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Plasma amino acid analysis in the differential diagnosis of jaundice
Plasma Amino Acid Analysis in the Differential Diagnosis of Jaundice
Herbert Freund, MD,* Cincinnati, Ohio J. Howard James, BS, Cincinnati, Ohio William Brenner, BS, Cincinnati, Ohio Josef E. Fischer, MD, Cincinnati, Ohio
The distinction between obstructive and hepatocellular jaundice (surgical and medical jaundice) may be one of the most difficult differential diagnoses, even with the use of current clinical and laboratory procedures. Accurate diagnosis will avoid unnecessary operation or undue delay of necessary surgery. Direct reading hyperbilirubinemia and elevated levels of liver enzymes can be present in both disorders, with a nonspecific pattern occurring in up to 40 percent of patients [I]. Tests and procedures that have been and still are used to differentiate surgical from medical jaundice are numerous, but none are completely reliable, and many require invasive diagnostic procedures or exploratory laparotomy [2]. Recent studies have focused on the plasma amino acid pattern as a sensitive and reliable indicator of hepatic function in patients with liver disease [3-51 or derangements in hepatic function associated with other pathologic processes [6]. The purpose of this study is to evaluate changes in plasma amino acids in hepatobiliary disorders and to compare the plasma amino acid pattern in obstructive and hepatocellular jaundice. The results, both in rats and in patients, suggest that in obstructive jaundice no major deviations from the normal plasma amino acid pattern occur. This pattern is easily distinguishable from the plasma amino acid pattern occurring in various forms of hepatitis, in which hyperaminoacidemia occurs with increased From the Department of Surgery, University of Cincinnati Medical Center and Cincinnati General Hospital, Cincinnati, Ohio. This study was supported in part by Grants AM-15347 and AM-19124 from the U.S. Public Health Service, Bethesda, Maryland. * Present address: Department of Surgery, Hebrew University Medical Center, Jerusalem, Israel. Presented at the 20th Annual Meeting of the Society for Surgery of the Alimentary Tract, New Orleans, Louisiana, May 22-23, 1979. Reprint requests should be addressed to Josef E. Fischer, MD, Department of Surgery, University of Cincinnati Medical Center, 231 Bethesda Avenue, Cincinnati, Ohio 45267.
142
levels of all amino acids except the branched chain amino acids, which remain at near-normal concentrations. Material and Methods Experimental
Twenty-four Sprague-Dawley rats weighing 250 to 300 g were divided into two groups of 12 rats each. Group I animals underwent ligation of the common bile duct under pentobarbital anesthesia. The bile duct was ligated just distal to the junction of the hepatic ducts and divided between ligatures. Two weeks after bile duct ligation the animals were killed by decapitation and blood was collected for determination of plasma amino acid patterns and bilirubin, serum glutamic oxaloacetic transaminase and alkaline phosphatase levels. Group II consisted of 12 rats receiving repeated intraperitoneal injections of D-( +)-galactosamine hydrochloride (Sigma Chemical). Animals received six doses of 250 mg/‘kg each during a 24 hour period (total of 1.5 g/kg) and were killed 24 hours after receiving the last dose [7]. Blood was collected for determination of amino acid patterns and bilirubin, serum glutamic oxaloacetic transaminase and alkaline phosphatase levels. Livers were excised and prepared for histopathologic examination. Clinical Fourteen patients admitted for the evaluation of jaundice were examined. The patients included 10 men and 4 women, aged 23 to 80 years (mean 59 f 6). Patients were divided into two groups based on the cause of jaundice. Group I consisted of seven patients with obstructive jaundice proved at surgery; five had malignant obstruction of the biliary system and two had choledocholithiasis. Group II consisted of seven patients with presumably previously normal livers and acute onset hepatitis of different causes: viral (three patients), halothane-induced (three patients) and toxic (one patient). Diagnosis was
The American
Journal of Surgery
Plasma
TABLE l
Results of Bilirubin and Liver Function Tests in Rats Subjected to Common Bile Duct Ligation (Obstructive Jaundice) and GalactosaniineInduced Hepatocellular Jaundice* Obstructive Jaundice
~___Bi irubin (mg/lOO ml) Serum glutamic oxaloacetic transaminase (p/ml) AlKaline phosphatase (j.Uml)
Acids, in Jaundice
o Hepatwllulor Jaundice 0 ObstructiveJaundice
r
0
Hepatocellular Jaundice
10.8 f 0.8+ 488 f 93
3.5 f 0.7 > 10,000~
958 f 88
304 f 31
* Values are expressed as mean f
2000
Amino
standard
error of the
mean + p <0.05. 1 p
TABLE II
Results of Bilirubin, Albumin, Cholesterol and Liver Function Tests in Seven Patients With Obstructive Jaundice a?d Seven Patients With Hepatocellular Jaundice*
Bilirubin (mg/ 100 ml) Serum glutamic oxaloacetic transaminase (p/ml) Alkaline phosphatase (p/ml) Albumin (g/100 ml) Cholesterol (mg/iOO ml)
* Values are expressed mean. +p <0.05.
