Atherosclerosis 231 (2013) 238e245
Contents lists available at ScienceDirect
Atherosclerosis journal homepage: www.elsevier.com/locate/atherosclerosis
Serum cholesterol and triglyceride reference ranges of twenty lipoprotein subclasses for healthy Japanese men and women Norihiro Furusyo a, *, Masumi Ai b, c, Mitsuyo Okazaki d, Hiroaki Ikezaki a, Takeshi Ihara a, Takeo Hayashi a, Satoshi Hiramine a, Kazuya Ura a, Takuji Kohzuma e, Ernst J. Schaefer b, Jun Hayashi a a
Department of General Internal Medicine, Kyushu University Hospital, 3-1-1 Maidashi, Higashi-Ku, Fukuoka 812-8582, Japan Lipid Metabolism Laboratory, Human Nutrition Research Center on Aging at Tufts University, and Tufts University School of Medicine, Boston, MA, USA Department of Insured Medical Care Management, Graduate School of Medicine, Tokyo Medical and Dental University, Tokyo, Japan d Professor Emeritus of Tokyo Medical and Dental University, Skylight Biotech Inc., Akita, Japan e Diagnostics Department, Asahi Kasei Pharma Corporation, Tokyo, Japan b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 15 May 2013 Received in revised form 27 August 2013 Accepted 9 September 2013 Available online 1 October 2013
Aim: This epidemiological study was done to generate normal ranges for the cholesterol and triglyceride levels in serum lipoprotein subclasses isolated from healthy adults based on gender and menopausal status. Methods: Cholesterol and triglyceride levels in 20 lipoprotein subclasses as separated by high performance liquid chromatography were measured in serum obtained from 825 fasting healthy subjects (267 men, 558 women). Results: For serum cholesterol, 13.7% was found in very low density lipoprotein (VLDL) subclasses, 55.6% in low density lipoprotein (LDL) subclasses, and 30.4% in high density lipoprotein (HDL) subclasses. For serum triglycerides, these values were 52.1%, 27.9%, and 17.4%, respectively. Levels of cholesterol in some VLDL subclasses were inversely correlated with the levels of some HDL subclasses, while for triglycerides, elevated levels in any one subclass were generally strongly associated with elevated levels in all other subclasses. Men had significantly higher large VLDL-cholesterol levels than women (P < 0.05), while women had significantly higher small VLDL-cholesterol levels than men (P < 0.001). Women had significantly higher large LDL- and large and medium HDL-cholesterol levels than men (P < 0.001). Men had significantly higher chylomicron (CM), large and medium VLDL-, and small LDL-triglyceride levels than women (P < 0.001). Women had significantly higher very large and large HDL-triglyceride levels than men (P < 0.01). Postmenopausal women had significantly higher CM, all VLDL, and large, medium and small LDL-cholesterol levels, and significantly higher all VLDL, LDL, and HDL-triglyceride levels than premenopausal women (P < 0.001). Conclusions: Our data document important gender and menopausal status differences in cholesterol and triglyceride subclass levels, as well as significant correlations between values in the various serum lipoprotein subclasses. Ó 2013 Elsevier Ireland Ltd. All rights reserved.
Keywords: High performance liquid chromatography Cholesterol Triglycerides Lipoprotein subclasses General population Gender Menopause
1. Introduction
Abbreviations: VLDL, very low density lipoproteins; LDL, low density lipoproteins; HDL, high density lipoproteins; HPLC, high performance liquid chromatography; CM, chylomicrons; CHD, coronary heart disease; NMR, nuclear magnetic resonance; KOPS, Kyushu and Okinawa Population Study; FPG, fasting plasma glucose; HbA1c, glycosylated hemoglobin A1c. * Corresponding author. Tel.: þ81 92 642 5909; fax: þ81 92 642 5916. E-mail address: [email protected] (N. Furusyo). 0021-9150/$ e see front matter Ó 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.atherosclerosis.2013.09.008
Coronary heart disease (CHD) due to atherosclerosis can be prevented by significant risk factor modification, including reductions in atherogenic lipoproteins by diet and medications. Lipid abnormalities play a critical role in the development of atherosclerosis [1]. Elevated LDL-cholesterol and decreased HDLcholesterol levels have been shown to be independent risk factors for CHD [2e4]. In a prospective study, high non HDL-cholesterol level was reported to be a CHD risk marker, independent of the plasma LDL-cholesterol level, as were a high triglyceride level, an
N. Furusyo et al. / Atherosclerosis 231 (2013) 238e245
