13C-Methacetin Breath Test For Monitoring Hepatic Function............

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13C-Methacetin Breath Test For Monitoring Hepatic Function in Cirrhotic Patients Before and After Liver Transplantation

A. Petrolati, D. Festi, G. De Berardinis, L. Colaiocco-Ferrante, D. Di Paolo, G. Tisone, M. Angelico Aliment Pharmacol Ther 18(8):785-790, 2003. © 2003 Blackwell Publishing

Posted 11/26/2003

Summary and Introduction

Summary

Background: In patients with chronic liver disease, the measurement of liver function is critical for monitoring disease progression, predicting the prognosis and choosing therapeutic strategies. The 13C-methacetin breath test is a simple, non-invasive diagnostic tool based on an inexpensive, non-toxic substance, which allows the accurate measurement of liver functional reserve.Aim: To investigate the 13C-methacetin breath test as a tool to monitor hepatic function in liver transplant candidates and recipients.Methods: Twenty-eight cirrhotic patients listed for orthotopic liver transplantation and 10 healthy controls were studied. The 13C-methacetin breath test (75 mg per os) was performed at baseline and at 12-week intervals. Intra-operative measurements were obtained during the liver transplantation procedure in nine patients. Results were expressed as the 13C-methacetin cumulative oxidation percentage 45 min after substrate ingestion.Results: The mean 13C-methacetin cumulative oxidation at 45 min was 16.4 ± 3.5% in healthy controls and 5.4 ± 4.2% in cirrhotic patients at the time of listing. In 11 patients who underwent successful liver transplantation, mean oxidation increased from 3.3 ± 1.6% before transplantation to 17.0 ± 5.2% at 6 months of follow-up. Variations in methacetine oxidation were closely related to the recovery of liver function. The mean intra-operative 13C-methacetin cumulative oxidation increased from 0.1% during the anhepatic phase to 3.7 ± 2.0% 2 h after reperfusion.Conclusions: The 13C-methacetin breath test is a simple and potentially useful tool for monitoring hepatic function in cirrhotic patients listed for liver transplantation, and during the intra-operative and post-operative phases.

Introduction

The Child-Pugh and Model for End-Stage Liver Disease (MELD) scoring systems are the most common prognostic models used to assesssurvival in cirrhotic patients listed for liver transplantation.[1,2] However, the clinical and biochemical parameters included in these scoring systems do not provide a satisfactory evaluation of liver functional reserve, thus limiting their overall accuracy. Currently, quantitative information on hepatic function can easily be obtained from breath tests based on the use of labelled substrates selectively metabolized within the liver.[3-5] Of the different tests investigated so far, the 13C-methacetin breath test (13C-MBT) appears to be particularly suitable for the assessment of the hepatic functional reserve.[6-9]13C-MBT employs a manageable, innocuous substance, which is less expensive than other molecules used for breath tests (i.e. galactose, phenylalanine),[10-12] and has a high rate of blood extraction.

A knowledge of liver functional reserve is important, not only in cirrhotic patients awaiting orthotopic liver transplantation, but also after transplantation for an early recognition of complications (such as primary non-function, early poor function of the graft, cellular rejection, etc.). We therefore investigated whether 13C-MBT could be employed as a useful, non-invasive and simple tool for monitoring the functional restoration of the transplanted organ. In this study, we analysed the behaviour of 13C-MBT in a group of cirrhotic patients before, during and after orthotopic liver transplantation in relation to time and disease severity.

Methods

Patients

Twenty-eight cirrhotic patients listed for orthotopic liver transplantation (20 males, 8 females; age range, 38-60 years) were included in the study. The indications for liver transplantation were virus-related liver cirrhosis in 23 patients (17 related to hepatitis C virus and six related to hepatitis B virus, one of whom had both hepatitis B and D virus infections), alcoholic cirrhosis in three patients and primary biliary cirrhosis and primary sclerosing cholangitis in one patient each. The Child-Pugh classification was as follows: class A, eight patients; class B, 13 patients; class C, seven patients. All patients in class A had already experienced one or more major complications of cirrhosis, such as variceal bleeding or spontaneous bacterial peritonitis. All patients were non-smokers at the time of the study and had normal pulmonary function tests. Sixteen patients underwent orthotopic liver transplantation between December 1999 and March 2001. All patients received their graft from cadaveric donors. One patient received a split liver (right). Three patients died within 2 months of transplantation (due to primary non-function, renal insufficiency and cerebral haemorrhage, respectively); the remainder are long-term survivors. Ten non-smoking healthy volunteers (six females, four males; median age, 30 years) acted as controls. Patients and controls provided written informed consent. Table 1 lists the characteristics of the studied population.

