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Technical Briefs |
1 Department of Physiology,
2 Cardiovascular Research Institute Maastricht, Maastricht University, PO Box 616, 6200 MD Maastricht, The Netherlands
Departments of
3
Clinical Biochemistry and
4 Medicine, Addenbrooke’s Hospital, Cambridge CB2 2QQ, United Kingdom
aauthor for correspondence: fax 31-43-3884166, e-mail glatz{at}fys.unimaas.nl
Serum tests of acute hepatocellular injury are commonly used to investigate the presence and monitor the progress of liver disease (1)(2). The release of cytoplasmic proteins from damaged hepatocytes into the vascular system follows tissue necrosis caused by, e.g., acetaminophen intoxication, ischemia and reperfusion injury, or rejection after liver transplantation. Although the hepatocytes are in direct contact with the vasculature and no interstitial barrier is between the two, smaller proteins appear earlier in the circulation than do larger ones (1) and would therefore increase earlier in serum above their upper reference value after an acute hepatocellular injury. 
-Glutathione S-transferase (-GST; 26 kDa) is a more sensitive and specific marker of hepatocellular damage (3)(4) than either alanine transaminase (ALT; 96 kDa) or aspartate transaminase. -GST is present in liver, kidney, and intestine; is released rapidly from damaged hepatocytes; and has a relatively short in vivo plasma half-life (3)(4). The use of -GST for the detection of hepatocellular injury secondary to acute rejection after liver transplantation improved the biochemical monitoring of patients and decreased mortality and morbidity (3). In search of even smaller and more specific cytoplasmic proteins for the detection of liver injury, we have studied the liver-type fatty acid-binding protein (L-FABP). FABPs are a family of 15-kDa proteins that are involved in the intracellular transport of long-chain fatty acids (5). To date, nine different FABPs have been identified and named according to the tissues in which they were first identified (5). L-FABP occurs mainly in the liver but, in small quantities, also in kidney and small intestine. In the hepatic lobule, L-FABP is expressed in hepatocytes in a declining portal-to-central gradient (5)(6). Extensive studies on heart-type FABP (H-FABP) have shown that this protein is released rapidly after myocardial injury and, in view of its low circulating concentrations in healthy individuals (7)(8), is a sensitive marker of myocardial infarction (9)(10), as is the intestinal FABP for intestinal injury (11). Because all FABP types have a comparable mass (5), we expected L-FABP to have a similar diagnostic capacity for acute liver damage.
The aim of our study was to compare the behavior of serum concentrations of L-FABP with ALT and -GST in association with episodes of acute hepatocellular damage. The population of patients chosen was a group of 21 liver transplant recipients (15 males and 6 females) who had episodes of acute hepatocellular rejection during their posttransplantation stay in the hospital. Rejection episodes were diagnosed either histologically or, when biopsy was contraindicated (n = 3), by previously published clinical criteria based on deteriorating liver function tests, increased prothrombin time, and an increase in the peripheral blood eosinophil count (12)(13). Immunosuppressive therapy consisted of treatment with steroids, azathioprine, and either tacrolimus or cyclosporine. After rejection, augmented immunosuppressive therapy was given in the form of three daily doses of 1 g of intravenous methylprednisolone. Blood samples were taken daily (usually in the early morning) up to 3 months after transplantation. The present study retrospectively considered the period from 4 days before to 3 days after either a biopsy-confirmed episode or the start of treatment for the episode. An increase in serum concentration or activity of at least 50% in relation to the previous daily value was taken to be significant. We chose the 50% value to exclude the influence of analytical variation on the interpretation of the changes in the daily values. Because L-FABP and -GST are excreted by the kidneys, we measured plasma creatinine to evaluate kidney function (14)(15).
To determine the biological variation of circulating L-FABP on the reference interval, plasma samples were obtained, as described previously (7)(8), from 80 healthy individuals (40 males and 40 females) to study the influence of age and gender and from another 12 healthy individuals (6 males and 6 females) to study the influence of circadian rhythm. L-FABP was measured with a sandwich ELISA developed in collaboration with HyCult Biotechnology (Uden, The Netherlands). No influence of different matrices (plasma or serum) or cross-reactivity with other types of FABP was noticed. The detection limit of the assay was 0.1 µg/L. With calibrators containing 2 and 20 µg/L L-FABP, the intra- and interassay CVs were <5% and <15%, respectively. -GST concentrations were measured by an enzyme immunometric assay obtained from Biotrin International. The CVs were 14% and 5.5% at -GST concentrations of 7.5 and 25 µg/L, respectively. ALT activity was measured on a DuPont Dimension multichannel analyzer according to the manufacturer’s instructions. The CV of the ALT method was <12%. Creatinine concentrations were measured by the Jaffe rate method and also on a DuPont Dimension multichannel analyzer.