Obstructive Jaundice
Hepatocellular Jaundice
17 f 4.4 138 f 27
22.6 f 4.7 942 f 480+
50f 17 2.8 f 0.3
54.6 f 3.2 2.7 f 0.3
218 f 104
96f31
as mean f
standard error of the
ked on the patient’s history, clinical characteristics livsr biopsy or autopsy findings. ‘lasma amino acid determinations were carried
and out
using a Beckman MB-121 amino acid analyzer on the supernatant of plasma rendered protein-free by precipitation with 5 percent sulfosalicylic acid. All values are expressed as mean f standard error of the mean, and statistical analyses are based on Student’s t test.
THR
LEU MET LYS
~s,‘IT,FwTfwJ
TAU GLU PRO
ALA
CfrN
ARG
(yGLYsTY;YSJ
L-
Figure 1. Plasma amino acid patterns ( expressed as percent of normal) in rats with hepatocellular jaundice and obstructive jaundice. ALA = alanine; ARG = arginine; ASP = aspartic acid; C/T = citrulline; GLU = glutamine; GL Y = giycine; HIS = histidine; ILE = isoleucine; LEU = leucihe; LYS = lysine; MET = methionine; ORN = ornithine; PHE = phenylalanine; PRO = proline; SER = serine; TAU = taurine; THR = threonine; TRP = tryptophan; TYR = tyrosine; VAL = valine.
The plasma free amino acid pattern in the two jaundiced groups differed markedly. While the obstructive jaundice group had an almost normal amino acid pattern, except for high levels of ornithine and low levels of arginine and aspartic acid, the hepatocellular jaundice group had grossly elevated levels of all amino acids except arginine, which was almost nondetectable (Figure 1).
Results Experimental
Both groups of rats (galactosamine-induced hepaGc necrosis and common bile duct ligation) had jaundice and elevated levels of serum glutamic oxaloacetic transaminase and alkaline phosphatase. Although t.he increase in alkaline phosphatase level was greater in the obstructive jaundice group, the difference was not statistically significant, whereas the level of serum glutamic oxaloacetic transaminase was significantly higher in the hepatocellular jaundice group than in the rat,s wit,h obstructive jaundice (Table I).
Volume
139,
January
1990
Bilirubin, albumin, cholesterol and liver enzyme levels in both groups of patients studied are presented in Table II. There were no statistically significant differences in bilirubin, cholesterol, albumin and alkaline phosphatase levels among patients with obstructive and hepatocellular jaundice. Serum glutamic loxaloacetic transaminase levels, were increased significantly more in the hepatocellular jaundice group than in the obstructive jaundice group. The plasma free amino acid patterns in the two groups of patients also differed markedly. The group with acute-onset hepatitis exhibited grossby elevated
143
Freund et al
o Hepotocellular Jaundice l Obstructive Jaundice
TAU
L!w~~~
GLU
rN;;_-&
PRO ALA
ORN ARG
CYS
C’T ,
Figure 2. Plasma amino acid patterns (expressed as percent of normal) in seven patients with acute hepatitis and seven patients with surgically proved obstructive jaundice. CYS = cystine; other abbreviations as in Figure 1.
