Hepatic Transaminase Activity in Alcoholic Liver Disease

GASTROENTEROLOGY 78:1389-1392, 1980

Hepatic Transaminase Activity in Alcoholic Liver Disease DANIEL S. MATLOFF, MITCHELL MARSHALL M. KAPLAN

J.

SELINGER, and

Gastroenterology Service, Department of Medicine, New England Medical Center Hospital, Boston, Massachusetts

Glutamic oxaloacetic transaminase (GOT) and glutamic pyruvic transaminase (CPT) activities were measured in percutaneous needle biopsy specimens of human liver tissue and compared with transaminase values in serum obtained on the day of biopsy. Hepatic CPT activity was significantly decreased in liver tissue of patients with alcoholic hepatitis and cirrhosis compared with the activity in individuals with normal livers (P < 0.05) and individuals with primary biliary cirrhosis (P < 0.05). The decreased hepatic CPT activity was not related to the presence of cirrhosis in biopsy specimens and was not increased by the addition of saturating amounts of pyridoxal phosphate to the assay mixture. Hepatic GOT was also slightly but significantly lowered in individuals with alcoholic liver disease (P <; 0.05). The GOT/CPT ratio in serum and liver tissue was increased only in individuals with alcoholic liver disease, but the increase did not reach statistical significance. The increased GOT/CPT ratio is · due primarily to the low activity of CPT in liver 'and serur;n. The less than expected elevation of CPT in serum of patients with alcoholic hepatitis reflects the diminished hepatic CPT activity and lesser amounts of this enzyme available to leak into serum from damaged hepatocytes. Alcoholic liver disease is characterized by distinct patterns of serum glutamic oxaloacetic transaminase

(SGOT) and serum glutamic pyruvic transaminase (SGPT) elevation. 1 ' 2 We have previously demonstrated that an SGOT /SGPT ratio greater than 2 is highly suggestive of alcoholic hepatitis or cirrhosis. 1 In addition, SGOT and SGPT values greater than 300 IU are rare in patients with alcoholic liver disease and correlate poorly with the degree of hepatic inflammation and liver cell necrosis seen on liver biopsy! Values may be minimally elevated in the face of extensive liver cell necrosis. The mechanisms responsible for these clinically useful observations are not clear. Various factors, including toxicity of ethanol to liver mitochondria, damage to nonhepatic tissue such as skeletal muscle, and vitamin deficiencies have been proposed as causes but not proven. The aim of the present study was to investigate the mechanism of the characteristic SGOT/SGPT pattern in patients with alcoholic liver disease. For this purpose, fragments of percutaneous liver biopsy material were obtained from patients with various types of liver disease, including alcoholic liver disease and the specific activity of glutamic oxaloacetic transaminase (GOT) and glutamic pyruvic transaminase (GPT) measured in liver tissue.

Materials and Methods Patients

Received September 14, 1979. Accepted January 20, 1980. Address requests for reprints to: Marshall M. Kaplan, M.D., Gastroenterology Service, New England Medical Center Hospital, 171 Harrison Avenue, Boston, Massachusetts 02111. This study was presented in part at the Annual Meeting of the American Gastroenterological Association, May, 1979, in New Orleans, Louisiana, and published in abstract form in GASTROENTEROLOGY (76:1195, 1979). This work was supported in part by Research Grant AM 10571 and Training Grant AM 07024 from the National Institutes of Health. © 1980 by the American Gastroenterological Association 0016-5085/80/061389-04$02.25

A 5-10-mg piece of liver, approximately 10-20% of the total specimen, was obtained prospectively from hospitalized patients undergoing diagnostic percutaneous liver biopsy. Informed consent was obtained from each patient. The remaining 80-90% of the specimen was sent for routine histology. All biopsies were performed using the 16-gauge, thin-walled Klatskin needle (Becton, Dickinson & Co., Orangeburg, N.Y., No. 1403). Within 6 hr of the biopsy, 5 cm 3 of .serum was obtained from each patient. All liver biopsies were reviewed by one of us (M. M.. Kaplan) without knowledge of the transaminase values in

1390

MA TLOFF ET AL.

