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Relationship between thrombopoietin serum levels and liver function in patients with chronic liver disease related to hepatitis C virus infection
THE AMERICAN JOURNAL OF GASTROENTEROLOGY © 2003 by Am. Coll. of Gastroenterology Published by Elsevier Inc.
Vol. 98, No. 11, 2003 ISSN 0002-9270/03/$30.00 doi:10.1016/S0002-9270(03)01698-8
Relationship Between Thrombopoietin Serum Levels and Liver Function in Patients With Chronic Liver Disease Related to Hepatitis C Virus Infection Edoardo Giannini, M.D., Federica Botta, M.D., Paolo Borro, M.D., Federica Malfatti, M.D., Alessandra Fumagalli, M.D., Emanuela Testa, M.D., Elena Podesta`, M.D., Bruno Chiarbonello, M.D., Simone Polegato, M.D., Mario Mamone, and Roberto Testa, M.D. Gastroenterology Unit, Department of Internal Medicine, University of Genoa, Genoa, Italy
OBJECTIVES: Thrombopoietin (Tpo) is an important regulator of megakaryocyte maturation and platelet production, and is mainly produced by the liver. A decrease in Tpo production is partly responsible for the thrombocytopenia observed in patients with chronic liver disease (CLD). The aim of this study was to evaluate the relationship between Tpo serum levels and liver function in patients with CLD related to hepatitis C virus (HCV) infection. METHODS: We studied 37 patients with various degrees of HCV-related CLD. Of the patients, 17 had chronic hepatitis and 20 liver cirrhosis. Liver function was evaluated in all patients by the following hepatic blood flow dependent and independent tests that explore various hepatic metabolic functions: carbon-13 (13C)–aminopyrine breath test (13CABT), 13C-galactose breath test (13C-GBT), and monoethylglycinexylidide (MEGX) test. Liver function tests results were correlated with Tpo serum levels. RESULTS: Tpo serum levels were significantly lower in patients with liver cirrhosis (88 ⫾ 23 pg/ml) as compared to those in patients with chronic hepatitis (128 ⫾ 55 pg/ml, p ⫽ 0.0031). However, they did not correlate with serum albumin, bilirubin, or prothrombin activity. Tpo serum levels showed a significant positive correlation with 13C-ABT results (hourly dose at 30 min, rs⫽ 0.489, p ⫽ 0.002; cumulative dose at 120 min, rs⫽ 0.425, p ⫽ 0.008). Moreover, they showed a fair, positive correlation with 13C-GBT hourly dose at 30 min (rs⫽ 0.366, p ⫽ 0.028), and a trend toward a positive correlation with the various MEGX test sampling times (MEGX15, rs⫽ 0.314, p ⫽ 0.059; MEGX30, rs⫽ 0.284, p ⫽ 0.088; and MEGX60, rs⫽ 0.320, p ⫽ 0.059). CONCLUSIONS: In this study we have shown that a progressive decline in liver function in patients with HCV-related CLD is paralleled by a decrease in Tpo production. The different correlations observed between Tpo and the various liver function tests suggests that this finding is mainly the result of a decrease in hepatic functional mass rather than dependent on alteration in splanchnic hemodynamic. (Am J Gastroenterol 2003;98:2516 –2520. © 2003 by Am. Coll. of Gastroenterology)
INTRODUCTION The recent description of thrombopoietin (Tpo) as a cytokine that regulates megakaryocyte maturation and platelet production has modified the historical concept of thrombocytopenia in patients with CLD (1–3). The main Tpo production site is the liver (4), and decreased Tpo production by the liver has been proposed as being an important concomitant cause of the reduction in platelet production in patients with CLD, although not all studies obtained similar results (5–9). Indeed, in patients with liver cirrhosis it has been shown that restoring liver function by means of orthotopic liver transplantation, although not by simple relief of portal hypertension alone, is accompanied by increased Tpo production followed by a rise in the platelet count (6, 10). It has recently been suggested that liver fibrosis may play some role in determining relatively low Tpo serum levels among patients with chronic hepatitis, although fibrosis per se may be a surrogate for both initial portal hypertension or decreased liver function (11). As far as this issue is concerned, however, the results seem to focus on the role that decreased liver function plays on Tpo production. In fact, some studies have shown that Tpo production correlates with disease stage and liver function in patients with various degrees of CLD, although we recently showed that Tpo serum levels are linked to liver function in patients with chronic hepatitis related to hepatitis C virus (HCV) infection (12–14). The carbon-13 (13C)–aminopyrine breath test (13C-ABT), monoethylglycinexylidide (MEGX) test, and 13C-galactose breath test (13C-GBT) are dynamic, quantitative liver function tests (15). The results of these tests are correlated with the various stages of CLD, although when the pharmacokinetics of the probes is taken into account, 13C-ABT and 13 C-GBT results are considered independent of liver blood flow, whereas MEGX test results are partly dependent on hepatic perfusion (16). Moreover, both 13C-ABT and MEGX test explore the cytochrome P-450 dependent microsomal liver function, whereas 13C-GBT depends on liver cytosolic galactokinase activity and its results are correlated with the hepatic functional mass (17, 18).
