Differential reactivities of four homogeneous assays for LDL-cholesterol in serum to intermediate-density lipoproteins and small dense LDL: Comparisons with the Friedewald equation
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.
References (41)
- et al.
Practical methods for plasma lipoprotein analysis
Adv Lipid Res
(1968) - et al.
Lipoprotein separation and low density lipoprotein molecular weight determination using high performance gel-filtration chromatography
J Lipid Res
(1983) - et al.
Increased frequency of lipoprotein disorders similar to type III hyperlipoproteinemia in survivors of myocardial infarction in Japan
Atherosclerosis
(1984) - et al.
Detailed analysis of serum lipids and lipoproteins from Japanese type III hyperlipoproteinemia with apolipoprotein E2/2 phenotype
Clin Chim Acta
(2004) - et al.
Characteristics of coronary artery disease and lipoprotein abnormalities in patients with heterozygous familial hypercholesterolemia associated with diabetes mellitus or impaired glucose tolerance
Atherosclerosis
(1997) - et al.
Intermediate-density lipoproteins and progression of coronary artery disease in hypercholesterolaemic men
Lancet
(1987) - et al.
Apoprotein B structure and receptor recognition of triglyceride-rich low density lipoprotein (LDL) in modified in small LDL, but not in triglyceride rich LDL of normal size
J Biol Chem
(1994) - et al.
Differences in LDL subspecies involve alterations in lipid composition and conformational changes in apolipoprotein B
J Lipid Res
(1996) - et al.
Dense low density lipoprotein subspecies with diminished oxidative resistance predominate in combined hyperlipidemia
J Lipid Res
(1993) Role of low-density lipoproteins in atherogenesis and development of coronary heart disease
Clin Chem
(1995)
Lipoproteins, cardiovascular disease and death. The Framingham study
Arch Interm Med
Expert panel on detection, evaluation, and treatment of high blood cholesterol in adults (Adult Treatment Panel III)
JAMA
Measurement of low-density-lipoprotein cholesterol
Measurement of high-density-lipoprotein cholesterol
National cholesterol education program recommendations for measurement of low-density lipoprotein cholesterol: executive summary. The national cholesterol education program working group on lipoprotein measurement
Clin Chem
Standardization of lipid and lipoprotein measurements
Estimation of the concentration of low-density lipoprotein cholesterol in plasma, without use of the preparative ultracentrifuge
Clin Chem
Quantitative determination of high-, low-, and very-low-density lipoproteins and lipoprotein (a) by agarose gel electrophoresis and enzymatic cholesterol staining
Clin Chem
Forcus on cholesterol research
Cited by (13)
Psychological well-being and green tea consumption are associated with lower pentosidine serum levels among elderly female residents in Japan
2019, Journal of Psychosomatic ResearchCitation Excerpt :Other blood substances in relation to lifestyle diseases, blood sugar (BS), HbA1c, triglyceride (TG), total cholesterol (TC), and high-density lipoprotein cholesterol (HDL-C) were measured in a commercial laboratory (SRL Inc., Tokyo, Japan). Low-density lipoprotein cholesterol (LDL-C) level was calculated using the Friedewald equation (LDL-C = TC–HDL-C–TG/5) [14]. LDL-C level was calculated because it can stay within tissues due to the accumulation of locally deposited reactive AGEs or can inhibit normal lipid clearance mechanisms due to the formation of AGE lipid particles [15].
Evaluation of Martin's equation for LDL-C estimation in type 2 diabetes mellitus Egyptian patients
2019, Clinica Chimica ActaCitation Excerpt :Roche direct LDL-C measurement method is standardized against the reference method and Nauck et al. [40] reported that Roche assay meets the currently established analytical performance goals, so it is useful for both diagnosis and management decision in hyperlipidemic patients. Yamashita et al. [41] found that the selective solubilization method as used in Roche kit exhibited the highest correlation with the β-quantification method. Further studies are recommended to evaluate a larger number of participants in multi-centers as ME can easily be incorporated in the laboratory data system with no additional cost.
Differences in reaction specificity toward lipoprotein X and abnormal LDL among 6 homogeneous assays for LDL-cholesterol
2015, Clinica Chimica ActaCitation Excerpt :Detailed procedures for the homogeneous LDL-C assays are shown in Supplemental data Tables S2 and S3. LDL-C was determined by the BQ method with some modifications [17]. After ultracentrifugation at a density of 1.006 kg/l, the bottom fraction was obtained by heparin-Mn precipitation method, and the TC concentration in the bottom fraction was measured using the enzymatic method (Kyowa Medex Co., Ltd., Tokyo, Japan).
A multicentre analysis of four low-density lipoprotein cholesterol direct assays in samples with extreme high-density lipoprotein cholesterol concentrations
2014, Clinica Chimica ActaCitation Excerpt :Although some studies have validated these assays against the reference methods, other authors have revealed significant discrepancies in the results obtained by them, especially in samples from diseased individuals [9,10]. This heterogeneity could be explained by the distinct determination principles and the different reactivities to lipoproteins described for some of the commercially available methods [11]. This issue constitutes a major problem for lipoprotein determination given that the cut-off points established by the NCEP are universal, and thus, it is crucial that all these methods offer transferable results between laboratories and over time.
A multicenter study on the precision and accuracy of homogeneous assays for LDL-cholesterol: Comparison with a beta-quantification method using fresh serum obtained from non-diseased and diseased subjects
2012, AtherosclerosisCitation Excerpt :Similar to Miller's study, our study showed that diseased subjects had greater discordance between LDL-C (H) and LDL-C (BQ) than non-diseased subjects [12]. Individual homogeneous assays have different determination principles [20] (Table S1) and different reactivities to lipoproteins [21–25]. In this study, the accuracies of Reagent-C, Reagent-D, Reagent-E, and Reagent-F were more susceptible to hypertriglyceridemic conditions (Fig. 1).
Methods for LDL-cholesterol determination: An update
2011, Medecine des Maladies Metaboliques