GASTROENTEROLOGYVol. 78, No. 6

GOT

GPT 150

~

!'::

w

f-

500

'

t

100

0

400

300

0'

rn

z

'::> 200

50

100

•.

NL

the liver tissue. Patients with the following diagnoses were included in the study: normal liver biopsies (11 patients whose diagnostic liver biopsies yielded histologically normal liver tissue), fatty liver with no history of alcohol abuse (8 patients), alcoholic liver disease (9 patients), primary biliary cirrhosis (13 patients), chronic active hepatitis (9 patients) and chronic persistent hepatitis (4 patients) . Diagnoses were based on clinical data and currently accepted histologic criteria.

FL

ALD

PBC

CAH

CPH

NL

FL

ALD

PBC

Statistical comparisons were carried out by analysis of variance-using a program written . fora Data General Corporation Nova 830 computer. Valu.~s are expressed as mean ± 1 SEM.

Results Liver GOT and GPT specific activities are given in Figure 1. There was no significant difference in the protein content of any · of the patient groups with protein expressed as micrograms/milligram wet weight liver. The specific activity of GPT in normal liver was 133 ± 18 U/g protein. Hepatic GPT activity in patients with alcoholic liver disease was 41% of normal (P < 0.05) and also significantly lower than that in patients with primary biliary cirrhosis (P < 0.05). Decreased h epatic GPT activity was found only in patients with alcoholic hepatitis and cirrhosis. The specific activity of GOT in normal liver was 495 ± 40 U/g protein. Tissue GOT activity in alco-

SERUM

LIVER 0..

CPH

Statistical Analysis

Liver tissue was immediately washed in ice-cold 0.25 M sucrose, weighed, and homogenized in 1.0 ml of 0.25 M sucrose in an all-glass Ten Broeck tissue grinder. The crude liver homogenate and serum samples were stored at -70°C. Glutamic oxabacetic transaminase was measured by the method of Karmen 3 and GPT measured by the method of Wroblewski and LaDue.• Both assays were modified to achieve optimal substrate concentrations.5 Final assay volumes were 1.0 ml. In additional studies, assays were repeated with the addition of pyridoxal phosphate, final concentration 0.1 mM. 6 Protein was measured by the method of Lowry et al.' One unit of enzyme was defined as that amount which converted 1 ~tmol of substrate to end product/min. Both assays were linear with respect to time and protein concentration. Enzyme activity was expressed as units/gram protein. SGOT and

f-

CAH

SGPT were measured in the hospital's Clinical .C hemistry laboratory by the Karmen method. .

Assays .

::0

2

6

(.!) _J

~

0

(f)

G)

'f-

0

(.!) _J

(f)

0

Figure 2. GOT /GPT ratios in liver and serum of patients with liver disease . The abbreviations are given in the legend of Figure 1. The liver and serum GOT /CPT ratio of each patient was calculated. The ratios shown in the figure are the mean of the individual ratios.

-o

::0

0 --i

0:: 0..

Figure 1. CPT and GOT activity in human liver tissue. NL = normal liver histology; FL = fatty liver with no history of et hanol abuse; ALD =alcoholic liver disease, both alcoholic hepatitis and cirrhosis; PBC = primary biliary cirrhosis; CAH = chronic active hepatitis; and CPH = chronic persistent hepatitis (n =number of patients).

c:
-~

0

~

3

~

mm

0::

NL

FL

ALD

PBC

CAH

CPH

~

NL

G)