AJG – November, 2003
In this study, we aimed to identify a correlation between TPO serum levels and liver function as evaluated by both hepatic blood flow dependent and independent tests that explore various hepatic metabolic functions in a cohort of patients with various degrees of CLD caused by HCV infection.
MATERIALS AND METHODS Study Patients We studied 37 patients with various degrees of CLD related to HCV infection. Of the patients, 17 had chronic hepatitis and 20 liver cirrhosis. Some of these patients had already been included in a previous study (6). Diagnosis was based on liver histology results in all chronic hepatitis patients, whereas the diagnosis of liver cirrhosis was made on histological (six patients) or on clinical (biochemical, ultrasonographic, and endoscopic results) bases in the remaining patients. At the time of the study none of the patients was receiving or had previously received interferon therapy. None of the patients reported consuming any alcohol. All patients were positive for anti-HCV antibodies detected by a third generation enzyme immunoassay containing HCV antigens from the viral core and from areas of the nonstructural NS3, NS4, and NS5 regions (Ortho HCV SAVe 3.0; Ortho, Raritan, NJ), and were positive for HCV-RNA in the serum as assessed by means of nested reverse transcription polymerase chain reaction. A peripheral blood sample was obtained from all patients to assess platelet count, aminotransferase, serum bilirubin, prothrombin actitvity, serum albumin, and Tpo. Thrombocytopenia was defined as a platelet count ⬍150 ⫻109/L. In patients with liver cirrhosis the severity of liver disease was scored according to Child-Pugh classification (19). Presence of splenomegaly (maximal spleen bipolar diameter ⬎120 mm) was estimated in all patients by means of ultrasonography. Quantitative Tpo Assessment Serum Tpo levels (in picograms per milliliter) were measured by means of an ELISA (Quantikine; R&D Systems Europe, Oxon, UK) according to the manufacturer’s instructions. The lower limit of detection of the kit is 15 pg/ml Tpo. Sera were collected in a serum separator tube and allowed to clot for approximately 45– 60 min; they were then centrifuged and rapidly stored at ⱕ20°C until Tpo was determined. In our laboratory the intra-assay coefficient of variation was 8.7%. Liver Function Liver function was evaluated by means of 13C-ABT, 13CGBT, and MEGX test in all patients. The MEGX test and 13 C-GBT were carried out on the same day; the 13C-ABT was performed 3 days later. All tests were performed as previously described (20 –22); the results are expressed according to standard criteria (20 –24).
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Briefly, MEGX test was performed as follows. Lidocaine was injected into a forearm vein over approximately 2–3 min, at a dosage of 1 mg/kg body weight; blood samples were then taken 15, 30, and 60 min after lidocaine injection (MEGX15, MEGX30, and MEGX60). MEGX serum concentration was measured at the three sampling times by means of the TDx fluorescence polarization immunoassay system, and was calculated as follows: MEGXt ⫺ MEGX0, where MEGXt is the MEGX concentration at sampling time and MEGX0 is at baseline. The 13C-ABT testing was carried out as follows: two basal breath samples were collected after an overnight (ⱖ12 h) fast, then 2 mg/kg of 13C aminopyrine (N-N-dimethyl13 C-aminopyrine; Euriso-Top Carbon 13 Breath Tests substrates, Saint Aubin, France; supplied by Cortex Italia, Milan, Italy) were dissolved in 200 ml of water and administered p.o.. Breath samples were collected every 30 min for 2 h after aminopyrine administration and were obtained as follows. Patients were asked to exhale for 10 s through a small plastic tube directly into a vial that was immediately sealed. The ratio of 13CO2 to 12CO2 was determined for each sample with an isotope ratio mass spectrometer (Breath Mat, Finnigan, Bremen, Germany), and the excess 13CO2 was calculated by the increase in the isotope ratio. The results were expressed as a percentage of the administered dose of 13C recovered hourly and in the cumulative percentage of administered dose of 13C recovered over time. The 13C-GBT evaluation was performed as follows: two basal breath samples were collected after an overnight fast, then 10 g/m2 body surface of 13C galactose (1 ⫺ 13C D-Galactose, Euriso-Top Carbon 13 Breath Tests Substrates; supplied by Cortex Italia) dissolved in 100 ml of sterile water were administered p.o. to the patients. The concentration of labeled galactose was 1%. This concentration has been proved to ensure significant isotopic CO2 enrichment while limiting the costs related to the labeled substrate (18). The amount of galactose we administered was chosen to saturate the galactose metabolic pathway so as to reflect the hepatic metabolic activity rather than liver blood flow (25). Breath samples were collected every 30 min for 3 h after 13C galactose administration and were obtained as for 13C-ABT. The results are expressed as a percentage of the administered dose of 13C recovered hourly and in the cumulative percentage of administered dose of 13 C recovered over time. Statistical Analysis Data are shown as the mean ⫾ SD. The differences between quantitative variables were evaluated with the Mann-Whitney U test. Differences in both Tpo serum levels and platelet counts among patients with chronic hepatitis, Child-Pugh class A, and class B plus C liver cirrhosis were evaluated by means of analysis of the variance. A p value ⬍0.05 for two-sided tests was considered statistically significant. The correlation between two variables was analyzed using the
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Table 1. Main Characteristics of the 37 Study Patients With AntiHCV Positive Chronic Liver Disease Variable
Unit
AST ALT Bilirubin Albumin Prothrombin activity Platelets Tpo Spleen diameter 13 C-ABT 13 C-ABT 13 C-GBT MEGX MEGX MEGX
IU/ml IU/ml mg/dl g/ml % n ⫻ 109/L pg/ml mm % dose/h at 30 min % dose cum at 120 min % dose/h at 30 min ng/ml at 15 min ng/ml at 30 min ng/ml at 60 min
Mean
SD
89 70 132 122 0.9 0.6 4.1 0.5 83 13 141 53 106 46 120 22 7.2 4.6 5.6 3.0 1.5 0.9 45 34 54 33 60 27
Spearman rank correlation test (rs). Statistical analysis was performed using the MedCalc 5.00 statistical software (MedCalc Software, Mariakerke, Belgium).
