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Tests of Liver Function
TESTS OF LIVER FUNCTION
lAMES F. WEIR THE diagnosis and management of diseases of the liver and biliary tract fundamentally rest on the evaluation of clinical data. The procedures have been established through long periods of clinical practice and have been reasonably satisfactory. There are certain instances in which a clinical diagnosis cannot be made with certainty. In some such cases, the patients have been denied the possible benefits of surgical exploration; in other cases surgical procedures have been employed to the detriment of the patient, particularly if parenchymatous disease of the liver has been present. During the past two or three decades, much new knowledge concerning the physiology of the liver has been developed and, as a corollary of this, attempts have been made to determine the nature, extent and significance of functional abnormalities in various types of disease of the liver or biliary tract. This has led to the development and use of many tests of liver function. In the use of such tests, attempts have been made to apply them to the differential diagnosis and management of such diseases with, unfortunately, an unwarranted halo of importance and significance. Actually, these tests are somewhat limited in value yet no apologies are needed for their judicious employment. Such tests presuppose a knowledge of the functions of the liver and there are many of these. Approximately thirty-three different, though related, functions of the liver cells have been listed and it is estimated that at least 100 chemical activities are known to occur in the liver. In view of this, the following question may be asked, "What is a liver function test?" One answer, elementary and facetious, might be, "Some laboratory procedure that reveals something wrong with the liver." That something is wrong is usually known before employment of the procedure, from clinical observation, such as enlargement of the liver, jaundice or ascites. This answer may be satisfactory in cases in which clinical evidence of disease is absent. Perhaps a better answer would be, "A procedure that shows a disturbance, and perhaps the degree of such disturbance, in some particular function of the liver." Obviously, in view of the many functions of the liver, its known large reserve and great regenerative capacity, no "miracle" test can be expeeted, that is, no single test can be anticipated that will determine deviations from normal of even a few major functions of the organ, let alone all of them. Some authors claim that the chief value of these tests is the detection of early functional disturbance or focal disease such as carcinoma or abscess. Because of the known large reserve capacity, severe and ex973
974
J AMES
F. WEIR
tensive damage is necessary before positive tests may be obtained in such conditions. Consequently, some physicians would say that the tests lack sensitivity. Others claim that the tests are too sensitive. Positive results may be obtained but no clinical signs of disease of the liver may be apparent or develop. Rafsky, and Newman, for example, have reported a considerable group of aged persons on whom positive tests were obtained in the absence of clinical evidence of disease of the liver. The Question may then be asked, "Is a positive test an indication of disease of the liver or only of a dysfunction (as diarrhea in a case of irrit:lhle colon), and if it is an indication nf dvsfunction. what is its significance?" Answer to this question is difficult to !live. The significance of minor deviations from normal are particularly difficult to evaluate. The results must always be interpreted in association with other availahle data. In some instances, the findings are significant; in others, correlation of the finding's is impossihle. If tests are to be used clinicallv, they must be readily performed, reliable, and give some worth-while information from a diagnostic, therapeutic or prognostic standpoint. In addition, the results must be capable of being- interpreted easily. Probablv about seventy-five to 100 tests for liver function have been devised. The large number of these that have been used and discarded is evidence of their failure to meet the criteria which I have mentioned. The multitude of tests and the claims and counterclaims made in regard to them have created a very confusing situation, particularly for the g'eneral practitioner. It would seem well, therefore, to consider a few of the more simple procedures and their practical application. In such a consideration, certain facts should be pointed out. First, diagnostic procedures should be distinguished from tests for liver function; thus, duodenal drainage and examination of the stools for bile are best classified as diagnostic procedures because they are primarily used to determine the patency of the bile ducts. Such procedures leave much to be desired. Second, there are two groups of cases in which tests of liver function are commonly used (1) cases of suspected liver disease in which jaundice is absent, and (2) cases of jaundice. In the first group, the dye excretion tests and van den Bergh test are particularly valuable. In the second group, it is necessary to determine whether the jaundice is hemolytic, obstructive or hepatogenous. Hemolytic jaundice is usually readily distinguished by clinical and hematologic studies, and the van den Bergh test is also helpful. The last two types of jaundice usually are distinguished by clinical study, chiefly by the history in regard to pain or exposure to infection and hepatotoxins, by physical evidence of abdominal tumors, and by employment of diagnostic procedures to determine the patency of bile ducts. Some aid in this distinction may be obtained from the use of some of the tests of liver function, such as the distribution of urobilinogen in the urine and feces, the van den
TESTS OF LIVER FUNCTION
975
Bergh test, the galactose tolerance test and the partition of cholesterol in the plasma. This, then, is the first practical use of tests of liver function, that is, assistance in the differential diagnosis of various types of jaundice. Their second use is for the confirmation of the presence of liver damage in cases in which it is suspected and the evaluation of the extent of liver damage in cases in which the presence of disease of the liver or biliary tract is known. The qualitative and quantitative determination of serum bilirubin by the van den Bergh test, dye excretion tests, hippuric acid synthesis, galactose tolerance test, blood prothrombin time, partition of cholesterol in the blood and the value for the protein in the blood are procedures most commonly used in the study of such conditions but they give information of variable value. Many of these are not truly functional tests but laboratory procedures which throw some light on one or another aspect of disease of the liver. In the interpretation of the results of such tests in the presence of jaundice, it should be remembered that there is seldom any case of jaundice pure in type. For example, severe hepatic damage may occur in cases of obstruction of the biliary tract or in hemolytic processes, and the tests may be valuable in these types of disease as well as in primary parenchymatous disease of the liver. Third, and finally, there is the question of progression of activity of disease of the liver or recovery. Repetition of some of these tests aids in the determination of this point. Some physicians claim that the cephalin-cholesterol flocculation test is of particular value as such an indicator. Some of these tests will now be considered individually and in more detail. The first of these is the van den Bergh test, which is used to determine the type and quantity of bilirubin in the serum. It also can De applied to the duodenal contents and urine. Various modifications of the original test have been developed. 