Evaluation of a Homogeneous Assay for Measuring LDL-cholesterol in Hyperlipidemic Serum Specimens

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1 8 Original Article Evaluation of a Homogeneous Assay for Measuring -cholesterol in Hyperlipidemic Serum Specimens Shizuya Yamashita, Mitsuhiro Nakamura, Hiroaki Koizumi, Hiroyuki Oku, Jose C. Sandoval, Kazumi Tsubakio-Yamamoto, Miyako Kawase, Daisaku Masuda, Masahiro Koseki, Fumihiko Matsuura, Iichiro Shimomura 3, Makoto Nishida, and Masato Ishigami 5 Department of Cardiovascular Medicine, Osaka University Graduate School of Medicine, Osaka, Japan. Daiichi Pure Chemicals Co., Ltd., Tokyo, Japan. 3 Department of Metabolic Medicine, Osaka University Graduate School of Medicine, Osaka, Japan. Health Care Center, Osaka University, Osaka, Japan. 5 Department of Biomedical Informatics, Division of Health Sciences, Osaka University Graduate School of Medicine, Osaka, Japan. Aim: Homogeneous assay reagents for the determination of low-density lipoprotein cholesterol (-C) have been available from several manufacturers. However, there has been considerable controversy due to uncertainty regarding their reactivity with intermediate-density lipoprotein (), which is detected at an especially high frequency in patients with type hyperlipemia. In this study, we examined the reactivity of a homogeneous assay, Cholestest (CT-), with hyperlipemic sera that were classified according to the WHO system. Methods: Sera from 6 normolipidemic and hyperlipidemic patients classified according to the WHO system were used for this study. All serum specimens were fractionated by the ultracentrifugation method of Hatch and Lees, and subjected to lipid and protein measurements. Results: The percent bias of values measured by CT- relative to those determined by the ultracentrifugation method was calculated and compared to the lipid/protein ratios of each lipoprotein fraction. Consequently, the coefficient of correlation between the bias and the Triglyceride/Total cholesterol (TG/TC) ratio in the fraction was 0.7. There were also correlations with the TG/TC ratio and the apo-lipoprotein B/Total Cholesterol ratio in the fraction and in the fraction, respectively. Conclusion: Further testing will be required in order to know more about the clinical condition of hyperlipidemic patients, since CT- may react differently with some beta lipoproteins having a diverse lipid/protein composition compared to those in normolipidemic specimens. J Atheroscler Thromb, 008; 5:8-86. Key words; Low-density lipoprotein, Hyperlipemia, Homogeneous assay, subclasses Introduction An epidemiologic investigation performed by a study group of the Japanese Ministry of Health, Labour and Welfare showed that low-density lipoprotein cholesterol (-C), which has been gaining general Address for correspondence: Shizuya Yamashita, Department of Cardiovascular Medicine, Osaka University Graduate School of Medicine, - Yamadaoka, Suita, Osaka , Japan. shizu@imed.med.osaka-u.ac.jp Received: June 7, 007 Accepted for publication: November 8, 007 recognition as a baddie, is far more closely related to ischemic heart disease than total cholesterol. Thus, C has been incriminated as a risk factor for coronary artery disease ). The Japan Atherosclerosis Society (JAS) Guidelines for Prevention of Atherosclerotic Cardiovascular Diseases issued in 007 give -C values as yardsticks for the diagnosis and treatment of dyslipidemia under the normal range of cholesterol values for Japanese as viewed from the standpoint of prevention and treatment of coronary artery disease and patients categorized by risk factors for coronary artery disease and goal values ).