RESULTS A total of 37 patients were studied. Of these, 30 were male and seven female, with a mean age of 53 ⫾ 12 yr. The main clinical, biochemical, and functional characteristics of the patients are shown in Table 1. Among the 20 cirrhotic patients, 14 were Child-Pugh class A, five in class B, and one in class C. In all, 21 patients had splenomegaly (i.e., spleen diameter ⬎120 mm), and 24 patients had thrombocytopenia (i.e., platelet count ⬍150 ⫻ 109)/L). Both of these features were more commonly seen in patients with liver cirrhosis. Tpo serum levels were significantly lower in patients with liver cirrhosis (88 ⫾ 23 pg/ml) as compared to patients with chronic hepatitis (128 ⫾ 55 pg/ml, p ⫽ 0.0031). Patients with Child-Pugh class B and C liver cirrhosis were grouped for clinical and statistical reasons, and Tpo serum levels were compared among patients with chronic hepatitis, Child-Pugh class A cirrhosis, and Child-Pugh class B plus C cirrhosis. This analysis showed that Tpo serum levels were significantly different among these three subgroups of patients (p ⫽ 0.019) (Fig. 1). Tpo serum levels varied significantly among patients with thrombocytopenia (88 ⫾ 25 pg/ml, five patients with chronic hepatitis, and 19 with liver cirrhosis) as compared to levels in patients with normal platelet counts (139 ⫾ 57 pg/ml, p ⫽ 0.0008). Platelet counts were significantly different among patients with chronic hepatitis, Child-Pugh class A liver cirrhosis, and class B plus C liver cirrhosis (p ⬍ 0.001). Spleen size (in millimeters) was inversely correlated with platelet count (rs ⫽ ⫺0.358, p ⫽ 0.032), whereas Tpo serum levels were no different among patients with (94 ⫾ 27 pg/ml) or without splenomegaly (116 ⫾ 55 pg/ml, p ⫽ 0.2973). Tpo serum levels were not correlated with serum albumin, bilirubin, or prothrombin activity.
Figure 1. Box and whisker plot of thrombopoietin serum levels in patients with chronic hepatitis, Child-Pugh class A (CIR A) and Child-Pugh class B plus C (CIR B and C) liver cirrhosis.
Tpo serum levels showed a significant, positive correlation with both 13C-ABT dose/h at 30 min (rs⫽ 0.489, p ⫽ 0.002), and 13C-ABT cumulative dose at 120 min (rs⫽ 0.425, p ⫽ 0.008) (Fig. 2). Moreover, they showed a fair positive correlation with 13C-GBT dose/h at 30 min (rs⫽ 0.366, p ⫽ 0.028) and a trend toward a positive correlation with the various MEGX test sampling times (MEGX15, rs⫽ 0.314, p ⫽ 0.059; MEGX30, rs⫽ 0.284, p ⫽ 0.088; and MEGX60, rs⫽ 0.320, p ⫽ 0.059).