1 Prior to two years ago, we employed the technic of Thannhauser and Andersen but since then the technic of Sepulveda and Osterberg has been used. This permits the quantitative determination separately of the indirect and direct reacting bilirubin in the serum in any given case. In health, the value for the bilirubin is less than 0.6 mg. per 100 C.c. of serum and it reacts indirectly with Ehrlich's diazo reagent. In pathologic states, such as pernicious anemia, congenital hemolytic icterus, other hemolytic conditions and familial hyperbilirubinemia, the reaction is characteristica,lly indirect. In these conditions the concentration of the bilirubin in the serum usually is variably increased. In many cases of cirrhosis, metastatic tumors of the liver and other pathologic conditions of tbe liver without evident jaundice, the reaction to the van den Bergh test may be indirect. This type of reaction does not exclude any of these pathologic processes. Pigmentation, such as that due to carotene and atabrine, must be distinguished from icterus. In cases in which pig-
976
JAMES F. WEIR
mentation is due to carotene or atabrine, the concentration of serum bilirubin is normal and the reaction is indirect. A direct reaction always is a pathologic finding and is commonly encountered in the various forms of obstructive and hepatogenous jaundice. Such a reaction also is present in the latent jaundice at times seen after biliary colic, in acute cholecystitis, in metastatic tumors of the liver and in many cases of chronic and even acute hepatitis. In the presence of a hemolytic process, a direct reaction indicates an associated hepatic or biliary disorder. Little attention from a clinical standpoint need be given to biphasic or delayed direct reactions. The quantitative determination shows the degree of retention of bilirubin and its fluctuations from time to time. In many instances estimations every few days are necessary to determine whether the jaundice is stationary, increasing or decreasing in severity. In obstructive jaundice due to stone in the common bile duct, the average value for the serum bilirubin is 10 to 15 mg. per 100 C.c. Fluctuations frequently occur and there is a general tendency for the bilirubinemia to decrease. In neoplastic obstruction of the common bile duct, average values are higher, that is 20 to 30 mg. per 100 C.c. Fluctuations seldom occur and the jaundice is persistent. In parenchymatous disease of the liver, the degree of icterus is variable. In some cases, very mild degrees of bilirubinemia are encountered. On the other hand, some of the highest concentrations (70 to 100 mg. per 100 c.c.) encountered are due to this type of disease of the liver. The higher concentrations are indicative of more serious disorganization of the liver. In obstructive jaundice, similarly high concentrations (that is, much above the average concentration) are indicative of an associated hepatic degeneration. The icterus index is a simple and approximate method of following the degree and variations of bilirubinemia in any case of icterus and is used in many laboratories. Dye excretion tests are frequently used in the determination of liver function. Various dyes may be used, such as phenoltetrachlorophthalein, tetraiodophenolphthalein, Bengal red and bromsulfalein. These tests depend on the rate of disappearance of the dye from the blood stream. It has been our custom at the Clinic to use bromsulfalein. We inject 5 mg. of the dye per kilogram of body weight of the patient, remove a specimen of blood in one hour and determine the amount of dye remaining in the blood with a block colorimeter. Normally, little remains in the blood at the end of one hour. The presence of 40 per cent or more indicates a high degree of retention. Various modifications of this technic have been made but I doubt their practical importance. This is one of the best tests of liver function in cases in which jaundice is not present. The presence of jaundice is a contraindication to the use of this test. When jaundice is present, the test merely shows the impairment to be expected, as indicated by retention of bilirubin and furnishes no additional information. The test is to be used in cases
TESTS OF LIVER
FU~CTTON
977
in which disease of the liver is suspected when jaundice is absent. These diseases include enlargement of the liver and spleen, suspected metastatic tumor or abscess, ascites and toxic hepatitis such as may occur in cases of exophthalmic go iter, toxemias of pregnancy, many infections, poisoning with various therapeutic and industrial chemicals and many other conditions. Normal values usually are obtained in abdominal enlargement due to ovarian cysts, peritoneal carcinomatosis and tuberculous peritonitis. In cardiac disease with decompensation and chronic passive congestion, a moderate to high degree of retention of the dye usually is encountered. Ascites due to portal obstruction from canalized portal thrombosis usually gives normal values but some disturbance of excretion of the dye occasionally is found. In cases of suspected metastatic tml10rs or abscess of the liver, and occasionally in cirrhosis the values may be normal. In cases of splenomegaly in which splenectomy is being considered, high degrees of retention may be encountered, indicating a high surgical risk and a poor prognosis. In some cases in which the patients are in reasonably good health mild degrees of retention of the dye occasionally are encountered but no definite disease of the liver can be demonstrated and no satisfactory cause for the retention can be found. In some instances, dehydration and starvation may be etiologic factors. In other instances, these are absent but, in spite of lack of any explanation, the findings are probably of some significance. The hippuric acid test depends on the ability of the liver to furnish glycine to permit the synthesis of hippuric acid from benzoic acid (administered as sodium benzoate). It is claimed to be a test of the detoxicating function of the liver. The presence of disease of the kidney should be excluded before this test is employed. The amount of hippuric acid excreted in the urine is determined gravimetrically. After administration of 6 gm. of sodium benzoate, at least 2.5 to 3 gm. as hippuric acid should appear in the urine in four hours. Less than 1.5 gm. is indicative of impairment of liver function. The test does not aid in distinguishing obstructive jaundice from hepatogenous jaundice. In cases in which jaundice is absent, the results parallel those obtained with the bromsulfalein test. An intravenous method of performing the test has been devised and is employed by many physicians. The galactose tolerance test is one of the sugar tolerance tests used to determine deviations of the normal function of the liver to metabolize carbohydrates. Galactose is chiefly utilized by the liver, and in the performance of the test it may be administered orally or intravenously. When the galactose is administered intravenously, the test is spoken of as the "galactose clearance test." In using the test it is necessary that the presence of diabetes mellitus be excluded. If oral administration is employed, 40 gm. of galactose in 500 C.c. of water is given and the urine is collected over a five hour period and examined for sugar. Normally, less than 3 gm. of sugar should be excreted. Excretion of more than 5 gm.