2 83 Table. Lipid and protein profiles of the subjects a b LCAT deficient CETP deficient n 6 TC FC Values are expressed as the. TG , HDLC C Apo A Apo A Apo B Apo C Apo C Apo E Lp(a) LCAT U/L CETP g/ml LPL ng/ml Various methods of measurement, such as ultracentrifugation, electrophoresis, and high performance liquid chromatography (HPLC), have been employed to determine the blood levels of -C. Ultracentrifugation, in particular, has been considered a standard method in the National Cholesterol Education Program (NCEP), and the Centers for Disease Control and Prevention (CDC) have been leading the effort to standardize the method for -C determination 3). However, the above methods are time-consuming and complicated, and thus not suitable for routine testing in clinical practice. To circumvent that problem, values obtained with Friedewald s equation are generally used as -C values ). In recent years, homogeneous assay reagents that are immune to the effect of dietary chylomicron and very low-density lipoprotein () have been developed by several manufacturers 5), though, there has been considerable controversy due to uncertainty regarding their reactivity with intermediate-density lipoprotein (), which is detected at an especially high frequency in patients with type hyperlipemia 6-8). In the current study, we determined the profiles of the lipoprotein fractions of hyperlipemic sera that were separated by ultracentrifugation and investigated the reactivity of Cholestest (CT-: Daiichi Pure Chemicals Co., Ltd.) with lipoprotein fractions from a variety of hyperlipidemic patients. Materials and Methods Materials In this study the materials were sera from 6 normolipidemic volunteers and hyperlipidemic patients who were referred to Osaka University Hospital. The patients sera separated from fasting blood were classified according to the WHO system 9, 0) based on the measured values for serum lipids and the results of poly-acrylamide gel electrophoresis. Ultracentrifugation All serum specimens were fractionated according to the method of Hatch and Lees ) into CM (chylomicron: d0.95 g/ml), (very-low-density lipoprotein; 0.95d.006 g/ml), (.006d.09 g/ml), (low-density lipoprotein;.09d.063 g/ml), and HDL (high-density lipoprotein;.063d. g/ml). Definition of the Measured Values by Ultracentrifugation Since the risk of coronary artery disease increases with increasing blood levels of these lipoprotein subclasses, the NCEP Recommendations for the Measurement of Low-Density Lipoprotein Cholesterol defines d.006 g/ml -lipoprotein as cholesterol in atherogenic lipoproteins 3). In this study, therefore, the values measured by the ultracentrifugation method were defined as cholesterol in the and fractions. Definition of Percent Bias Percent bias is defined as in percentage terms, the relative absolute difference between the values obtained by the test method (Cholestest ) and those obtained by the comparative method (ultracentrifugation). Lipid and Protein Measurements The following 5 measurements were performed for the serum specimens and each fraction obtained by ultracentrifugation. Serum total cholesterol (TC), free cholesterol (FC), triglyceride (TG), high-density lipoprotein cholesterol (HDL-C), and -C were determined by homogeneous assay, apolipoproteins (apo) A-, A-, B, C-, C-, and E were determined by turbidimetric immunoassay, lipoprotein (a) (Lp(a))

3 8 Fig.. Correlation plot. -C concentrations of 6 normolipidemic volunteers and hyperlipidemic patients were measured using CT- and the ultracentrifugation method. CM HDL serum CM HDL Fig.. Disc gel electrophoresis of the CETP-deficient specimen. Each sample fractionated by ultracentrifugation was applied. HDL was found in the lower layer of the fraction (a dot circle). was determined by latex turbidimetric immunoassay, and lecithin: cholesterol acyl-transferase (LCAT), CETP, and lipoprotein lipase (LPL) were determined by enzyme-linked immunosorbent-assay. The lipid and protein profiles of the subjects are shown in Table. The assay principle of CT- is as follows. a) In the first reaction phase, non- cholesterol is exposed by the effect of a specific surfactant and removed by a coupling enzyme system. The enzymes are incapable Table. Correlation coefficients (r) between the percent bias of CT- to -C