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 α-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, γ-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). Summary of results.1 All results are expressed in mmol/L, except for total proteins, albumin, and apolipoproteins (g/L); bilirubin (μmol/L); enzymatic activities (μkat/L); and prothrombin time (%). P <0.001, with respect to HDL-C measured by ultracentrifugation. HDL-C/apo A–I: ratio of HDL-C concentration measured by ultracentrifugation to plasma apo A–I. IDL, intermediate-density lipoprotein; ALT, alanine aminotransferase; ALP, alkaline phosphatase; GGT, γ-glutamyltransferase. P <0.001, with respect to control subjects. Summary of results.1 All results are expressed in mmol/L, except for total proteins, albumin, and apolipoproteins (g/L); bilirubin (μmol/L); enzymatic activities (μkat/L); and prothrombin time (%). P <0.001, with respect to HDL-C measured by ultracentrifugation. HDL-C/apo A–I: ratio of HDL-C concentration measured by ultracentrifugation to plasma apo A–I. IDL, intermediate-density lipoprotein; ALT, alanine aminotransferase; ALP, alkaline phosphatase; GGT, γ-glutamyltransferase. P <0.001, with respect to control subjects. 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. Relationship between the bias and the ratio of HDL-C to apo A-I in the three homogeneous HDL-C methods. (•), 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. 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. Centre de Recerca Biomèdica, Hospital Universitari de Sant Joan, Catalunya, Spain fax 34-77-312569, e-mail jcamps@correu.grupsgs.com 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.
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Camps et al. (1999) studied this question.