levels of all amino acids, particularly methionine, phenylalanine, lysine, glutamine, proline, tyrosine and aspartic acid, except for the branched chain amino acids, which were present at normal levels (Figure 2). The group with obstructive jaundice presented with an essentially normal amino acid pattern except for mild increases in methionine, aspartic acid and glutamine levels (Figure 2); these levels, however, were significantly lower than in the hepatitis patients. Comments
Biliary tract obstruction is a common clinical condition requiring surgical intervention for relief. The presenting symptom is usually jaundice, which may also be the presenting symptom of hepatocellular damage. Although it is important to differentiate extrahepatic obstruction from hepatocellular causes of jaundice and to avoid unnecessary surgery, it is equally important not to delay surgery. The correct approach to the jaundiced patient is to arrive at as accurate a diagnosis as possible and, if necessary, to expeditiously relieve the obstruction. Surgical (obstructive) jaundice can usually be distinguished from medical (hepatocellular) jaundice by the relatively simple means of history, physical examination and liver function tests. However, these tests fail to differentiate extrahepatic obstruction from hepatocellular disease in up to 40 percent of patients [I], in whom the distinction requires more extensive diagnostic procedures. In an attempt to reduce the number of jaundiced patients in need of
144
complex and expensive diagnostic procedures, we investigated the use of the plasma amino acid pattern as a simple, relatively inexpensive and noninvasive addition to the initial history, physical examination and liver function tests performed in every jaundiced patient. Characteristic alterations in plasma amino acid patterns have been well documented in chronic liver disease, hepatic insufficiency of different causes and hepatic encephalopathy [3,4,8-l 01, which is not surprising since the liver is the primary site for the metabolism of many amino acids. The amino acid pattern in patients with chronic liver disease and experimental animals with chronic hepatic insufficiency generally includes elevated levels of the aromatic amino acids phenylalanine and tyrosine, free but not necessarily total tryptophan, methionine, histidine, glutamine and aspartic acid, combined with low levels (as low as 40 to 50 percent of normal) of the branched chain amino acids [3,4,8-131. In acute hepatic failure due to hepatitis or hepatic necrosis, all amino acids were markedly elevated except for the branched chain amino acids, which were present at near-normal levels. The greatest increase was noted in the aromatic amino acids phenylalanine and tyrosine (up to 700 percent of normal) as well as in methionine, glutamate and aspartate [4,13]. In this category of liver disease the severity of amino acid abnormalities correlated well with increases in serum glutamic oxaloacetic transaminase [4], indicating that the major source of the hyperaminoacidemia is destroyed hepatic parenchyma [5]. Because hepatic parenchymal damage occurs only late in the course of biliary obstruction, it is to be expected that obstructive jaundice will result in only mild abnormalities in plasma amino acid patterns, as was seen in experimental animals. The rats with centrolobular hepatic necrosis secondary to D-(+)galactosamine presented with an amino acid pattern similar to the one describe.d in dogs [13] and humans [4] with acute hepatic failure, with all amino acid levels increased significantly. In contrast, the group subjected to common bile duct ligation had a nearnormal amino acid pattern. The two groups of patients studied presented a similar, although not identical, relation. Patients with hepatocellular jaundice exhibited increased levels of all plasma amino acids except for the branched chain amino acids (leucine, isoleucine and valine) and taurine. Mean levels of methionine, phenylalanine, lysine, aspartic acid, proline and tyrosine were elevated to 400 percent to 1,000 percent of normal. In comparison, the obstructive jaundice group presented an essentially normal amino acid pattern with
The American
Journal
of Surgery
Plasma
only mild increases (up to maximum of 200 percent of normal) in methionine, phenylalanine, aspartic acid and glutamine levels. From our animal experiments and clinical data, there appear to be very clear qualitative and quantitative differences in the plasma amino acid patterns of hepatocellular and obstructive jaundice, with the latter having an almost normal pattern. The results suggest that the plasma amino acid pattern may be a useful adjunct in the differential diagnosis of jaundice, decreasing the number of patien1.s who need invasive diagnostic procedures or exploratory laparotomy. Summary
Distinguishing between obstructive (surgical) and hepatocellular (medical) jaundice is sometimes impossible using the relatively simple diagnostic means of’ history, physical examination and liver function tests. In an attempt to reduce the number of jaundiced patients in need of complex and expensive diagnostic procedures, we investigated the use of the plasma amino acid pattern in the diagnosis of jaundice. *Jaundiced rats with galactosamine-induced hepatitis and seven patients with acute onset hepati :is presented a plasma amino acid pattern in which most all amino acid levels were elevated except for arginine in the rat and branched chain amino acids in the patients. Rats jaundiced due to common bile duct ligat.ion and seven patients with -obstructive jaundice proved at surgery exhibited a near-normal amino acid pattern. These experimental and clinical data demonst,rat,e very clear qualitative and quanti :ative differences in plasma amino acid patterns of ht>patotellular and obstruct,ive jaundice, with the latter rxhihit,ing an almost-normal pattern. We suggest. the use of’ the plasma amino acid pattern as a useful adjunct in t,he differential diagnosis of medical and surgical jaundice. References 1. Stern RB, Knill-Jones RP, Williams R. Pitfalls in the diagnosis of jaundice due to carcinoma of the pancreas or biliary tree. Br Med J 1973; 1:533-5. 2. Eiseman B, Escajeda D. Modern diagnosis of jaundice. Arch Sura 1978: 113:555-6. 3. Fischer JE, Funovics JM, Aguirre A, James JH, Keane JM, Wesdorp RIG, et al. The role of plasma amino acids in hepatic encephalopathy. Surgery 1975; 78:276-90. 4. Rosen HM, Yoshimura N, Hodgman JM, Fischer JE. Plasma amino acid patterns in hepatic encephalopathy of differing etiology. Gastroenterology 1977; 72:483-7. 5. Fischer JE, Rosen HM, Ebeid AM, James JH, Keane JM, Soeters PB. The effect of normalization of plasma amino acids on hepatic encephalopathy in man. Surgery 1976; 80:77-91. 6. Freund H. Ryan JA, Fischer JE. Amino acid derangement in
Volume
139,
January
1980
Amino
Acids
in Jaundice
patients with sepsis. Ann Surg 1978; 188:423-30.