978
.T AMES
F. WEIR
is indicative of severe hepatic damage. In acute hepatitis, positive tests may be obtained early (less than two weeks). In obstructive jaundice, the test may become positive at the end of two weeks. In our experience at the Clinic, the test has been found mildly positive in 50 per cent of cases of malignant obstruction and in 25 per cent of cases of obstruction due to stone or cicatricial stricture. Normal results do not exclude the possibility of parenchymatous origin of jaundice. The prothrombin time should be determined in all cases of jaundice, particularly if the disease is surgical, in which case it should be determined during the preoperative and postoperative periods. In the laboratories of the Clinic, the method of Quick, as modified by Magath,2 is used. With this method, the normal values are eighteen to twenty seconds. Elevation of the prothrombin time may be due to deficient intake, deficient absorption or deficient utilization of vitamin K. Deficiency of intake is seldom a factor in jaundiced conditions. A deficiency of absorption is common in the acholia of jaundice. It also occurs in intestinal disorders such as those associated with sprue. Deficiency of utilization of vitamin K is encountered chiefly in parenchymatous disease of the liver. In this disease, the vitamin K is not converted into prothrombin, and if the damage to the liver is severe, administration of vitamin K will not control the hypoprothrombinemia and its associated hemorrhagic manifestations. The degree of this failure is an indication of hepatic insufficiency. Data accumulated in the past two years indicate that larger doses than former.ly employed may be advisable when small doses are ineffective. Vitamin K should be administered in all cases of jaundice, whether or not the prothrombin time is prolonged. This is particularly true in cases in which operation is to be performed and the administration should be continued during the postoperative period. Another test is the determination of the distribution of urobilinogen in the feces and urine. Much of the recent work in this field has been done by Watson and by Steigmann and Dyniewicz. 5 • 6 The methods are tedious and complicated but Watson has simplified the procedure to a great extent. Normally, much of the bilirubin of bile entering the intestine is changed to urobilinogen. Much of this is absorbed into the blood stream, passes to the liver and is reconverted to bilirubin. Under normal conditions, 30 to 200 mg. of urobilinogen is excreted in the feces during twenty-four hours. Small amounts appear in the urine (0.2 to 3 mg. in twenty-four hours). Persistent absence of urobilinogen from the feces (and urine) indicates that bile is not entering the intestine, and hence complete obstruction of the biliary passages. This is most frequently seen in cases of malignant tumors but occasionally occurs from a stone impacted in the common bile duct and from complete cicatricial stricture of the bile ducts. A marked increase in urobilinogen in the feces (up to 3,600 mg. in twenty-four hours) is
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979
encountered in the various hemolytic processes such as congenital hemolytic icterus and pernicious anemia. In diseases of the liver, such as acute hepatitis and active cirrhotic processes, the normal conversion of absorbed urobilinogen to bilirubin is interfered with and excessive amounts of urobilinogen appear in the urine. In such diseases, there is frequently a decreased amount of bilirubin entering the intestine and, consequently, less urobilinogen can be formed. Nevertheless, increased urinary urobilinogen is the usual finding. In inactive cirrhosis without jaundice, variable amounts of urobilinogen are found in the feces and urine. In these circumstances, interpretation of the results should be made with cauti,m. Summarizing, one may say that the presence of the substance in the feces indicates that the bile passages are not occluded and that excessive amounts in the urine indicate some impairment of liver function or the presence of an associated hemolytic process. Determination of the concentration of several of the constituents of the blood often gives valuable information on sufficiency or insufficiency of some of the functions of the liver . .The protein content of the serum normally is from 6 to 8 gm. per 100 c.c. In chronic parenchymatous disease of the liver and at times in the latter stages of acute parenchymatous disease, the concentration of the proteins of the blood is reduced. This is particularly true of the albumin fraction, thus reversing the albumin-globulin ratio. The value for the globulin may be increased. The decrease in concentration of the albumin apparently is due to failure of its fabrication by the liver rather than to deficiency of intake or absorption or an abnormal loss. The hypo-albuminemia produces a lowered colloid osmotic pressure of the blood and is a factor in the formation of edema and ascites, which frequently are encountered in chronic disease of the liver. Although hypo-albuminemia is frequently encountered in disease of the liver, there are many instances, particularly those in which the condition is of short or moderate duration, in which normal values for protein do exist and, in rare instances, an increased concentration of protein may be noted. Abnormalities of the partition of fats in the plasma are at times encountered in various types of jaundice. However, little is known of the significance of many of these changes. In general, there is a mild to moderate increase in all the fats of the plasma in obstructive jaundice. This is particularly true of cholesterol and cholesterol esters. The concentration of these two components may be decreased in acute parenchymatous disease of the liver and the ester fraction may be entirely absent. Such absence or a marked decrease of this fraction has been used as a prognostic sign, and often indicates a fatal outcome. Marked increase in the concentration of all of the plasma lipids occurs in certain rare chronic diseases of the liver, such as xanthomatous biliary cirrhosis as well as in other chronic metabolic disorders, such xanthoma tuberosum. In considering these changes in plasma lipids, it
