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Lipoprotein(a)-cholesterol: A significant component of serum cholesterol
Clinica Chimica Acta 412 (2011) 1783–1787
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Clinica Chimica Acta j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / c l i n c h i m
Lipoprotein(a)-cholesterol: A significant component of serum cholesterol Kiyoko Kinpara a, 1, Hiroshi Okada b, 1, Akiko Yoneyama a, Minoru Okubo c, d, Toshio Murase c, d,⁎ a
Department of Clinical Laboratory, Toranomon Hospital, Japan Development Group, Diagnostic Business Department, Sanwa Kagaku Kenkyusho Co. Ltd, Japan c Department of Endocrinology and Metabolism, Toranomon Hospital, Japan d Okinaka Memorial Institute for Medical Research, Japan b
a r t i c l e
i n f o
Article history: Received 4 April 2011 Received in revised form 26 May 2011 Accepted 31 May 2011 Available online 13 June 2011 Keywords: Lipoprotein(a) Lipoprotein(a)-cholesterol The Friedewald formula
a b s t r a c t Background: Lipoprotein(a) [Lp(a)] is known to be a cholesterol-rich lipoprotein, however, the contribution of Lp(a)-cholesterol [Lp(a)-C] to the serum cholesterol and LDL-C levels has not yet been fully evaluated. Methods: We determined the serum Lp(a)-C in 55 subjects with serum Lp(a) concentrations ranging from 9 to 129 mg/dl. To measure the serum Lp(a)-C concentrations, we developed an immunoaffinity gel assay; serum was incubated with Sepharose 4B gel coupled with immunoglobulin G (IgG) prepared from a polyclonal antiLp(a) goat antiserum. After separating Lp(a) from other lipoproteins, we determined the serum Lp(a)-C concentrations. Validation of the assay showed satisfactory results in terms of the specificity and reproducibility. Results: The mean cholesterol content of Lp(a), determined as Lp(a)-C/Lp(a), was 29.5± 10.4%. The serum Lp(a)C values were found to be highly correlated with the serum Lp(a) mass (r= 0.923, p b 0.001). At serum Lp(a) levels of over 50 mg/dl, the contribution of Lp(a)-C to the serum total cholesterol was 10.2%. Further, the Friedewald formula overestimated the serum LDL-C by 20.4%. Conclusions: Lp(a) contains approximately 30% cholesterol in each molecule. In subjects with markedly elevated serum Lp(a) concentrations, the Lp(a)-C values should be taken into account when evaluating the serum LDL-C. © 2011 Elsevier B.V. All rights reserved.
1. Introduction Lipoprotein(a) [Lp(a)] is an emerging risk factor for atherosclerotic cardiovascular disease [1–5]. Lp(a) is composed of one copy of LDL linked by a disulfide bond to the glycoprotein apolipoprotein(a) [1,6,7]; thus, each molecule of Lp(a) contains some cholesterol. However, the contribution of Lp(a)-cholesterol [Lp(a)-C] to the total serum cholesterol concentrations is not yet well understood. We recently demonstrated that markedly increased serum Lp(a) (≥100 mg/dl) concentrations are associated with a 5-fold or greater increase in the risk of coronary heart disease (CHD) [8]. During an analysis to identify the independent risk factors for CHD, we considered that the serum Lp(a)-C values would be considerably increased in association with increased serum Lp(a) concentrations, and that, therefore, cholesterol content of Lp(a) should be taken into account when evaluating the serum LDL-C and total cholesterol. This prompted us to examine the cholesterol content of Lp(a). A few previous studies have shown that the Lp(a) molecule contains appreciable amounts of cholesterol. In previous studies, Abbreviations: Lp(a), lipoprotein(a); Lp(a)-C, lipoprotein(a)-cholesterol; CHD, coronary heart disease. ⁎ Corresponding author at: Okinaka Memorial Institute for Medical Research, Toranomon 2-2-2, Minato-ku, Tokyo 105-8470, Japan. Tel.: +81 3 3588 1111; fax: +81 3 3588 0356. E-mail address: [email protected] (T. Murase). 1 These two authors contributed equally to this work. 0009-8981/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.cca.2011.05.036
serum Lp(a)-C concentrations were determined by analyzing the chemical composition of isolated Lp(a) [9–12] or using chemical reagents that precipitate [13] or absorb [14] serum Lp(a). In the present study, we developed an immunoaffinity gel assay that separates Lp(a) from other cholesterol-containing lipoproteins. Using this assay, we determined the serum Lp(a)-C concentrations and characterized the relationship between serum Lp(a)-C concentrations and the Lp(a) mass.
