Measurement of liver function for patients with cirrhosis by 13C-methacetin breath test compared with Child-Pugh score and routine liver function tests

13C-methacetin breath test was used for the evaluation of liver function, as for quantitative data could be achieved using this method, it had the characteristics of safety, quantification, and repetition and got recognition gradually through the world.1,2 We began this 13C-methacetin test to assess liver function of patients with cirrhosis from January 2002. The aim of this study was to explore the characteristic of this test for liver function evaluation and explore the correlation of this method with some clinical liver biochemical parameters and Child-Pugh score. METHODS Clinical data Twenty-two healthy volunteers served as control group from January 2002 to December 2005, 15 men, 7 women, age 28—73 years, mean 48 years. A total of 68 inpatients with cirrhosis at the same period served as test group, 39 men, 29 women, age 32 — 78 years, mean 53 years. All were confirmed as cirrhosis by liver CT, ultrasound examination, liver function test and/or liver biopsy. According to the Child-Pugh classification, 19 cases in Child A, 21 cases in Child B, 28 cases in Child C. Operational method 13C-methacetin breath test was performed after at least 8 hours of fasting, through the straw breathed air was blown up to the bottom of the test tube, the sample was marked as initial phase. 13CO2 abundance was detected using breath mass spectrometer. Seventy-five mg 13C-methacetin was dissolved in 100 ml of plain boiled water, and then the water was taken by the volunteers and patients, then at different time of 10, 20, 30, 40, 50, 60, 80, 100 and 120 minutes, the 13CO2 abundance was detected as the method detecting the sample at initial phase. Instruments Breath-Mat breath mass spectrometer was offered by German Finnigan-Mat Company, 13C-methacetin (99% in purity) was applied by Beijing Jiahuibin Medical Technology Ltd., China. Data achievement d values at different time were achieved with automatic isotope mass spectrometer (ISMS). The change of 13CO2 abundance (Δ) = d values of different time —initial d value. According to these results, curves of delt over baseline (DOB), metabolic velocity (MV) and cumulative percentage (CUM) were got, three indexes of MVmax30, CUM30, CUM120 were got. MVmax30 = the ratio of peak 13CO2 metabolic velocity before 30 minutes to normal value (27); CUM30 = the ratio of cumulative 13CO2 abundance before 30 minutes to normal value (12); CUM120 = the ratio of cumulative 13CO2 abundance before 120 minutes to normal value (28). Statistical analysis Data are expressed as mean ± standard deviation (SD). Statistical analysis was made by SPSS version 12. One-way analysis of variance (ANOVA) was used for multiple comparisions. If variance was equal, difference among groups was compared using Bonferroni method; if not, then q test was used. The correlation of breath test parameters with some hepatic biochemical indexes and Child-Pugh score was made using Pearson correlation method. P< 0.05 was considered statistically significant. RESULTS DOB curve The peaks of DOB at groups of control, Child A, Child B, Child C were (20.96 ± 1.41) %, (8.77 ± 1.19), (7.19 ± 0.93) ‰, (3.23 ± 0.91) ‰, respectively. The time the peak appeared was at (20.1 ±0.5) minutes, (23.6 ± 6.9) minutes, (41.5 ± 11.9) minutes, (60.9 ± 18.7) minutes, respectively. DOB peaks were significantly different among all groups, the time the peak appeared was significantly different among groups of Child A, B and C (P<0.05) (Fig. 1).Fig. 1.: DOB curveMV curve The peaks of MV at groups of control, Child A, Child B, Child C were (31.28 ± 1.40) %, (11.38 ± 1.97) %, (7.67 ± 1.46) %, (3.49 ± 2.49) %, respectively. The time the peak appeared was at (9.10 ± 0.89) minutes, (22.3 ± 6.0) minutes, (45.3 ± 6.8) minutes, (68.8 ± 15.0) minutes, respectively. MV peaks were significantly different among all groups (P<0.05), and the time the peak appeared was significantly different among groups of Child A, B and C (P<0.05) (Fig. 2).Fig. 2: . MV curveCUM curve CUM30 at groups of control, Child A, Child B, Child C were (11.10 ± 1.35) %, (3.86 ± 1.73) %, (2.15 ± 0.64)%, (0.85 ± 0.38)%, respectively; CUM120 were (33.32 ± 2.94) %, (20.96 ± 4.60)%, (18.86 ± 5.45)%, (7.37 ± 2.60)%, respectively. CUM30 was significantly different among all groups. CUM120 was not significantly different between groups of Child A and B (P>0.05), while it was significantly different among all the other groups (P<0.05) (Fig. 3).Fig. 3.: CUM curveParameters in relation to some hepatic biochemical indexes and Child-Pugh score MVmax30, CUM30 and CUM120 were positively correlated to serum albumin (ALB) (P<0.001), while negatively correlated to serum total bilirubin (TBIL), prothrombin time (PT) and Child-Pugh