measured by ultracentrifugation and the values for TC, TG, apo-b, TG/TC, apo B/TC and apo B/TG TC TG Apo B TG/TC Apo B/TC Apo B/TG d g/ml d g/ml Fraction of acting on. b) The structure of particles is disrupted by another surfactant, and subsequently the enzymatic reaction with is initiated in the second reaction phase. The absorbance measured at the time of the reaction is proportional to the -C concentration in the specimen. Results d g/ml d g/ml Comparison of CT- with the Ultracentrifugation Method In the current study, the values determined with CT- were compared with those obtained using the ultracentrifugation method. There was generally a good correlation between the methods, except for one CETP-deficient, 3) specimen (ultracentrifugation: 80. ; CT-: 5.7 ). The coefficient of correlation and the equation of linear regression between these two methods were R and y (CT- ).037x (ultracentrifugation).53, respectively (Fig. ). When the disc gel electrophoresis of ultracentrifuged fractions of the CETP-deficient specimen was performed, contamination by HDL (in the lower layer) was found in the fraction (Fig. ). Lipid/protein Ratios and Bias of CT- Relative to Ultracentrifugation The percent bias of values measured by CT- relative to those determined by the ultracentrifugation method was calculated and compared to the lipid/ protein ratios of each lipoprotein fraction to examine the tendency for deviation from the values measured by ultracentrifugation. In addition, since a CETPdeficient specimen was not completely separated by the ultracentrifugation method, and since the TG values of specimens in exceeded the specified dynamic range (up to,500 ), they were not included in

4 85 Table 3. Mean percent bias of CT- to ultracentrifugation method Type a b n 6 Mean value obtained by CT- () Mean value obtained by ultracentrifugation method () Mean absolute value of the difference between CT- and the ultracentrifugation method () Mean percent bias of CT- to ultracentrifugation method 37.9% 8.7%.0% 5.% 0.% 0.8% 3.5% Table. Lipid Compositions of fractionated lipoprotein specimens Fraction (d g/ml) (d g/ml) (d g/ml) Classification a b a b a b TC () TG () Apo B () TG/TC Apo B/TC Apo B/TG the statistical analysis. It was found that there was no correlation between the percent bias of values measured by CT- relative to those obtained by the ultracentrifugation method and the values for TC, TG, and apo-b in any fraction. However, there was a very close correlation between the bias and the TG/TC ratio in the fraction having a specific gravity of.006 to.09. There were also correlations with the TG/TC ratio and the apo-b/tc ratio in the fraction and the fraction, having a specific gravity of.09 to.063 and.006 to.063, respectively (Table ). When specimens were classified according to the WHO system, the percent bias for the specimens with a relatively high cholesterol content in the fraction obtained from patients with type hyperlipidemia was 5.%, with little difference from the bias of 3.5% for normolipidemic (volunteer) specimens. Although the percent bias of values measured by CT- versus the ultracentrifugation method was 37.9% for type hyperlipidemic specimens, this was assumed to be due to the small denominator (Table 3). Furthermore, it was indicated that the TG/TC ratios in the and fractions and the apo-b/tc ratios in the fraction of type and hyperlipidemic specimens, that is, the structures of -lipoprotein, were related to the reactivity of CT- (Table ).

5 86 Discussion In the current study, the reactivity of CT- with hyperlipidemic serum specimens was determined. When hyperlipidemia was classified according to the WHO system, the values obtained with CT- were well consistent with those generated by the ultracentrifugation method for type specimens containing both and. In addition, though the number of type a and b specimens examined was small, Kajinami et al. reported that CT- was correlated with the ultracentrifugation method for twenty-six (6) type a and twenty-three (3) type b specimens ). However, caution is warranted in regard to the discrepancy observed with a CETP-deficient specimen and some hyperlipidemic specimens. It was found that a CETP-deficient specimen was not completely separated by the ultracentrifugation method in the aresent