980
J AMES
F. WEIR
is necessary that the presence of these metabolic diseases, as well as that of diabetes mellitus and nephrosis be excluded. Hypolipemia is also encountered in sprue and certain rare diseases of the pancreas. Elevation of the concentration of blood urea may occur if involvement of the kidneys develops in the course of disease of the biliary tract or liver. Occasionally, a lowering of the concentration of blood urea occurs in cases in which long-standing and chronic disease of the liver is approaching its terminal stage. Lowering of the concentration of blood sugar and associated hypoglycemic symptoms occasionally may be found in cases of chronic disease of the liver in the absence of any demonstrable pancreatic disorder. The concentration of serum calcium may be lowered in chronic obstructive jaundice, such as that due to cicatricial stenosis of the common bile duct. This apparently is due to deficiency of absorption of calcium caused by the acholia. Osteoporosis and spontaneous fractures (particularly of the vertebrae) are usually accompaniments. The concentration of plasma chlorides and the carbon dioxide combining power of the plasma are altered in cases of external biliary fistula especially if the fistula is complete or nearly complete, and in cases in which choloresis is present. Dehydration, hypochloremia, acidosis with their accompanying symptoms ensue. Several serologic tests based on alterations of the blood proteins, particularly the globulin fraction, have been developed. These include the Takata-Ara, the colloidal gold, and Hanger's cephalin-cholesterol flocculation tests. Only the last-mentioned test has any support at present. It has been advocated as a means of distinguishing obstructive jaundice from parenchymatous jaundice but these claims have not been substantiated. There is some evidence that it may be of some value in the estimation of the activity of hepatic disease. The laboratory procedures and tests of liver function that I have mentioned are the ones that we have commonly employed at the Clinic in the past few years. It is still our firm conviction that the diagnosis and management of diseases of the biliary tract and liver are largely based on clinical grounds and that the results of the various tests are rather limited in value. The tests do not distinguish obstructive jaundice from hepatogenous jaundice although they may furnish some assistance in such distinction. They do not distinguish acute disease of the liver from the chronic type. They do not indicate the cause of cellular disintegration. However, some estimate of functional capacity of the liver in several respects can be made from data afforded by several of the tests, particularly with their judicious repetition. They afford some aid in following the clinical course of disease of the liver, in determining the risk of surgical procedures, in directing preoperative and postoperative treatment and nonsurgical treatment, and in evaluating the prognosis. Although these tests have limitations, the data obtained are usually significant. Interpretation of results may be difficult, especially if deviations from normal are slight. Positive
TESTS OF LIVER FUNCTION
tests are much more significant than negative ones. Negative resuits du not necessarily exclude the existence of disease of the liver or disturbances of hepatic function. Furthermore in some cases, it seems impossible to correlate all the findings adequately. In such cases, particularly, clinical data should be followed and at a later date the tests may be repeated. Finally, the tests do have a value in teaching the physiology of the liver and in research. REFERENCES
1. Magath, T. B.: The serum bilirubin test. (Editorial.) J. Lab. & Clin. Med. 18: 974-980 (June) 1933.
2. Magath, T. B.: Technic of the prothrombin time determination. Am. J. Clin. Path. (Tech. Suppl.) 3:187-189 (Sept.) 1939. 3. Rafsky, H. A. and Newman, Bernard: Liver function tests in the aged. (The serum cholesterol partition, bromsulphalein, cephalin-flocculation and oral and intravenous hippuric acid tests.) Am. J. Digest. Dis. 10:66--69 (Feb.)
1943. 4. Sepulveda, B. and Osterberg, A. E.: Serum bilirubin: Its clinical importance and a recommended procedure for the determination of indirect and direct values. Proc. Staff Meet., Mayo Clin. 18:252-258 (July 28) 1943. 5. Steigmann, Frederick and Dyniewicz, Josephine M.: Studies of urobilinogen: I. The daily urobilinogen excretion in urine and feces in health and disease: an evaluation of Watson's and Sparkman's methods. Gastroenterology 1 :743-764 (Aug.) 1943. 6. Steigmann, Frederick and Dyniewicz, Josephine M.: Studies of urobilinogen: n. Quantitative urobilinogen determinations in the differential diagnosis of jaundice. Gastroenterology 1:855-875 (Sept.) 1943. 7. Thannhauser, J. S. and Andersen, E.: Methodik der quantitativen Bilirubinbestimmung im menschlichen Serum; iiber die Ehrlich-Proscher'sche Reaktion. Deutsches Arch. f. klin. Med. 137:179-186, 1921. 8. 'Varson, C. J.: Studies of urobilinogen. I. An improved method for the quantitative estimation of urobilinogen in urine and feces. Am. J. Clin. Path. 6:458-475 (Sepf.) 1936.