score (P<0.001), and they were not correlated to serum alanine transaminase (ALT) and aspartate transaminase (AST) (P>0.05) (Table).Table: Correlation of breath test parameters with some hepatic biochemical indexes and Child-Pugh scoreSide effect No side effects such as abdominal pain, abdominal swell and other signs were observed in all the volunteers and patients, hepatic and renal functions were not significantly changed after oral taking methacetin. DISCUSSION Child-Pugh classification was ordinarily used for liver function evaluation, however, this classification could not evaluate liver function accurately. It could be influenced by the subjectivity of some parameters (i.e., degree of ascites or hepatic encephalopathy).3 Only dynamic parameters could evaluate liver function quantitatively, timely and accurately. Several currently used dynamic excretion tests such as sulfobromophthalein dyes and indocyanine green, etc, have been shown to be unpractical in clinic for need of repeated blood samples, risk for anaphylactic reactions, need of long time, trauma, etc, though they were accurate to some extent in evaluating liver function. Recently, 13C-methacetin breath test has been paid more attention in the world for liver function evaluation because of its safety, quantification, accuracy and repetition.4,5 With the progress of liver transplantation, liver function evaluation before transplantation became necessary. 13C-methacetin breath test was used for liver function evaluation before and after liver transplantation.6,7 The mechanism of 13C-methacetin breath test for liver function evaluation is that when 13C-methacetin was orally taken, it was absorbed into blood. In smooth endoplasmic reticulum of hepatocyte, demethylation of zero point occurred in monooxygenases systems of cytochrome P450, nicotinamide-adenine dinucleotide phosphate (NADPH), then formaldehyde was produced, which was oxidized to 13CO2 and expired. Rate and abundance of 13CO2 was detected with breath mass spectrometer, which could reflect function of monooxygenases system of hepatic cell.8 Results from present study show that methacetin could discriminate liver function not only between healthy subjects and liver disease patients, but also between different stages of cirrhosis patients. DOB and MV curves show the peaks were obvious in control group, the peak appeared at 20 minutes after methacetin administration. In the test group, peaks of DOB and MV curves decreased with deterioration of liver function, the appearance of the peaks lag with deterioration of liver function. It showed that with deterioration of liver function, demethyled capacity of hepatocellular microsome weakened, so peaks of DOB and MV curves decreased and lag. With comprehensive analysis of the peak value and the time the peak appeared, we think that methacetin could guide clinical scale of liver function, at the same time, normal liver function and abnormal liver function could be discriminated according to peak value of DOB and MV curve. It was reported that aminopyrine breath test was not influenced by age,9 in this study 13C-methacetin breath test was not influenced by age, according with other report.10 The values of MVmax30, CUM30 and CUM120 of 13C-methacetin breath test were significantly different among control and test groups, except CUM120 in Child A and B, perhaps because CUM120 is the ratio of 13CO2 expired in the 120 minutes to the normal value, mainly reflecting hepatic compensatory function. When liver function was in Child A and B, hepatic compensatory function was not significantly different between Child A and B, so CUM120 was not significantly different. MVmax30 and CUM30 mainly reflect hepatic compensatory function, so it was closely related with Child-Pugh score and was significantly different among all the groups. MVmax30, CUM30 and CUM120 were positively correlated with serum ALB, negatively correlated with serum TBIL, PT and Child-Pugh score. It was reported that liver function could not be compensated only when the liver function was damaged more than 60 — 70 percent.11 Our study showed that MVmax30, CUM30 and CUM120 were correlated with serum TBIL, ALB, PT and Child-Pugh score, indicating that 13C-methacetin breath test could reflect compensation of liver function, especially reflect liver compensatory function quantitatively. It was a beneficial complementarity for liver function evaluation. No side effect related with 13C-methacetin occurred in all the volunteers and patients, and hepatic and renal functions did not change significantly after oral taking of 13C-methacetin, in accordance with other report.12 In conclusion, 13C-methacetin breath test could quantitatively evaluate compensatory function of liver, thus it is a safe and effective method for evaluating liver function of patients with cirrhosis.