometrically (2) (28) (29) (41) and colorimet- rically (4) (45) (52) (56). Spectrophotometrically, SGO—T is assayed by employing a double enzyme system. GO-T is coupled to the enzymatic oxidation of reduced diphosphopyridine nucleotide, using either malic dehydrogenase or lactic dehydrogenase (2) (22) (29) (#1). All of the spectrophotometric techniques measure SGo-T by estimating the rate of the enzymatic reaction rather than any of the end products of the tran- samination. Spectrophotometrically, SGO-T activity is * HareaTter refered to in this paper as GO-T expressed as units per milliliter of serum per minute. One unit equals a decrease in optical density of 0.001 mq under the conditions specified in the literature. Although simple, rapid and accurate, these techniques require malic or lactic dehydrogenase and these reagents are expensive, unstable and not generally available. The colorimetric method estimates SGOJT activity by measuring the amount of pyruvic acid formed under standard incubation conditions (A) (33) (A5). The oxalacetic acid formed by transamination is converted by aniline citrate to pyruvic acid. This method is simple and required no enzymes as reagents, but it is somewhat less accurate than other methods. Colorimetrically, SGO—T is expressed as units per milliliter of serum. One unit equals the formation of 1v of pyruvate under the conditions speci- fied (A5). ‘ When SGP-T is determined colorimetrically the sub- strate is changed to L-alanine and pyruvate determined without the use of aniline citrate (#5). The maximal activity of GO-T in cardiac musculature suggested that alterations in metabolic behavior of this _tissue might reflect itself in changes in enzymatic levels in the blood. In 1953 the presence of GO-T was sought for and demonstrated in whole blood, serum and plasma (29). The normal range for activity was established (29), and elevations in some patients with clinical diagnosis of myocardial infarction were observed (33). Early in the use of SGO-T as a diagnostic aid many investigators noted that necrosis in other tissues resulted in an elevated level of the SGO-T. The possibility of the SGO-T determination as a diagnostic aid to liver con- ditions was postulated. Virus hepatitis in mice has been associated with rises in SGO-T activity. The rise in SGO-T activity appears to be associated with the size of the virus ino- culum, the blood virus titer and the degree of liver necrosis. A rise in the SGO-T of mice has also been noted in partial hepatectomy. This was probably due to trauma of hepatic tissue (18). Rats and dogs have been used by investigators as laboratory animals in experiments with toxic hepatitis due to carbon tetrachloride. Levels of activity have been reported that are 50 to 1,000 times the normal SGO-T levels (39) (64) (65) (66) (60). There appeared to be a correlation between the extent of centrilobular zonal necrosis and the height of the SGO-T activity attained. Whether Judged by the gross or microscopic picture the highest levels were seen in the animals with the most severely damaged livers (39). Neither the myocardium nor kidneys of the rats with carbon tetrachloride poison- ing showed any gross or microscopic damage (39). Two days after exposure to carbon tetrachloride elevations in SGO-T were regularly observed in the canine. 0n the sixth-day following administration of the toxic agent practically normal levels of SGO-T were found in surviving dogs (17). Common-duct occlusion experimentally produced in dogs has resulted in temporary elevations in SGO-T activity. Elevated levels returned to normal within a week following the relief of the biliary tract obstruction (36). Experimental ligation of the common duct in rats was rapidly followed by a rise in the SGO-T activity. Sham operated controls showed no significant changes in SGO—T activity. Six hours following duct ligation a peak level was reached after which a recession took place de- spite continuation of the obstruction. Upon release of the common duct obstruction the SGO-T rapidly returned to normal (9). Cirrhosis and hepatic tumors experimentally produced in rats with butter yellow 18 have been shown to