13C-Methacetin Breath Test

13C-MBT was performed after at least 8 h of fasting. Before administration of the substrate, a basal air sample was taken to evaluate the amount of 13C present at baseline (International Standard Ratio = 0.1%).[10] 13C-MBT was performed after the oral administration of 75 mg of 13C-methacetin (CIL, Andover, MA, USA) diluted in 50 cm3 of water. Air samples were obtained by slow expiration through a straw into a test-tube, after a deep inspiration. Each air sample was collected twice, for greater reliability in the case of a test-tube breach or insufficient volume. After substrate administration, air sample collections were performed at 15, 30 and 45 min. Breath analysis was performed using gas isotope ratio mass spectrometry (ABCA 400, Europe Scientifics, Crewe, UK) and the results, expressed as a ratio between the measured 13C and the standard, were corrected for the body surface area to obtain the oxidation percentage.[11] Healthy controls underwent 13C-MBT twice at 12-week intervals. Cirrhotic patients on the waiting list for orthotopic liver transplantation were scheduled to be tested regularly at 12-week intervals. Nine patients were tested only once or twice (six because orthotopic liver transplantation was performed before the next defined time interval and three due to death during the waiting time). The remaining patients on the waiting list were tested at least three times. Of the 16 patients who underwent orthotopic liver transplantation, 13 were re-tested 1, 2, 4, 12 and 24 weeks after transplantation and the three who died were re-tested only until week 4. Nine transplant recipients were also tested during the surgical procedure. In all of these patients, the substrate was administered through a nasogastric tube at the time of first surgical incision and the air sample collections were obtained from the oro-tracheal ventilation tube 15 min before substrate administration, at the end of the initial 15 min of the anhepatic phase and 5, 30, 60, 90 and 120 min after graft reperfusion.

Results

Pre-Orthotopic Liver Transplantation Measurements

Figure 1 depicts the mean 13C-methacetin cumulative oxidation percentage 45 min after substrate ingestion. In healthy controls, the oxidation percentage was 17.5 ± 2.8% at baseline and 18.8 ± 4.3% after 12 weeks (P = 0.30). In cirrhotic patients, the baseline mean oxidation percentage was 5.4 ± 4.2%. This value was markedly influenced by the Child-Pugh score (class A, 8.7%; class B, 3.7%; class C, 2.7%) (Figure 1).

Figure 1. Mean 13C-methacetin oxidation percentages in control subjects (ctrl) and cirrhotic patients classified according to the Child-Pugh score at enrolment.

The oxidation percentage was closely related to the clinical status. For example, patients who died whilst on the waiting list were all classified as Child-Pugh class C and their mean 13C-methacetin oxidation percentages were 0.1%, 3.5% and 3.0%, respectively. Patients who remained longer on the waiting list without having significant clinical changes (all classified as Child-Pugh class B, score 9, or C), and who underwent 13C-MBT at least twice, showed mean oxidation percentages of 2.5 ± 0.9% at the first measurement and 2.2 ± 1.3% at the second. Eleven patients on the waiting list who underwent 13C-MBT three times before transplantation (all classified as Child-Pugh class A or B, scores 7 or 8) showed an increase in the 13C-methacetin oxidation percentage from 6.1 ± 4.0% at the first measurement to 7.2 ± 3.9% at the third assay (P = 0.016). The average MELD score in these patients was 11.5 ± 2.9 at the first breath test and 12.6 ± 3.5 at the last test before liver transplantation.

Pre- vs. Post-Orthotopic Liver Transplantation Measurements

The average 13C-methacetin oxidation percentage increased from 3.3 ± 1.6% at the last determination before orthotopic liver transplantation to 15.1 ± 5.4% 1 month after the procedure (P = 0.0001) and to 17.0 ± 5.2% at 6 months of follow-up (P = 0.0002). The oxidation percentage increased progressively during the post-orthotopic liver transplantation follow-up in all patients. Only one patient showed a transient decrease (from 15.1% at month 3 to 10.4% at month 6) concomitant with a cytomegalovirus infection. The 13C-methacetin oxidation percentage was significantly (P = 0.0001) related to the decrease in serum bilirubin (from 5.9 ± 6.0 mg/dL at 1 month to 1.3 ± 0.6 mg/dL at 6 months after orthotopic liver transplantation) and the increase in serum albumin (from 3.3 ± 0.6 g/L to 4.7 ± 0.7 g/L). A summary of the 13C-MBT results in cirrhotic patients before and after orthotopic liver transplantation is shown in Figure 2. Figure 3 depicts the results observed in the patient transplanted with a split liver, in whom the 13C-methacetin oxidation rate was closely associated with clinical recovery.