lAMES F. WEIR THE diagnosis and management of diseases of the liver and biliary tract fundamentally rest on the evaluation of clinical data. The procedures have been established through long periods of clinical practice and have been reasonably satisfactory. There are certain instances in which a clinical diagnosis cannot be made with certainty. In some such cases, the patients have been denied the possible benefits of surgical exploration; in other cases surgical procedures have been employed to the detriment of the patient, particularly if parenchymatous disease of the liver has been present. During the past two or three decades, much new knowledge concerning the physiology of the liver has been developed and, as a corollary of this, attempts have been made to determine the nature, extent and significance of functional abnormalities in various types of disease of the liver or biliary tract. This has led to the development and use of many tests of liver function. In the use of such tests, attempts have been made to apply them to the differential diagnosis and management of such diseases with, unfortunately, an unwarranted halo of importance and significance. Actually, these tests are somewhat limited in value yet no apologies are needed for their judicious employment. Such tests presuppose a knowledge of the functions of the liver and there are many of these. Approximately thirty-three different, though related, functions of the liver cells have been listed and it is estimated that at least 100 chemical activities are known to occur in the liver. In view of this, the following question may be asked, "What is a liver function test?" One answer, elementary and facetious, might be, "Some laboratory procedure that reveals something wrong with the liver." That something is wrong is usually known before employment of the procedure, from clinical observation, such as enlargement of the liver, jaundice or ascites. This answer may be satisfactory in cases in which clinical evidence of disease is absent. Perhaps a better answer would be, "A procedure that shows a disturbance, and perhaps the degree of such disturbance, in some particular function of the liver." Obviously, in view of the many functions of the liver, its known large reserve and great regenerative capacity, no "miracle" test can be expeeted, that is, no single test can be anticipated that will determine deviations from normal of even a few major functions of the organ, let alone all of them. Some authors claim that the chief value of these tests is the detection of early functional disturbance or focal disease such as carcinoma or abscess. Because of the known large reserve capacity, severe and ex973
974
J AMES
F. WEIR
tensive damage is necessary before positive tests may be obtained in such conditions. Consequently, some physicians would say that the tests lack sensitivity. Others claim that the tests are too sensitive. Positive results may be obtained but no clinical signs of disease of the liver may be apparent or develop. Rafsky, and Newman, for example, have reported a considerable group of aged persons on whom positive tests were obtained in the absence of clinical evidence of disease of the liver. The Question may then be asked, "Is a positive test an indication of disease of the liver or only of a dysfunction (as diarrhea in a case of irrit:lhle colon), and if it is an indication nf dvsfunction. what is its significance?" Answer to this question is difficult to !live. The significance of minor deviations from normal are particularly difficult to evaluate. The results must always be interpreted in association with other availahle data. In some instances, the findings are significant; in others, correlation of the finding's is impossihle. If tests are to be used clinicallv, they must be readily performed, reliable, and give some worth-while information from a diagnostic, therapeutic or prognostic standpoint. In addition, the results must be capable of being- interpreted easily. Probablv about seventy-five to 100 tests for liver function have been devised. The large number of these that have been used and discarded is evidence of their failure to meet the criteria which I have mentioned. The multitude of tests and the claims and counterclaims made in regard to them have created a very confusing situation, particularly for the g'eneral practitioner. It would seem well, therefore, to consider a few of the more simple procedures and their practical application. In such a consideration, certain facts should be pointed out. First, diagnostic procedures should be distinguished from tests for liver function; thus, duodenal drainage and examination of the stools for bile are best classified as diagnostic procedures because they are primarily used to determine the patency of the bile ducts. Such procedures leave much to be desired. Second, there are two groups of cases in which tests of liver function are commonly used (1) cases of suspected liver disease in which jaundice is absent, and (2) cases of jaundice. In the first group, the dye excretion tests and van den Bergh test are particularly valuable. In the second group, it is necessary to determine whether the jaundice is hemolytic, obstructive or hepatogenous. Hemolytic jaundice is usually readily distinguished by clinical and hematologic studies, and the van den Bergh test is also helpful. The last two types of jaundice usually are distinguished by clinical study, chiefly by the history in regard to pain or exposure to infection and hepatotoxins, by physical evidence of abdominal tumors, and by employment of diagnostic procedures to determine the patency of bile ducts. Some aid in this distinction may be obtained from the use of some of the tests of liver function, such as the distribution of urobilinogen in the urine and feces, the van den
TESTS OF LIVER FUNCTION
975
Bergh test, the galactose tolerance test and the partition of cholesterol in the plasma. This, then, is the first practical use of tests of liver function, that is, assistance in the differential diagnosis of various types of jaundice. Their second use is for the confirmation of the presence of liver damage in cases in which it is suspected and the evaluation of the extent of liver damage in cases in which the presence of disease of the liver or biliary tract is known. The qualitative and quantitative determination of serum bilirubin by the van den Bergh test, dye excretion tests, hippuric acid synthesis, galactose tolerance test, blood prothrombin time, partition of cholesterol in the blood and the value for the protein in the blood are procedures most commonly used in the study of such conditions but they give information of variable value. Many of these are not truly functional tests but laboratory procedures which throw some light on one or another aspect of disease of the liver. In the interpretation of the results of such tests in the presence of jaundice, it should be remembered that there is seldom any case of jaundice pure in type. For example, severe hepatic damage may occur in cases of obstruction of the biliary tract or in hemolytic processes, and the tests may be valuable in these types of disease as well as in primary parenchymatous disease of the liver. Third, and finally, there is the question of progression of activity of disease of the liver or recovery. Repetition of some of these tests aids in the determination of this point. Some physicians claim that the cephalin-cholesterol flocculation test is of particular value as such an indicator. Some of these tests will now be considered individually and in more detail. The first of these is the van den Bergh test, which is used to determine the type and quantity of bilirubin in the serum. It also can De applied to the duodenal contents and urine. Various modifications of the original test have been developed. 