Bc, sugar-conjugated bilirubins (sum of mBc and dBc); DBIL, direct diazo-reacting bilirubin (sum of Bc and Bd); Bu, unconjugated bilirubin, with the. IX-alpha ...
CLIN. CHEM. 36/1, 9-i4
(1990)
Delta and Conjugated Bilirubin as Complementary Markers of Early Rejection in Liver-Transplant Recipients T.-W. Wu,1 G. A. Levy, Samuel Ylu, J.-X. Au, P. D. Grelg, S. M. Strasberg, M. Etties, M. Abecassis, R. A. Superina, B. Langer, L. M. Blendis, M. J. Phillips, and B. R. Taylor
For 51 liver allograft recipients, we evaluated whether serum profiles of delta (Bd) and conjugated bilirubins (Be) could be used to diagnose rejection during the first 30-50 postoperative days, in comparison with histology as the “gold standard.” Daily measurements of aspartate aminotransferase, alkaline phosphatase, total bilirubin, Bd, and Bc were made, the last two by liquid chromatography. In 34 patients without any biochemical or histological evidence of rejection, within seven to 10 postoperative days Bd increased to >40-50% of total bilirubin, while Bc decreased to 50%, while Bd remained 50% total bilirubin. Treatment of rejection resulted in a prompt reversal of these trends. Individually promising as prognostic adjuncts to existing liver-function markers, Bd and Bc also complemented each other, rendering the diagnosis of liver rejection much more incisive. Liver patients
transplantation with acute
is an accepted form of therapy for end-stage liver disease (1). Two major barriers to a successful transplant are rejection and infection, both of which are strongly influenced by the amount and type of immunosuppressants administered. In liver recipients’ immediate postoperative period, increases in
and chronic
enzymes do not adequately distinguish between rejection and infection (2). Often it is difficult for the clinician to determine if abnormalities in liver function reflect rejection. There are as yet no noninvasive tests that are either predictive of or specific for rejection. Even liver biopsy, often considered a “gold standard” in assessing graft rejection, is not totally definitive (3), and it is too invasive for routine use. Delta bilirubin (Bd) was first identified as a bilirubin
linked covalently to albumin
Liver Transplant
Program,
(4, 5), Concentrations
University
of Toronto,
of Bd
Toronto,
Canada. ‘Address for correspondence: Department of Clinical Biochemistry, University of Toronto and Toronto General Hospital ES 3-404B, 200 Elizabeth St., Toronto, Canada M5G 2C4. Portions of this manuscript were presented at the Joint Conference of the Canadian Society of Clinical Chemists/Canadian Association of Medical Biochemists/AACC (Upstate New York) on June 5, 1989, where the presenter (S.Y.) received the Young Investigator’s Award. 2Nonstandard abbreviations: Bd, delta bilirubin, biliprotein or bili-albumin covalent complex; mBc, mono-sugar conjugated bilirubin; dBc, di-sugar conjugated bilirubin; Bc, sugar-conjugated bilirubins (sum of mBc and dBc); DBIL, direct diazo-reacting bilirubin (sum of Bc and Bd); Bu, unconjugated bilirubin, with the IX-alpha isomer as the predominant species; TBIL, total bilirubin (sum of Bu, Bc, and Bd); AST, aspartate aminotransferase (EC 2.6.1.1); and ALP, alkaline phosphatase (EC 3.1.3.1). Received June 23, 1989; accepted August 17, 1989.
increase rapidly along with those of the sugar-conjugated bilirubins (Bc) in patients’ sera during the early phase of various (especially obstructive) hepatobiliary disorders. However, Bd is the last bile pigment to clear from the blood when jaundice subsides (6). Significantly, Bd [as percent of total bilirubin (TBIL)] always fails to rise above 30-40% during unresolved hepatic failure (7). In such cases, Bc often remains above 50% of TBIL until or near the 4eath of the patient (8). These correlations between Bd and Bc and patient outcome are more apparent when the bilirubins are plotted as percentage proportions of TBIL than as absolute concentrations (6-8), and are frequently supported by tissue pathology but not by the changes such as aspartate aminotransferase
in key liver enzymes (AST) and alkaline
(ALP) (7, 8). Thus Bd and Bc appear to be and complementary markers of liver function or dysfunction. The present study was undertaken to ascertain whether determinations of Bd and Bc would be useful in identifying phosphatase unusually
sensitive
rejection in patients undergoing orthotopic liver transplant. We studied patients with various liver diseases (see
below), and blood samples were collected and analyzed blinded fashion. Results were analyzed retrospectively.
in a
Materials and Methods 51 adult patients (35 women, 16 liver transplantation at the University of Toronto between September i985 and November 1988. The average age of the patients was 43 ± 12 Patients.
We studied
men) undergoing
years.
Their
orthotopic
disorders
fell within
the following
categories
(number of patients in parentheses):
chronic active hepatitis with cirrhosis (9), fulminant hepatitis (10), primary biliary cirrhosis (ii), hepatocellular carcinoma (4), sclerosing cholangitis (3), Wilson’s Disease (2), alcoholic cirrhosis (2), cryptogenic cirrhosis (3), hemochromatosis (2), autoimmune hepatitis (1), cholangiocarcinoma (2), and alphai-antitrypsin
deficiency (2).
Bilirubin
determinations. Bilirubin was determined by “high-performance” liquid chromatography (HPLC) accord-
ing to Lauff and Wu (9). By this method total bilirubin
in
serum is resolved into four fractions in order of elution: delta bilirubin (Bd), dicomjugated bilirubin (dBc), monoconjugated bilirubin (mBc), and unconjugated bilirubin (Bu). Conjugated bilirubin (Bc) refers to the sum of mBc and dBc. The percentage of Bc and Bd is calculated based on the percentage of the total peak area, as previously described (9). Results represent the average of duplicate determinations per specimen. Sera were analyzed within 1-3 h of acquisition and were preserved at 4#{176}C in the dark until analysis.
Assays. Sera were collected daily from patients and analyzed quantitatively in a Hitachi 737 analyzer for AST, ALP, glucose,
electrolytes,
urea nitrogen,
creatinine,
amy-
lase, biirubin (total and direct), calcium, and phosphate. This paper mainly deals with results for AST, ALP, TBIL, Bc, and Bd. Direct bilirubin (DBIL), where shown, was the CLINICAL CHEMISTRY, Vol. 36, No. 1, 1990
9
sum of Bc + Bd (7, 8). Histological
examination
and
of rejection.
assessment
Percutaneous liver biopsies were done approximately weekly on all patients and at times of suspected rejection as indicated by increases in AST, total bilirubin, or ALP. The decision to biopsy or treat rejection was made without knowledge of Bd or Bc results. The biopsy specimens were fixed by immersion into a 100 milL solution of formalin (Fisher Scientific Co., Fairlawn, NJ) in 0.1 molJL phosphate-buffered isotonic saline (pH 7.4). After fixation, the
positive and negative predictive values were determined as described by Haynes (11) and were calculated based on changes in Bd, Bc, AST, and ALP. Results
consecutive days is also indicative of hepatic dysfunction, which in most cases is accompanied by a stable Bd fraction remaining at
