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Technical Briefs |
1 Laboratorio Analisi Chimico Cliniche 1, Azienda Ospedaliera ‘Spedali Civili’, 25125 Brescia, Italy
aauthor for correspondence: fax 39-030-3995369, e-mail panteghi{at}bshosp.osp.unibs.it
5'-Nucleotidase (5'-ribonucleotide phosphohydrolase; EC 3.1.3.5; 5NT), an intrinsic membrane glycoprotein present as an ectoenzyme in a wide variety of mammalian cells, hydrolyzes 5'-nucleotides to their corresponding nucleosides (1). Despite its ubiquitous distribution, serum concentrations of 5NT appear to reflect hepatobiliary disease with considerable specificity (2). Dixon and Purdom (3) first observed that serum 5NT was clinically useful for differential diagnosis of hepatobiliary and osseous diseases, the enzyme activity being increased only in hepatobiliary diseases. This finding has been confirmed by several authors (4)(5)(6)(7).
Assay of 5NT activity may have value as an addition to measurement of nonspecific total alkaline phosphatase (AP) in patients with suspected hepatobiliary disease (8)(9)(10)(11)(12), and abnormal 5NT activity is routinely interpreted by some clinicians as evidence of a hepatic origin of increased AP activity in serum. However, a direct comparison of 5NT and the liver form of AP (L-AP) in a clinical setting has not been reported (13).
The aim of this study was to evaluate the efficiency of a 5NT assay in detecting abnormal concentrations of L-AP in serum and to verify the possibility of replacing total AP determinations and the analytically cumbersome L-AP assay with 5NT in the evaluation of patients with suspected liver disease.
Fresh serum (leftover) samples were obtained from 100 adults (60 women and 40 men; median age, 66 years; range, 21–91 years), consecutively hospitalized in the Divisions of General Medicine and General Surgery of our regional medical center, who showed increased catalytic concentrations of total AP in routine biochemical examinations performed at hospital admission. Total AP was determined with the IFCC-recommended method at 37 °C on a DAXTM chemistry analyzer [Bayer Diagnostics; between-day CV <3%; upper reference limit (URL), 85 U/L]. Procedures followed were in accordance with the Helsinki Declaration of 1975 as revised in 1996.
5NT activity was determined at 37 °C with a Mega® analyzer (Dade Behring) by a kinetic method (Enzyline® 5'NU; BioMerieux). The previously calculated URL in our laboratory was 8 U/L (12). L-AP activity was determined by an electrophoretic procedure with neuraminidase added to the sample 15 min before electrophoresis (14). Gels were scanned by densitometry, and L-AP was calculated by the product of the total AP activity and the percentage of the stain appearing in the L-AP region. The URL for L-AP in our laboratory was 50 U/L, with no sex-related differences in the reference distribution (14). The total imprecision (CV) of the 5NT and L-AP assays, as monitored in our routine practice with pooled human serum internal quality-control material, was <5.4% for 5NT (mean activity, 9 U/L) and <4.3% for L-AP (mean activity, 53 U/L), respectively (data from fiscal year 1999). All statistical analyses, including ROC curve analysis and calculation of the area under the curve, were performed using MedCalc® for Windows (MedCalc Software).
L-AP was increased in 57 (57%) hyperphosphatasemic patients (median value, 77 U/L; range, 51–746 U/L). Fig. 1
shows the ROC curve obtained when we compared 5NT activities in samples collected from patients with and without increased L-AP. When we used the URL (8 U/L) as cutoff, 5NT showed a sensitivity of 47% [95% confidence interval (CI), 34–61%], a specificity of 86% (95% CI, 72.1–94.7%), and an efficiency of 64% (95% CI, 54–73%) for demonstrating the presence of increased L-AP. The area under the curve [± SE; 0.76 (95% CI, 0.66–0.84) ± 0.047] confirmed the relatively low accuracy of 5NT in identifying increased L-AP in serum.
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5NT was increased in six patients with normal L-AP (Table 1
). Five of the six showed clear evidence of liver disease. The only patient with no apparent hepatic involvement (an elderly woman with a chronic obstructive pulmonary disease) showed slightly increased 5NT with a concurrent L-AP value equal to the URL.
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The results of this study indicate that a high number of individuals (53%) in whom L-AP activity is increased in serum may simultaneously show a normal 5NT. Of importance is the fact that increased 5NT in the serum of patients with normal L-AP identified with high probability the presence of liver disease, confirming that any increase in 5NT activity that is more than marginal is highly specific for hepatobiliary disease (15), at least in patients with increased AP.
We cannot suggest replacement of the analytically cumbersome L-AP assay with 5NT measurements. On the contrary, there is a need for a simple, direct, and inexpensive method to determine L-AP. The dissociation of the two enzyme activities supports the utility of determining both L-AP and 5NT for diagnosis of diseases of the liver, especially if one assumes that increased L-AP indicates liver disease in all cases.
Acknowledgments
We gratefully acknowledge the technical assistance of Francesca Stefini. This work was presented in part at the 51st AACC National Meeting, July 25–29, 1999, New Orleans, LA [Clin Chem 1999;45(Suppl 6):A56].
References
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