elevated total/HDL-cholesterol ratio, and the LDL/HDL-cholesterol ratio [5]. The recommended method for measuring plasma serum lipoprotein levels is to obtain a blood sample after an overnight fasting, measure serum total cholesterol and triglycerides, then measure HDL-cholesterol after removing other lipoproteins by precipitation. LDL-cholesterol is then calculated using the Friedewald formula [6]. The formula works well as long as the triglyceride level is not >400 mg/dL. When this is the case, the LDL-cholesterol level must be measured directly using either ultracentrifugation or one of several commercially available enzymatic methods. Lipoprotein subclasses, characterized by variations in their density, particle size, and chemical composition, are clinically important [7e11]. Patients with established CHD are much more likely than control subjects to have increased levels of small dense LDL and decreased levels of large HDL particles [7e11]. Many techniques have been used for lipoprotein subclass analysis: analytical ultracentrifugation [7], sequential separation at various densities, rate zonal ultracentrifugation [13], and density-gradient ultracentrifugation with a swing rotor [14] or a vertical rotor [15]. Lipoprotein particle size analysis is usually done by non-denaturing gradient gel electrophoresis [16]. Alternative techniques include the use of the Lipoprint LDL system, which is based on a nongradient polyacrylamide gel electrophoretic method [17], or nuclear magnetic resonance (NMR), in which particle concentration is used to evaluate lipoprotein subclasses [18]. However, with the exception of newer direct methods for measuring small dense LDL cholesterol, all these techniques are laborious and not available in routine clinical laboratories [4]. In contrast to gradient gel electrophoresis techniques, the HPLC method used in this study allows for direct quantitation of the cholesterol and triglyceride content of the individual lipoprotein subclasses [19e24]. No studies have published reference intervals for the serum lipoprotein subclasses as measured in healthy individuals. For this reason, this large, community-based population study was done to measure 20 serum lipoprotein subclasses of cholesterol and triglycerides by the HPLC to provide reference intervals and to determine gender and menopausal status differences in the serum lipoprotein profile of Japanese adults. 2. Materials and methods 2.1. Study population This study is part of the Kyushu and Okinawa Population Study (KOPS) survey of vascular events associated with lifestyle-related diseases [25e27]. Eligible participants were 1062 residents aged 20 who took part in free public physical examinations between 2010 and 2011. An interview was done to gather information about personal medical history, family medical history, smoking and drinking behaviors, and menopausal status. Of the participants, 237 were excluded because they were being treated for CHD, dyslipidemia (on cholesterol-lowering medication), diabetes (on antidiabetes medicines including insulin injections, hemoglobin A1c (HbA1c) 6.5% and/or fasting plasma glucose (FPG) 7.0 mmol/L (126 mg/dL)), thyroid disease (on hormone medications), chronic liver disease, nephropathy (estimated glomerular filtration rate 90 nm and 75 nm in diameter. VLDL subclasses: large VLDL (VLDL1 to VLDL-3, 64, 53.6 and 44.5 nm in diameter), medium VLDL (VLDL-4, 36.8 nm), and small VLDL (VLDL-5, 31.3 nm). LDL subclasses: large LDL (LDL-1, 28.6 nm), medium LDL (LDL-2, 25.5 nm), small LDL (LDL-3, 23.0 nm) and very small LDL (LDL-4 to LDL-6, 20.7, 18.6 and 16.7 nm). HDL subclasses: very large HDL (HDL-1 and HDL-2, 15.0 and 13.5 nm), large HDL (HDL-3, 12.1 nm), medium HDL (HDL-4, 10.9 nm), small HDL (HDL-5, 9.8 nm) and very small HDL (HDL-6 and HDL-7, 8.8 and 7.6 nm). Total serum cholesterol and triglyceride levels were calculated as the sum of the 20 subclasses, and the major lipoprotein classes, CM, VLDL, LDL and HDL were the sum of their lipoprotein subclasses, CM-1 and -2, VLDL-1 to -5, LDL-1 to -6, and HDL-1 to -7, respectively. The HDL-7 triglyceride levels were affected by the colored materials in plasma such as bilirubin. We previously compared this method with ultracentrifugation, and very high correlations were obtained [20]. Each of the ultracentrifugally isolated fractions consisted of several subclasses, as follows: VLDL fraction, large VLDL (10%e30%), medium VLDL (45%), small VLDL (10%e25%); IDL fraction, small VLDL
6.1 8.4 17.4 0.80 0.60 0.79 23.3 18.5 24.1 3.48 1.97 3.97 23 (5.7) 19.5 16.1 20.4 3.12 1.71 3.41 30 (19.5)
5.7 8.8 14.3 0.80 0.59 0.85
62.0 22.4 81.2 125.6 73.9 5.2 5.5 6.3 74.5