Figure 2. Mean 13C-methacetin oxidation percentages in cirrhotic patients before orthotopic liver transplantation (OLT) and during follow-up after liver transplantation. At 6 months of follow-up, there was a significant (P = 0.0002) increase in the mean 13C-methacetin oxidation percentage compared with baseline.

Figure 3. 13C-Methacetin oxidation percentage at 45 min during follow-up after orthotopic liver transplantation (OLT) in the patient who received a split liver.

Intra-Operative Measurements

In the nine patients in whom 13C-MBT was performed intra-operatively during the transplantation procedure, the mean 13C-methacetin oxidation percentage was 1.5 ± 0.6% at the beginning of the operation, decreased to 0.1% during the anhepatic phase and then progressively increased to reach 3.7 ± 2.0% 2 h after graft reperfusion. Figure 4 depicts the individual 13C-methacetin oxidation percentage patterns obtained before orthotopic liver transplantation and at different time points during the intra-operative phase.

Figure 4. 13C-Methacetin oxidation percentages in nine cirrhotic patients tested before orthotopic liver transplantation (OLT) at 12-week intervals and during OLT until 120 min after graft reperfusion.

Discussion

The most commonly used prognostic tests for the evaluation of survival in cirrhotic patients listed for liver transplantation, the Child-Pugh and MELD scoring systems, are unable to assess quantitatively the hepatic functional mass.[1-4] Several tests are available for the evaluation of the hepatic functional mass through the measurement of hepatic blood flow as well as mitochondrial and microsomal hepatic activity. These include the galactose elimination capacity,[12,13] indocyanine green clearance[14] and monoethylglycinexylide lignocaine (lidocaine) metabolite formation.[11] The latter test was proposed almost 10 years ago as a simple dynamic liver function test to assess the severity of chronic liver disease and to judge patient priority in waiting lists for orthotopic liver transplantation.[15] This was due to the high sensitivity of serum levels of lignocaine (lidocaine) and its metabolite, monoethylglycinexylide, in differentiating between cirrhotic patients and controls (77.8 ± 25 ng/mL vs. 35.6 ± 30 ng/mL; P <0.05),[16] and evaluating the prognosis. Patients with serum levels of less than 10 ng/mL had a reported life expectancy no longer than 1 year. However, compared with the galactose elimination capacity, this test gives a worse discrimination between patients with mild liver disease and those with cirrhosis and, in addition, the monoethylglycinexylide concentration is dependent on the blood flow, is influenced by food intake (especially proteins) and tends to be lower in females under 50 years of age. The galactose elimination capacity also offers an estimate of the functional liver cell mass, providing a functional measure of the P-450 enzyme system selectively based on the phosphorylation of galactose in the hepatocyte cytoplasm. On the other hand, indocyanine green is a substance absorbed exclusively by the liver and immediately excreted unchanged in bile. Therefore, the indocyanine green clearance is influenced by both liver perfusion and biliary secretory capacity. All of these tests are invasive and require intravenous substrate administration and serial blood samples in order to evaluate hepatic function.

Breath tests, on the other hand, are easily carried out because they do not require blood sampling or the intravenous administration of endogenous compounds. They are based on the oral administration of a carbon isotope-labelled substrate, followed by the analysis of labelled CO2 in expired air.[5,6] In recent years, several clinical studies have investigated breath tests as a tool for the evaluation of hepatic function. The aminopyrine breath test has been used at some centres,[17] but shows several drawbacks, including a slow clearance rate of the administered compound and the risk of serious side-effects, such as agranulocytosis.[18] Another breath test involves the use of phenylalanine, which assesses amino acid metabolism;[19] however, this test seems to have low accuracy in discriminating between patients with liver disease.[8,9]