DISCUSSION In this study, we have shown that decreased TPO production in patients with various degrees of CLD related to HCV infection is linked to decreased hepatocellular functioning mass. In particular, we have shown that Tpo serum levels correlate with the results of hepatic blood flow-independent quantitative liver function tests. Moreover, we observed that the decrease in Tpo production is not reflected by changes in the common tests used for liver function, such as serum albumin, bilirubin, or prothrombin activity. In this study we confirmed previous results by showing that Tpo serum levels have a positive correlation with 13C-ABT results (14). Furthermore, we showed that Tpo serum levels correlate with 13C-GBT, and that they showed a trend toward correlation with MEGX test results. 13 C-ABT and MEGX test are dynamic quantitative liver function tests. Both tests explore cytochrome P-450 dependent liver microsomal function, although they differ in that the former is independent of liver blood flow, whereas the latter is partly dependent on hepatic perfusion. 13C-GBT results evaluate the cytosolic function of galactokinase enzyme. When the amount of administered galactose saturates the galactose metabolic pathway as in this study, the results of the test reflect the hepatic metabolic activity rather than
AJG – November, 2003
Figure 2. Scatter diagram and correlation line between serum thrombopoietin levels and both 13C aminopyrine breath test hourly dose at 30 min (a) and cumulative dose at 120 min (b) in the 37 study patients.
liver blood flow. Therefore, the findings of this study seem to suggest that the decrease in Tpo production that occurs as liver disease worsens is mainly the result of a decrease in hepatic functional mass rather than being dependent on alteration in splanchnic hemodynamic. With regard to this point, the lack of correlation with common liver tests is not surprising because these tests, although clinically useful for staging the CLD, are not so good in pointing out the modifications in such a subtle parameter as Tpo. The important correlation that was observed between Tpo and 13C-ABT further underscores this finding, inasmuch as this liver function test was able to identify even minimal alterations in liver function, such as those exerted by external agents, those existing between normal subjects and chronic hepatitis patients with minimal fibrosis, or the difference in liver function between patients with advanced liver fibrosis and well compensated cirrhosis (24, 26 –28). Our findings confirm a series of experimental and clinical data. In fact, animal studies showed that Tpo is mainly
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produced by the liver (4, 29), whereas in humans, relatively low levels of circulating Tpo levels are commonly observed in patients with parenchymal liver disease and impaired liver function. It was also shown that liver transplantation restores the correct feedback between platelets and Tpo (5, 6). Kato et al. (12) showed that if we take into consideration liver weight Tpo m-RNA levels correlate with disease progression from chronic hepatitis to liver cirrhosis, whereas Okubo et al. (13) first documented the existence of a correlation between Tpo and quantitative liver function tests in patients with CLD. These latter investigators also suggested that because the expression of Tpo m-RNA is similar in different stages, Tpo serum levels are likely the reflection of the total mass of functional liver. Finally, we recently showed that Tpo serum levels correlate with 13C-ABT in a subset of patients with chronic hepatitis related to HCV. Thus, there may be several clinical applications of these results. In fact, there could be a number of perturbing elements on liver function in patients with CLD, and the possible effects on Tpo production and therefore its reflection on platelet count may be of interest (30). Moreover, R-hu-Tpo as well as other drugs aimed at restoring liver function can have hematological implications and may be of both clinical and research interest. In conclusion, in this study we have shown that a progressive decline in liver function in patients with HCVrelated CLD is paralleled by a decrease in Tpo production. Although our findings are motivating, we believe that both a decrease in Tpo production by the liver and platelet pooling within the spleen may be responsible for thrombocytopenia that is observed in patients with advanced disease. Nevertheless, we believe that these finding may be clinically relevant because of the important clinical implications of thrombocytopenia in these patients. Reprint requests and correspondence: Roberto Testa, M.D., Gastroenterology Unit, Department of Internal Medicine, University of Genoa, Viale Benedetto XV, n°6, 16132 Genoa, Italy. Received Dec. 19, 2002; accepted Apr. 17, 2003.
