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Technical Briefs |
a address correspondence to this author at: C/.Sant Joan s/n, 43201-Reus, Catalunya, Spain
Despite the obvious clinical advantages, the measurement of HDL-cholesterol (HDL-C) by reliable and easy-to-perform methods is not yet completely free of problems. Several reports have described homogeneous (direct) assays for HDL-C that are readily adaptable to automated analyzers as online procedures (1)(2)(3). These methods have proved to be effective and inexpensive tools for the routine screening of HDL-C in large populations. However, in a recent article (4) we observed that one of these techniques significantly undervalued the concentrations of HDL-C in patients with liver cirrhosis, a condition in which alterations in lipoprotein structure and composition are commonly found (5). Although HDL-C is not a clinically important determination in liver cirrhosis, our finding may have consequences for research groups investigating lipoprotein metabolism and its alterations.
The aims of the present study were (a) to compare three different techniques for homogeneous HDL-C measurement with a reference method [single vertical-spin ultracentrifugation (SVS)] in a group of patients with cirrhosis; and (b) to investigate whether there was a relationship between the method biases and abnormal composition of lipoproteins.
The study was performed on 58 control subjects and 37 patients with liver cirrhosis. Control subjects were chosen randomly from the routine health and safety-at-work checks conducted in several industrial companies in our area. Excluded were those subjects with clinical or laboratory evidence of diabetes, neoplasia, renal disease, hepatic damage, and cardiovascular disease. Cirrhotic patients were diagnosed by liver biopsy and proceeded from the outpatient clinics of the Hospital Universitari de Sant Joan de Reus. The etiology of cirrhosis was alcoholic in 23 patients (62%), viral in 12 (32%), and cryptogenic in 2 (6%). Twelve of the 23 alcoholic cirrhotic patients had quit alcohol consumption at least 3 months prior to the study. The other 11 patients had continued drinking. In three patients, cirrhosis was associated with diabetes mellitus. All procedures were in accordance with the ethics standards of our Institution. Blood samples were drawn in the fasted state into glass tubes containing EDTA; the plasma was separated by centrifugation at 1500g for 25 min and stored at -20 °C for batched analysis.
Three homogeneous HDL-C assays were used. All three methods contained
an initial reagent to block lipoproteins other than HDL and a second
reagent to measure HDL-C by slight modifications of the CHOD/PAP
technique (6). In the method that used polymers, polyanions,
and detergent (PPD), reagent 1 was a mixture of polyanions and
synthetic polymers, forming LDL-, VLDL-, and
chylomicron-polymer-polyanion and HDL-polymer complexes (Daichii;
supplied in Spain by ITC Diagnostics, Izasa, Barcelona, Spain). The
method that uses polyethylene glycol-modified enzymes (PEGME;
Boehringer Mannheim, Mannheim, Germany) used 
-cyclodextrin sulfate
as a sequestering agent of apolipoprotein (apo) B-containing
lipoproteins and modified enzymes that specifically react with HDL-C.
The method that uses antibodies (AB; Sigma Diagnostics, St. Louis, MO)
used anti-human apo B antibodies to bind lipoproteins other than HDL.
SVS ultracentrifugation was performed as published previously
(7). Cholesterol, triglycerides, and phospholipids in
lipoprotein fractions and in patients' plasma were determined by
standard methods (ITC Diagnostics, Izasa). Plasma concentrations of apo
A-I and B were analyzed by immunoturbidimetry (Biokit, Izasa). apo
measurements were calibrated according to the IFCC standard
(8). Liver-related tests were also measured in the plasma of
all subjects by standard techniques (ITC Diagnostics, Izasa). These
tests included total protein, albumin, total and esterified bilirubin,
alanine aminotransferase, alkaline phosphatase,
-glutamyltransferase, and prothrombin time. All measurements were
performed on an Ilab® 900 automatic analyzer
(Instrumentation Laboratories), except for prothrombin time, which was
performed on an ACL 1000 automated coagulation analyzer
(Instrumentation Laboratories). Results are presented as means and
ranges. The presence of lipoprotein X in the plasma of cirrhotic
patients was tested qualitatively by agarose gel electrophoresis
(9). Bias between the homogeneous assays and the reference
method was calculated as the homogeneous HDL-C result minus the
ultracentrifugation method result. Differences between means were
estimated by the Student t-test. The association between
variables was measured by linear regression analysis. Statistical
significance was set at P <0.05.