In this study, we present evidence that 13C-MBT offers remarkable efficiency in monitoring hepatic function in patients with advanced liver cirrhosis listed for orthotopic liver transplantation. In addition, 13C-MBT appears to be a potentially useful tool for assessing the recovery of hepatic function immediately after graft reperfusion and in the first weeks after liver transplantation. Methacetine is a manageable substance with numerous advantages as a breath test substrate: it has a low cost, is safe and has a rapid clearance.[20,21]

Our findings confirm that the 13C-MBT results are related to the Child-Pugh score, as reported previously,[7-9] suggesting that the test may also be used in the prognostic assessment of liver transplant candidates. This may be of particular interest in judging the priority within transplant lists. A sudden or progressive decrease in oxidation percentage could allow the identification of patients whose liver function is deteriorating, even in the absence of clinical symptoms, and thus those who deserve priority. Conversely, stable 13C-MBT results could identify patients on the waiting list who do not require urgent treatment. As 13C-MBT can be repeated easily over time, it could become part of the routine periodic assessment of patients awaiting transplantation, in addition to conventional liver function tests and imaging techniques. Obviously, further prospective investigation of 13C-MBT is required for this purpose, in relation to the current criteria used to evaluate priority for transplantation (United Network for Organ Sharing, MELD, etc.). Notably, 13C-MBT is not influenced by subjective criteria, as is the Child-Pugh scoring system, or by changes in blood flow, as occurs with other breath tests (i.e. the galactose breath test), suggesting that it may provide additional information.

The strict relationship between hepatic function and methacetine metabolism is emphasized by our observations obtained during the intra-operative phase of liver transplantation. We found that the 13C-MBT oxidation rate decreased almost to zero during the anhepatic phase, thus truly reflecting the absence of any liver metabolism. Later, it increased rapidly, reaching 3.7% 2 h after graft reperfusion. Although the intra-operative data could have been partly biased by the concomitant anaesthesia, the unpredictable methacetine absorption after substrate administration through a nasogastric tube and the air sample collection from the oro-tracheal ventilation tube, we believe that 13C-MBT may be useful to monitor intra-operative hepatic function in the high-risk phase occurring immediately after reperfusion.

Finally, the increase observed in the 13C-methacetin oxidation percentage in the initial months following successful liver transplantation appears to be a simple and reliable tool for the follow-up of these patients: at 6 months of follow-up, the 13C-methacetin oxidation percentage had increased almost five times compared with that pre-transplantation; this increase was associated with an improvement in conventional laboratory parameters. Therefore, 13C-MBT could be employed in the post-operative follow-up of transplanted patients, when events such as primary non-function or early poor function can occur; in these conditions, characterized by severe hepatic functional derangement, which is often difficult to quantify, 13C-MBT may prove to be particularly advantageous, as it is a simple, safe and rapid semi-quantitative diagnostic tool.

In conclusion, the results of the present study support the use of 13C-MBT in the routine follow-up of cirrhotic patients awaiting orthotopic liver transplantation as well as in transplant recipients. More studies are necessary to improve the breath test accuracy, especially during the intra-operative phase of liver transplantation.

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Tables

Table 1. Demographic and clinical characteristics of the studied population

Cirrhotic patients(n = 28)

Healthy controls(n = 10)

Gender (male/female)

20/8

4/6

Median age (years)

52

28

Number of 13C-MBT

3

2

Child-Pugh class A

8 (28%)

N/A

Child-Pugh class B

13 (46%)

N/A

Child-Pugh class C

7 (26%)

N/A

Pre-OLT deaths

3 (10%)

N/A

OLT

16 (57%)

N/A

Post-OLT deaths

3 (18%)

N/A

13C-MBT, 13C-methacetin breath test; N/A, not applicable; OLT, orthotopic liver transplantation.

References

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Reprint Address

Dr M. Angelico, Chair of Gastroenterology, Department of Public Health, University of Rome 'Tor Vergata', Via di Tor Vergata 135, 00133 Rome, Italy. E-mail: angelico@...

A. Petrolati*, D. Festi†, G. De Berardinis†, L. Colaiocco-Ferrante†, D. Di Paolo*, G. Tisone‡, and M. Angelico**Gastroenterology Unit, Department of Public Health, University of Rome Tor Vergata Medical School, Rome, Italy; †Department of Medicine and Ageing, G. d'Annunzio University, Chieti, Italy; ‡Liver Transplantation Centre, Department of Surgery, University of Rome Tor Vergata Medical School, Rome, Italy