be accom- panied by elevation of SGO-T activity (39). From the experimental data the following theory was postulated, ”Any process causing death of enough liver cells should theoretically increase the serum transaminase activity" (38). Investigators have reported many compounds which gave rise to elevated SGOJT levels due to toxic hepatitis; chlorpromazine (7) (63), salicylate (35), cinchophen (36), azasirine (36), pyrazinamide (63). SGobT increased pro- portionaiiy with (the continued administration of these drugs when they proved to be hepatotoxic. However, elevations of toxic hepatitis were greater when caused by carbon tetrachloride than when caused by any of the above mentioned drugs (39) (60) (6A) (65) (66). Discon- tinuance of the heptotoxic drug resulted in a rapid fall of SGOJT toward normal (36). Acute hepatic diseases have been shown to be asso- ciated with rises in SGC-T activity. In many instances the serial changes and/or levels of SGO-T activity were sufficiently characteristic to assist in differential diagnosis of liver disease (7) (8) (19) (38) (39) (58) (60) (63) (66). The highest levels of SGO-T activity were observed in acute toxic hepatitis due to carbon tetrachloride, and in patients with acute infectious or homologous serum hepatitis (7) (66). The elevations following carbon tetra- chloride exposure occurred within 2n hours and have been reported to reach levels of 27,000 units (66). After cessation of exposure to the toxin the SGO—T rapidly returned to normal values. Acute infectious and homologous serum hepatitis resulted in alterations of SGO-T to levels of 13 to 40 times greater than normal (8) (IA) (37) (43) (58) (60) (63) (65) (66) (33). The SGO-T rise in virus hepatitis began during the prodromal phase of the disease (8) (60) (63) (66), and reached its peak elevation when the patients were the sickest as Judged clinically (7) (66). With clinical evidence of improvement the hepatitis was accom- panied by a fall of SGO-T toward normal (7) (38) (66). Relapse and/or development of chronic hepatitis was reflected in continued abnormal SGO-T levels (60) (63) (66). The theory has been suggested that serial determina- tions of SGO-T during the course of hepatitis reflected the liver damage better than conventional liver function tests (66). Because it does reflect liver cell injury rather than function it does not necessarily correlate with the routinely employed liver function tests (7) (38) (39) (66). There was a correlation between the extent of damage to hepatic cells and the degree of elevation of SGO-T (15) (63). SGO-T may be useful as a guide in following the progress of restoration of hepatocellular integrity follow- ing acute injury (39). The determination of the SGO-T activity will become a useful tool in studying the natural course of hepatic diseases (15). Epidemiological data has shown that SGO-T levels in persons exposed to infectious hepatitis rose one to four weeks before other clinical laboratory evidence of liver injury becAme evident (60). In homologous serum hepatitis the SGO-T may be elevated 8 to 1% days following exposure to contaminated serum (A9). This permits diagnosis of hepatitis in the preicteric stage and aids in controlling epidemics (60). Infectious mononucleosis unless associated with hepa- titis was accompanied by normal SGOJT levels. Increased levels of SGO—T accompanying infectious mononucleosis appeared to be related quantitatively to the severity of the hepatitis (7) (60) (63) (65). .Elevations of SGO-T in the range of 50 to 250 units have been reported in active or decompensated Laennec's cirrhosis (7) (8) (65). Cirrhosis complicated by acute hepatitis has been reported to present SGO-T alterations characteristic of acute hepatitis but of prolonged eleva- tions (58) (65). Active cirrhosis presents much the same picture (58) (65). Biliary tract obstruction, either extrahepatic or intrahepatic, has been accompanied by elevations in SGO-T as high as 300 units (7) (8) (14) (19) (38) (58) (65). Upon relief of biliary obstruction the SGO-T level fell to normal within 7 to 14 days (65). "Acute and chronic hepatic disease is associated with quantitative and serial