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Serum laminin and N-terminal type III procollagen in chronic alcoholic liver disease
292
CORRESPONDENCE
The treatment of dogs with repeated IV calcium injection (a) increased the sensitivity of acinar cells to cerulein and urecholine and the potentiation by cerulein of the water and bicarbonate secretion in response to secretin; in contrast, it decreased the sensitivity to secretin alone: (b) induced an inhibition of water and bicarbonate secretion with urecholine stimulation; and (c) induced a significant inhibition of calcium secretion after cerulein injection (4). These findings findings enlighten the mechanism of action of hypercalcemia, the biochemical composition of protein plugs described for the first time after calcium injection, and the persistence of calcium-induced modification of pancreatic secretion. This could have some importance in the understanding of calciuminduced pancreatitis and should be mentioned. HENRISARI&S Hdpital Ste-Marguerite Marseilles, France
1. Frich TW, Hailemariam S, Heitz PU, Largiader F, Goodale RL. Acute hypercalcemia induces acinar cell necrosis and intraductal precipitates in the pancreas of cat and guinea pig. Gastroenterology 1990;98:1675-1681. 2. Noel-Jorand MC, Verine HJ, Sarles H. Dose-dependent and long lasting effects of repeated intravenous injections of calcium on the canine secretin-stimulated pancreatic juice secretion. Eur J Clin Invest 1981;11:25-31. 3. Noel-Jorand MC, Colomb E, Astier JP, Sarles H. Persisting modifications of dogs’ basal exocrine pancreatic secretion after repeated intravenous calcium injections. Eur J Clin Invest 1981;11:249-256. 4. NoelJorand MC, Schmidt, Devaux, Sarles H. Biomed Pharmacother 1983;37:129-135. Reply.
The experiments of Dr. Sarles et al. were of very much importance in the design and the interpretation of our experiments (1). Although precipitates were not seen in the histologic slides of dog pancreas after calcium injection (2), the findings of plugs in the pancreatic juice illustrate important changes in secretory activity of the pancreas with a possible implication to the genesis of chronic or acute pancreatitis (3). However, neither these experiments nor our results give any direct clue as to whether intraductal precipitates are the cause or the result of changes eventually leading to acute pancreatitis. In the cat, acute hypercalcemia has been shown to increase pancreatic duct permeability (4). Acute pancreatitis developed after perfusion of the pancreatic duct with activated pancreatic enzymes. These findings suggest that early events of hypercalcemia-associated acute pancreatitis take place in the ductal system. On the other hand, preliminary ultrastructural studies of cat and guinea pig pancreas in hypercalcemia revealed acinar cell changes similar to early findings in the hyperstimulation pancreatitis model (5,6). Because an increase in extracellular calcium levels is able to increase the secretory activity of the acinar cell (2,3,7), it can be speculated that hypercalcemia-induced pancreatitis represents a form of hyperstimulation pancreatitis. Clearly, more research is needed to conclusively answer this question. THOMASFRICK,M.D. Department of Surgery University of Ziirich Hospital 8091 Ziirich, Switzerland
1. Frick TW, Hailemariam S, Heitz PU, Largiader F, Goodal RL. Acute hypercalcemia induces acinar cell necrosis and intraductal precipitates in the pancreas of cat and guinea pig. Gastroenterology 1990;98:1675-1681.
GASTROENTEROLOGY Vol. 100, No. 1
2. Noel-Jorand MC, Verine HJ, Sarles H. Dose-dependent and long lasting effects of repeated intravenous injections of calcium on the canine secretin-stimulated pancreatic juice secretion. Eur J ClinInvest 1981;11:25-31. 3. Noel-Jorand MC, Colomb E, Astier JP, Sarles H. Persisting modifications of dogs’ basal exocrine pancreatic secretion after repeated intravenous calcium injections. Eur J Clin Invest 1981;11:249-256. 4. Cates MC, Singh SM, Peick AL, Harvey MH, Reber HA. Acute hypercalcemia, pancreatic duct permeability, and pancreatitis in cats. Surgery 1988;104:137-141. 5. Frick TW, Runge W, Sibley RK, Satterberg TL, Goodale RL. Effect of acute hypercalcemia on guinea pig pancreas (abstr). Dig Dis Sci 1985;30:972. 6. Frick TW, Runge W, Sibley RK, Satterberg TL, Goodale RL. Calcium infusion and pancreatic ultrastructure in the cat (abstr). Dig Dis Sci 1985;30:971. 7. Layer P, Hotz J. Eysselein VE, Jansen JBMJ, Lamers CBHW, Schmitz-Moormann HP, Gdbell H. Effects of acute hypercalcemia on exocrine pancreatic secretion in the cat. Gastroenterology 1985;88:1168-1174.