7. Keppler D, Lesch R, Reutter W, Decker K. Expelrimental hepatitis induced by D-Galactosamine. Exp Mol Pathol 1968; 9:279-90. 8. lob V, Coon WW, Sloan H. Altered clearance of free amino acids from plasma of patients with cirrhosis of the liver. J Surg Res 1966; 6:233-g. 9. Wu CT, Bollman G, Butt HR. Changes in free amino acids in the plasma during hepatic coma. J Clin Invest 1955; 34:845-
9. 10. lber FL, Rosen H. Levenson SM. Chalmers TC. The plasma amino acids in patients with liver failure. J Lab Clin Med 1957; 50:417-25. 11. Smith AR, Rossi-Fanelli F, Ziparo V, James JH, Perelle BA, Fischer JE. Alterations in plasma and CSF amino acids, amines and metabolites in hepatic coma. Ann Surg 1978; 187:343-50. 12. Svec MH. Freeman S. Effect of imoaired heoatic circulation on plasma free amino acids of iogs. Am’J Physiol 1949; 159: 357-64. 13. Aguire AM, Yoshimura N, Westman J, Fischer JE. Plasma amino acids in dogs with two experimental forms of liver damage. J Surg Res 1974; 16:339-45.
Discussion
Bernard Gardner (Brooklyn, NY): You showed some very exciting differences in the levels in these two groups of patients. Do you have any information on .the number of determinations that may be used t,o derive standard deviations or p values? Laurie
M. Blendis (Toronto, Canada): Some years ago told us that amino acid patterns in portasystemic encephalopathy were specific and highly related to the etiology of that disease. Since then we have llearned that these patterns are not specific, that they can be found in nonencephalopathic patients and that they may be related neither to the disease nor its complications but merely to nutritional intake. Thus I have a deja uu feeling about this paper. Were the differences in amino acid patterns in these patients with liver disease related to (1) total calorie intake, (2) total protein intake, (3) the relation of protein to calorie or (4) the relation to the actual amino acid intake? In the diagnosis of obstructive jaundice, clinical and ultrasound tests can be most helpful, and although they are invasive, they should not be dangerous if performed by physicians familiar with them. What is the cost of performing a very difficult estimation of amino acids requiring expensive amino analyzers, and how do the results compare with simple endoscopic retrograde cholangiography or percutaneous cholangiography?
Dr. Fischer
George L. Blackburn (Boston, MA): I wonder if Dr. Fischer could isolate one of the amino acids that might reflect the entire pattern, and perhaps suggest some sort of challenge test, such as a clearance test, that might help differentiate the two types of liver disease. I noted that the groups were quite jaundiced. I think bilirubin levels were 20. Would amino acid profiles be as sensitive if either obstructive
disease or hepatitis
were less advanced?
145
Freund et al
Josef E. Fischer (closing): We gave a preliminary report on this particular technique, and as we continue to evaluate more patients, the results seem to be valid. One of the problems with amino acid analysis is the significant variation in results among laboratories. Although Dr. Blendis stated that the ljlasma amino acid pattern in hepatic encephalopathy does not indicate different disease states in encephalopathy, I as well as others would strongly disagree with him. Caloric intake and other nutritional influences on plasma amino acid patterns are well known. Gi,ven those variations, it is perfectly clear that even among the minor variations, many of which emerged in the dispute over Charlie Lieber’s use of alpha amino butyric acid, one thing that is most important-a major influence as opposed to the comparatively minor influence of caloric intake or starvation-is hepatic function. On a scale of 10, caloric intake may rate 1 or 2, but the sick liver may rate ‘7 to 10.
146
Dr. Blackburn’s questions are very cogent, but I think we will have to wait for more data before we can answer them. As we explore the problem of recognizing patterns of plasma amino acids in different diseases, it appears that one can take a whole group of amino acids and through discriminant and variant analysis, using a fairly high powered computer, focus in on one, two or possibly four amino acids that appear more important than the others. Dr. Freund has done this in sepsis, as reported in his article in the November issue of Annals of Surgery, and with a larger group of patients I feel we will have enough data to similarly report on jaundice. Superficial examination of the data suggests that tyrosine remains an important amino acid, as Dr. Hal Conn described as early as 1967. The cost of plasma amino acid analysis is $62, and I doubt that anyone can perform endoscopic retrograde cholangiopancreatography for that amount.