elevations of SGOJT'which are sufficiently characteristic to permit diagnostic differentiation" (60). Both primary and secondary carcinomas of the liver were associated with elevations in SGO-T. Neoplastic involvement of the liver appeared to be very sensitively indicated by the SGO-T levels (7) (8) (58) (65), except in the lymphomas and leukemias (65) where normal values were found. The absence of elevated SGO-T levels did not exclude the possibility of hepatic metastases (8). In bone disease, either malignant or nonmalignant, IO the SGO-T values were normal unless there was also hepatic involvement (65). Although kidney tissue contains a high level of GO-T, elevations of SGO-T in renal disease were found in only a few cases (47) (48) (58) (59). Most of the patients showing elevated SGO-T levels with renal disease had accompanying hepatic damage (59). Acute pancreatitis has been associated with eleva- tion in SGO-T (8) (14) (43) (58). The reason for this elevation was not clear at the time but was thought to be due to hepatic factors involved with the acute pan- creatitis (8). Patients with prolonged shock for periods greater than 24 hours showed elevated SGO-T levels (8) (58). In those patients observed at post-mortem following prolonged shock, well developed centrilobular liver cell necrosis was present in each case. This necrosis was thought to have caused the elevations in SGO-T. With shorter periods of shock the SGO-T levels appeared to be unrelated to the vascular collapse and primarily related to the underlying disease (8). Skeletal muscle necrosis due to severe trauma, in- fectious processes or following surgery was associated with elevated SGO-T levels (8) (37) (42) (43). Elevations seen with very rapid arrhythmias were probably due to the centrilobular liver cell necrosis secondary to diminished cardiac output (8). II Because there was such a lack of Specificity in the SGO-T activity in tissue necrosis, other serum enzyme systems were studied in an attempt to locate ones more specific for individual tissue. SGP-T was one such enzyme studied. SGP-T was claimed to be more sensitive than the SGO-T in depicting acute hepatocellular necrosis (62) (63). The relative differences in enzyme concentration of the tissues involved was reported to reflect itself in the relative differences found in the serum concentrations. The following table of GO—T and Glutamic Pyruvic Transa- minase* values for human tissue was taken from the litera- mme(6n. Comparison 2£_Glutamic Oxalacetic Transaminase and Glutamic Pyruvic Transaminase in Normal Adult Tissue Homogenates (61) TISSUE GO-T GP-T Units/gm. wet tissue Heart 156,000 7,100 Liver 142,000 44,000 Skeletal Muscle 99,000 4,800 Kidney 91,000 19,000 Pancreas 28,000 2,000 Spleen 14,000 1,200 Lung ', 10,000 700 Serum 2O 16 * Hereafter refered to in this paper as GP-T I2 Experimental animals were used to test the effect of liver damage on SGP-T levels. Common duct ligation in rats resulted in an increased SGP-T level. This level reached its peak six hours following the operation and continued to recede toward normal despite the con- tinuation of the obstruction (9). The use of carbon tetrachloride poisoning in dogs to cause liver damage resulted in greatly elevated SOP-T levels (17). Since elevation of SGP-T was usually accompanied by elevations in SGO-T it was suggested by many investigators that the two be run simultaneously. Many disease states have been reported in the litera- ture evaluating SGO-T and SGP-T measurements as diagnostic aids. Whereas SGO-T was greatly elevated in myocardial infarction SGP-T was not unless the SGO-T rise was greater than 150 to 200 units. This was explained by the lower concentration of GP-T in heart muscle as compared to 004T (61) (62). In very rapid arrhythmias SOP-T was elevated along with the SGO-T. Necropsy of one such case showed evidence of extensive liver cell necrosis (9). Viral hepatitis has been associated with elevations of SGP-T greater than SGO-T (9) (13) (61) (62). By the simultaneous measurement of SGO-T and SGP-T a diagnosis of viral hepatitis was reported to be possible. The normal ratio of SGO-T to SGP-T was usually greater