2. Subjects and methods 2.1. Subjects We enrolled outpatients (men, n = 33; women, n = 22) visiting the Toranomon Hospital (Tokyo, Japan) who agreed to our evaluation of their serum lipid profiles (mean age, 63 ± 13 y; age range, 23–84 y). In our earlier experiences [8] the distribution of the serum Lp(a) concentrations showed skewing toward lower values in the Japanese population, with 90% of the population having values b30 mg/dl and this distribution was consistent with the serum concentration profile reported for whites [1]. However, since the aim of the current study was to determine the cholesterol content of Lp(a), we intentionally selected subjects with relatively high serum Lp(a) concentrations. The study was conducted with the approval of the institutional review board of Toranomon Hospital.
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The 55 participants were classified into 3 groups according to their lipid profiles and underlying conditions: group 1: normal serum lipid profile, defined as serum total cholesterol b240 mg/dl and serum triglyceride b150 mg/dl (n= 17); group 2: hyperlipidemia, defined as serum cholesterol N240 mg/dl, serum triglyceride N150 mg/dl, or treatment with lipid-lowering agents (n = 26); group 3: type 2 diabetes mellitus, diagnosed based on the criteria established by the World Health Organization [15] (n= 12). In group 2, hyperlipidemia could be further divided into hypercholesterolemia (n = 16), including familial hypercholesterolemia (n= 6), diagnosed based on the characteristic features (i.e., LDL cholesterol N200 mg/dl, thickening of the Achilles tendon [N9 mm], and history of marked hypercholesterolemia or CHD in first-degree relatives), mixed hyperlipidemia (n= 6), and hypertriglyceridemia (n= 4). In group 3, 7 participants with diabetes mellitus also had hyperlipidemia.
LDL-C” (after subtracting Lp(a)-C) were evaluated by the paired t-test. Values of p b 0.05 were considered significant. 3. Results 3.1. Assay conditions To determine the effect of the incubation time on the adsorption of Lp(a), we incubated each serum specimen (150 μl) with the immunoaffinity gel (715 μl aliquots containing 0.5 mg IgG protein) for various
A
100.0 90.0 80.0
Adsorption rate (%)
2.2. Determination of the serum Lp(a) and lipid concentrations Serum Lp(a) concentrations were determined by a latex immunosorbent assay using polyclonal anti-Lp(a) goat serum [Inotec Lp(a), Sanwa, Tokyo] [16]. Serum concentrations of total cholesterol, triglyceride and high-density lipoprotein cholesterol were determined using routine laboratory methods.
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2.3. Determination of the serum concentration of Lp(a)-C by immunoaffinity gel
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2.3.1. Preparation of the immunoaffinity gel IgG (10 mg) prepared from a polyclonal anti-Lp(a) goat antiserum (SHIMA Laboratories Co. LTD) was coupled to cyanogen bromideactivated Sepharose 4B (1.67 g; Pharmacia Fine Chemicals, Uppsala, Sweden), according to the manufacturer's instructions. The gel was suspended in 0.01 mol/l Tris–HCl buffer (final volume, 10 ml) containing 0.15 mol/l NaCl (pH 8.0), and then 715-μl aliquots (0.5 mg protein) were divided into tubes and stored in a refrigerator until the Lp(a)-C measurement. It was confirmed in a preliminary study that the gel can be stored at 4 °C for at least 2 months.