Contents lists available at ScienceDirect
Atherosclerosis journal homepage: www.elsevier.com/locate/atherosclerosis
Serum cholesterol and triglyceride reference ranges of twenty lipoprotein subclasses for healthy Japanese men and women Norihiro Furusyo a, *, Masumi Ai b, c, Mitsuyo Okazaki d, Hiroaki Ikezaki a, Takeshi Ihara a, Takeo Hayashi a, Satoshi Hiramine a, Kazuya Ura a, Takuji Kohzuma e, Ernst J. Schaefer b, Jun Hayashi a a
Department of General Internal Medicine, Kyushu University Hospital, 3-1-1 Maidashi, Higashi-Ku, Fukuoka 812-8582, Japan Lipid Metabolism Laboratory, Human Nutrition Research Center on Aging at Tufts University, and Tufts University School of Medicine, Boston, MA, USA Department of Insured Medical Care Management, Graduate School of Medicine, Tokyo Medical and Dental University, Tokyo, Japan d Professor Emeritus of Tokyo Medical and Dental University, Skylight Biotech Inc., Akita, Japan e Diagnostics Department, Asahi Kasei Pharma Corporation, Tokyo, Japan b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 15 May 2013 Received in revised form 27 August 2013 Accepted 9 September 2013 Available online 1 October 2013
Aim: This epidemiological study was done to generate normal ranges for the cholesterol and triglyceride levels in serum lipoprotein subclasses isolated from healthy adults based on gender and menopausal status. Methods: Cholesterol and triglyceride levels in 20 lipoprotein subclasses as separated by high performance liquid chromatography were measured in serum obtained from 825 fasting healthy subjects (267 men, 558 women). Results: For serum cholesterol, 13.7% was found in very low density lipoprotein (VLDL) subclasses, 55.6% in low density lipoprotein (LDL) subclasses, and 30.4% in high density lipoprotein (HDL) subclasses. For serum triglycerides, these values were 52.1%, 27.9%, and 17.4%, respectively. Levels of cholesterol in some VLDL subclasses were inversely correlated with the levels of some HDL subclasses, while for triglycerides, elevated levels in any one subclass were generally strongly associated with elevated levels in all other subclasses. Men had significantly higher large VLDL-cholesterol levels than women (P < 0.05), while women had significantly higher small VLDL-cholesterol levels than men (P < 0.001). Women had significantly higher large LDL- and large and medium HDL-cholesterol levels than men (P < 0.001). Men had significantly higher chylomicron (CM), large and medium VLDL-, and small LDL-triglyceride levels than women (P < 0.001). Women had significantly higher very large and large HDL-triglyceride levels than men (P < 0.01). Postmenopausal women had significantly higher CM, all VLDL, and large, medium and small LDL-cholesterol levels, and significantly higher all VLDL, LDL, and HDL-triglyceride levels than premenopausal women (P < 0.001). Conclusions: Our data document important gender and menopausal status differences in cholesterol and triglyceride subclass levels, as well as significant correlations between values in the various serum lipoprotein subclasses. Ó 2013 Elsevier Ireland Ltd. All rights reserved.
Keywords: High performance liquid chromatography Cholesterol Triglycerides Lipoprotein subclasses General population Gender Menopause
1. Introduction
Abbreviations: VLDL, very low density lipoproteins; LDL, low density lipoproteins; HDL, high density lipoproteins; HPLC, high performance liquid chromatography; CM, chylomicrons; CHD, coronary heart disease; NMR, nuclear magnetic resonance; KOPS, Kyushu and Okinawa Population Study; FPG, fasting plasma glucose; HbA1c, glycosylated hemoglobin A1c. * Corresponding author. Tel.: þ81 92 642 5909; fax: þ81 92 642 5916. E-mail address: [email protected] (N. Furusyo). 0021-9150/$ e see front matter Ó 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.atherosclerosis.2013.09.008
Coronary heart disease (CHD) due to atherosclerosis can be prevented by significant risk factor modification, including reductions in atherogenic lipoproteins by diet and medications. Lipid abnormalities play a critical role in the development of atherosclerosis [1]. Elevated LDL-cholesterol and decreased HDLcholesterol levels have been shown to be independent risk factors for CHD [2e4]. In a prospective study, high non HDL-cholesterol level was reported to be a CHD risk marker, independent of the plasma LDL-cholesterol level, as were a high triglyceride level, an
N. Furusyo et al. / Atherosclerosis 231 (2013) 238e245