than 1.0 (9) (13). If the ratio was less than 1.0 the diagnosis of viral hepatitis was proposed (13). I3 In infectious mononucleosis elevations in SGP-T were associated only with involvement of the liver. The rise in SGP-T was less that that associated with viral hepatitis or homologous serum hepatitis (61) (62). In hepatic cirrhosis the elevation in SGP-T was variable. In patients with active cirrhosis the SGP-T and SGO-T levels were both elevated. In patients with inactive cirrhosis the SGP-T and SGO-T were both within the normal range (61) (62). In obstructive Jaundice, in a limited number of observations, the elevations of SGP-T were so variable that conclusions could not be drawn (9) (61) (62). SGP-T level elevations have been noted in metastatic cancer of the liver. The levels attained were usually close to the normal levels, and of little diagnostic value (61). Although the SGP-T has been reported to be elevated in hepatic disease its specificity as an indicator of hepatic disease when used alone was questioned (9). The procedure of using both the SGO-T and SGP-T simultaneously to help in differentiating acute from chronic liver disease has been suggested (61). The SGP-T should have its great- est use as an adjunct in the interpretation of an elevated SGO-T level (9). The use of the transaminase levels to aid in the dif- ferential diagnosis of liver disease in no way relieved the physician of the necessity of interpreting the test in the light of the total clinical setting (58). MATERIALS AND METHODS Nine calves’were obtained from local sales barns at about one week of age. The calves were of mixed breeding mainly from dairy animals. Both males and females were represented. N0 experimental work was started until the calves were Judged to be clinically normal. Due to various origins all calves were treated prophylactically for calf scours immediately upon arrival. One-quarter gram of oral tetracycline hydrochloride* was given per day for one week. When experimental work was begun the calves ranged in age from two to six months, and in weight from 50 to 172 kilo- grams . Fecal examinations for parasitic infestation were run on arrival and immediately preceeding the experimental work. The sugar flotation method of fecal examination was used (10). At least two urine examinations were made before pro- ceeding with experimental work. The urine examination included the following tests: Specific gravity by urino- meter**, pH by paper strip**, urine bilirubin***, urine albumin***, urine occult blood***, and urine acetone***. Urine was collected without catherization by stimulation of external genitalia. * Charles Pfizer a Co. Inc., Brooklyn, N.Y. 25 Gm. Oxytetracycline HCl ** Squibb, Chicago Apparatus Co., Chicago, Ill. **“ Ames Co. Inc., Elkhart, Ind. I5 Blood was drawn from the external Jugular vein using a 16 gauge needle. Thirty milliliters of blood for serum was collected in two tubes to eliminate the possibility of hemolysis occurring in any one complete sample. Upon clotting, this blood was incubated for one hour at 37° Centigrade and then centrifuged. Serum was then removed and transferred to clean tubes. All samples were refrigerated until examination, and no samples were stored longer than 24 hours before determination of any tests requiring serum other than electrophoresis (7) (67). Blood for cell counts was collected in citrated vials. Both red and white cell counts were run by standard dilu- tion techniques and counted in a bright line hemacytometer (10). Differential white blood cell counts were made on a thin blood smear, stained with Wright's stain and counted as described in the literature (23). Hematocrit values were determined by allowing freshly drawn blood to flow into heparinized capillary tubes, seal- ing one end with a gas flame, centrifuging for five minutes at 11,000 r.p.m., and then evaluating as percent packed cell volume. Biopsy specimens were taken using a Vim-Silverman punch biopsy needle, and a technique modified from the literature (55). (Figure I and II) A liberal area around the eleventh and twelfth rib on the right side was clipped. The area was washed with soap and water and then