Serum Laminin and N-Terminal Type III Procollagen in Chronic Alcoholic Liver Disease Dear Sir: We have read with interest the article by Niemela et al. (1) dealing with the relationships between N-terminal type III procollagen (III-PC), serum laminin, and type IV collagen, and the severity of chronic alcoholic liver disease. We here present our data, which provide similar information to that of Niemell’s group, as we have conducted a similar prospective study of 50 alcoholic patients, 39 men and 11 women, 30 cirrhotics and 22 noncirrhotics, histologically diagnosed by percutaneous liver biopsy. The following parameters were histomorphometritally determined using a WIDS II image analyzer: total amount of fibrosis, as the area of the fibrous tracts (Van Giesson stain) in relation to the area of the section of the biopsy cylinder (at 265~); and total amount of fat (mean area of fat droplets x no. of fat droplets in the biopsy cylinder section/total area of cylinder, at 3625x). We also graded the intensity of polymorphonuclear infiltrate (0, + = absent: + +, + + + = present), the intensity of mononuclear infiltrate (0, + = absent; ++, +++ = present), presence or not of Mallory’s hyalin and satelitosis, and the intensity of necrosis (0, + = absent; ++, +++ = present). We have also recorded (at admission of the patients to the hospital) the clinical and biological parameters listed in Table 1, categorized our patients according to Child’s classification with Pugh’s modification (2), and determined serum levels of III-PC and laminin by radioimmunoanalysis (Behring, Marburg, Germany). As can be seen in Table 1, we found a similar degree of correlation between III-PC and laminin as Niemela did, and also a good correlation with an index of disease severity as the ChildPugh score. The parameter most strongly related to laminin was prothrombin activity, but the narrow relation between laminin and the degree of fibrosis is also noteworthy. Indeed, the amount of liver fibrosis is a criterion of severity, with prognostic value regarding mortality (3). The good relationships between III-PC and parameters derived from progressive advance of the disease are perfectly in accordance with the results obtained by our group in a study of 77 patients-different from those here presented-in which III-PC values > 38 ng/mL were associated to a likelihood of mortality of
CORRESPONDENCE
January 1991
Table 1. Correlation Between N-Terminal Type III Procollagen and Laminin and Clinical and Biological Parameters III PC (n = 50) Hemoglobin MCV GGT ALT AST Alkaline phosphatase LDH Bilirubin Prothrombin Serum albumin III-PC Fibrosis (%) Fat (%) Necrosis Polymorphonuclear infiltrate Mononuclear infiltrate Hyalin Satelitosis Ascites Encephalopathy Jaundice Cirrhosis Pugh’s score Child’s classification
r = r = r = r = r = r= r= r= r= r= -
-0.16 0.36 0.42” 0.14 0.22 0.54" 0.25 0.51"
r= r= t= t= t= t=
0.46" 0.16 1.11 3.85' 0.24 0.52 0.8 3.4" 1.72 3.55" 3.63" 0.55' 10.72'
t t t t t
= = = = =
r= F =
0.59”
-0.17
Laminin (n = 50) r = r= r= r= r= r= r = r= r= r = r= r= r= t=
-0.24 0.34' 0.42“ 0.05 0.23 0.47 0.03 0.47" 0.64" -0.55" 0.66" 0.52" -0.20 1.03 t = 3.15" t = 0.70 t = 0.64 t = 0.1 t = 3.90’
r = 1.77 t = 4.20" t = 5.58" r = 0.66" F = 16.93”
MCV, mean corpuscular volume; GGT, y-glutamyl transferase; ALT, alanine aminotransferase; AST, aspartate aminotransferase; LDH, lactate dehydrogenase. “P < 0.01. bP < 0.05. 'P < 0.001.
