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Technical Briefs |
1
Centre de Recerca Biomèdica, Hospital Universitari de Sant Joan, 43201-Reus, Catalunya, Spain
a address correspondence to this author at: C/. Sant Joan s/n, 43201-Reus, Catalunya, Spain: fax 34-977-312569, e-mail jcamps{at}grupsgs.com
In recent years, several methods for the direct measurement of HDL-cholesterol (HDL-C) and LDL-cholesterol (LDL-C) have been introduced (1)(2)(3)(4)(5). These methods have been effective and inexpensive tools for the routine screening of large populations because they prevent the need for complex ultracentrifugation procedures. In addition, the direct methods for LDL-C are better than indirect calculation by the Friedewald formula because they can be used accurately in hypertriglyceridemic patients and in nonfasting subjects (4). However, it is still being debated whether these methods are also valuable under more challenging conditions than those found in the general population (4), i.e., pathological circumstances in which there are abnormalities in lipoprotein structure and/or composition.
In a recent study, we observed that three different homogeneous methods significantly undervalue HDL-C concentrations in patients with liver cirrhosis. We suggested that this could be related to the abnormal lipoprotein composition in these subjects (6)(7). A new type of homogeneous method, based on the elimination principle (Randox Laboratories Ltd., Crumlin, UK), has been developed for measuring HDL-C and LDL-C. The manufacturer states that lipoproteins with abnormal compositions may be correctly identified and measured with these methods. The present study therefore aimed to compare the elimination methods for determining homogeneous HDL-C and LDL-C with single vertical-spin ultracentrifugation in a group of patients with liver cirrhosis.
The study was carried out on 44 control subjects and 55 cirrhotic patients. Control subjects were chosen randomly from the routine health and safety-at-work checks conducted in several companies in our area. Cirrhotic patients were diagnosed by liver biopsy and came from the outpatient clinics of the Hospital Universitari de Sant Joan de Reus. 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 4 °C for analytical determinations, which were performed in <3 days.
The homogeneous methods for HDL-C and LDL-C determination were used according to the manufacturer’s instructions. They involved releasing the undesirable lipoprotein components by means of several components included in reagent 1 (selective ionic strength buffers, cholesterol esterase, cholesterol oxidase, and catalase). Subsequently, the desired lipoprotein fraction was released by specific surfactants in reagent 2, and cholesterol was measured by the CHOD/PAP method. The intra- and interassay CVs were <2.5% and <4.7%, respectively. Single vertical-spin ultracentrifugation was performed as published previously (8). This technique was chosen as a reference method because it minimizes in vitro apolipoprotein dissociation from lipoprotein particles (9), an aspect of crucial importance in patients with altered lipoprotein composition. Cholesterol in the lipoprotein fractions and in the patients’ plasma was determined by standard methods (ITC Diagnostics). Plasma concentrations of apolipoprotein (apo) A-I and B were analyzed by immunoturbidimetry (Biokit) and calibrated according to the IFCC standard (10).
Liver-related tests were also measured in the plasma of all subjects by
standard techniques (ITC Diagnostics). Total protein, albumin,
bilirubin, alanine aminotransferase, alkaline phosphatase, and
-glutamyltransferase were also measured in the plasma of all
subjects by standard techniques. All measurements were performed on an
ILab® 900 automatic analyzer (Instrumentation
Laboratories). Results are presented as the mean ± SD. The
presence of lipoprotein X in the plasma of cirrhotic patients was
tested qualitatively by agarose gel electrophoresis (11).
The agreement between the homogeneous and the ultracentrifugation
methods was estimated by the Bland-Altman graphical procedure
(12). Statistical significance was set at P
<0.05.
The results of the analytical determinations are shown in Table 1
. Cirrhotic patients presented with a significant decrease in
total plasma cholesterol as well as in LDL-C, intermediate-density
lipoprotein-cholesterol (IDL-C), and VLDL-cholesterol (VLDL-C). These
alterations were similar to those reported previously in this type of
patient (13). As expected, several hepatic function indices
were significantly altered.