REFERENCES 1. Aster RH. Pooling of platelets in the spleen: Role in the pathogenesis of “hypersplenic” thrombocytopenia. J Clin Invest 1966;45:645–57. 2. Kaushansky K. Thrombopoietin: The primary regulator of megakaryocyte and platelet production. Thromb Haemost 1995;74:521–5. 3. Peck-Radosavljevic M. Thrombocytopenia in liver disease. Can J Gastroenterol 2000;14(suppl D):60 –6D. 4. Wolber E-M, Jelkmann W. Thrombopoietin: The novel hepatic hormone. News Physiol Sci 2002;17:6 –10. 5. Peck-Radosavljevic M, Zacherl J, Meng YG, et al. Is inadequate thrombopoietin production a major cause of thrombocytopenia in cirrhosis of the liver? J Hepatol 1997;27:127–31. 6. Goulis J, Chau TN, Jordan S, et al. Thrombopoietin concentrations are low in patients with cirrhosis and thrombocytope-
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20. Testa R, Caglieris S, Risso D, et al. Monoethylglycinexylidide formation measurement as a hepatic function test to assess severity of chronic liver disease. Am J Gastroenterol 1997;92: 2268 –73. 21. Fasoli A, Giannini E, Botta F, et al. 13CO2 excretion in breath of normal subjects and cirrhotic patients after 13C-aminopyrine oral load. Comparison with MEGX test in functional differentiation between chronic hepatitis and liver cirrhosis. Hepato-Gastroenterology 2000;47:234 –8. 22. Giannini E, Fasoli A, Borro P, et al. Impairment of cytochrome P-450 dependent liver activity in cirrhotic patients with Helicobacter pylori infection. Aliment Pharmacol Ther 2001;15:1975–82. 23. Schoeller DA, Baker AL, Monroe PS, et al. Comparison of different methods of expressing results of the aminopyrine breath test. Hepatology 1982;2:455–62. 24. Giannini E, Fasoli A, Chiarbonello B, et al. 13C-aminopyrine breath test to evaluate severity of disease in patients with chronic hepatitis C virus infection. Aliment Pharmacol Ther 2002;16:717–25. 25. Mion F, Ge´ loe¨ n A, Rousseau M, et al. Mechanism of carbon tetrachloride autoprotection: An in vivo study based on 13Caminopyrine and 13C-galactose breath tests. Life Sci 1994;54: 2093–8. 26. Opekun AR Jr, Klein PD, Graham DY. [13C]Aminopyrine breath test detects altered liver metabolism caused by lowdose oral contraceptives. Dig Dis Sci 1995;40:2417–22. 27. Van Vlierberghe H, Van Durme F, Verdievel H, et al. Influence of low-dose oral contraceptives, alcohol, and grapefruit on [13C]aminopyrine breath test. Dig Dis Sci 2001;46:133–9. 28. Herold C, Heinz R, Niedobitek G, et al. Quantitative testing of liver function in relation to fibrosis in patients with chronic hepatitis B and C. Liver 2001;21:260 –5. 29. Shimada Y, Kato T, Ogami K, et al. Production of thrombopoietin (TPO) by rat hepatocytes and hepatoma cell lines. Exp Hematol 1995;23:1388 –9. 30. Peck-Radosavljevic M, Wichlas M, Pidlich J, et al. Blunted thrombocytopenia response to interferon alfa-induced thrombocytopenia during treatment for hepatitis C. Hepatology 1998;28:1424 –9.
Vol. 98, No. 11, 2003 ISSN 0002-9270/03/$30.00 doi:10.1016/S0002-9270(03)01698-8
Relationship Between Thrombopoietin Serum Levels and Liver Function in Patients With Chronic Liver Disease Related to Hepatitis C Virus Infection Edoardo Giannini, M.D., Federica Botta, M.D., Paolo Borro, M.D., Federica Malfatti, M.D., Alessandra Fumagalli, M.D., Emanuela Testa, M.D., Elena Podesta`, M.D., Bruno Chiarbonello, M.D., Simone Polegato, M.D., Mario Mamone, and Roberto Testa, M.D. Gastroenterology Unit, Department of Internal Medicine, University of Genoa, Genoa, Italy
OBJECTIVES: Thrombopoietin (Tpo) is an important regulator of megakaryocyte maturation and platelet production, and is mainly produced by the liver. A decrease in Tpo production is partly responsible for the thrombocytopenia observed in patients with chronic liver disease (CLD). The aim of this study was to evaluate the relationship between Tpo serum levels and liver function in patients with CLD related to hepatitis C virus (HCV) infection. METHODS: We studied 37 patients with various degrees of HCV-related CLD. Of the patients, 17 had chronic hepatitis and 20 liver cirrhosis. Liver function was evaluated in all patients by the following hepatic blood flow dependent and independent tests that explore various hepatic metabolic functions: carbon-13 (13C)–aminopyrine breath test (13CABT), 13C-galactose breath test (13C-GBT), and monoethylglycinexylidide (MEGX) test. Liver function tests results were correlated with Tpo serum levels. RESULTS: Tpo serum levels were significantly lower in patients with liver cirrhosis (88 ⫾ 23 pg/ml) as compared to those in patients with chronic hepatitis (128 ⫾ 55 pg/ml, p ⫽ 0.0031). However, they did not correlate with serum albumin, bilirubin, or prothrombin activity. Tpo serum levels showed a significant positive correlation with 13C-ABT results (hourly dose at 30 min, rs⫽ 0.489, p ⫽ 0.002; cumulative dose at 120 min, rs⫽ 0.425, p ⫽ 0.008). Moreover, they showed a fair, positive correlation with 13C-GBT hourly dose at 30 min (rs⫽ 0.366, p ⫽ 0.028), and a trend toward a positive correlation with the various MEGX test sampling times (MEGX15, rs⫽ 0.314, p ⫽ 0.059; MEGX30, rs⫽ 0.284, p ⫽ 0.088; and MEGX60, rs⫽ 0.320, p ⫽ 0.059). CONCLUSIONS: In this study we have shown that a progressive decline in liver function in patients with HCV-related CLD is paralleled by a decrease in Tpo production. The different correlations observed between Tpo and the various liver function tests suggests that this finding is mainly the result of a decrease in hepatic functional mass rather than dependent on alteration in splanchnic hemodynamic. (Am J Gastroenterol 2003;98:2516 –2520. © 2003 by Am. Coll. of Gastroenterology)