disinfect- ed with alcohol. A local infiltrative anesthetic of ten milliliters of four percent procaine was used in the subcutaneous and muscular tissues. Most of this anes- thetic was placed between the eleventh and the twelfth rib, about nine to ten centimeters below the vertebral- rib Junction. A one centimeter incision was made through the skin with a scalpel blade. With a sharp thrust the biopsy stylet and outer needle were inserted in an antero— ventral direction toward the left olecranon process. When hepatic tissue was entered and movement of the needle cor- responded to diaphragmatic movements the stylet was removed. The inner biopsy needle was inserted with a rotating motion. The outer needle was forced over the inner needle for a distance of two centimeters and the entire unit was then removed. A topical antibiotic powder was applied to the incision. Animals were observed for an hour for any signs of internal hemorrhage or unusual distress. Biopsy specimens were taken immediately preceeding carbon tetrachloride administration and during the subse- quent 24, 72 and 144 hour periods. Specimens were placed in isotonic formalin for fixing. They were later embedded in paraffin, sectioned, and stained with hematoxylin and eosin for microscopic examination. Daily clinical examination included observations on appetite, temperature and stethoscopic auscultation of thoracic and abdominal cavities. Total serum protein was determined using the Hitachi hand protein refractometer*. Electrophoresis was run on * National Instrument Company, Baltimore, Md. I7 serum samples on a paper electrophoreses system'. Six ten-thousandths milliliter of serum was run for 16 hours at 2.5 milliampere constant current using a veronal buffer of 8.6 pH and 0.075 ionic strength. Paper strips were analyzed on an analytrol following the procedure for human serum* (25). SGO-T and SGP—T were determined colorimetrically (50) on a Bausch and Lomb Spectronic 20 Colorimeter. Only serum samples which had been refrigerated no longer than 24 hours and which showed no hemolysis were used (7) (45) (50). An incubation temperature of 37° Centigrade was used. Serum bilirubin was determined by the colorimetric method as described in the literature (23). Both the one minute and the thirty minute bilirubin were determined. When possible, samples were run on the day collected, but never were they stored longer than overnight before pro- cessing (67). Prothrombin clotting times were determined on the six month old calves before any biopsy specimens were taken (23). Liver poisoning was accomplished by varying doses of carbon tetrachloride given by stomach tube. Twenty-five milliliters of mineral oil were given following the carbon tetrachloride to carry the total dose of toxin to the sto- mach. Control animals were given twenty-five milliliters of mineral oil by stomach tube. Dosages were arbitrarily selected due to the lack of literature on this subject on the bovine. * Spinco moaelfRfPaper Electrophoresis System, Beckman Spinco Division. I8 Because of the absence of reports in the literature concerning normal values in the bovine for either SGO-T or SGPJT, it was necessary to establish normal values. Random samples on the experimental calves were used to establish these normals. During the course of the experi- mental work two reports of normal values in the bovine were published (12) (32). some FIGURE I A B C D Slylel Inner needle Outer needle C'. Mognificollon 10 show detail of outer needle Inner needle in place within ouler needle 0'. Magnification of needle llps in"D". I9 .9898 832020 :32: Ease. no.9... a. 0:38. COB S¢u>o 7‘ “— mosque gs ouaneq who“. won“ onetouou btocou OAEBFVA £08 . - o.‘ 3-,... u. “34“”, glam“: 3'." )Irm , J'§®,L\\ “ab" mm, “.9 u. a». .l. H- «in: §°-‘-" #11”: 2| x... renew E33 m2_>om mo”. 