SERUM FREE PROLINE AND FREE HYDROXYPROLINE IN PATIENTS WITH CHRONIC LIVER DISEASE

Transcription

1 GASTROENTEROLOGY 68: , 1975 Copyright 1975 by The Williams & Wilkins Co. Vol. 68, No.5, Part 1 Printed in U.S.A. SERUM FREE PROLINE AND FREE HYDROXYPROLINE IN PATIENTS WITH CHRONIC LIVER DISEASE JU.AN MANUEL MATA, M.D., DAVID KERSHENOBICH, M.D., ENRIQUETA VILLARREAL, M.D. AND MARCOS RoJKIND, M.D. Departmento de Gastroenterolog't.a, Instituto Nacional de Ia Nutricibn; and Departamento de Biologta Celular, Centro de Investigacibn y de Estudios, Avanzados del I.P.N., Mexico Serum free proline and free hydroxyproline were determined in 71 patients with liver disease and in 62 control subjects. The group with liver disease included 60 patients with liver cirrhosis and 11 with chronic active liver disease. Forty-five of the cirrhotic patients were alcoholics, 9 of which were studied during an episode of alcoholic hepatitis. The control group consisted of 24 healthy volunteers, 19 primary malnourished patients, and 19 severely ill patients without liver disease. The values obtained in normal subjects were quite constant; no differences related to sex or age were detected. In patients with malnutrition, and especially in severely ill patients, the proline values were always below the normal limit. Patients with nonalcoholic cirrhosis or chronic active liver disease had serum proline and hydroxyproline values similar to those of normal subjects. However, the patients with alcoholic liver cirrhosis had proline and hydroxyproline values significantly higher than the normal group. Furthermore, in patients with alcoholic hepatitis the serum free proline values were significantly higher than in the other groups. The results suggest that alcohol might have a direct effect on proline metabolism or facilitate its release from the liver cell. One of the distinctive features of collagen is its amino acid composition, with a high content of the imino acids proline and hydroxyproline. 1 Hydroxyproline is almost specific for collagen in mammals and its levels in serum and urine reflects the extent of collagen metabolism in tissues with a high metabolic turnover of this protein. 2 Under pathological conditions in which active collagen synthesis takes place, there is also a change in the pool of free proline. In the livers of rats or of patients with cirrhosis the pool of free proline Received November 26, Accepted November 5, Address requests for reprints to: Dr. Marcos Rojkind, Departamento de Biologia Celular, Centro de Investigaci6n y de Estudios Avanzados del I.P.N., Apartado Postal , Mexico 14, D.F. increases proportionally to the extent of liver fibrosis. a-s From these and other data, 6 7 it has been suggested that in conditions of increased fibrogenesis, such as liver cirrhosis 3 or bone formation in fetal rat calvaria, 6 7 the pool of free proline or the amount of prolyl trna may play an important role in the regulation of collagen biosynthesis. Early attempts to determine modifications in the levels of free proline in sera of cirrhotic patients failed to show any differences as compared to normal subjects. 4 However, in cirrhotic patients with a history of chronic alcoholism, a slight deviation from the main values was observed (J. M. Mata, unpublished observation). The aim of this is to determine whether the levels of free proline and hydroxypro- 1265