INTRODUCTION The recent description of thrombopoietin (Tpo) as a cytokine that regulates megakaryocyte maturation and platelet production has modified the historical concept of thrombocytopenia in patients with CLD (1–3). The main Tpo production site is the liver (4), and decreased Tpo production by the liver has been proposed as being an important concomitant cause of the reduction in platelet production in patients with CLD, although not all studies obtained similar results (5–9). Indeed, in patients with liver cirrhosis it has been shown that restoring liver function by means of orthotopic liver transplantation, although not by simple relief of portal hypertension alone, is accompanied by increased Tpo production followed by a rise in the platelet count (6, 10). It has recently been suggested that liver fibrosis may play some role in determining relatively low Tpo serum levels among patients with chronic hepatitis, although fibrosis per se may be a surrogate for both initial portal hypertension or decreased liver function (11). As far as this issue is concerned, however, the results seem to focus on the role that decreased liver function plays on Tpo production. In fact, some studies have shown that Tpo production correlates with disease stage and liver function in patients with various degrees of CLD, although we recently showed that Tpo serum levels are linked to liver function in patients with chronic hepatitis related to hepatitis C virus (HCV) infection (12–14). The carbon-13 (13C)–aminopyrine breath test (13C-ABT), monoethylglycinexylidide (MEGX) test, and 13C-galactose breath test (13C-GBT) are dynamic, quantitative liver function tests (15). The results of these tests are correlated with the various stages of CLD, although when the pharmacokinetics of the probes is taken into account, 13C-ABT and 13 C-GBT results are considered independent of liver blood flow, whereas MEGX test results are partly dependent on hepatic perfusion (16). Moreover, both 13C-ABT and MEGX test explore the cytochrome P-450 dependent microsomal liver function, whereas 13C-GBT depends on liver cytosolic galactokinase activity and its results are correlated with the hepatic functional mass (17, 18).
AJG – November, 2003
In this study, we aimed to identify a correlation between TPO serum levels and liver function as evaluated by both hepatic blood flow dependent and independent tests that explore various hepatic metabolic functions in a cohort of patients with various degrees of CLD caused by HCV infection.
MATERIALS AND METHODS Study Patients We studied 37 patients with various degrees of CLD related to HCV infection. Of the patients, 17 had chronic hepatitis and 20 liver cirrhosis. Some of these patients had already been included in a previous study (6). Diagnosis was based on liver histology results in all chronic hepatitis patients, whereas the diagnosis of liver cirrhosis was made on histological (six patients) or on clinical (biochemical, ultrasonographic, and endoscopic results) bases in the remaining patients. At the time of the study none of the patients was receiving or had previously received interferon therapy. None of the patients reported consuming any alcohol. All patients were positive for anti-HCV antibodies detected by a third generation enzyme immunoassay containing HCV antigens from the viral core and from areas of the nonstructural NS3, NS4, and NS5 regions (Ortho HCV SAVe 3.0; Ortho, Raritan, NJ), and were positive for HCV-RNA in the serum as assessed by means of nested reverse transcription polymerase chain reaction. A peripheral blood sample was obtained from all patients to assess platelet count, aminotransferase, serum bilirubin, prothrombin actitvity, serum albumin, and Tpo. Thrombocytopenia was defined as a platelet count ⬍150 ⫻109/L. In patients with liver cirrhosis the severity of liver disease was scored according to Child-Pugh classification (19). Presence of splenomegaly (maximal spleen bipolar diameter ⬎120 mm) was estimated in all patients by means of ultrasonography. Quantitative Tpo Assessment Serum Tpo levels (in picograms per milliliter) were measured by means of an ELISA (Quantikine; R&D Systems Europe, Oxon, UK) according to the manufacturer’s instructions. The lower limit of detection of the kit is 15 pg/ml Tpo. Sera were collected in a serum separator tube and allowed to clot for approximately 45– 60 min; they were then centrifuged and rapidly stored at ⱕ20°C until Tpo was determined. In our laboratory the intra-assay coefficient of variation was 8.7%. Liver Function Liver function was evaluated by means of 13C-ABT, 13CGBT, and MEGX test in all patients. The MEGX test and 13 C-GBT were carried out on the same day; the 13C-ABT was performed 3 days later. All tests were performed as previously described (20 –22); the results are expressed according to standard criteria (20 –24).