0.0
2.4. Statistical analysis Results were expressed as mean ± standard deviation (SD) or range. Linear regression analysis was performed to evaluate the association between the serum concentrations of Lp(a)-C and Lp(a). Comparisons between groups were performed by two-tailed Student's t-test. The differences between the serum LDL-C as calculated by the Friedewald formula (“calculated LDL-C”) and the “corrected
0
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Incubation time (min)
B
60.0
Lp(a)-C concentrations (mg/dl)
2.3.2. Measurement of serum Lp(a)-C To determine the optimal conditions for measurement of the serum Lp(a)-C, each serum specimen (0–250 μl) was mixed with immunoaffinity gel (715 μl) and incubated for 0–180 min under gentle shaking at room temperature. The mixture was then centrifuged at 3000 rpm for 15 min, and the Lp(a) concentration in the supernatant was determined. The supernatant Lp(a) concentration was corrected for serum dilution during incubation with the gel by simultaneous measurement of the serum protein as the internal reference. Serum Lp(a)-C adsorption was calculated as ([Serum Lp(a)supernatant Lp(a)] × 100)/Serum Lp(a). To determine the serum Lp(a)-C concentrations in the study participants, each serum specimen (150 μl) was mixed with immunoaffinity gel (715 μl) and incubated under gentle shaking for 120 min at room temperature. The mixture was then centrifuged at 3000 rpm for 15 min, and the cholesterol concentration in the supernatant was determined after correction for the serum dilution. Serum Lp(a)-C concentration was determined by subtracting the supernatant cholesterol concentration from the serum total cholesterol.
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Sample volume ( µ I) Fig. 1. (A) Relationship between serum Lp(a) adsorption by the gel and the incubation period. Each serum specimen (150 μl) was mixed with immunoaffinity gel (715 μl) and incubated under gentle shaking for 0–180 min at room temperature. The mixture was then centrifuged at 3000 rpm for 15 min, and the Lp(a) concentration in the supernatant was determined; the supernatant Lp(a) concentration was corrected for serum dilution during incubation with the gel by simultaneous measurement of serum protein as the internal reference. Serum Lp(a) adsorption was calculated as ([Serum Lp(a)-supernatant Lp(a)] × 100)/Serum Lp(a). Results are expressed as mean ± SD of 3 samples: Sample 1, 63.4 mg/dl Lp(a); Sample 2, 86.6 mg/dl Lp(a); Sample 3, 90.3 mg/dl Lp(a). (B) Relationship between serum volume and serum Lp(a)-C concentration. This was evaluated using 2 serum samples [○, Lp(a) 63.4 mg/dl; ●, Lp(a) 86.6 mg/dl] run in parallel. Increasing volumes of serum were mixed with immunoaffinity gel (715 μl) and incubated under gentle shaking for 120 min at room temperature. The mixtures were then centrifuged at 3000 rpm for 15 min, and the cholesterol concentrations in the supernatant were determined; the supernatant cholesterol concentration was corrected for serum dilution during incubation with the gel by simultaneous measurement of serum protein as the internal reference. Serum Lp(a)-C concentration was calculated as (Serum cholesterol–Supernatant cholesterol).
K. Kinpara et al. / Clinica Chimica Acta 412 (2011) 1783–1787
3.2. Validation of the Lp(a)-C assay 3.2.1. Specificity Recovery of non-Lp(a)-associated cholesterol from the gel was determined by incubating serum specimens containing a very small amount of Lp(a) (1.4 mg/dl) with the gel. We found that 99.7% of the total cholesterol (mean of five replicate experiments) could be recovered after incubation with the gel, indicating the lack of crossreactivity with non-Lp(a)-associated cholesterol. After incubation of the serum specimens with the gel and centrifugation, the supernatant Lp(a)-C concentrations were below the detection sensitivity of 1 mg/ dl in all the samples.
40
Lp(a)- C concentration (mg/dl)
durations. We found that the serum Lp(a)-C adsorption was greater than 80% at 30 min and reached a plateau at approximately 2 h (Fig.1, A). Therefore, in subsequent experiments, incubation was carried out for 2 h. To determine the effect of serum volume on the adsorption of the serum Lp(a)-C, increasing volumes of serum were incubated with the immunoaffinity gel, and we found that the adsorption of Lp(a) increased in a linear serum-volume-dependent manner. The serum concentration of Lp(a)-C could be measured up to N50 mg/dl (Fig. 1, B).