2 1266 MATA ETAL. Vol. 68, No.5, Part 1 line in serum of patients with alcoholic liver cirrhosis reflect the metabolic state of the fibrous tissue in the liver. Experimental Procedure Patients A total of 133 subjects were studied and divided into six groups: control, cirrhotics, chronic active liver disease, alcoholic hepatitis, severely ill patients without liver disease (acute apendicitis, pancreatitis, diabetic coma), and malnourished patients. The first group (controls) consisted of 24 healthy volunteers whose ages ranged from 16 to 70 years with a mean of 34.5 years. The group of cirrhotic patients was divided in two subgroups: nonalcoholics (15 patients) and alcoholics (45 patients). The diagnosis of cirrhosis was established clinically and by laboratory methods. Histological confirmation was obtained in only 17 of the cirrhotic patients. In the remaining cases a liver biopsy was not performed due to clotting abnormalities, mainly increased prothrombin time in 11 cases, increased partial thromboplastine time in 8 cases, and severe plaquetopenia in three cases. The criteria for selection of patients with chronic active liver disease were similar to those described by Alarc6n-Segovia et al. and required the persistence of jaundice, elevation of SGOT above 200 U, and increased serum globulin above 2 g continuously during a period of 4 months or intermittently during 1 year. All patients had histological evidence of piecemeal necrosis, lymphocyte and plasma cell infiltrates, and distortion of liver architecture. None of them had received treatment with corticosteroids or immunosuppresive drugs prior to this study. The patients were on a low collagen diet, without gelatin and fibrous meat. To determine the daily variation of serum free proline and hydroxyproline, serum samples of a control subject with the low collagen diet were analyzed for both imino acids during 28 consecutive days. The maximal variation for serum free proline was ±0.010 ~mole per ml and for serum free hydroxyproline ± ~mole per ml. The number of patients and their age distribution in each group are shown in table 1. Methods Serum free proline was determined by the method of Rojkind and Gonzalez. Duplicate samples were oxidized for 20 min with 1 ml of chloramine-t solution prepared according to Woessner. 10 The reaction was stopped with 0.5 ml. of 2 N thiosulfate followed by addition TABLE 1. Age and sex distribution of patients and normal subjects Group Males Females Age range years Normal subjects (34) Malnourished (47) Severely ill (53) Chronic active liver disease (39) Nonalcoholic cirrhotics (57) Alcoholic cirrhotics (48) Alcoholic hepatitis (49) Numbers in parentheses are means. of 1.0 ml of 1 N NaOH. The solution was saturated with NaCl and the product of proline oxidation was extracted into 6 ml of toulene by shaking in a Vortex mixer. The extraction of the oxidation product was almost 100% after two successive extractions with toluene. Up to 1 ml of toluene was incubated at 100 C for 60 min with 3 ml of glacial acetic acid and 3.0 ml of 3% ninhydrin in a mixture of acetic and phosphoric acids. The colorimetric reaction with ninhydrin was read at 515 nm.u To determine serum free hydroxyproline, the reaction mixture was extracted twice more with 6 ml of toluene. The water layer obtained after oxidation with chloramine and extraction of proline was incubated at 100 C for 30 min to convert the oxidation product of hydroxyproline into a pyrrol. Mter cooling the sample, the pyrrol was extracted into 6 ml of toluene. Appropriate aliquots of the toluene layer were used for pyrrol determination with p dimethylaminobenzaldehyde. 12 Results As can be seen in table 2, the amount of proline in the sera of normal subjects is J.Lmole per ml, with a standard error of ± This value is slightly higher than the values previously reported after purification of proline by column chromatography. Although the amount of hydroxyproline in serum is very low, the high sensitivity of the method allows the determination of this imino acid. The average value of hydroxyproline obtained in 24 different sera (each analysis performed in

3 May 1975 PROLINE AND HYDROXYPROLINE IN LIVER DISEASE 1267 TABLE 2. Serum free proline and serum free hydroxyproline in patients with liver cirrhosisa Normal subjects (24) Nonalcoholic cirrhotics (15) Alcoholic cirrhotics (36) Chronic active liver disease (11) Alcoholic hepatitis (9) Malnourished (19) Group' Proline Hydroxyproline!Lmole/ml ± ± ± (P < 0.005) ± ± (P < 0.005) ± Severely ill (19) ± (P < 0.005)!Lmole/ml ± ± ± (P < ) ± ± (P < 0.005) ± ± a Values are means ± standard error. P values were obtained by the Student's t-test. Only significant P values are shown. Numbers in parentheses are total number of subjects in each group. e o.1 Ill ""' Cll ~ 0.6 ::1. z I- 1:2 0.4 I- z w u 03 z 0 u. w 0.2 ~ z ::::i ~ lo a::: a.. ". * r l. f: =..,. ~... A NORMALS MALNOURISHED SEVERELY CHRONIC ACTIVE NON ALCOHOLIC ALCOHOLIC ALCOHOLIC ILL LIVER DISEASES CIRRHOSIS CIRRHOSIS HEPATITIS FIG. 1. Serum free proline concentration in different diseases., <" 3: duplicate) was Jlmole per ml ± In view of the reproducibility of results with a very low standard error, the values were considered adequate. No differences related to age or sex were seen. Serum proline and hydroxyproline values in nonalcoholic cirrhotic patients and those with chronic active hepatitis were similar to the values obtained in normal volunteers. In malnourished subjects both proline and hydroxyproline values were below normal, although the differences were not statistically significant. In the severely ill patients, the serum proline values were lower than normal (P < 0.005). In patients with alcoholic liver cirrhosis serum proline and hydroxyproline were significantly increased. Furthermore, in a small group of patients that entered the hospital with alcoholic hepatitis, the serum proline and hydroxyproline values were the highest. The dispersion of the individual proline values is shown in figure 1. Discussion It has already been shown that changes in the metabolism of bone and skin collagen are accompanied by changes in the amount of hydroxyproline excreted in the