The Mann–Whitney test for unpaired values was used to asses the influence of age, gender, and circadian rhythm on the values from the healthy donor groups, with P <0.05 regarded as significantly different. A paired Wilcoxon signed-rank test was used to assess any difference between the increases in serum L-FABP, ALT, and -GST. All data are expressed as medians, with the 25th–75th percentiles.
Healthy individuals had a median plasma L-FABP concentration of 9.46 µg/L, and the 99th percentile was 17.5 µg/L. In contrast to H-FABP (5), there was no significant influence of age or gender on L-FABP concentrations in healthy individuals between 21 and 70 years of age: females, 8.54 (7.14–11.06) µg/L; males, 9.87 (8.9–12.13) µg/L. However, similar to H-FABP (5)(14), there was a significant increase (P <0.01) in plasma L-FABP in healthy individuals during the night (Table 1
). This may be explained by a reduced glomerular filtration rate at night, which decreases the clearance of L-FABP from the circulation. The presence of a circadian variation in serum L-FABP would not have influenced our results significantly, because daily blood samples were routinely collected during a narrow time window in the morning.
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In the patient population, until the day of rejection, a significant (>50%) increase in serum L-FABP was observed in association with all 21 acute rejection episodes studied, whereas -GST increased in association with 19 (91%) and ALT in association with only 9 (42%) episodes. L-FABP increased by a median of 2.0 days (range, -1 to 4 days) earlier than the day that acute rejection was diagnosed, whereas -GST increased 1.5 days (range, -2 to 3 days) and ALT 0 days (range, -3 to 1 day) earlier. Notably, serum L-FABP increased earlier (P = 0.038) than -GST. The cumulative number of patients with a significant (>50%) increase in serum marker protein concentrations or activities relative to the day of diagnosis of rejection is shown in Fig 1
. (Figures showing the L-FABP, -GST, and ALT concentrations in the intervals just before rejection or initiation of treatment are available with the online version of this Technical Brief at http://www.clinchem.org/content/vol48/issue11.)
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A significant impairment of renal function leads to an increased half-life of L-FABP in the circulation (14). In our study, 13 transplant recipients had mild to marked impairment of renal function (creatine clearance range, 23.5–68.7 mL/min; median, 55.9 mL/min). Although this did not appear to hamper the ability of L-FABP to detect acute hepatocellular damage by serial sampling in these patients, care should be exercised in the interpretation of L-FABP values in patients with renal impairment.
This is the first study showing that L-FABP is a promising biochemical marker for the early detection of hepatocellular injury and that, in this small group of liver transplant patients, L-FABP performs as well if not better than -GST. We believe that a combination of the low molecular mass of L-FABP and its relative abundance in the liver (2.8 mg/g wet weight; unpublished data) are the contributing factors to the relatively large and rapid increases in circulating L-FABP in association with acute liver injury, thus making L-FABP a sensitive marker. More frequent blood sampling at the time of a suspected rejection may enhance this sensitivity even more. The development of biosensor (16)(17) and turbidimetric assays (18) for FABPs should simplify the use of L-FABP and encourage its future application in routine clinical practice.
Acknowledgments
We thank J.P. Chapelle for providing blood samples to establish reference values and D. van der Voort for graphical assistance. This study was supported by the Ministry of Economic Affairs of The Netherlands (Grant BTS 97.188).
References
-glutathione S-transferase concentration monitoring after liver transplantation. Transplantation 1997;64:1446-1452.[ISI][Medline]
[Order article via Infotrieve]
- and 
-glutathione-S-transferase will improve monitoring after liver transplantation. Transplant Proc 1997;29:2827-2829.[ISI][Medline]
[Order article via Infotrieve]
-glutathione S-transferase. Ann Clin Biochem 1995;32:575-583.
The following articles in journals at HighWire Press have cited this article:
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  A. Xu, Y. Wang, J. Y. Xu, D. Stejskal, S. Tam, J. Zhang, N. M.S. Wat, W. K. Wong, and K. S.L. Lam Adipocyte Fatty Acid-Binding Protein Is a Plasma Biomarker Closely Associated with Obesity and Metabolic Syndrome Clin. Chem., March 1, 2006; 52(3): 405 - 413. [Abstract] [Full Text] [PDF]
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, ALT; 
, 
, L-FABP.