Thus, we agree with Niemell et al., as with 60% at 600 days (4). other authors (5-lo), that both laminin and III-PC are good indicators of the severity of chronic alcoholic liver disease, and are also related to the classical markers of alcoholism, mean corpuscular volume and y-glutamyl transferase. E.GONZALBZ-REIMERS F.SANTOLARIA-FERNANDEZ M.M.BRAJIN-RODRIGUEZ N.BATISTA-LOPEZ Departamenta de Medicina Interna Hospital Universitariode Canarias La Laguna, Tenerife Canary Islands, Spain
Niemela 0, Risteli J, Blake JE, Risteli L, Compton KV, Orrego H. Markers of fibrogenesis and basement membrane formation in alcoholic liver disease. Relation to severity, presence of hepatitis, and alcohol intake. Gastroenterology 1990;98:1612-1619. Infante-Rivard C, Esnaola S, Villeneuve JP. Clinical and statistical validity of conventional prognostic factors in predicting short-term survival among cirrhotics. Hepatology 1987;7:660664. GonzBlez-Reimers E, Brajin-Rodriguez MM, Batista-L6pez N, et al. Hepatocyte and nuclear areas and fatty infiltration of the liver
293
in chronic alcoholic liver disease. Drug Alcohol Depend 1988;22: 195-203. 4. Gonzilez-Reimers E, Brajin-Rodriguez MM, Rodriguez-Moreno F, et al. Clinical and prognostic value of serum procollagen levels in chronic alcoholic liver disease. Drug Alcohol Depend 1990;25:91-95. 5. Rhode H, Vargas L, Hahn EG, Kalbfleishch H, Bruguera M, Timpl R. Radioimmunoassay for type III collagen peptide and its application to human liver disease. Eur J Clin Invest 1979;9:451459. 6. Huerta G, Allende MT, Rodrigo L, Ruibal A. Comportamiento de la laminina sdrica en pacientes hepatbpatas. Experiencia preliminar. Med Clin (Bare) 1989;93:597. Niemell 0, Risteli L, Sotaniemi EA, Risteli J. Aminoterminal propeptide of type III procollagen in serum in alcoholic liver disease. Gastroenterology 1983; 85:254-259. Sato S, Nouchi T, Worner TM, Lieber CS. Liver fibrosis in alcoholics. Detection by fab radioimmunoassay of serum procollagen III peptides. JAMA 1986;256:1471-1473. Frei A, Zimmermann A, Weigand K. The N-terminal propeptide of collagen type III in serum reflects activity and degree of fibrosis in patients with chronic liver disease. Hepatology 1984;4:830-834. 10. Annoni G, Colombo M, Cantaluppi MC, Khlat B, Lampertico P, Rojkind M. Serum type III procollagen peptide and laminin (Lam-PI) detect alcoholic hepatitis in chronic alcohol abusers. Hepatology 1989;9:693-697. Reply. It is gratifying to us that Dr. Gonzalez-Reimers et al., who have followed a similar line of research to our own, are reporting similar findings. The main interest of our paper is that it shows that the serum markers of connective tissue metabolism are related to the clinical and morphological severity of alcoholic liver disease, the presence of alcoholic hepatitis, and the degree of alcohol intake. Gonzalez-Reimers et al. also show a correlation between serum laminin, PIIINP, and the total amount of fibrosis in a biopsy specimen. Indeed, the severity of cirrhosis has often been attributed to the presence of fibrotic nodules or septa. However, these abnormalities can coexist with a completely asymptomatic clinical state. Clinically, a wide spectrum of cirrhosis exists, and we believe that the amount of liver fibrosis per se is not necessarily a criterion of disease severity. The morphological abnormalities coexisting with cirrhosis that are significantly related to mortality are the presence of Mallory bodies, necrosis, and inflammation. In our work we show a correlation between these morphological indices and the markers of fibrogenesis and basement membrane formation. It should also be noted that the serum concentrations of various markers of connective tissue metabolism are a complicated sum of biological events related to biosynthesis and degradation of connective tissue as well as liver endothelial cell function. Therefore, at this time it may not be a realistic application to use serum values of the connective tissue components to mark the degree of existing fibrosis. Gonzalez-Reimers et al. also suggest that the markers of connective tissue are related to biochemical markers of alcohol use (GGT and MCV). Since the classical biochemical tests for alcohol consumption are known to be ineffective for such purposes, we also investigated the relationship between the markers of connective tissue metabolism and the degree of actual alcohol consumption as assessed by the daily urinary alcohol. A close relationship emerged between the markers of connective tissue metabolism and alcohol intake. If this observation continues to be confirmed, it may eventually lead to a better understanding of the role of ethanol itself