1 Prior to two years ago, we employed the technic of Thannhauser and Andersen but since then the technic of Sepulveda and Osterberg has been used. This permits the quantitative determination separately of the indirect and direct reacting bilirubin in the serum in any given case. In health, the value for the bilirubin is less than 0.6 mg. per 100 C.c. of serum and it reacts indirectly with Ehrlich's diazo reagent. In pathologic states, such as pernicious anemia, congenital hemolytic icterus, other hemolytic conditions and familial hyperbilirubinemia, the reaction is characteristica,lly indirect. In these conditions the concentration of the bilirubin in the serum usually is variably increased. In many cases of cirrhosis, metastatic tumors of the liver and other pathologic conditions of tbe liver without evident jaundice, the reaction to the van den Bergh test may be indirect. This type of reaction does not exclude any of these pathologic processes. Pigmentation, such as that due to carotene and atabrine, must be distinguished from icterus. In cases in which pig-
976
JAMES F. WEIR
mentation is due to carotene or atabrine, the concentration of serum bilirubin is normal and the reaction is indirect. A direct reaction always is a pathologic finding and is commonly encountered in the various forms of obstructive and hepatogenous jaundice. Such a reaction also is present in the latent jaundice at times seen after biliary colic, in acute cholecystitis, in metastatic tumors of the liver and in many cases of chronic and even acute hepatitis. In the presence of a hemolytic process, a direct reaction indicates an associated hepatic or biliary disorder. Little attention from a clinical standpoint need be given to biphasic or delayed direct reactions. The quantitative determination shows the degree of retention of bilirubin and its fluctuations from time to time. In many instances estimations every few days are necessary to determine whether the jaundice is stationary, increasing or decreasing in severity. In obstructive jaundice due to stone in the common bile duct, the average value for the serum bilirubin is 10 to 15 mg. per 100 C.c. Fluctuations frequently occur and there is a general tendency for the bilirubinemia to decrease. In neoplastic obstruction of the common bile duct, average values are higher, that is 20 to 30 mg. per 100 C.c. Fluctuations seldom occur and the jaundice is persistent. In parenchymatous disease of the liver, the degree of icterus is variable. In some cases, very mild degrees of bilirubinemia are encountered. On the other hand, some of the highest concentrations (70 to 100 mg. per 100 c.c.) encountered are due to this type of disease of the liver. The higher concentrations are indicative of more serious disorganization of the liver. In obstructive jaundice, similarly high concentrations (that is, much above the average concentration) are indicative of an associated hepatic degeneration. The icterus index is a simple and approximate method of following the degree and variations of bilirubinemia in any case of icterus and is used in many laboratories. Dye excretion tests are frequently used in the determination of liver function. Various dyes may be used, such as phenoltetrachlorophthalein, tetraiodophenolphthalein, Bengal red and bromsulfalein. These tests depend on the rate of disappearance of the dye from the blood stream. It has been our custom at the Clinic to use bromsulfalein. We inject 5 mg. of the dye per kilogram of body weight of the patient, remove a specimen of blood in one hour and determine the amount of dye remaining in the blood with a block colorimeter. Normally, little remains in the blood at the end of one hour. The presence of 40 per cent or more indicates a high degree of retention. Various modifications of this technic have been made but I doubt their practical importance. This is one of the best tests of liver function in cases in which jaundice is not present. The presence of jaundice is a contraindication to the use of this test. When jaundice is present, the test merely shows the impairment to be expected, as indicated by retention of bilirubin and furnishes no additional information. The test is to be used in cases
TESTS OF LIVER
FU~CTTON
977
in which disease of the liver is suspected when jaundice is absent. These diseases include enlargement of the liver and spleen, suspected metastatic tumor or abscess, ascites and toxic hepatitis such as may occur in cases of exophthalmic go iter, toxemias of pregnancy, many infections, poisoning with various therapeutic and industrial chemicals and many other conditions. Normal values usually are obtained in abdominal enlargement due to ovarian cysts, peritoneal carcinomatosis and tuberculous peritonitis. In cardiac disease with decompensation and chronic passive congestion, a moderate to high degree of retention of the dye usually is encountered. Ascites due to portal obstruction from canalized portal thrombosis usually gives normal values but some disturbance of excretion of the dye occasionally is found. In cases of suspected metastatic tml10rs or abscess of the liver, and occasionally in cirrhosis the values may be normal. In cases of splenomegaly in which splenectomy is being considered, high degrees of retention may be encountered, indicating a high surgical risk and a poor prognosis. In some cases in which the patients are in reasonably good health mild degrees of retention of the dye occasionally are encountered but no definite disease of the liver can be demonstrated and no satisfactory cause for the retention can be found. In some instances, dehydration and starvation may be etiologic factors. In other instances, these are absent but, in spite of lack of any explanation, the findings are probably of some significance. The hippuric acid test depends on the ability of the liver to furnish glycine to permit the synthesis of hippuric acid from benzoic acid (administered as sodium benzoate). It is claimed to be a test of the detoxicating function of the liver. The presence of disease of the kidney should be excluded before this test is employed. The amount of hippuric acid excreted in the urine is determined gravimetrically. After administration of 6 gm. of sodium benzoate, at least 2.5 to 3 gm. as hippuric acid should appear in the urine in four hours. Less than 1.5 gm. is indicative of impairment of liver function. The test does not aid in distinguishing obstructive jaundice from hepatogenous jaundice. In cases in which jaundice is absent, the results parallel those obtained with the bromsulfalein test. An intravenous method of performing the test has been devised and is employed by many physicians. The galactose tolerance test is one of the sugar tolerance tests used to determine deviations of the normal function of the liver to metabolize carbohydrates. Galactose is chiefly utilized by the liver, and in the performance of the test it may be administered orally or intravenously. When the galactose is administered intravenously, the test is spoken of as the "galactose clearance test." In using the test it is necessary that the presence of diabetes mellitus be excluded. If oral administration is employed, 40 gm. of galactose in 500 C.c. of water is given and the urine is collected over a five hour period and examined for sugar. Normally, less than 3 gm. of sugar should be excreted. Excretion of more than 5 gm.