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Briefly, MEGX test was performed as follows. Lidocaine was injected into a forearm vein over approximately 2–3 min, at a dosage of 1 mg/kg body weight; blood samples were then taken 15, 30, and 60 min after lidocaine injection (MEGX15, MEGX30, and MEGX60). MEGX serum concentration was measured at the three sampling times by means of the TDx fluorescence polarization immunoassay system, and was calculated as follows: MEGXt ⫺ MEGX0, where MEGXt is the MEGX concentration at sampling time and MEGX0 is at baseline. The 13C-ABT testing was carried out as follows: two basal breath samples were collected after an overnight (ⱖ12 h) fast, then 2 mg/kg of 13C aminopyrine (N-N-dimethyl13 C-aminopyrine; Euriso-Top Carbon 13 Breath Tests substrates, Saint Aubin, France; supplied by Cortex Italia, Milan, Italy) were dissolved in 200 ml of water and administered p.o.. Breath samples were collected every 30 min for 2 h after aminopyrine administration and were obtained as follows. Patients were asked to exhale for 10 s through a small plastic tube directly into a vial that was immediately sealed. The ratio of 13CO2 to 12CO2 was determined for each sample with an isotope ratio mass spectrometer (Breath Mat, Finnigan, Bremen, Germany), and the excess 13CO2 was calculated by the increase in the isotope ratio. The results were expressed as a percentage of the administered dose of 13C recovered hourly and in the cumulative percentage of administered dose of 13C recovered over time. The 13C-GBT evaluation was performed as follows: two basal breath samples were collected after an overnight fast, then 10 g/m2 body surface of 13C galactose (1 ⫺ 13C D-Galactose, Euriso-Top Carbon 13 Breath Tests Substrates; supplied by Cortex Italia) dissolved in 100 ml of sterile water were administered p.o. to the patients. The concentration of labeled galactose was 1%. This concentration has been proved to ensure significant isotopic CO2 enrichment while limiting the costs related to the labeled substrate (18). The amount of galactose we administered was chosen to saturate the galactose metabolic pathway so as to reflect the hepatic metabolic activity rather than liver blood flow (25). Breath samples were collected every 30 min for 3 h after 13C galactose administration and were obtained as for 13C-ABT. The results are expressed as a percentage of the administered dose of 13C recovered hourly and in the cumulative percentage of administered dose of 13 C recovered over time. Statistical Analysis Data are shown as the mean ⫾ SD. The differences between quantitative variables were evaluated with the Mann-Whitney U test. Differences in both Tpo serum levels and platelet counts among patients with chronic hepatitis, Child-Pugh class A, and class B plus C liver cirrhosis were evaluated by means of analysis of the variance. A p value ⬍0.05 for two-sided tests was considered statistically significant. The correlation between two variables was analyzed using the
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Table 1. Main Characteristics of the 37 Study Patients With AntiHCV Positive Chronic Liver Disease Variable
Unit
AST ALT Bilirubin Albumin Prothrombin activity Platelets Tpo Spleen diameter 13 C-ABT 13 C-ABT 13 C-GBT MEGX MEGX MEGX
IU/ml IU/ml mg/dl g/ml % n ⫻ 109/L pg/ml mm % dose/h at 30 min % dose cum at 120 min % dose/h at 30 min ng/ml at 15 min ng/ml at 30 min ng/ml at 60 min
Mean
SD
89 70 132 122 0.9 0.6 4.1 0.5 83 13 141 53 106 46 120 22 7.2 4.6 5.6 3.0 1.5 0.9 45 34 54 33 60 27
Spearman rank correlation test (rs). Statistical analysis was performed using the MedCalc 5.00 statistical software (MedCalc Software, Mariakerke, Belgium).
RESULTS A total of 37 patients were studied. Of these, 30 were male and seven female, with a mean age of 53 ⫾ 12 yr. The main clinical, biochemical, and functional characteristics of the patients are shown in Table 1. Among the 20 cirrhotic patients, 14 were Child-Pugh class A, five in class B, and one in class C. In all, 21 patients had splenomegaly (i.e., spleen diameter ⬎120 mm), and 24 patients had thrombocytopenia (i.e., platelet count ⬍150 ⫻ 109)/L). Both of these features were more commonly seen in patients with liver cirrhosis. Tpo serum levels were significantly lower in patients with liver cirrhosis (88 ⫾ 23 pg/ml) as compared to patients with chronic hepatitis (128 ⫾ 55 pg/ml, p ⫽ 0.0031). Patients with Child-Pugh class B and C liver cirrhosis were grouped for clinical and statistical reasons, and Tpo serum levels were compared among patients with chronic hepatitis, Child-Pugh class A cirrhosis, and Child-Pugh class B plus C cirrhosis. This analysis showed that Tpo serum levels were significantly different among these three subgroups of patients (p ⫽ 0.019) (Fig. 1). Tpo serum levels varied significantly among patients with thrombocytopenia (88 ⫾ 25 pg/ml, five patients with chronic hepatitis, and 19 with liver cirrhosis) as compared to levels in patients with normal platelet counts (139 ⫾ 57 pg/ml, p ⫽ 0.0008). Platelet counts were significantly different among patients with chronic hepatitis, Child-Pugh class A liver cirrhosis, and class B plus C liver cirrhosis (p ⬍ 0.001). Spleen size (in millimeters) was inversely correlated with platelet count (rs ⫽ ⫺0.358, p ⫽ 0.032), whereas Tpo serum levels were no different among patients with (94 ⫾ 27 pg/ml) or without splenomegaly (116 ⫾ 55 pg/ml, p ⫽ 0.2973). Tpo serum levels were not correlated with serum albumin, bilirubin, or prothrombin activity.