CORRESPONDENCE
The treatment of dogs with repeated IV calcium injection (a) increased the sensitivity of acinar cells to cerulein and urecholine and the potentiation by cerulein of the water and bicarbonate secretion in response to secretin; in contrast, it decreased the sensitivity to secretin alone: (b) induced an inhibition of water and bicarbonate secretion with urecholine stimulation; and (c) induced a significant inhibition of calcium secretion after cerulein injection (4). These findings findings enlighten the mechanism of action of hypercalcemia, the biochemical composition of protein plugs described for the first time after calcium injection, and the persistence of calcium-induced modification of pancreatic secretion. This could have some importance in the understanding of calciuminduced pancreatitis and should be mentioned. HENRISARI&S Hdpital Ste-Marguerite Marseilles, France
1. Frich TW, Hailemariam S, Heitz PU, Largiader F, Goodale RL. Acute hypercalcemia induces acinar cell necrosis and intraductal precipitates in the pancreas of cat and guinea pig. Gastroenterology 1990;98:1675-1681. 2. Noel-Jorand MC, Verine HJ, Sarles H. Dose-dependent and long lasting effects of repeated intravenous injections of calcium on the canine secretin-stimulated pancreatic juice secretion. Eur J Clin Invest 1981;11:25-31. 3. Noel-Jorand MC, Colomb E, Astier JP, Sarles H. Persisting modifications of dogs’ basal exocrine pancreatic secretion after repeated intravenous calcium injections. Eur J Clin Invest 1981;11:249-256. 4. NoelJorand MC, Schmidt, Devaux, Sarles H. Biomed Pharmacother 1983;37:129-135. Reply.
The experiments of Dr. Sarles et al. were of very much importance in the design and the interpretation of our experiments (1). Although precipitates were not seen in the histologic slides of dog pancreas after calcium injection (2), the findings of plugs in the pancreatic juice illustrate important changes in secretory activity of the pancreas with a possible implication to the genesis of chronic or acute pancreatitis (3). However, neither these experiments nor our results give any direct clue as to whether intraductal precipitates are the cause or the result of changes eventually leading to acute pancreatitis. In the cat, acute hypercalcemia has been shown to increase pancreatic duct permeability (4). Acute pancreatitis developed after perfusion of the pancreatic duct with activated pancreatic enzymes. These findings suggest that early events of hypercalcemia-associated acute pancreatitis take place in the ductal system. On the other hand, preliminary ultrastructural studies of cat and guinea pig pancreas in hypercalcemia revealed acinar cell changes similar to early findings in the hyperstimulation pancreatitis model (5,6). Because an increase in extracellular calcium levels is able to increase the secretory activity of the acinar cell (2,3,7), it can be speculated that hypercalcemia-induced pancreatitis represents a form of hyperstimulation pancreatitis. Clearly, more research is needed to conclusively answer this question. THOMASFRICK,M.D. Department of Surgery University of Ziirich Hospital 8091 Ziirich, Switzerland
1. Frick TW, Hailemariam S, Heitz PU, Largiader F, Goodal RL. Acute hypercalcemia induces acinar cell necrosis and intraductal precipitates in the pancreas of cat and guinea pig. Gastroenterology 1990;98:1675-1681.
GASTROENTEROLOGY Vol. 100, No. 1
2. Noel-Jorand MC, Verine HJ, Sarles H. Dose-dependent and long lasting effects of repeated intravenous injections of calcium on the canine secretin-stimulated pancreatic juice secretion. Eur J ClinInvest 1981;11:25-31. 3. Noel-Jorand MC, Colomb E, Astier JP, Sarles H. Persisting modifications of dogs’ basal exocrine pancreatic secretion after repeated intravenous calcium injections. Eur J Clin Invest 1981;11:249-256. 4. Cates MC, Singh SM, Peick AL, Harvey MH, Reber HA. Acute hypercalcemia, pancreatic duct permeability, and pancreatitis in cats. Surgery 1988;104:137-141. 5. Frick TW, Runge W, Sibley RK, Satterberg TL, Goodale RL. Effect of acute hypercalcemia on guinea pig pancreas (abstr). Dig Dis Sci 1985;30:972. 6. Frick TW, Runge W, Sibley RK, Satterberg TL, Goodale RL. Calcium infusion and pancreatic ultrastructure in the cat (abstr). Dig Dis Sci 1985;30:971. 7. Layer P, Hotz J. Eysselein VE, Jansen JBMJ, Lamers CBHW, Schmitz-Moormann HP, Gdbell H. Effects of acute hypercalcemia on exocrine pancreatic secretion in the cat. Gastroenterology 1985;88:1168-1174.