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y = 0.273x + 0.666 r = 0.923 n = 55
35 30 25 20 15 10 5 0
0
20
40
60
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100
120
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Lp(a) concentration(mg/dl) Fig. 2. Relationship between serum Lp(a) and Lp(a)-C concentrations.
3.4. Serum Lp(a), Lp(a)-C, Lp(a)-C/Lp(a), and Lp(a)-C/TC ratio in the three subject groups 3.2.2. Reproducibility Within-assay reproducibility was estimated by using 2 serum specimens (each specimen, 5 replicate experiments), one with moderate elevation of the Lp(a) concentration (63.4 mg/dl) and one with marked elevation of the Lp(a) concentration (129.8 mg/dl). The serum Lp(a)-C concentrations of these specimens were 22.0 ± 1.4 and 36.6 ± 1.1 mg/dl (CV 6.4% and 3.1%), respectively. Inter-day assay error was evaluated by comparing the assay results of 2 serum samples (Lp(a) concentrations, 67.7 and 89.7 mg/dl) on 7 different days. The serum Lp(a)-C concentrations in these samples were 22.1 ± 1.0 and 28.6 ± 0.85 mg/dl (CV 5.0% and 3.0%), respectively.
The serum concentrations of lipids, Lp(a), Lp(a)-C, Lp(a)-C/ Lp(a), and Lp(a)-C/TC ratio are shown in Table 1. The serum Lp(a) and Lp(a)-C concentrations were similar in the three groups of subjects. In the present study, 20 of the 55 subjects (36%) were taking lipid-lowering agents, typically a statin. The Lp(a)-C/Lp(a) ratio in the subjects under treatment with statins (25.8 ± 8.4%, n = 16) was similar to that in the subjects not taking statins (31.3 ± 8.6%, n = 35; p = 0.086). The mean contributions of the serum Lp(a)-C to the serum TC and non-HDL-C were 6.3 and 8.6%, respectively, and, at serum Lp(a) concentrations of N50 mg/dl, these values increased to 10.2 ± 3.8 and 14.2 ± 5.9%, respectively.
3.3. Cholesterol content of Lp(a)
3.5. Contribution of Lp(a)-C to conventional “calculated LDL-C”
To determine the serum Lp(a) concentrations, we carried out immunoaffinity gel analysis by incubating 150 μl serum of each study subject for 2 h; the mean Lp(a) concentration in the 55 subjects was determined to be 45 ± 28 mg/dl (range, 9–129 mg/dl) (Table 1). As shown in Fig. 2, the serum Lp(a)-C concentration and serum Lp(a) mass were correlated (r = 0.923, p b 0.001). The mean cholesterol content of Lp(a), determined as Lp(a)-C/Lp(a), was 29.5 ± 10.4%. The cholesterol contents of Lp(a) according to the serum Lp(a) concentrations were 30.3 ± 14.1 (0–30 mg/dl Lp(a); n = 23), 29.8 ± 8.3 (3060 mg/dl Lp(a); n = 15), and 28.3 ± 5.6% (≥60 mg/dl Lp(a); n = 17). Thus, the Lp(a)-C/Lp(a) ratio was not affected by the serum Lp(a) concentration, remaining constant at least up to a serum Lp(a) concentration of 129 mg/dl.
The differences between the “calculated LDL-C” and “corrected LDLC” [after subtracting Lp(a)-C from the “calculated LDL”] increased as the serum Lp(a) concentrations increased, and reached the mean value of 21 mg/dl at serum Lp(a) concentrations of N50 mg/dl (Table 2). Further, when the value of the [(“calculated LDL-C”-“corrected LDL-C)/“corrected LDL-C”] × 100 was calculated, the “calculated LDL-C” was 20.4% higher than the “corrected LDL-C”, indicating that the “calculated LDL” overestimated the serum LDL-C by 20.4%. 4. Discussion We demonstrated that cholesterol constitutes approximately 30% of each Lp(a) molecule and that the serum Lp(a)-C concentrations
Table 1 Serum concentrations of lipids, Lp(a) and Lp(a)-cholesterol in the study subjects.