4 1268 MATA ETAL. Vol. 68, No.5, Part I urine. 2 Under conditions in which collagen metabolism is maintained constant in bone and skin, it is still possible to detect modifications in the serum values of proline and hydroxyproline in patients with liver disease. The results obtained in this communication show that free proline and free hydroxyproline are significantly increased in alcoholic cirrhotics. Furthermore, in patients with alcoholic hepatitis, the serum values of free proline and free hydroxyproline are the highest. In several patients with high serum values of both imino acids who stopped drinking alcohol the free imino acids decreased significantly. These results suggest, as previously reported, that ethanol by itself may play an important role not only in the pathogenesis of cell damage but also m fibrogenesis Rojkind and Diaz de Le6n 3 and Kershenobich et al. 4 have suggested that the amount of free proline in the liver, or its specific trna, available for acylation may play an important role in regulation of collagen biosynthesis. Accordingly, if ethanol is producing increased levels of proline in the sera of the cirrhotic patients, this could be accompanied by a concomitant increase in free proline in the liver, and this in turn could be a trigger for fibrogenesis. This is supported by the fact that livers of rats made cirrhotic by chronic administration of carbon tetrachloride, or livers of patients with cirrhosis, have increased levels of free proline. Furthermore, there is also a direct correlation between the pool of the proline and collagen deposition. 3 4 Although one could speculate that the increase of proline in serum could be due to a direct toxic effect of ethanol on the liver with cell destruction and leakage of free proline, this does not hold true for hydroxyproline. It has been demonstrated that hydroxyproline is formed after proline has been incorporated into the protein. 15 This result indicates that hydroxyproline is derived chiefly from collagen catabolism. 16 Accordingly, the increase in free hydroxyproline in the sera suggests that the changes in free imino acids reflect an increase in collagen catabolism, perhaps secondary to an increase in the rate of synthesis. As already shown, normal livers do not synthesize collagen at measurable rates, whereas cirrhotic livers do. 17 REFERENCES 1. Rojkind M: Molecular structure of the fibrous components of the connective tissue, chap 1, Molecular Pathology of Connective Tissues. Edited by R Perez-Tamayo, M Rojkind. New York, Marcel Dekker Inc, 1973, p Sjoerdsma A, Udenfriend S, Keiser H, et al: Hydroxyproline and collagen metabolism. Clinical implications. Ann Intern Med 63: , Rojkind M, Diaz de Leon L: Collagen biosynthesis in cirrhotic rat liver slices. Biochim Biophys Acta 217: , Kershenobich D, Fierro FJ, Rojkind M: The relationship between the free pool of proline and collagen content in human liver cirrhosis. J Clin Invest 49: , Chvapil M, Ryan JN: The pool of free proline in acute and chronic liver injury and its effect on the synthesis of collagen and globular proteins. Agents Actions 3:38-44, Phang JM, Finerman GAM, Singh B, et al: Compartmental analysis of collagen synthesis in fetal rat calvaria. I. Perturbation of proline transport. Biochim Biophys Acta 230: , Finerman GAM, Downing S, Rosenberg LE: Amino acid transport in bone. II. Regulation of collagen synthesis by perturbation of proline transport. Biochim Biophys Acta 135: , Alarcon-Segovia D, Kershenobich D, Velazquez F, et al: Hepatopatias cronicas activas. Clasificacion y criterios diagnosticos. Rev Invest Clin 24: , Rojkind M, Gonzalez E: An improved method for determining specific radioactivities of proline-"c and hydroxyproline-"c in collagen and in noncollagenous proteins. Anal Biochem 57:1-7, Woessner JF Jr: The determination of hydroxyproline in tissue and protein samples containing small proportions of this iminoacid. Arch Biochem Biophys 93: , Troll W, Linsley J: A photometric method for the determination of proline. J Bioi Chern 215: , Prockop DJ, Udenfriend S: A specific method for the analysis of hydroxyproline in tissues and urine. Anal Biochem 1: , Chen T, Zetterman R, Leevy CM: Sensitized lymphocyte and hepatic fibrogenesis. Gastroenterology 65:532, 1973

5 May 1975 PROLINE AND HYDROXYPROLINE IN LIVER DISEASE Walker F, Shand J: Influence of alcohol on collagen synthesis in vitro. Lancet 29: , Lazarides EL, Lukens LN, Infante AA: Collagen polysomes: Site of hydroxylation of proline residues. J Mol Bioi 58: , Adams E: Metabolism of proline and of hydroxyproline. In International Review of Connective Tissues Research. Edited by DA Hall, DS Jackson. New York, Academic Press, 1970, p Huberman A, Recio A, Rojkind M: Collagen biosynthesis in normal and cirrhotic rat liver slices. Proc Soc Exp Bioi Med 131: , 1969