Serum Laminin and N-Terminal Type III Procollagen in Chronic Alcoholic Liver Disease Dear Sir: We have read with interest the article by Niemela et al. (1) dealing with the relationships between N-terminal type III procollagen (III-PC), serum laminin, and type IV collagen, and the severity of chronic alcoholic liver disease. We here present our data, which provide similar information to that of Niemell’s group, as we have conducted a similar prospective study of 50 alcoholic patients, 39 men and 11 women, 30 cirrhotics and 22 noncirrhotics, histologically diagnosed by percutaneous liver biopsy. The following parameters were histomorphometritally determined using a WIDS II image analyzer: total amount of fibrosis, as the area of the fibrous tracts (Van Giesson stain) in relation to the area of the section of the biopsy cylinder (at 265~); and total amount of fat (mean area of fat droplets x no. of fat droplets in the biopsy cylinder section/total area of cylinder, at 3625x). We also graded the intensity of polymorphonuclear infiltrate (0, + = absent: + +, + + + = present), the intensity of mononuclear infiltrate (0, + = absent; ++, +++ = present), presence or not of Mallory’s hyalin and satelitosis, and the intensity of necrosis (0, + = absent; ++, +++ = present). We have also recorded (at admission of the patients to the hospital) the clinical and biological parameters listed in Table 1, categorized our patients according to Child’s classification with Pugh’s modification (2), and determined serum levels of III-PC and laminin by radioimmunoanalysis (Behring, Marburg, Germany). As can be seen in Table 1, we found a similar degree of correlation between III-PC and laminin as Niemela did, and also a good correlation with an index of disease severity as the ChildPugh score. The parameter most strongly related to laminin was prothrombin activity, but the narrow relation between laminin and the degree of fibrosis is also noteworthy. Indeed, the amount of liver fibrosis is a criterion of severity, with prognostic value regarding mortality (3). The good relationships between III-PC and parameters derived from progressive advance of the disease are perfectly in accordance with the results obtained by our group in a study of 77 patients-different from those here presented-in which III-PC values > 38 ng/mL were associated to a likelihood of mortality of
CORRESPONDENCE
January 1991
Table 1. Correlation Between N-Terminal Type III Procollagen and Laminin and Clinical and Biological Parameters III PC (n = 50) Hemoglobin MCV GGT ALT AST Alkaline phosphatase LDH Bilirubin Prothrombin Serum albumin III-PC Fibrosis (%) Fat (%) Necrosis Polymorphonuclear infiltrate Mononuclear infiltrate Hyalin Satelitosis Ascites Encephalopathy Jaundice Cirrhosis Pugh’s score Child’s classification
r = r = r = r = r = r= r= r= r= r= -
-0.16 0.36 0.42” 0.14 0.22 0.54" 0.25 0.51"
r= r= t= t= t= t=
0.46" 0.16 1.11 3.85' 0.24 0.52 0.8 3.4" 1.72 3.55" 3.63" 0.55' 10.72'
t t t t t
= = = = =
r= F =
0.59”
-0.17
Laminin (n = 50) r = r= r= r= r= r= r = r= r= r = r= r= r= t=
-0.24 0.34' 0.42“ 0.05 0.23 0.47 0.03 0.47" 0.64" -0.55" 0.66" 0.52" -0.20 1.03 t = 3.15" t = 0.70 t = 0.64 t = 0.1 t = 3.90’
r = 1.77 t = 4.20" t = 5.58" r = 0.66" F = 16.93”
MCV, mean corpuscular volume; GGT, y-glutamyl transferase; ALT, alanine aminotransferase; AST, aspartate aminotransferase; LDH, lactate dehydrogenase. “P < 0.01. bP < 0.05. 'P < 0.001.