Total NL HL DM
n (M/F)
Age (yr)
TC (mg/dl)
TG (mg/dl)
HDL-C (mg/dl)
Lp(a) (mg/dl)
Lp(a)-C (mg/dl)
Lp(a)-C/Lp(a) (%)
Lp(a)-C/TC (%)
55(33/22) 17(12/5) 26(15/11) 12(6/6)
63 ± 13 58 ± 14 66 ± 13⁎ 64 ± 10
210 ± 39 189 ± 37 223 ± 35⁎⁎ 209 ± 39
121 ± 57 98 ± 32 128 ± 67 137 ± 58⁎
53 ± 15 51 ± 14 57 ± 18 48 ± 8
45 ± 28 49 ± 32 47 ± 29 33 ± 17
13 ± 8 13 ± 9 14 ± 9 10 ± 5
29.5 ± 10.4 27.4 ± 8.9 30.8 ± 7.5 30.0 ± 14.8
6.3 ± 4.1 6.9 ± 4.4 6.5 ± 4.4 4.7 ± 2.5
Data represent means ± SD. NL, normolipidemia; HL, Hyperlipidemia; DM, diabetes mellitus; TC, total cholesterol; TG, triglyceride; HDL-C, high density lipoprotein cholesterol; Lp(a), lipoprotein(a); and Lp(a)C, lipoprotein(a) cholesterol. ⁎ p b 0.05 (vs. NL). ⁎⁎ P b 0.01 (vs. NL).
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Table 2 Comparison of “conventional calculated LDL-C” and “corrected LDL-C” values. Lp(a) values mg/dl)
n
Calculated LDL-C (mg/dl)
Corrected LDL-C (mg/dl)
Difference (range) (mg/dl)
Difference§ (%)
Total 0–b30 30–b50 50≤
55 24 11 20
132 ± 32 128 ± 38 140 ± 36 134 ± 25
120 ± 33 122 ± 38 128 ± 35 112 ± 25
12.8 ± 2.7⁎ (3–36) 6.2 ± 2.7⁎ (3–11) 11.7 ± 2.7⁎ (7–16) 21.0 ± 6.9⁎ (11–36)
11.9 ± 8.9 5.8 ± 3.5 9.7 ± 2.6 20.4 ± 9.0
Results are expressed as mean ± SD. §[(“calculated LDL-C” -“corrected LDL-C”)/“corrected LDL-C”] × 100. Differences between “calculated LDL-C” and “corrected LDL-C” were evaluated with paired t test t test. ⁎ p b 0.001.
were significantly correlated with the serum Lp(a) mass over the range of 9 to 129 mg/dl. Our results indicate that the contribution of Lp(a)-C to the serum cholesterol should be taken into account in clinical subjects, especially those with markedly increased serum Lp(a) concentrations. Lp(a) is known to be a cholesterol-rich lipoprotein [9–12]; however, Lp(a)-C is not typically assessed in clinical studies. Previously, we studied subjects with markedly increased serum Lp(a) concentrations (i.e., N100 mg/dl) [8] and patients with type 2 diabetes mellitus [17]. During an analysis conducted to identify the independent risk factors for CHD, we considered that in subjects with markedly increased serum Lp(a) concentrations, the serum concentrations of LDL-C may be overestimated when calculated using the Friedewald formula [18], because Lp(a)-C is also included in the calculation of the serum LDL-C in this formula. Further, Lp(a) and LDL-C cannot be evaluated as independent variables. Therefore, we attempted to determine the actual cholesterol content of Lp(a). Earlier studies that analyzed the chemical composition of isolated Lp(a) reported that cholesterol constitutes 25% to 35% of each Lp(a) molecule [9–12]. However, no convenient method was available to evaluate the serum Lp(a)-C in patients, because preparative ultracentrifugation was necessary to isolate the lipoproteins. Thus, few studies have evaluated serum Lp(a)-C, and its clinical significance remains unclear. Li et al. measured the serum Lp(a)-C concentrations by precipitating Lp(a) with polyethylene glycol 6000, and reported that serum LDL-C was overestimated in subjects with serum Lp(a) concentrations N30 mg/dl [13]. Seman et al. measured the serum Lp(a)-C concentrations in the Framingham study population using lectin, a substance shown to non-specifically trap Lp(a), and reported that increased Lp(a)-C concentrations were an independent risk factor for CHD [19]. However, the clinical