Tests of graft metabolic function functioning mass and blood flow

Rational assessment of liver function by the measurement of bilirubin, albumin and the prothrombin time is limited by the relative lack of sensitivity of these tests and their inability to quantify the functional reserve of the liver. Simple, cheap and reliable tests of liver metabolic function, functioning hepatic mass or liver blood flow have long been the 'Holy Grail' of liver transplant units. Methods that showed early promise involve the intravenous administration of model drugs that are metabolized exclusively by the hepatic cytochrome P450 cyclo-oxygenase (CYP 450) enzyme systems. These permit the quantitative assessment of any impairment in either the rate of clearance of the parent drug (e.g. antipyrine and caffeine clearance tests) or formation of one of its metabolites (e.g. formation of monoethylgly-cinexylidide [MEGX] from lignocaine).

The MEGX test has been proposed as a measure of metabolic function, but, since lignocaine is a high extraction drug, its metabolism will be predominantly influenced by blood flow. Thus, the total systemic clearance of lignocaine (CLs) is essentially equivalent to the drug's hepatic clearance (CLH) which equates with the product of the hepatic blood flow (QH) and the hepatic extraction ratio (E):

In standard pharmacokinetic modelling, this product is related to the unbound fraction of the drug (fub) and the intrinsic hepatic clearance of the unbound drug (CLuint) according to the following equation:

QH X fub X CLuint

With very high extraction drugs, intrinsic clearance is not limiting and the clearance of the drug is predominantly influenced by delivery of the drug to the liver. Indeed, it has been shown that MEGX concentrations in patients with congestive heart failure were significantly lower than those measured in healthy subjects [1].

Measurement of antipyrine clearance is still regarded by many as the ideal test of hepatic CYP 450 function. The drug is well tolerated, it is almost completely absorbed after an oral dose, it is not bound to plasma proteins to any great extent, its volume of distribution is equivalent to total body water and it is mainly eliminated by hepatic metabolism. Unlike lignocaine, antipyrine is a poorly extracted compound and so its clearance is independent of liver blood flow - its clearance being largely constrained by the availability of CYP 450 enzymes. Furthermore, the

Phenytoin Healthy Day 7 Cirrhosis therapy controls LTx

Figure 42.1 Antipyrine clearance estimated in patients receiving phenytoin therapy, in healthy subjects, in patients 7 days after liver transplantation (LTx) and in patients with severe liver disease (cirrhosis). Data summarized from population distributions derived from multiple sources including unpublished personal observations in the liver transplant group [2, 3]. Clearance measurements were significantly different between all groups (p<0.001).

three major metabolites are each formed via different CYP 450 enzyme pathways and antipyrine can provide a 'broad spectrum' model substrate of hepatic metabolism. However, enzyme-inducing (e.g. phenytoin) or -inhibiting factors can have a profound influence on antipyrine clearance in humans (Figure 42.1) and must be controlled for in any comparisons between populations of patients.

The elimination of an intravenous dose of galactose from the blood by the liver is linear with time and can be used to estimate the galactose elimination capacity. This correlates well with other measures of hepatocellular function such as the aminopyrine breath test - a measure of cytochrome P450-dependent demethyla-tion of isotopically labelled (13C or 14C) aminopyrine to carbon dioxide - but has the advantage that it measures a single aspect of hepatocellular function. Hepatic functional imaging with a 99mtechnitium-labelled ligand of the asialoglycoprotein receptor (where internalization of galactose-terminated glycoproteins occurs) has been shown to correlate with conventional markers and scoring systems of liver dysfunction and could provide a sensitive measure of functioning hepatic mass. Removal of intravenous doses of bromosulphathalein, and the safer indocyanin green, by hepatic parenchymal cells has been used to measure both hepatic uptake and blood flow, but the latter, potentially very useful, application, unfortunately involves hepatic venous catheterization. The arterial ketone body ratio (AKBR) has been proposed to indicate hepatic mitochondrial redox state and changes in AKBR following revascularization are claimed to reflect the viability and subsequent clinical outcome of living related donor grafts.