29583 H l mmDOC laily rectal temperatures never rose over 1030 Fahrenheit. At no time did the calves appear depressed or give any indications of being clinically 111. At the beginning of the experimental work all temperatures were 220 Fahrenheit. At no time were there any indications of increased pneumonic sounds of abnormal character. All fecal examinations were negative for parasitic ova on initial examinations and remained negative through- out the duration of the experiment. The results of urine examinations for control and experimental animals are placed in Chart I and II. The only abnormalities were three transient elevations in the urine albumin. These elevations were a trace as determined by the information provided by the supply company. The rest of the urine samples were all within the ranges of normal as reported in the literature (10). The prothrombin clotting times ranged from 19.2 to 22.6 seconds. The results from the six month old calves were placed in Chart III. A total of 26 biopsy specimens were taken during the course of the experiment. The technique as described in the materials and methods adequately locates the liver for needle punch biopsy. On five different occasions it was necessary to re-enter the animal due to the absence of the liver tissue when the needle was withdrawn from the animal. 23 Discomfort was not noted in animals once the intercostal muscles were entered, although animal #6 showed a mild discomfort and dyspnea following biopsy on the first day. This subsided in less than one-half hour and no recurrences of a similar nature were noted. The biopsy specimen ob- tained was usually five millimeters in length and one-half millimeter in diameter. Opinions formed upon histological examination of the biopsy specimens were confirmed by the Michigan State University Pathology Department*. Two calves two months of age received ten milliliters of carbon tetrachloride orally by stomach tube. The biopsy specimens taken 2h hours following showed generalized changes. The normal cord cell arrangement was no longer present, and there was cloudy swelling, fatty changes, pyknotic nuclei and hydropic changes throughout the lobule. The biopsy specimens taken before and 1&4 hours fol- lowing carbon tetrachloride administration were normal. Biopsy specimens from control calves were normal. The biopsy specimens taken before experimentation with the five calves six months of age were Judged to be normal. Specimens taken during experimentation never showed general- ized changes as seen in the two-month old calves. Twenty-four hours following the administration of 17 milliliters of carbon tetrachloride orally the specimens from calf #5 showed extensive areas of zonal coagulation *lDr. Ralph D: Earner, MSU Pathology Department, E. Lansing, M1Chigan o 24 necrosis with hemorrhage. Surrounding the zonal necrosis was a rim of cloudy swelling, vacuolation and both fatty and hydropic changes. Both calves 4n and #6 showed cloudy swelling and small areas of focal necrosis, lymphatic infiltration and small amounts of hemorrhage in the 24 hour specimen. By the end of lhh hours no changes from normal were evident. In calves receiving 35 milliliters of carbon tetra- chloride the 24 hour specimen showed zonal necrosis, some cloudy swelling and in calf M7 some hydropic changes. Calf 45 after receiving 100 milliliters of carbon tetrachloride showed necrosis, pyknotic nuclei, and cloudy swelling in an hours. The normal hepatic architecture was lost in the 48 hour specimen. All pathological changes were gonein the 1&4 hour specimen. The SGO-T normals were run on two groups of calves. The first group consisted of four calves, all about two months old. There were three males; two Holstein and one Holstein-Angus cross, and one Holstein female. The weight range was from 50 kilograms for the Angus cross to 75 kilo- grams for one Holstein male. A total of 25 SGO-T samples were run on these calves to establish normal values. The results showed a range of 10 to 76 units, the average is 54 units with a standard deviation of 14.6 units (Chart Iv). The SOP-T normals were determined on the same calves by 30 samples. The range in units was from 3 to 22; the average was 1h.l units with a standard deviation of h.0 units (Chart IV) . 