usefulness of polyethylene glycol 6000 and lectin was not assessed further. In the present study, we developed an immunoaffinity gel assay which separates Lp(a) from other cholesterol-containing lipoproteins. The assay was found to be satisfactory in terms of the specificity and reproducibility, and, therefore, to be suitable for clinical application. Our findings suggest that the cholesterol content of Lp(a) molecules does not vary depending on the underlying disease or condition. This result is consistent with earlier reports suggesting that most lipidlowering agents, including statins (HMG-CoA reductase inhibitors), exert no effect on the serum Lp(a) concentrations [1]. Apo(a) is heterogeneous in size, and the size of apo(a) determines the serum Lp(a) concentrations [1,20]. In the present study, we did not perform apo(a) phenotyping and therefore did not determine the effect of Lp(a) size heterogeneity on the serum Lp(a)- C concentration. However, no differences in the serum Lp(a)C/Lp(a) ratio were detected among subjects grouped according to the serum Lp(a) concentrations. Our study reveals that in patients with markedly increased serum Lp(a) concentrations (N50 mg/dl), serum low-density lipoprotein cholesterol (LDL-C) value may be overestimated when calculated
using the Friedewald formula, because the “calculated” LDL-C includes significant amounts of Lp(a)-C as well. Accurate measurement of the serum LDL-C is important in order to establish appropriate diagnostic and therapeutic targets for the serum LDL-C. Clinical studies typically do not evaluate serum Lp(a)-C and “calculated LDL-C” as independent risk factors, because Lp(a)-C is considered as a part of the calculated LDL-C. However, our results indicate that the contribution of Lp(a)-C to serum cholesterol should be taken into account, especially in cases with marked elevation of the serum Lp(a) concentrations. The distribution of the serum Lp(a) concentrations in the population is known to show skewing towards lower values, with 90% of the population having values less than 30 mg/dl [1,8], and subjects with marked elevation of serum Lp(a) concentrations being thought to be rather rare. Thus, the cholesterol contents of Lp(a) have not attracted any clinical attention so far. However, subjects whose serum Lp(a) concentrations N50 mg/dl seem to be not so rare, especially in some specific patient subgroups. In our experience, the percentage of subjects with serum Lp(a) concentrations of over 50 mg/dl was 8.8% in patients with type 2 diabetes mellitus (31/352 cases)(17) and 15.6% (20/128) in patients with familial hypercholesterolemia. This may strengthen the importance of this study. In summary, we determined the cholesterol content of Lp(a) using an immunoaffinity gel assay developed by us, and demonstrated that Lp(a) contains approximately 30% cholesterol in each molecule. The serum Lp(a)-C values were found to be highly correlated with the serum Lp(a) mass. Most previous studies have overlooked Lp(a)-C when analyzing serum cholesterol, however, our results indicate that the contribution of Lp(a)-C to the serum cholesterol should be taken into account in clinical subjects, especially in subjects with markedly increased serum Lp(a) concentrations. References [1] Utermann G. Lipoprotein(a). In: Scriber CR, Beaudet AL, Aly WS, Vallo O, editors. The metabolic and molecular bases of inherited diseases. 