Most of these functional tests have been applied in the context of liver transplantation, but usually for assessing pretransplant residual liver function, the donor graft or function immediately after transplantation. The MEGX test has probably received the most attention because an automated assay is available for the measurement of the lignocaine metabolite and only a single blood sample is required. In a prospective multivariate analysis of 102 potential liver donors, it was shown that MEGX testing offered limited incremental value in predicting early donor graft function when used in conjunction with traditional clinical, laboratory and histo-logical criteria but that it may be of value in predicting graft survival after liver transplantation [4]. Similarly, a Cox proportional hazard model has shown that the indocyanin green elimination constant on day 1 after 50 liver transplants was a better predictor of liver-related graft outcome than conventional liver function tests. The elimination constant correlated with the severity of preservation injury, intensive care unit and hospital stay, prolonged liver dysfunction and septic complications [5]. The galactose elimination capacity has been used in a prospective study to predict the survival of 194 patients with cirrhosis and to identify those who might benefit most from urgent transplantation. Only Child-Pugh score, creati-nine, varices and galactose elimination capacity were independent predictors of mortality in a multivariate analysis [6].

In most institutions, the application of such functional tests for the longitudinal monitoring of liver transplant recipients has not been a practical option as they usually involve expensive and complicated procedures, and reference ranges are difficult to establish in patients receiving polydrug therapy and who differ in sex and age, as well as dietary and smoking habits. Consequently, very few serial studies have been done to compare prospectively the clinical value of these quantitative liver function tests after transplantation and such studies have been largely confined to comparisons of new and conventional markers of biliary epithelial or hepatocyte damage. The biliary, endothelial and hepatocyte damage incurred by acute allograft rejection has probably received most attention. Although the influence of isolated rejection episodes on long-term outcome is debatable, a prospective study of 170 consecutive liver transplant recipients with a mean follow up of 3.7 ±0.2 years showed that, in comparison with those who experienced only one or no rejection episodes, the 30 patients who experienced recurrent acute rejection had greater impairment of liver function tests, including lower indocyanin green clearance, and more severe histological damage [7]. It was proposed that the 50% of patients who experience rejection should receive heavier immunosuppression, leaving the remainder on a lifelong light maintenance immunosuppressive regimen.

Much attention has been paid to the development of improved methods for detecting the onset of acute rejection on the unsubstantiated premise that if it could

-7 -6 -5 -4 -3 -2 -1 0 1 2 3 4 5 Day relative to rejection

Figure 42.2 The cumulative frequency of >50% increases in plasma hyaluronic acid (HA), alanine transaminase (ALT), alkaline phosphatase (ALP) and bilirubin (BI LI) in the 7 days before and 5 days after the clinical diagnosis (vertical broken line) of 21 episodes of acute liver allograft rejection. The median time to the first >50% increase in HA was 2.5 days (95% confidence interval -3.5 to -1.0 days) prior to the diagnosis of rejection - 1 or more days earlier than the conventional liver function tests.

be treated earlier there would be less sequellar damage to the allograft. Reflecting the characteristic histopathological features of rejection, surrogate markers of rejection have conventionally included the transaminases and bilirubin. More novel markers of vascular injury have included increased nitric oxide production [8] and reduced clearance of plasma hyaluronic acid [9]. In the author's experience, increases in plasma hyaluronate are a very early and sensitive indicator of acute rejection (Figure 42.2) - a greater than 50% increase in concentration occurring in association with 20 of 21 acute rejection episodes. This probably does largely reflect endothelial damage as the hepatic endothelium has specific receptors for hyaluronic acid and this is the major route of its elimination. However, changes in production of hyaluronate in extrahepatic tissues and changes in liver blood flow could also influence the plasma concentration. Although there is a simple enzyme-linked immunosorbent assay method for quantitating hyaluronic acid (Chugai Diagnostics, Japan), the method has not been widely adopted for monitoring transplant recipients. Significant decreases in aminopyrine clearance have also been found coinciding with acute rejection [10], but tests of this type are not a practical option for routine monitoring.

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