The American Journal of Surgery
Herbert Freund, MD,* Cincinnati, Ohio J. Howard James, BS, Cincinnati, Ohio William Brenner, BS, Cincinnati, Ohio Josef E. Fischer, MD, Cincinnati, Ohio
The distinction between obstructive and hepatocellular jaundice (surgical and medical jaundice) may be one of the most difficult differential diagnoses, even with the use of current clinical and laboratory procedures. Accurate diagnosis will avoid unnecessary operation or undue delay of necessary surgery. Direct reading hyperbilirubinemia and elevated levels of liver enzymes can be present in both disorders, with a nonspecific pattern occurring in up to 40 percent of patients [I]. Tests and procedures that have been and still are used to differentiate surgical from medical jaundice are numerous, but none are completely reliable, and many require invasive diagnostic procedures or exploratory laparotomy [2]. Recent studies have focused on the plasma amino acid pattern as a sensitive and reliable indicator of hepatic function in patients with liver disease [3-51 or derangements in hepatic function associated with other pathologic processes [6]. The purpose of this study is to evaluate changes in plasma amino acids in hepatobiliary disorders and to compare the plasma amino acid pattern in obstructive and hepatocellular jaundice. The results, both in rats and in patients, suggest that in obstructive jaundice no major deviations from the normal plasma amino acid pattern occur. This pattern is easily distinguishable from the plasma amino acid pattern occurring in various forms of hepatitis, in which hyperaminoacidemia occurs with increased From the Department of Surgery, University of Cincinnati Medical Center and Cincinnati General Hospital, Cincinnati, Ohio. This study was supported in part by Grants AM-15347 and AM-19124 from the U.S. Public Health Service, Bethesda, Maryland. * Present address: Department of Surgery, Hebrew University Medical Center, Jerusalem, Israel. Presented at the 20th Annual Meeting of the Society for Surgery of the Alimentary Tract, New Orleans, Louisiana, May 22-23, 1979. Reprint requests should be addressed to Josef E. Fischer, MD, Department of Surgery, University of Cincinnati Medical Center, 231 Bethesda Avenue, Cincinnati, Ohio 45267.
142
levels of all amino acids except the branched chain amino acids, which remain at near-normal concentrations. Material and Methods Experimental
Twenty-four Sprague-Dawley rats weighing 250 to 300 g were divided into two groups of 12 rats each. Group I animals underwent ligation of the common bile duct under pentobarbital anesthesia. The bile duct was ligated just distal to the junction of the hepatic ducts and divided between ligatures. Two weeks after bile duct ligation the animals were killed by decapitation and blood was collected for determination of plasma amino acid patterns and bilirubin, serum glutamic oxaloacetic transaminase and alkaline phosphatase levels. Group II consisted of 12 rats receiving repeated intraperitoneal injections of D-( +)-galactosamine hydrochloride (Sigma Chemical). Animals received six doses of 250 mg/‘kg each during a 24 hour period (total of 1.5 g/kg) and were killed 24 hours after receiving the last dose [7]. Blood was collected for determination of amino acid patterns and bilirubin, serum glutamic oxaloacetic transaminase and alkaline phosphatase levels. Livers were excised and prepared for histopathologic examination. Clinical Fourteen patients admitted for the evaluation of jaundice were examined. The patients included 10 men and 4 women, aged 23 to 80 years (mean 59 f 6). Patients were divided into two groups based on the cause of jaundice. Group I consisted of seven patients with obstructive jaundice proved at surgery; five had malignant obstruction of the biliary system and two had choledocholithiasis. Group II consisted of seven patients with presumably previously normal livers and acute onset hepatitis of different causes: viral (three patients), halothane-induced (three patients) and toxic (one patient). Diagnosis was
The American
Journal of Surgery
Plasma
TABLE l
Results of Bilirubin and Liver Function Tests in Rats Subjected to Common Bile Duct Ligation (Obstructive Jaundice) and GalactosaniineInduced Hepatocellular Jaundice* Obstructive Jaundice
~___Bi irubin (mg/lOO ml) Serum glutamic oxaloacetic transaminase (p/ml) AlKaline phosphatase (j.Uml)
Acids, in Jaundice
o Hepatwllulor Jaundice 0 ObstructiveJaundice
r
0
Hepatocellular Jaundice
10.8 f 0.8+ 488 f 93
3.5 f 0.7 > 10,000~
958 f 88
304 f 31
* Values are expressed as mean f
2000
Amino
standard
error of the
mean + p <0.05. 1 p
TABLE II
Results of Bilirubin, Albumin, Cholesterol and Liver Function Tests in Seven Patients With Obstructive Jaundice a?d Seven Patients With Hepatocellular Jaundice*
Bilirubin (mg/ 100 ml) Serum glutamic oxaloacetic transaminase (p/ml) Alkaline phosphatase (p/ml) Albumin (g/100 ml) Cholesterol (mg/iOO ml)
* Values are expressed mean. +p <0.05.