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The analytical results suggested that the cirrhosis was mainly hepatocellular because although the increase in esterified bilirubin in some patients may suggest cholestasis, alkaline phosphatase activity was never more than twice the upper reference limit and lipoprotein X was not detected in any of the patients’ plasma.
Correlation and Bland-Altman plots for the elimination and
ultracentrifugation methods for HDL-C and LDL-C are shown in Fig. 1
. The mean difference for HDL-C measurements was 0.13 ±
0.14 mmol/L in control subjects and 0.18 ± 0.22 mmol/L in
patients with cirrhosis. These differences were not related to the
average HDL-C concentration. The limits of agreement were quite high,
i.e., values showed a considerable degree of dispersion above or below
the mean; however, the elimination method was superior to the
previously published homogeneous assays, in which biases of -2.0
mmol/L were frequently observed in cirrhotic patients (7).
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The mean difference for LDL-C determinations was -1.18 ± 0.46 mmol/L in control subjects, which indicates a high negative bias for the elimination method. Several authors have already observed a marked lack of agreement between the homogeneous assays and ultracentrifugation procedures in LDL-C determination. The reported biases are sometimes positive (14)(15), but they are more frequently negative (4)(5)(16)(17)(18). Some of the biases are very high and similar to those found in the present investigation (16)(17). Studies comparing homogeneous assays with the ß-quantification of LDL have suggested an explanation of overestimation of the ultracentrifugation procedure because it includes IDL-C together with LDL-C (5)(16)(17)(18). This was not the case in the present investigation because our ultracentrifugation procedure efficiently separated IDL from LDL.
Surprisingly, the situation in patients with liver cirrhosis was very different. In these subjects, the bias was not as strongly negative as in the control group and was clearly positive at high average LDL-C concentrations. There was a direct relationship between bias and LDL-C concentrations in cirrhotic patients (r = 0.61; P <0.001) but not in the control subjects (r = 0.08; P, not significant). This difference between the two groups was also observed in the correlation study, in which the regression lines were different in control and cirrhotic subjects. The reason for this disagreement is unknown but may be related to the abnormal synthesis of lipoproteins in these patients (19). Liver diseases are known to alter lipoprotein structure and composition. VLDL and HDL are synthesized in the liver as nascent lipoproteins. Nascent VLDL is rich in apo B and poor in apo C and E. On the other hand, nascent HDL is rich in apo C and E and poor in apo A. In blood, nascent VLDL is converted into the mature lipoprotein by lecithin:cholesterol acyl transferase, an enzyme also synthesized by the liver. This enzyme transfers apo C and E from HDL to VLDL. In liver diseases, however, lecithin:cholesterol acyl transferase deficiency means that apo C and E are not transferred to VLDL, which leads to the accumulation of nascent VLDL. A misidentification of apo B-rich nascent VLDL as LDL by reagent 1 is likely to account for the positive bias observed with the elimination method.
Therefore, although homogeneous LDL assays have demonstrated that they are useful in routine analyses, they fail to measure LDL-C accurately in extreme pathological situations in which profound structural and/or compositional lipoprotein alterations are likely to be observed.
Acknowledgments
We thank Randox Laboratories Ltd., Crumlin, UK for kindly donating the reagents. N.F. received a grant from the Generalitat de Catalunya (FI/FIAP-98).
References
-cyclodextrin. Clin Chem 1995;41:717-723.
The following articles in journals at HighWire Press have cited this article:
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  W. G. Miller, P. P. Waymack, F. P. Anderson, S. F. Ethridge, and E. C. Jayne Performance of Four Homogeneous Direct Methods for LDL-Cholesterol Clin. Chem., March 1, 2002; 48(3): 489 - 498. [Abstract] [Full Text] [PDF]
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G. R. Warnick, M. Nauck, and N. Rifai Evolution of Methods for Measurement of HDL-Cholesterol: From Ultracentrifugation to Homogeneous Assays Clin. Chem., September 1, 2001; 47(9): 1579 - 1596. [Abstract] [Full Text] [PDF]
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).