I

f.li FL

ALD

PBC

CAH

CPH

-o --i

June 1980

holic hepatitis and cirrhosis was 71% of normal (P < 0.05). Tissue GOT activity was also reduced in patients with chronic active hepatitis (75% of normal), but the difference did not reach statistical significance (P < 0.2). Of the 9 patients with alcoholic liver disease, 6 had acute alcoholic hepatitis without cirrhosis, and 3 had unequivocal cirrhosis. Equally low tissue GPT values were found in the alcoholic hepatits patients without cirrhosis (48 ± 15 U/g protein) as in those with cirrhosis (69 ± 22 U/g protein). Hepatic GPT activity in patients with chronic hepatitis and primary biliary cirrhosis whose liver biopsies demonstrated unequivocal cirrhosis were similar to values in those patients without cirrhosis. Hence, cirrhosis per se did not account for the lowered hepatic GPT activity. The addition of saturating amounts of the GPT and GOT cofactor, pyridoxal phosphate, to the assay mixtures did not increase GPT or GOT activity in liver tissue of patients with alcoholic liver disease and chronic active hepatitis. Thus, the lowered GPT activity in patients with alcoholic liver disease was not due to a deficiency of this cofactor in the assay mixture or in liver tissue. Table llists the mean SGOT and SGPT values in each group of patients. Figure 2 demonstrates the GOT /GPT ratios in serum and liver tissue of patients with the different types of liver disease. Serum and tissue ratios are increased only in patients with alcoholic liver disease, compared with those with normal liver histology and other types of liver disease, but the difference is statistically significant only in serum (P < 0.025).

Discussion This study demonstrates that liver GPT activity is selectively decreased in patients with alcoholic liver disease compared with individuals with normal livers and other types of liver disease. The decreased liver GPT activity is specific for alcoholic liver disease and not due to chronicity of liver disease or cirrhosis per se. Hepatic GOT activity is also decreased in alcoholic liver disease but to a lesser extent than GPT. In addition, the diminished hepatic GOT activity is not as specific, because it is also decreased in patients with chronic hepatitis. Although there was no statistical difference between the normal and chronic active hepatitis group, a true difference may have been missed because of the small sample size and wide variation in individual values. These data help explain the characteristics serum transaminase pattern in patients with alcoholic hepatitis and cirrhosis, only modest elevations of SGOT and SGPT accompanied by an elevated SGOT/SGPT ratio. In alcoholic hepatitis and cir-

HEPATIC TRANSAMINASES IN ALCOHOLIC HEPATITIS

Table 1.

1391

Serum Transaminase Values in Patients with Liver Disease

Normal liver biopsies Fatty liver Alcoholic liver disease Primary biliary cirrhosis Chronic active hepatitis Chronic persistent hepatitis

No.

SCOT

SGPT

11

35 ± 7 38±4 107 ± 30 58.5 ± 16 202 ± 70 88± 13

55± 18 40 ± 12 41 ±9 81 ± 24 281 ± 105 91 ± 8

8 9 13 9 4

rhosis, the less than expected elevations of SGOT and SGPT reflect diminished hepatic activity of these enzymes. There is, accordingly, less enzyme available for leakage into serum from damaged or destroyed hepatocytes. The cause of the lowered hepatic GPT activity in patient with alcoholic liver disease is not known. However, available data suggest that it may be related to chronic pyridoxal phosphate deficiency. Lumeng and Li have shown that pyridoxal phosphate deficiency is common in patients with chronic alcohol abuse." They measured serum pyridoxal phosphate levels in 66 alcoholic subjects without evidence of liver or hematologic disease and found lowered levels in 35. Their data suggested that the lowered levels were due to increased catabolism of pyridoxal phosphate mediated not by alcohol, but by acetaldehyde, a product of alcohol metabolism. 8 Decreased levels of pyridoxal phosphate have also been found in both serum and liver tissue of patients with alcoholic fatty liver and cirrhosis. 9 ' 10 Deficient dietary intake in alcoholics may also contribute to the decreased serum pyridoxal phosphate concentration in this population, but there is little data bearing on this. Although there are no studies directly relating serum or hepatic pyridoxal phosphate levels to hepatic GPT activity in humans, data in rats do suggest such a relationship. Ludwig and Kaplowitz have recently reported that hepatic GPT activity was lowered by 47% in rats maintained for 8 wk on a vitamin Be deficient diet. 11 Vitamin Be deficiency appeared to inhibit GPT synthesis since addition of pyridoxal phosphate to assay mixtures did not increase GPT activity. We obtained similar results in our patients. Addition of excess amounts of pyridoxal phosphate to GPT assays in human liver homogenates did not increase GPT activity. That pyridoxal phosphate deficiency in rats can be induced by chronic alcohol feeding is suggested by the studies of Henley et al. 12 They noted that ethanol, given as the sole source of fluid in rats on an otherwise adequate diet, lowered hepatic GPT activity significantly compared with pair fed rats, P < 0.001. After 24 wk on such a diet, the hepatic GOT /GPT ratio was significantly in-