Obstructive Jaundice
Hepatocellular Jaundice
17 f 4.4 138 f 27
22.6 f 4.7 942 f 480+
50f 17 2.8 f 0.3
54.6 f 3.2 2.7 f 0.3
218 f 104
96f31
as mean f
standard error of the
ked on the patient’s history, clinical characteristics livsr biopsy or autopsy findings. ‘lasma amino acid determinations were carried
and out
using a Beckman MB-121 amino acid analyzer on the supernatant of plasma rendered protein-free by precipitation with 5 percent sulfosalicylic acid. All values are expressed as mean f standard error of the mean, and statistical analyses are based on Student’s t test.
THR
LEU MET LYS
~s,‘IT,FwTfwJ
TAU GLU PRO
ALA
CfrN
ARG
(yGLYsTY;YSJ
L-
Figure 1. Plasma amino acid patterns ( expressed as percent of normal) in rats with hepatocellular jaundice and obstructive jaundice. ALA = alanine; ARG = arginine; ASP = aspartic acid; C/T = citrulline; GLU = glutamine; GL Y = giycine; HIS = histidine; ILE = isoleucine; LEU = leucihe; LYS = lysine; MET = methionine; ORN = ornithine; PHE = phenylalanine; PRO = proline; SER = serine; TAU = taurine; THR = threonine; TRP = tryptophan; TYR = tyrosine; VAL = valine.
The plasma free amino acid pattern in the two jaundiced groups differed markedly. While the obstructive jaundice group had an almost normal amino acid pattern, except for high levels of ornithine and low levels of arginine and aspartic acid, the hepatocellular jaundice group had grossly elevated levels of all amino acids except arginine, which was almost nondetectable (Figure 1).
Results Experimental
Both groups of rats (galactosamine-induced hepaGc necrosis and common bile duct ligation) had jaundice and elevated levels of serum glutamic oxaloacetic transaminase and alkaline phosphatase. Although t.he increase in alkaline phosphatase level was greater in the obstructive jaundice group, the difference was not statistically significant, whereas the level of serum glutamic oxaloacetic transaminase was significantly higher in the hepatocellular jaundice group than in the rat,s wit,h obstructive jaundice (Table I).
Volume
139,
January
1990
Bilirubin, albumin, cholesterol and liver enzyme levels in both groups of patients studied are presented in Table II. There were no statistically significant differences in bilirubin, cholesterol, albumin and alkaline phosphatase levels among patients with obstructive and hepatocellular jaundice. Serum glutamic loxaloacetic transaminase levels, were increased significantly more in the hepatocellular jaundice group than in the obstructive jaundice group. The plasma free amino acid patterns in the two groups of patients also differed markedly. The group with acute-onset hepatitis exhibited grossby elevated
143
Freund et al
o Hepotocellular Jaundice l Obstructive Jaundice
TAU
L!w~~~
GLU
rN;;_-&
PRO ALA
ORN ARG
CYS
C’T ,
Figure 2. Plasma amino acid patterns (expressed as percent of normal) in seven patients with acute hepatitis and seven patients with surgically proved obstructive jaundice. CYS = cystine; other abbreviations as in Figure 1.