25 A second series of normal values was determined for six month old calves. This consisted of five calves; two Holstein females, two Holstein males and one Guernsey male. The weight range at the time experimental work was begun was lh5 to 172 kilograms. SGO-T for the second group was determined on 36 samples with the following results. The range was 12 to 95 units, the average value being 59.3 units and the standard deviation 2h.2 units (Chart V). The same group of six month old calves was used to establish normals for SGPiT. The results were a range of 4 to #0 units, an average value of 11.68 units and a stan- dard deviation of 8.82 units (Chart V). Graph I shows the results following the oral adminis- tration of ten milliliters of carbon tetrachloride to a 70 kilogram male Holstein calf,l63,and its effect on the SGOJT levels. One day following the administration of the toxin the SGO-T level rose from the normal range to l,h80 units. An abnormal level was noted until the tenth day after exposure to the toxin. The SOP-T levels for the same animal are shown in Graph II. The elevation in SGPAT level was noted two days after the toxin was given by stomach tube. The peak level reached was 80 units four days following the administration of the toxin; three days later the level had again descended to the normal range. ‘A second animal, 16%, demonstrated a reaction similar to 163. Following the administration of ten milliliters 28 of carbon tetrachloride, both SGO-T and SGP-T were elevated. Outstanding SGO-T elevation was noted in the 2” hour samile following toxin administration. The peak level, occurring in 2“ hours, was 1,960 units. Again ten days was required for SGO-T to decline to normal values. The values declined rapidly 2h hours following the peak elevation and then receded to normal ranges more slowly (Graph III). The SGP-T elevations as shown in Graph IV for animal 16“ were somewhat different than those observed on animal 163. The peak elevation again was not noted until the fourth day following toxin administration. Two days after the peak level was reached the values were at a normal level. The total duration of elevated levels was shorter than observed in animal 163. The six-month old calves used in the experimental work were the same as those described in the establish- ment of normals for SGO-T and SGP-T. Seventeen milliliters of carbon tetrachloride were administered to a 150 kilogram Holstein female calf, 44. This oral dosage of carbon tetrachloride was followed by an elevation to 300 units #8 hours later. The SGO-T level was elevated for a period of 48 hours (Graph VI). The SGP-T was never significantly elevated. calf #5 also received 17 milliliters of carbon tetra- chloride orally. This was a 162 kilogram Guernsey male. Twenty-four hours following the toxin 3 level of l,h80 units of SGO-T was reached. The SGP-T level was elevated for 27 four days (Graph VII). No elevation was noted in the SGP-T level of this calf. Thirty-five milliliters of carbon tetrachloride were administered orally to a six month old Holstein female, #3, weighing 150 kilograms. The toxin was administered on the ninth day as shown in Graph V. Two days later an elevation in SGO-T was noted to a peak of 96 units. The following day the level fell to 38 SGO-T units. On the third day post-administration a second rise to 1&8 units was noted that lasted 24 hours and then fell to normal levels. The SGP-T levels on the same calf, #3, showed a pattern similar to that of the SGO-T in its lack of any great eleva- tions. The third day following the toxin a SGP-T level of 36 units was seen which declined to normal in 2% hours. A similar protocol was followed on a male Holstein calf of six months of age weighing 172 kilograms. Thirty- five milliliters of carbon tetrachloride were administered on the eighth day as shown in Graph IX. The SGO-T levels on calf #7 rose sharply to 176 units on the day following the carbon tetrachloride, but the rise was only transitory and fell to normal levels in 2# hours. A second rise to 124 units was seen on the third day which also proved to be transitory and rapidly returned to normal. The SGP-T levels on calf #7 were never significantly elevated. A Holstein male, six months old, and weighing 170 kilograms, #6, was given a total dose of 35 milliliters of 28 carbon tetrachloride, but administered in divided doses on the fourth and eighth day as shown in Graph