8nd edn. NewYork: McGraw-Hill; 2001. p. 2753–89. [2] Danesh J, Collins R, Peto R. Lipoprotein(a) and coronary heart disease: meta-analysis of prospective studies. Circulation 2000;102:1082–5. [3] Kamstrup PR, Tybjærg-Hansen, Steffensen R, Nordestgaard BG. Genetically elevated lipoprotein(a) and increased risk of myocardial infarction. JAMA 2009;301:2331–9. [4] Erqou S, Kaptoge, Perry PL, et al. Lipoprotein(a) concentration and the risk of coronary heart disease, stroke, and nonvascular mortality. JAMA 2009;302: 412–23. [5] Nordestgaard BG, Chapman MJ, Ray K, et al. Lipoprotein(a) as a cardiovascular risk factor: current status. Eur Heart J 2010;31:2844–53. [6] Berg K. A new serum type system in man: the Lp(a) system. Acta Pathol Microbiol Scand 1963;59:362–82. [7] Scanu AM, Fless GM. Lipoprotein(a). Heterogeneity and biological relevance. J Clin Invest 1990;85:1709–15. [8] Murase T, Okubo M, Amemiya-Kudo M, et al. Impact of markedly elevated serum lipoprotein(a) concentrations (≥ 100mg/dl) on the risk of coronary heart disease. Metabolism 2007;56:1187–91. [9] Gaubatz JW, Heideman C, Gotto Jr A, Morrisett JD, Dahlen GH. Human plasma lipoprotein[a]: structural properties. J Biol Chem 1983;256:4582–9. [10] Fless GM, Rolih CA, Scanu AM. Heterogeneity of human plasma lipoprotein (a): isolation and characterization of the lipoprotein subspecies and their apoproteins. J Biol Chem 1984;259:11470–6. [11] Kostner GM, Ibovnik A, Holzer H, Grillhofer H. Preparation of a stable fresh frozen primary lipoprotein[a] (Lp[a]) standard. J Lipid Res 1999;40:2255–63. [12] Marcovina SM, Albers JJ, Scanu AM, et al. Use of a reference material proposed by the International Federation of Clinical and Laboratory Medicine to evaluate analytical methods for the determination of plasma lipoprotein(a). Clin Chem 2000;46:1956–67. [13] Li KM, Wilcken DEL, Dudman NPB. Effect of serum lipoprotein(a) on estimation of low-density lipoprotein cholesterol by the Friedewald formula. Clin Chem 1994;40:571–3. [14] Seman LJ, Jenner JL, McNamara JR, Schaefer EJ. Quantification of lipoprotein(a) in plasma by assaying cholesterol in lecithin-bound plasma fraction. Clin Chem 1994;40:400–3. [15] World Health Organization. Report of a WHO consultation, Part 1: diagnosis and classification of diabetes mellitus and itsu complication. Geneva: World Health Organization; 1999. [16] Hiraga T, Shimada M, Okubo M, Nakanishi K, Kobayashi T, Murase T. Lipoprotein (a) is an independent risk factor for multiple cerebral infarction. Atherosclerosis 1996;122:29–32.
K. Kinpara et al. / Clinica Chimica Acta 412 (2011) 1783–1787 [17] Murase T, Okubo M, Amemiya-Kudo M, Ebara T, Mori Y. Impact of elevated serum lipoprotein (a) concentrations on the risk of coronary heart disease in patients with type 2 diabetes mellitus. Metabolism 2008;57:791–5. [18] Friedewald WT, Levy RI, Fredrickson DS. Estimation of the concentration of lowdensity lipoprotein cholesterol in plasma, without use of the preparative ultracentrifuge. ed.Clin Chem 1972;18:499–502.
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[19] Seman LJ, DeLuca C, Jenner JL, et al. Lipoprotein(a)-cholesterol and coronary heart disease in the Framingham Heart Study. Clin Chem 1999;45:1039–46. [20] Clarke R, Peden JF, Hopewell JC, et al. Genetic variants associated with Lp(a) lipoprotein concentration and coronary disease. N Engl J Med 2009;361: 2518–28.