978
.T AMES
F. WEIR
is indicative of severe hepatic damage. In acute hepatitis, positive tests may be obtained early (less than two weeks). In obstructive jaundice, the test may become positive at the end of two weeks. In our experience at the Clinic, the test has been found mildly positive in 50 per cent of cases of malignant obstruction and in 25 per cent of cases of obstruction due to stone or cicatricial stricture. Normal results do not exclude the possibility of parenchymatous origin of jaundice. The prothrombin time should be determined in all cases of jaundice, particularly if the disease is surgical, in which case it should be determined during the preoperative and postoperative periods. In the laboratories of the Clinic, the method of Quick, as modified by Magath,2 is used. With this method, the normal values are eighteen to twenty seconds. Elevation of the prothrombin time may be due to deficient intake, deficient absorption or deficient utilization of vitamin K. Deficiency of intake is seldom a factor in jaundiced conditions. A deficiency of absorption is common in the acholia of jaundice. It also occurs in intestinal disorders such as those associated with sprue. Deficiency of utilization of vitamin K is encountered chiefly in parenchymatous disease of the liver. In this disease, the vitamin K is not converted into prothrombin, and if the damage to the liver is severe, administration of vitamin K will not control the hypoprothrombinemia and its associated hemorrhagic manifestations. The degree of this failure is an indication of hepatic insufficiency. Data accumulated in the past two years indicate that larger doses than former.ly employed may be advisable when small doses are ineffective. Vitamin K should be administered in all cases of jaundice, whether or not the prothrombin time is prolonged. This is particularly true in cases in which operation is to be performed and the administration should be continued during the postoperative period. Another test is the determination of the distribution of urobilinogen in the feces and urine. Much of the recent work in this field has been done by Watson and by Steigmann and Dyniewicz. 5 • 6 The methods are tedious and complicated but Watson has simplified the procedure to a great extent. Normally, much of the bilirubin of bile entering the intestine is changed to urobilinogen. Much of this is absorbed into the blood stream, passes to the liver and is reconverted to bilirubin. Under normal conditions, 30 to 200 mg. of urobilinogen is excreted in the feces during twenty-four hours. Small amounts appear in the urine (0.2 to 3 mg. in twenty-four hours). Persistent absence of urobilinogen from the feces (and urine) indicates that bile is not entering the intestine, and hence complete obstruction of the biliary passages. This is most frequently seen in cases of malignant tumors but occasionally occurs from a stone impacted in the common bile duct and from complete cicatricial stricture of the bile ducts. A marked increase in urobilinogen in the feces (up to 3,600 mg. in twenty-four hours) is
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979
encountered in the various hemolytic processes such as congenital hemolytic icterus and pernicious anemia. In diseases of the liver, such as acute hepatitis and active cirrhotic processes, the normal conversion of absorbed urobilinogen to bilirubin is interfered with and excessive amounts of urobilinogen appear in the urine. In such diseases, there is frequently a decreased amount of bilirubin entering the intestine and, consequently, less urobilinogen can be formed. Nevertheless, increased urinary urobilinogen is the usual finding. In inactive cirrhosis without jaundice, variable amounts of urobilinogen are found in the feces and urine. In these circumstances, interpretation of the results should be made with cauti,m. Summarizing, one may say that the presence of the substance in the feces indicates that the bile passages are not occluded and that excessive amounts in the urine indicate some impairment of liver function or the presence of an associated hemolytic process. Determination of the concentration of several of the constituents of the blood often gives valuable information on sufficiency or insufficiency of some of the functions of the liver . .The protein content of the serum normally is from 6 to 8 gm. per 100 c.c. In chronic parenchymatous disease of the liver and at times in the latter stages of acute parenchymatous disease, the concentration of the proteins of the blood is reduced. This is particularly true of the albumin fraction, thus reversing the albumin-globulin ratio. The value for the globulin may be increased. The decrease in concentration of the albumin apparently is due to failure of its fabrication by the liver rather than to deficiency of intake or absorption or an abnormal loss. The hypo-albuminemia produces a lowered colloid osmotic pressure of the blood and is a factor in the formation of edema and ascites, which frequently are encountered in chronic disease of the liver. Although hypo-albuminemia is frequently encountered in disease of the liver, there are many instances, particularly those in which the condition is of short or moderate duration, in which normal values for protein do exist and, in rare instances, an increased concentration of protein may be noted. Abnormalities of the partition of fats in the plasma are at times encountered in various types of jaundice. However, little is known of the significance of many of these changes. In general, there is a mild to moderate increase in all the fats of the plasma in obstructive jaundice. This is particularly true of cholesterol and cholesterol esters. The concentration of these two components may be decreased in acute parenchymatous disease of the liver and the ester fraction may be entirely absent. Such absence or a marked decrease of this fraction has been used as a prognostic sign, and often indicates a fatal outcome. Marked increase in the concentration of all of the plasma lipids occurs in certain rare chronic diseases of the liver, such as xanthomatous biliary cirrhosis as well as in other chronic metabolic disorders, such xanthoma tuberosum. In considering these changes in plasma lipids, it
