Elsevier

Clinica Chimica Acta

Volume 410, Issues 1–2, 8 December 2009, Pages 31-38
Clinica Chimica Acta

Differential reactivities of four homogeneous assays for LDL-cholesterol in serum to intermediate-density lipoproteins and small dense LDL: Comparisons with the Friedewald equation

https://doi.org/10.1016/j.cca.2009.09.010Get rights and content

Abstract

Background

In routine clinical laboratory testing and numerous epidemiological studies, LDL-cholesterol (LDL-C) has been estimated commonly using the Friedewald equation. We investigated the relationship between the Friedewald equation and 4 homogeneous assays for LDL-C.

Methods

LDL-C was determined by 4 homogeneous assays [liquid selective detergent method: LDL-C (L), selective solubilization method: LDL-C (S), elimination method: LDL-C (E), and enzyme selective protecting method: LDL-C (P)]. Samples with discrepancies between the Friedewald equation and the 4 homogeneous assays for LDL-C were subjected to polyacrylamide gel electrophoresis and the β-quantification method.

Results

The correlations between the Friedewald equation and the 4 homogeneous LDL-C assays were as follows: LDL-C (L) (r = 0.962), LDL-C (S) (r = 0.986), LDL-C (E) (r = 0.946) and LDL-C (P) (r = 0.963). Discrepancies were observed in sera from type III hyperlipoproteinemia patients and in sera containing large amounts of midband and small dense LDL on polyacrylamide gel electrophoresis. LDL-C (S) was most strongly correlated with the β-quantification method even in sera from patients with type III hyperlipoproteinemia.

Conclusions

Of the 4 homogeneous assays for LDL-C, LDL-C (S) exhibited the closest correlation with the Friedewald equation and the β-quantification method, thus reflecting the current clinical databases for coronary heart disease.

Introduction

Numerous clinical studies have shown an independent relationship between increases in LDL-cholesterol (LDL-C) concentrations and risk of coronary heart disease (CHD) [1], [2]. According to the National Cholesterol Education Program (NCEP)-Adult Treatment Panel III, the diagnosis and management of adult patients with hypercholesterolemia is largely based on the concentration of LDL-C [3].

A wide variety of methods have been used for determining LDL-C in serum. These methods include sequential and density-gradient ultracentrifugation [4], β-quantification [5], [6], [7], [8], Friedewald equation [9], electrophoresis [10], HPLC [11] and homogeneous assay [12]. As a reference procedure for LDL-C, the CDC has adopted a variation of the multi-step β-quantification procedure used by the Lipid Research Clinics [13], which combines separation by ultracentrifugation and chemical precipitation. The β-quantification procedure for LDL-C has been also recommended by the NCEP Lipoprotein Measurement Working Group [7]. However, the β-quantification method requires a relatively large volume of serum, special equipment, and is a time-consuming procedure; therefore, it is not well suited for routine testing in hospitals and clinics. Although the Friedewald equation is the most commonly used technique in clinical laboratories for the estimation of LDL-C, it cannot be accurately estimated when plasma triglycerides (TG) > 4.52 mmol/l (400 mg/dl) or when specimens are collected in the non-fasting state [5], [14]. In most clinical studies investigating the relationship between LDL-C and CHD, including the Framingham Heart Study and the study of the prediction of CHD, LDL-C was estimated by the Friedewald equation [15], [16], [17]. In the NCEP-Adult Treatment Panel III study, the recommended level of LDL-C for the prevention of CHD was also estimated by the Friedewald equation. Like the β-quantification method, LDL-C estimated by the Friedewald equation includes cholesterols in lipoprotein (a) and intermediate-density lipoproteins (IDL) [5], [7], [18], which are atherogenic apolipoproptein (apo) B-containing lipoproteins, and there is a close correlation between the β-quantification method and the Friedewald equation [5], [19].

In recent years, convenient homogeneous assays for LDL-C that are less influenced by TG have been developed and are widely performed at clinical laboratories. As mentioned above, LDL-C, which has been shown to be a risk factor for CHD by clinical studies, is estimated by the Friedewald equation, and some studies have stated that LDL-C measured by homogeneous assays for LDL-C need to reflect the current clinical databases for CHD risks as assessed by the Friedewald equation [7]. Clinicians have also expressed the need for a homogeneous assay for LDL-C reflecting LDL-C as estimated by the Friedewald equation [7]. Most homogeneous assays for LDL-C have received CDC certification from the Cholesterol Reference Method Laboratory Network (CRMLN) [12]. Because different homogeneous assays for LDL-C employ different measurement principles, discrepancies have been reported with samples having IDL, lipoprotein (a) and abnormal lipoproteins associated with liver dysfunction, such as lipoprotein X or lipoprotein Y [20], [21], [22].

We investigated the correlations between the Friedewald equation and 4 homogeneous assays developed in Japan and to determine whether the homogeneous assays for LDL-C reflect the clinical databases for CHD where LDL-C was estimated by the Friedewald equation. Samples with discrepancies between the Friedewald equation and homogeneous assays for LDL-C were analyzed by polyacrylamide gel electrophoresis (PAGE) and the β-quantification method.

Section snippets

Samples

With approval from the Ethics Review Board of Osaka University Hospital, human serum samples were from 156 patients (age: 30–65 years) with <4.52 mmol/l (400 mg/dl) TG and from 5 patients (age: 58–75 years) with type 2 diabetes mellitus. Informed consent was obtained from all patients. After 12 h of fasting, blood samples were collected in tubes without anticoagulant. Blood samples were stored at 4 °C and were measured within 3 days using a Hitachi 7170 analyzer.

Total cholesterol (TC), TG and HDL-cholesterol (HDL-C) assays

TC and TG were measured by an enzymatic

Comparison between the Friedewald equation and homogeneous assays for LDL-C

The 4 homogeneous assays for LDL-C were compared with the Friedewald equation in fasting serum samples from 156 patients and 4 diabetic patients with < 4.52 mmol/l TG. As shown in Fig. 1, LDL-C (S) was correlated most closely with LDL-C (F), with a correlation coefficient of 0.986 (P < 0.001), and a regression line of y = 0.975x +0.01 mmol/l, the extent of agreement decreasing in the order of LDL-C (P) (r = 0.963, P < 0.001, y = 0.923x + 0.11 mmol/l) > LDL-C (L) (r = 0.962, P < 0.001, y = 0.964x + 0.08 mmol/l) > LDL-C (E) (r

Discussion

In most clinical studies investigating the relationship between LDL-C and CHD, LDL-C was estimated by the Friedewald equation or the β-quantification method [15], [16], [17]. Although LDL-C is generally considered to be a major risk factor for progression of atherosclerosis, the traditional measurement of LDL-C includes measurement of IDL [18]. Like the β-quantification method, LDL-C estimated by the Friedewald equation includes cholesterols in lipoprotein (a) and IDL [5], [7], [18], which are

Acknowledgments

We thank all patients and study participants. We also thank Dr. Masakazu Nakamura (Osaka Medical Center for Health Science and Promotion in Osaka, Japan, CDC/CRMLN Lipid Reference Laboratory) for the measurement of the β-quantification method.

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