1392

GASTROENTEROLOGY Vol. 78, No.6

MATLOFF ET AL.

creased in the alcohol fed rats, due entirely to the decrease in hepatic GPT activity. These investigators did not measure pyridoxal phosphate metabolism. However, extrapolation of data derived from alcoholic patients suggests that there may be a relationship between chronic alcohol abuse, pyridoxal phosphate deficiency, and lowered levels of hepatic GPT activity. 8 Unfortunately, our data do not provide a definite answer. We did not measure serum or hepatic vitamin B6 content in our patients and did not perform liver biopsies in alcoholic patients without liver disease. It should be possible, however, to test this hypothesis in prospective studies and determine if large amounts of supplemental vitamin B6 will restore the hepatic and serum transaminase pattern to normal.

References 1. Cohen JA, Kaplan MM: The SCOT/SGPT ratio-An indicator of alcoholic liver disease. Am J Dig Dis 24:835-838, 1979 2. DeRitis, F, Coltorti M, Giusti G: Serum transaminase activities in liver disease. Lancet 1:685, 1972 3. Karmen A: A note on the spectrophotometric assay of glutamic-oxaloacetric transaminase in human blood serum. J Clin Invest 34:131, 1955

4. Wroblewski F, LaDue JS: Serum glutamic pyruvic transaminase in cardiac and hepatic disease. Proc Soc Exp Bioi Med 9:569, 1956 5. Bergmeyer HA, Bernt E: Glutamate-oxaloacetate transaminase and glutamate-pyruvate transaminase. In: Methods of Enzymatic Analysis. Edited by HU Bergmeyer. New York and London, Academic Press, 1965, p 837-853 6. H¢rder M, Moore RE, Bowers GN Jr: Aspartate aminotransferase activity in human serum. Factors to be considered in supplementation with pyridoxal 5' -phospate in vitro. Clin Chern 22 :1876, 1976 7. Lowry OH, Rosebrough NJ, Farr AI, et al: Protein measurement with the Falin phenol reagent. J Bioi Chern 193:265, 1951 8. Lumeng L, Li TK: Vitamin B6 metabolism in chronic alcohol abuse. Pyridoxal phosphate synthesis and degradation in human erythrocytes. J Clin Invest 53:693, 1974 9. Hines JD, Love DS: Determination of serum and blood pyridoxal phosphage concentrations with purified rabbit skeletal muscle apophosphorylase b. J Lab Clin Med 73:343, 1969 10. Frank 0, Luisada-Opper A, Sorrell MF, et al: Vitamin deficits in severe alcoholic fatty liver of man calculated from multiple reference units. Exp Mol Pathol 15:191, 1971 11. Ludwig S, Kaplowitz N: Effect of pyridoxine deficiency (B 6 -D) on serum and liver transaminase (T) in experimental liver injury. Gastroenterology 76:1290, 1979 12. Henley KS, Wiggins HS, Hirschowitz GI, et al: The effect of oral ethanol on glutamic pyruvic and glutamic oxaloacetic transaminase activity in the rat liver. QJ Stud Alcohol 19:54, 1958