Figure 1. Box and whisker plot of thrombopoietin serum levels in patients with chronic hepatitis, Child-Pugh class A (CIR A) and Child-Pugh class B plus C (CIR B and C) liver cirrhosis.
Tpo serum levels showed a significant, positive correlation with both 13C-ABT dose/h at 30 min (rs⫽ 0.489, p ⫽ 0.002), and 13C-ABT cumulative dose at 120 min (rs⫽ 0.425, p ⫽ 0.008) (Fig. 2). Moreover, they showed a fair positive correlation with 13C-GBT dose/h at 30 min (rs⫽ 0.366, p ⫽ 0.028) and a trend toward a positive correlation with the various MEGX test sampling times (MEGX15, rs⫽ 0.314, p ⫽ 0.059; MEGX30, rs⫽ 0.284, p ⫽ 0.088; and MEGX60, rs⫽ 0.320, p ⫽ 0.059).
DISCUSSION In this study, we have shown that decreased TPO production in patients with various degrees of CLD related to HCV infection is linked to decreased hepatocellular functioning mass. In particular, we have shown that Tpo serum levels correlate with the results of hepatic blood flow-independent quantitative liver function tests. Moreover, we observed that the decrease in Tpo production is not reflected by changes in the common tests used for liver function, such as serum albumin, bilirubin, or prothrombin activity. In this study we confirmed previous results by showing that Tpo serum levels have a positive correlation with 13C-ABT results (14). Furthermore, we showed that Tpo serum levels correlate with 13C-GBT, and that they showed a trend toward correlation with MEGX test results. 13 C-ABT and MEGX test are dynamic quantitative liver function tests. Both tests explore cytochrome P-450 dependent liver microsomal function, although they differ in that the former is independent of liver blood flow, whereas the latter is partly dependent on hepatic perfusion. 13C-GBT results evaluate the cytosolic function of galactokinase enzyme. When the amount of administered galactose saturates the galactose metabolic pathway as in this study, the results of the test reflect the hepatic metabolic activity rather than
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Figure 2. Scatter diagram and correlation line between serum thrombopoietin levels and both 13C aminopyrine breath test hourly dose at 30 min (a) and cumulative dose at 120 min (b) in the 37 study patients.
liver blood flow. Therefore, the findings of this study seem to suggest that the decrease in Tpo production that occurs as liver disease worsens is mainly the result of a decrease in hepatic functional mass rather than being dependent on alteration in splanchnic hemodynamic. With regard to this point, the lack of correlation with common liver tests is not surprising because these tests, although clinically useful for staging the CLD, are not so good in pointing out the modifications in such a subtle parameter as Tpo. The important correlation that was observed between Tpo and 13C-ABT further underscores this finding, inasmuch as this liver function test was able to identify even minimal alterations in liver function, such as those exerted by external agents, those existing between normal subjects and chronic hepatitis patients with minimal fibrosis, or the difference in liver function between patients with advanced liver fibrosis and well compensated cirrhosis (24, 26 –28). Our findings confirm a series of experimental and clinical data. In fact, animal studies showed that Tpo is mainly
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produced by the liver (4, 29), whereas in humans, relatively low levels of circulating Tpo levels are commonly observed in patients with parenchymal liver disease and impaired liver function. It was also shown that liver transplantation restores the correct feedback between platelets and Tpo (5, 6). Kato et al. (12) showed that if we take into consideration liver weight Tpo m-RNA levels correlate with disease progression from chronic hepatitis to liver cirrhosis, whereas Okubo et al. (13) first documented the existence of a correlation between Tpo and quantitative liver function tests in patients with CLD. These latter investigators also suggested that because the expression of Tpo m-RNA is similar in different stages, Tpo serum levels are likely the reflection of the total mass of functional liver. Finally, we recently showed that Tpo serum levels correlate with 13C-ABT in a subset of patients with chronic hepatitis related to HCV. Thus, there may be several clinical applications of these results. In fact, there could be a number of perturbing elements on liver function in patients with CLD, and the possible effects on Tpo production and therefore its reflection on platelet count may be of interest (30). Moreover, R-hu-Tpo as well as other drugs aimed at restoring liver function can have hematological implications and may be of both clinical and research interest. In conclusion, in this study we have shown that a progressive decline in liver function in patients with HCVrelated CLD is paralleled by a decrease in Tpo production. Although our findings are motivating, we believe that both a decrease in Tpo production by the liver and platelet pooling within the spleen may be responsible for thrombocytopenia that is observed in patients with advanced disease. Nevertheless, we believe that these finding may be clinically relevant because of the important clinical implications of thrombocytopenia in these patients. Reprint requests and correspondence: Roberto Testa, M.D., Gastroenterology Unit, Department of Internal Medicine, University of Genoa, Viale Benedetto XV, n°6, 16132 Genoa, Italy. Received Dec. 19, 2002; accepted Apr. 17, 2003.
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