Thus, we agree with Niemell et al., as with 60% at 600 days (4). other authors (5-lo), that both laminin and III-PC are good indicators of the severity of chronic alcoholic liver disease, and are also related to the classical markers of alcoholism, mean corpuscular volume and y-glutamyl transferase. E.GONZALBZ-REIMERS F.SANTOLARIA-FERNANDEZ M.M.BRAJIN-RODRIGUEZ N.BATISTA-LOPEZ Departamenta de Medicina Interna Hospital Universitariode Canarias La Laguna, Tenerife Canary Islands, Spain
Niemela 0, Risteli J, Blake JE, Risteli L, Compton KV, Orrego H. Markers of fibrogenesis and basement membrane formation in alcoholic liver disease. Relation to severity, presence of hepatitis, and alcohol intake. Gastroenterology 1990;98:1612-1619. Infante-Rivard C, Esnaola S, Villeneuve JP. Clinical and statistical validity of conventional prognostic factors in predicting short-term survival among cirrhotics. Hepatology 1987;7:660664. GonzBlez-Reimers E, Brajin-Rodriguez MM, Batista-L6pez N, et al. Hepatocyte and nuclear areas and fatty infiltration of the liver
293
in chronic alcoholic liver disease. Drug Alcohol Depend 1988;22: 195-203. 4. Gonzilez-Reimers E, Brajin-Rodriguez MM, Rodriguez-Moreno F, et al. Clinical and prognostic value of serum procollagen levels in chronic alcoholic liver disease. Drug Alcohol Depend 1990;25:91-95. 5. Rhode H, Vargas L, Hahn EG, Kalbfleishch H, Bruguera M, Timpl R. Radioimmunoassay for type III collagen peptide and its application to human liver disease. Eur J Clin Invest 1979;9:451459. 6. Huerta G, Allende MT, Rodrigo L, Ruibal A. Comportamiento de la laminina sdrica en pacientes hepatbpatas. Experiencia preliminar. Med Clin (Bare) 1989;93:597. Niemell 0, Risteli L, Sotaniemi EA, Risteli J. Aminoterminal propeptide of type III procollagen in serum in alcoholic liver disease. Gastroenterology 1983; 85:254-259. Sato S, Nouchi T, Worner TM, Lieber CS. Liver fibrosis in alcoholics. Detection by fab radioimmunoassay of serum procollagen III peptides. JAMA 1986;256:1471-1473. Frei A, Zimmermann A, Weigand K. The N-terminal propeptide of collagen type III in serum reflects activity and degree of fibrosis in patients with chronic liver disease. Hepatology 1984;4:830-834. 10. Annoni G, Colombo M, Cantaluppi MC, Khlat B, Lampertico P, Rojkind M. Serum type III procollagen peptide and laminin (Lam-PI) detect alcoholic hepatitis in chronic alcohol abusers. Hepatology 1989;9:693-697. Reply. It is gratifying to us that Dr. Gonzalez-Reimers et al., who have followed a similar line of research to our own, are reporting similar findings. The main interest of our paper is that it shows that the serum markers of connective tissue metabolism are related to the clinical and morphological severity of alcoholic liver disease, the presence of alcoholic hepatitis, and the degree of alcohol intake. Gonzalez-Reimers et al. also show a correlation between serum laminin, PIIINP, and the total amount of fibrosis in a biopsy specimen. Indeed, the severity of cirrhosis has often been attributed to the presence of fibrotic nodules or septa. However, these abnormalities can coexist with a completely asymptomatic clinical state. Clinically, a wide spectrum of cirrhosis exists, and we believe that the amount of liver fibrosis per se is not necessarily a criterion of disease severity. The morphological abnormalities coexisting with cirrhosis that are significantly related to mortality are the presence of Mallory bodies, necrosis, and inflammation. In our work we show a correlation between these morphological indices and the markers of fibrogenesis and basement membrane formation. It should also be noted that the serum concentrations of various markers of connective tissue metabolism are a complicated sum of biological events related to biosynthesis and degradation of connective tissue as well as liver endothelial cell function. Therefore, at this time it may not be a realistic application to use serum values of the connective tissue components to mark the degree of existing fibrosis. Gonzalez-Reimers et al. also suggest that the markers of connective tissue are related to biochemical markers of alcohol use (GGT and MCV). Since the classical biochemical tests for alcohol consumption are known to be ineffective for such purposes, we also investigated the relationship between the markers of connective tissue metabolism and the degree of actual alcohol consumption as assessed by the daily urinary alcohol. A close relationship emerged between the markers of connective tissue metabolism and alcohol intake. If this observation continues to be confirmed, it may eventually lead to a better understanding of the role of ethanol itself