elevated total/HDL-cholesterol ratio, and the LDL/HDL-cholesterol ratio [5]. The recommended method for measuring plasma serum lipoprotein levels is to obtain a blood sample after an overnight fasting, measure serum total cholesterol and triglycerides, then measure HDL-cholesterol after removing other lipoproteins by precipitation. LDL-cholesterol is then calculated using the Friedewald formula [6]. The formula works well as long as the triglyceride level is not >400 mg/dL. When this is the case, the LDL-cholesterol level must be measured directly using either ultracentrifugation or one of several commercially available enzymatic methods. Lipoprotein subclasses, characterized by variations in their density, particle size, and chemical composition, are clinically important [7e11]. Patients with established CHD are much more likely than control subjects to have increased levels of small dense LDL and decreased levels of large HDL particles [7e11]. Many techniques have been used for lipoprotein subclass analysis: analytical ultracentrifugation [7], sequential separation at various densities, rate zonal ultracentrifugation [13], and density-gradient ultracentrifugation with a swing rotor [14] or a vertical rotor [15]. Lipoprotein particle size analysis is usually done by non-denaturing gradient gel electrophoresis [16]. Alternative techniques include the use of the Lipoprint LDL system, which is based on a nongradient polyacrylamide gel electrophoretic method [17], or nuclear magnetic resonance (NMR), in which particle concentration is used to evaluate lipoprotein subclasses [18]. However, with the exception of newer direct methods for measuring small dense LDL cholesterol, all these techniques are laborious and not available in routine clinical laboratories [4]. In contrast to gradient gel electrophoresis techniques, the HPLC method used in this study allows for direct quantitation of the cholesterol and triglyceride content of the individual lipoprotein subclasses [19e24]. No studies have published reference intervals for the serum lipoprotein subclasses as measured in healthy individuals. For this reason, this large, community-based population study was done to measure 20 serum lipoprotein subclasses of cholesterol and triglycerides by the HPLC to provide reference intervals and to determine gender and menopausal status differences in the serum lipoprotein profile of Japanese adults. 2. Materials and methods 2.1. Study population This study is part of the Kyushu and Okinawa Population Study (KOPS) survey of vascular events associated with lifestyle-related diseases [25e27]. Eligible participants were 1062 residents aged 20 who took part in free public physical examinations between 2010 and 2011. An interview was done to gather information about personal medical history, family medical history, smoking and drinking behaviors, and menopausal status. Of the participants, 237 were excluded because they were being treated for CHD, dyslipidemia (on cholesterol-lowering medication), diabetes (on antidiabetes medicines including insulin injections, hemoglobin A1c (HbA1c) 6.5% and/or fasting plasma glucose (FPG) 7.0 mmol/L (126 mg/dL)), thyroid disease (on hormone medications), chronic liver disease, nephropathy (estimated glomerular filtration rate 90 nm and 75 nm in diameter. VLDL subclasses: large VLDL (VLDL1 to VLDL-3, 64, 53.6 and 44.5 nm in diameter), medium VLDL (VLDL-4, 36.8 nm), and small VLDL (VLDL-5, 31.3 nm). LDL subclasses: large LDL (LDL-1, 28.6 nm), medium LDL (LDL-2, 25.5 nm), small LDL (LDL-3, 23.0 nm) and very small LDL (LDL-4 to LDL-6, 20.7, 18.6 and 16.7 nm). HDL subclasses: very large HDL (HDL-1 and HDL-2, 15.0 and 13.5 nm), large HDL (HDL-3, 12.1 nm), medium HDL (HDL-4, 10.9 nm), small HDL (HDL-5, 9.8 nm) and very small HDL (HDL-6 and HDL-7, 8.8 and 7.6 nm). Total serum cholesterol and triglyceride levels were calculated as the sum of the 20 subclasses, and the major lipoprotein classes, CM, VLDL, LDL and HDL were the sum of their lipoprotein subclasses, CM-1 and -2, VLDL-1 to -5, LDL-1 to -6, and HDL-1 to -7, respectively. The HDL-7 triglyceride levels were affected by the colored materials in plasma such as bilirubin. We previously compared this method with ultracentrifugation, and very high correlations were obtained [20]. Each of the ultracentrifugally isolated fractions consisted of several subclasses, as follows: VLDL fraction, large VLDL (10%e30%), medium VLDL (45%), small VLDL (10%e25%); IDL fraction, small VLDL
6.1 8.4 17.4 0.80 0.60 0.79 23.3 18.5 24.1 3.48 1.97 3.97 23 (5.7) 19.5 16.1 20.4 3.12 1.71 3.41 30 (19.5)
5.7 8.8 14.3 0.80 0.59 0.85
62.0 22.4 81.2 125.6 73.9 5.2 5.5 6.3 74.5