The results of the analytical determinations are summarized in Table 1
. Cirrhotic patients appeared to be a very heterogeneous group,
with hepatic function indices ranging from within the health-related
reference intervals to frankly altered values. Although the presence of
cholestasis might be suggested by an increase in esterified bilirubin
in some of the patients, alkaline phosphatase activity was never higher
than twice the upper reference limit, and lipoprotein X was not
detected in any of the patients' plasma. Lipoprotein composition was
also very heterogeneous in cirrhotic patients, with values above and
below those of the control subjects for all the indices measured both
in plasma and in SVS-obtained lipoprotein fractions. This is not
surprising because lipoprotein concentrations and composition in
cirrhotic patients are known to be affected by a variety of
derangements, including the degree of hepatocellular damage, the
possible associated cholestasis, the etiology of the disease, and
alcohol intake (5)(10). The three homogeneous
assays significantly (P <0.001) underestimated HDL-C
concentrations compared with SVS ultracentrifugation. The differences
were PEGME > PPD > AB. Differences >10% were observed in
20 patients by the AB method, in 33 patients by the PPD method, and in
all of the patients by the PEGME method. Linear regression analysis
found significant (P <0.001) associations between HDL
measured by SVS and the homogeneous assays, but in cirrhotic patients,
the correlation coefficients were not very high and the slopes of the
regression lines were low for all the homogeneous methods
(Controls: PPD, r = 0.93; y =
0.87x + 0.14; PEGME, r = 0.98;
y = 0.95x + 0.04; AB, r =
0.96; y = 0.95x + 0.07. Cirrhotic patients:
PPD, r = 0.62; y = 0.40x +
0.47; PEGME, r = 0.82; y =
0.49x + 0.01; AB, r = 0.90;
y = 0.56x + 0.45).
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For the three methods, we observed significant (P
<0.001) associations between the bias and the ratio of HDL-C (measured
by ultracentrifugation) to plasma apo A-I in cirrhotic patients (Fig. 1
). We did not observe any significant association between the
bias and HDL phospholipids or triglycerides, or between the bias and
any component of the other lipoproteins.
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This study shows that homogeneous assays do not acceptably measure HDL-C concentration in patients with liver cirrhosis. This lack of accuracy does not depend mainly on the physicochemical fundamentals of the assays. Rather, it is related to abnormalities in lipoprotein composition, such as the cholesterol/apoprotein A ratio in the HDL particle. Liver diseases are known to alter HDL structure and composition profoundly. HDL is synthesized in the liver as nascent HDL. This lipoprotein is rich in nonesterified cholesterol and apo E and C, and is relatively poor in apo A. In blood, nascent HDL is converted into mature HDL by lecithin:cholesterol acyl transferase, an enzyme that is also synthesized by the liver. This enzyme esterifies cholesterol and allows HDL to transfer apo C and E to VLDL. At the same time, HDL obtains apo A from chylomicrons [see Ref. (5) for a review]. Mature HDL, then, is rich in cholesterol esters and apo A and has completely lost apo C and E. In liver diseases, however, lecithin:cholesterol acyl transferase deficiency implies that a substantial amount of the circulating HDL is nascent HDL. The relationship found in our study between the bias and the ratio HDL-C/apo A-I suggests that apo A-poor nascent HDL is not identified correctly by the homogeneous assays, although the exact molecular mechanism of this failure cannot be determined by our investigation. Differences in hydrated density, net charge, size, or shape between nascent and mature HDL may explain an incorrect reaction with reagent 1 (1).
We conclude that homogeneous assays must not be used to measure HDL-C in liver cirrhosis and that reference methods such as SVS ultracentrifugation should still be the techniques of choice for groups investigating alterations in lipoprotein metabolism.
Acknowledgments
This study was supported in part by a grant from FIS (90/0918). Natàlia Ferré was awarded a grant from Fundació Privada Reddis (1998). We thank Izasa S.A. (Barcelona, Spain), Boehringer Mannheim (Mannheim, Germany), and Sigma Diagnostics (St. Louis, MO) for kindly donating the reagents.
Footnotes
Centre de Recerca Biomèdica, Hospital Universitari de Sant Joan, Catalunya, Spain
fax 34-77-312569, e-mail jcamps{at}correu.grupsgs.com
References
-cyclodextrin. Clin Chem 1995;41:717-723.
The following articles in journals at HighWire Press have cited this article:
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  F. Gomez, J. Camps, J. M. Simo, N. Ferre, and J. Joven Agreement Study of Methods Based on the Elimination Principle for the Measurement of LDL- and HDL-Cholesterol Compared with Ultracentrifugation in Patients with Liver Cirrhosis Clin. Chem., August 1, 2000; 46(8): 1188 - 1191. [Full Text] [PDF]
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| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
), control subjects; (
), cirrhotic patients. Correlation
coefficients for cirrhotic patients were as follows: A,
r = 0.67; B, r =
0.68; C, r = 0.65; P
<0.001. AB, method using antibodies;
PEGME, method using polyethylene glycol-modified
enzymes; PPD, method using polyanions, polymers, and
detergent; UC, ultracentrifugation.