980
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F. WEIR
is necessary that the presence of these metabolic diseases, as well as that of diabetes mellitus and nephrosis be excluded. Hypolipemia is also encountered in sprue and certain rare diseases of the pancreas. Elevation of the concentration of blood urea may occur if involvement of the kidneys develops in the course of disease of the biliary tract or liver. Occasionally, a lowering of the concentration of blood urea occurs in cases in which long-standing and chronic disease of the liver is approaching its terminal stage. Lowering of the concentration of blood sugar and associated hypoglycemic symptoms occasionally may be found in cases of chronic disease of the liver in the absence of any demonstrable pancreatic disorder. The concentration of serum calcium may be lowered in chronic obstructive jaundice, such as that due to cicatricial stenosis of the common bile duct. This apparently is due to deficiency of absorption of calcium caused by the acholia. Osteoporosis and spontaneous fractures (particularly of the vertebrae) are usually accompaniments. The concentration of plasma chlorides and the carbon dioxide combining power of the plasma are altered in cases of external biliary fistula especially if the fistula is complete or nearly complete, and in cases in which choloresis is present. Dehydration, hypochloremia, acidosis with their accompanying symptoms ensue. Several serologic tests based on alterations of the blood proteins, particularly the globulin fraction, have been developed. These include the Takata-Ara, the colloidal gold, and Hanger's cephalin-cholesterol flocculation tests. Only the last-mentioned test has any support at present. It has been advocated as a means of distinguishing obstructive jaundice from parenchymatous jaundice but these claims have not been substantiated. There is some evidence that it may be of some value in the estimation of the activity of hepatic disease. The laboratory procedures and tests of liver function that I have mentioned are the ones that we have commonly employed at the Clinic in the past few years. It is still our firm conviction that the diagnosis and management of diseases of the biliary tract and liver are largely based on clinical grounds and that the results of the various tests are rather limited in value. The tests do not distinguish obstructive jaundice from hepatogenous jaundice although they may furnish some assistance in such distinction. They do not distinguish acute disease of the liver from the chronic type. They do not indicate the cause of cellular disintegration. However, some estimate of functional capacity of the liver in several respects can be made from data afforded by several of the tests, particularly with their judicious repetition. They afford some aid in following the clinical course of disease of the liver, in determining the risk of surgical procedures, in directing preoperative and postoperative treatment and nonsurgical treatment, and in evaluating the prognosis. Although these tests have limitations, the data obtained are usually significant. Interpretation of results may be difficult, especially if deviations from normal are slight. Positive
TESTS OF LIVER FUNCTION
tests are much more significant than negative ones. Negative resuits du not necessarily exclude the existence of disease of the liver or disturbances of hepatic function. Furthermore in some cases, it seems impossible to correlate all the findings adequately. In such cases, particularly, clinical data should be followed and at a later date the tests may be repeated. Finally, the tests do have a value in teaching the physiology of the liver and in research. REFERENCES
1. Magath, T. B.: The serum bilirubin test. (Editorial.) J. Lab. & Clin. Med. 18: 974-980 (June) 1933.
2. Magath, T. B.: Technic of the prothrombin time determination. Am. J. Clin. Path. (Tech. Suppl.) 3:187-189 (Sept.) 1939. 3. Rafsky, H. A. and Newman, Bernard: Liver function tests in the aged. (The serum cholesterol partition, bromsulphalein, cephalin-flocculation and oral and intravenous hippuric acid tests.) Am. J. Digest. Dis. 10:66--69 (Feb.)
1943. 4. Sepulveda, B. and Osterberg, A. E.: Serum bilirubin: Its clinical importance and a recommended procedure for the determination of indirect and direct values. Proc. Staff Meet., Mayo Clin. 18:252-258 (July 28) 1943. 5. Steigmann, Frederick and Dyniewicz, Josephine M.: Studies of urobilinogen: I. The daily urobilinogen excretion in urine and feces in health and disease: an evaluation of Watson's and Sparkman's methods. Gastroenterology 1 :743-764 (Aug.) 1943. 6. Steigmann, Frederick and Dyniewicz, Josephine M.: Studies of urobilinogen: n. Quantitative urobilinogen determinations in the differential diagnosis of jaundice. Gastroenterology 1:855-875 (Sept.) 1943. 7. Thannhauser, J. S. and Andersen, E.: Methodik der quantitativen Bilirubinbestimmung im menschlichen Serum; iiber die Ehrlich-Proscher'sche Reaktion. Deutsches Arch. f. klin. Med. 137:179-186, 1921. 8. 'Varson, C. J.: Studies of urobilinogen. I. An improved method for the quantitative estimation of urobilinogen in urine and feces. Am. J. Clin. Path. 6:458-475 (Sepf.) 1936.