levels of all amino acids, particularly methionine, phenylalanine, lysine, glutamine, proline, tyrosine and aspartic acid, except for the branched chain amino acids, which were present at normal levels (Figure 2). The group with obstructive jaundice presented with an essentially normal amino acid pattern except for mild increases in methionine, aspartic acid and glutamine levels (Figure 2); these levels, however, were significantly lower than in the hepatitis patients. Comments
Biliary tract obstruction is a common clinical condition requiring surgical intervention for relief. The presenting symptom is usually jaundice, which may also be the presenting symptom of hepatocellular damage. Although it is important to differentiate extrahepatic obstruction from hepatocellular causes of jaundice and to avoid unnecessary surgery, it is equally important not to delay surgery. The correct approach to the jaundiced patient is to arrive at as accurate a diagnosis as possible and, if necessary, to expeditiously relieve the obstruction. Surgical (obstructive) jaundice can usually be distinguished from medical (hepatocellular) jaundice by the relatively simple means of history, physical examination and liver function tests. However, these tests fail to differentiate extrahepatic obstruction from hepatocellular disease in up to 40 percent of patients [I], in whom the distinction requires more extensive diagnostic procedures. In an attempt to reduce the number of jaundiced patients in need of
144
complex and expensive diagnostic procedures, we investigated the use of the plasma amino acid pattern as a simple, relatively inexpensive and noninvasive addition to the initial history, physical examination and liver function tests performed in every jaundiced patient. Characteristic alterations in plasma amino acid patterns have been well documented in chronic liver disease, hepatic insufficiency of different causes and hepatic encephalopathy [3,4,8-l 01, which is not surprising since the liver is the primary site for the metabolism of many amino acids. The amino acid pattern in patients with chronic liver disease and experimental animals with chronic hepatic insufficiency generally includes elevated levels of the aromatic amino acids phenylalanine and tyrosine, free but not necessarily total tryptophan, methionine, histidine, glutamine and aspartic acid, combined with low levels (as low as 40 to 50 percent of normal) of the branched chain amino acids [3,4,8-131. In acute hepatic failure due to hepatitis or hepatic necrosis, all amino acids were markedly elevated except for the branched chain amino acids, which were present at near-normal levels. The greatest increase was noted in the aromatic amino acids phenylalanine and tyrosine (up to 700 percent of normal) as well as in methionine, glutamate and aspartate [4,13]. In this category of liver disease the severity of amino acid abnormalities correlated well with increases in serum glutamic oxaloacetic transaminase [4], indicating that the major source of the hyperaminoacidemia is destroyed hepatic parenchyma [5]. Because hepatic parenchymal damage occurs only late in the course of biliary obstruction, it is to be expected that obstructive jaundice will result in only mild abnormalities in plasma amino acid patterns, as was seen in experimental animals. The rats with centrolobular hepatic necrosis secondary to D-(+)galactosamine presented with an amino acid pattern similar to the one describe.d in dogs [13] and humans [4] with acute hepatic failure, with all amino acid levels increased significantly. In contrast, the group subjected to common bile duct ligation had a nearnormal amino acid pattern. The two groups of patients studied presented a similar, although not identical, relation. Patients with hepatocellular jaundice exhibited increased levels of all plasma amino acids except for the branched chain amino acids (leucine, isoleucine and valine) and taurine. Mean levels of methionine, phenylalanine, lysine, aspartic acid, proline and tyrosine were elevated to 400 percent to 1,000 percent of normal. In comparison, the obstructive jaundice group presented an essentially normal amino acid pattern with
The American
Journal
of Surgery
Plasma
only mild increases (up to maximum of 200 percent of normal) in methionine, phenylalanine, aspartic acid and glutamine levels. From our animal experiments and clinical data, there appear to be very clear qualitative and quantitative differences in the plasma amino acid patterns of hepatocellular and obstructive jaundice, with the latter having an almost normal pattern. The results suggest that the plasma amino acid pattern may be a useful adjunct in the differential diagnosis of jaundice, decreasing the number of patien1.s who need invasive diagnostic procedures or exploratory laparotomy. Summary
Distinguishing between obstructive (surgical) and hepatocellular (medical) jaundice is sometimes impossible using the relatively simple diagnostic means of’ history, physical examination and liver function tests. In an attempt to reduce the number of jaundiced patients in need of complex and expensive diagnostic procedures, we investigated the use of the plasma amino acid pattern in the diagnosis of jaundice. *Jaundiced rats with galactosamine-induced hepatitis and seven patients with acute onset hepati :is presented a plasma amino acid pattern in which most all amino acid levels were elevated except for arginine in the rat and branched chain amino acids in the patients. Rats jaundiced due to common bile duct ligat.ion and seven patients with -obstructive jaundice proved at surgery exhibited a near-normal amino acid pattern. These experimental and clinical data demonst,rat,e very clear qualitative and quanti :ative differences in plasma amino acid patterns of ht>patotellular and obstruct,ive jaundice, with the latter rxhihit,ing an almost-normal pattern. We suggest. the use of’ the plasma amino acid pattern as a useful adjunct in t,he differential diagnosis of medical and surgical jaundice. References 1. Stern RB, Knill-Jones RP, Williams R. Pitfalls in the diagnosis of jaundice due to carcinoma of the pancreas or biliary tree. Br Med J 1973; 1:533-5. 2. Eiseman B, Escajeda D. Modern diagnosis of jaundice. Arch Sura 1978: 113:555-6. 3. Fischer JE, Funovics JM, Aguirre A, James JH, Keane JM, Wesdorp RIG, et al. The role of plasma amino acids in hepatic encephalopathy. Surgery 1975; 78:276-90. 4. Rosen HM, Yoshimura N, Hodgman JM, Fischer JE. Plasma amino acid patterns in hepatic encephalopathy of differing etiology. Gastroenterology 1977; 72:483-7. 5. Fischer JE, Rosen HM, Ebeid AM, James JH, Keane JM, Soeters PB. The effect of normalization of plasma amino acids on hepatic encephalopathy in man. Surgery 1976; 80:77-91. 6. Freund H. Ryan JA, Fischer JE. Amino acid derangement in
Volume
139,
January
1980
Amino
Acids
in Jaundice
patients with sepsis. Ann Surg 1978; 188:423-30.