study. Additionally, as the fraction obtained by beta-quantification recommended by Centers for Disease Control and Prevention contains Apo-E rich HDL 5), the ultracentrifugation method seemed to be unfavorable. Regarding the reactivity of CT- to Apo-E-rich HDL, furthermore, Akizawa et al. have reported that CT- did not react to HDL particles from patients with CETP deficiency 6). In an attempt to identify the factors responsible for the discrepancy in the results, lipoproteins were fractionated according to their specific gravity, and the protein and lipid contents of each fraction were measured to determine the relationship between the profiles of the lipoprotein fractions and the bias of values measured by CT- versus the ultracentrifugation method. It was found that the TG/TC ratio or the apo-b/tc ratio in the and/or fraction was related to the bias. In other words, the discrepancy seemed to be caused by qualitative changes in the or structure in a variety of hyperlipidemic patients. In the time, it has reported that the cross-reactivity of CT- with separated by ultracentrifugation was 7 percent 7). In conclusion, since CT- may differ in reactivity with some beta lipoproteins having a diverse lipid/protein composition compared to those in normolipidemic specimens, further testing will be required in order to know more about the clinical condition of hyperlipidemic patients. References ) MHLW Study Group on Primary Hyperlipemia, 988 ) Japan Atherosclerosis Society: Japan Atherosclerosis Society (JAS) Guidelines for Prevention of Atherosclerotic Cardiovascular Diseases, 007 3) National Cholesterol Education Program Recommendations for Measurement of Low-Density Lipoprotein Cholesterol: Executive Summary. Clin Chem, 995; :- 0 ) Friedewald WT: Estimation of serum low density lipoprotein. Clin Chem, 97; 8:99 5) Kanno T, Sakurabayashi I, Saito Y, Bujo H, Yamada N, Sekiguchi M, Kubono K, Kita T, Matsuzawa Y, Yamashita S, Katayama Y, and Fujita S: Evaluation of new assay for determination of -cholesterol. Jpn J Med Pharm Sci, 997; 37: ) Eto M, Saito M, Kaku K, Sasaki H, and Miida T: Controversial point of cholesterol assay. J Lipoprotein Res Soc, 000; :39- (in Japanese) 7) Saito N, Kudo A, Katsura T, and Takahashi N: -cholesterol method is the point an issue. Japanese J Medical Technology, 007; 56:3-36 (in Japanese) 8) J. L. Beaumont, L. A. Carlson, and G. R. Cooper: Bull. WHO, 970; 3:89 9) D. S. Fredrickson and R. S. Lees: A System for Phenotyping Hyperlipoproteinemia. Circulation, 965; 3:3-37 0) D. S. Fredrickson, R. I. Levy, and R. S. Lees: Fat transport in lipoproteins an integrated approach to mechanism and disorders. N Engl J Med, 967; 76:3- ) F. T. Hatch and R. S. Lees: Practical methods for plasma lipoprotein analysis. Adv Lipid Res, 968; 6:-68 ) Yamashita S, Matsuzawa Y, Okazaki M, Kako H, Yasugi T, Akioka H, Hirano K, and Tarui S: Small polydisperse low density lipoproteins in familial hyperalphalipoproteinemia with complete deficiency of cholesteryl ester transfer activity. Atherosclerosis, 988; 70:7-3) Koizumi J, Mabuchi H, Yoshimura A, Michishita I, Takeda M, Itoh H, Sakai Y, Sakai T, Ueda K, and Takeda R: Deficiency of serum cholesteryl-ester transfer activity in patients with familial hyperalphalipoproteinaemia. Atherosclerosis, 985; 58:75-86 ) Kajinami K, Todo Y, Emoto Y, Nohara A, and Mabuchi H: Clinical application of direct measurement for cholesterol levels. Japanese J Clinical Laboratory Automation, 000; 5:6-9 5) Koba A, Tanahashi Y, Kimura T, Obuchi K, Makise J, and Kijima Y: Evaluation of direct assay for low density lipoprotein-cholesterol and knowledge in cholesterol ester transfer protein deficiency. Japanese J Medical Technology, 999; 8:9-33 (in Japanese) 6) Akizawa K, Simamura H, Akita H, Kei S, Takahashi Y, Yanagiuchi H, Fujiwara H, Ishizuka S, and Chiba H: Heterogeneous reactivities in homogeneous -cholesterol assays of serum lipoproteins from patients with complete cholesteryl ester transfer protein deficiency. Jpn J Med Pharm Sci, 000; 3:37- (in Japanese) 7) Sakaue T, Hirano T, Yoshino G, Sakai K, Takeuchi H, and Adachi M: Reactions of direct -cholesterol assays with pure fraction and : comparison of three homogenous methods. Clinica Chemica Acta, 000; 95:97-06

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