VIII. A response in the SGO-T level was noted in 2h hours after the first 17 milliliters of carbon tetrachl)ride. A peak level of 611 units was reached and elevated levels were seen for three and one-half days. The second dose of carbon tetrachloride resulted in levels that were elevated but still within the normal range. The SGP-T levels were not changed following either the first or second dosage of carbon tetrachloride. Eleven days after normal SGO-T levels were reached animal 45 received 100 milliliters of carbon tetrachloride orally (Graph VII). The response to this dosage of carbon tetrachloride was an elevation of SGO-T on the second day of 280 units. The SGP-T level was elevated for 24 hours to 68 units. Both SGO-T and SGP-T levels were within the normal range for the remainder of the experiment. Erythrocyte, leucocyte and differential leucocyte counts are reported in Chart VI for the two-month old calves and in Chart VII for the six-month old calves. - Paper electrophoresis usually resulted in separation of the following bovine serum protein fractions: albumin, alpha globulin, beta globulin, and gamma globulin. A peak at the point of application on the paper strip was identi- fied as fibrinogen and foreign protein. In those samples in which gamma globulin was identifiable separately from the fibrinogen it was reported separately. Otherwise, 29 the two fractions were considered as one called fibrinogen and gamma globulin. The range of values are shown in Charts VIII, IX and X. A total of 45 samples were run on the six—month old calves spaced from the beginning to the end of the experimental work. The serum bilirubin test gave very variable results. The daily variations were so great that normals for indi- vidual calves could not be established. The results are shown in Chart x. The serum bilirubin test was discontinued after completion of the experimental work on the two-month old calves and not utilized on the six-month old calves. 30 m><0 FINI— Kmaxm 1 a q .. _ _ 4 a _ H . _ d a (A _ v N w. m. ¢_ 2 N. Z 0. m 0 x. 0 n oucm#m.z.10< z_x0h lllll l'lllllllllllllllll 304mm 02¢ mtz: 0s. m02(m J44<¢0 uo_¢0410<¢bu._. zommdo wmmhjjnzx 0— 5(1 1<¢00..=x Ob zfihmJO: IhZOZ 03% n0. m4<0 0:23 00¢. H Iddmo 00ml 00*... 0001 000 1 000.1 700w m Pi: ooo~l 3| GRAPH II ~80 UN'TS CALF I63 SGP-T Two MONTH HOLSTEIN 7o KILOGRAN MALE -70 no MILLILITERS CARBON TETRA- CHLORIDE ORALLY —60 1 —so -40 —3o - _ _ _ _ _ _ _ $939915. 3559.5 - .29 _ I9. 3?. _ -9317? _____________ ZQ___ngflL__§_gw_c_e___2o umrs AND sELow L10 TOXIN ADMINISTERED I 2 3 4 s s 7 s 9 IO M l2 l 1 I L 1 J L l 1 1 1 J EXPERIMENT DAYS 32 o. n. V. m. N. 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H omo.H auHssnw oHuHooam so . m0 0 Ham mm 02.5: .38 m . has ucolahoqu mopamo 0H0 sumosooza spassom eoHsmcHamwm ocHsp H Bmdmo CHART II Urine Examination Results Six-month old calves Experiment day Calf number Specific gravity pH Bilirubin Occult blood Acetone Albumin Experiment day Calf number Specific gravity pH Bilirubin Occult blood Acetone Albumin Experiment day Calf number Specific gravity. pH Bilirubin Occult blood Acetone Albumin Experiment day Calf number Specific gravity pH Bilirubin Occult blood Acetone Albumin Experiment day Calf number Specific gravity pH Bilirubin Occult blood Acetone Albumin *N = Negative 143 1.035 7.5 N' N N N 143 ”$222 143 1.030 7-5 2222 143 2222 143 1.035 7.5 2222 2222 m 1.037 .3 22226 144 2222 1414 1 .036 «I 22226 «Ma 0 H 2222U'IOKON 2222 «Ma 1 1 0 HP 2222\J'ICD-¥='1-J 2222 :w zzzzfibcw H \J1 m bum \D 5 #U'l 10 any In 147 1.039 7-5 2222 147 2222 147 1.030 7-5 2222 147 2222 147 1.035 7-5 2222 4| 42 CHART III Prothrombin Clotting Times Results on six-month old calves Calf number 43 44 45 46 47 Determination 1 20.0 21.0 19.2 21.6 20.1 2 20.6 22.2 19.4 22.6 19.4 3 19.5 21.6 20.0 22.0 21.8 (all times reported in seconds) CHART IV Normal Transaminase Values Two-month old calves Number of. Range in Average plus Samples Units Standard Deviation sac-r 25 10-76 54.0 t 14.6 SGP-T 30 3-22 14.1 i 4.0 CHART V Normal Transaminase Values Six-month old calves Number of Range in Average plus Samples Units. 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