7. Keppler D, Lesch R, Reutter W, Decker K. Expelrimental hepatitis induced by D-Galactosamine. Exp Mol Pathol 1968; 9:279-90. 8. lob V, Coon WW, Sloan H. Altered clearance of free amino acids from plasma of patients with cirrhosis of the liver. J Surg Res 1966; 6:233-g. 9. Wu CT, Bollman G, Butt HR. Changes in free amino acids in the plasma during hepatic coma. J Clin Invest 1955; 34:845-
9. 10. lber FL, Rosen H. Levenson SM. Chalmers TC. The plasma amino acids in patients with liver failure. J Lab Clin Med 1957; 50:417-25. 11. Smith AR, Rossi-Fanelli F, Ziparo V, James JH, Perelle BA, Fischer JE. Alterations in plasma and CSF amino acids, amines and metabolites in hepatic coma. Ann Surg 1978; 187:343-50. 12. Svec MH. Freeman S. Effect of imoaired heoatic circulation on plasma free amino acids of iogs. Am’J Physiol 1949; 159: 357-64. 13. Aguire AM, Yoshimura N, Westman J, Fischer JE. Plasma amino acids in dogs with two experimental forms of liver damage. J Surg Res 1974; 16:339-45.
Discussion
Bernard Gardner (Brooklyn, NY): You showed some very exciting differences in the levels in these two groups of patients. Do you have any information on .the number of determinations that may be used t,o derive standard deviations or p values? Laurie
M. Blendis (Toronto, Canada): Some years ago told us that amino acid patterns in portasystemic encephalopathy were specific and highly related to the etiology of that disease. Since then we have llearned that these patterns are not specific, that they can be found in nonencephalopathic patients and that they may be related neither to the disease nor its complications but merely to nutritional intake. Thus I have a deja uu feeling about this paper. Were the differences in amino acid patterns in these patients with liver disease related to (1) total calorie intake, (2) total protein intake, (3) the relation of protein to calorie or (4) the relation to the actual amino acid intake? In the diagnosis of obstructive jaundice, clinical and ultrasound tests can be most helpful, and although they are invasive, they should not be dangerous if performed by physicians familiar with them. What is the cost of performing a very difficult estimation of amino acids requiring expensive amino analyzers, and how do the results compare with simple endoscopic retrograde cholangiography or percutaneous cholangiography?
Dr. Fischer
George L. Blackburn (Boston, MA): I wonder if Dr. Fischer could isolate one of the amino acids that might reflect the entire pattern, and perhaps suggest some sort of challenge test, such as a clearance test, that might help differentiate the two types of liver disease. I noted that the groups were quite jaundiced. I think bilirubin levels were 20. Would amino acid profiles be as sensitive if either obstructive
disease or hepatitis
were less advanced?
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Freund et al
Josef E. Fischer (closing): We gave a preliminary report on this particular technique, and as we continue to evaluate more patients, the results seem to be valid. One of the problems with amino acid analysis is the significant variation in results among laboratories. Although Dr. Blendis stated that the ljlasma amino acid pattern in hepatic encephalopathy does not indicate different disease states in encephalopathy, I as well as others would strongly disagree with him. Caloric intake and other nutritional influences on plasma amino acid patterns are well known. Gi,ven those variations, it is perfectly clear that even among the minor variations, many of which emerged in the dispute over Charlie Lieber’s use of alpha amino butyric acid, one thing that is most important-a major influence as opposed to the comparatively minor influence of caloric intake or starvation-is hepatic function. On a scale of 10, caloric intake may rate 1 or 2, but the sick liver may rate ‘7 to 10.
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Dr. Blackburn’s questions are very cogent, but I think we will have to wait for more data before we can answer them. As we explore the problem of recognizing patterns of plasma amino acids in different diseases, it appears that one can take a whole group of amino acids and through discriminant and variant analysis, using a fairly high powered computer, focus in on one, two or possibly four amino acids that appear more important than the others. Dr. Freund has done this in sepsis, as reported in his article in the November issue of Annals of Surgery, and with a larger group of patients I feel we will have enough data to similarly report on jaundice. Superficial examination of the data suggests that tyrosine remains an important amino acid, as Dr. Hal Conn described as early as 1967. The cost of plasma amino acid analysis is $62, and I doubt that anyone can perform endoscopic retrograde cholangiopancreatography for that amount.
The American Journal of Surgery