Contents lists available at ScienceDirect
Clinica Chimica Acta j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / c l i n c h i m
Lipoprotein(a)-cholesterol: A significant component of serum cholesterol Kiyoko Kinpara a, 1, Hiroshi Okada b, 1, Akiko Yoneyama a, Minoru Okubo c, d, Toshio Murase c, d,⁎ a
Department of Clinical Laboratory, Toranomon Hospital, Japan Development Group, Diagnostic Business Department, Sanwa Kagaku Kenkyusho Co. Ltd, Japan c Department of Endocrinology and Metabolism, Toranomon Hospital, Japan d Okinaka Memorial Institute for Medical Research, Japan b
a r t i c l e
i n f o
Article history: Received 4 April 2011 Received in revised form 26 May 2011 Accepted 31 May 2011 Available online 13 June 2011 Keywords: Lipoprotein(a) Lipoprotein(a)-cholesterol The Friedewald formula
a b s t r a c t Background: Lipoprotein(a) [Lp(a)] is known to be a cholesterol-rich lipoprotein, however, the contribution of Lp(a)-cholesterol [Lp(a)-C] to the serum cholesterol and LDL-C levels has not yet been fully evaluated. Methods: We determined the serum Lp(a)-C in 55 subjects with serum Lp(a) concentrations ranging from 9 to 129 mg/dl. To measure the serum Lp(a)-C concentrations, we developed an immunoaffinity gel assay; serum was incubated with Sepharose 4B gel coupled with immunoglobulin G (IgG) prepared from a polyclonal antiLp(a) goat antiserum. After separating Lp(a) from other lipoproteins, we determined the serum Lp(a)-C concentrations. Validation of the assay showed satisfactory results in terms of the specificity and reproducibility. Results: The mean cholesterol content of Lp(a), determined as Lp(a)-C/Lp(a), was 29.5± 10.4%. The serum Lp(a)C values were found to be highly correlated with the serum Lp(a) mass (r= 0.923, p b 0.001). At serum Lp(a) levels of over 50 mg/dl, the contribution of Lp(a)-C to the serum total cholesterol was 10.2%. Further, the Friedewald formula overestimated the serum LDL-C by 20.4%. Conclusions: Lp(a) contains approximately 30% cholesterol in each molecule. In subjects with markedly elevated serum Lp(a) concentrations, the Lp(a)-C values should be taken into account when evaluating the serum LDL-C. © 2011 Elsevier B.V. All rights reserved.
1. Introduction Lipoprotein(a) [Lp(a)] is an emerging risk factor for atherosclerotic cardiovascular disease [1–5]. Lp(a) is composed of one copy of LDL linked by a disulfide bond to the glycoprotein apolipoprotein(a) [1,6,7]; thus, each molecule of Lp(a) contains some cholesterol. However, the contribution of Lp(a)-cholesterol [Lp(a)-C] to the total serum cholesterol concentrations is not yet well understood. We recently demonstrated that markedly increased serum Lp(a) (≥100 mg/dl) concentrations are associated with a 5-fold or greater increase in the risk of coronary heart disease (CHD) [8]. During an analysis to identify the independent risk factors for CHD, we considered that the serum Lp(a)-C values would be considerably increased in association with increased serum Lp(a) concentrations, and that, therefore, cholesterol content of Lp(a) should be taken into account when evaluating the serum LDL-C and total cholesterol. This prompted us to examine the cholesterol content of Lp(a). A few previous studies have shown that the Lp(a) molecule contains appreciable amounts of cholesterol. In previous studies, Abbreviations: Lp(a), lipoprotein(a); Lp(a)-C, lipoprotein(a)-cholesterol; CHD, coronary heart disease. ⁎ Corresponding author at: Okinaka Memorial Institute for Medical Research, Toranomon 2-2-2, Minato-ku, Tokyo 105-8470, Japan. Tel.: +81 3 3588 1111; fax: +81 3 3588 0356. E-mail address: [email protected] (T. Murase). 1 These two authors contributed equally to this work. 0009-8981/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.cca.2011.05.036
serum Lp(a)-C concentrations were determined by analyzing the chemical composition of isolated Lp(a) [9–12] or using chemical reagents that precipitate [13] or absorb [14] serum Lp(a). In the present study, we developed an immunoaffinity gel assay that separates Lp(a) from other cholesterol-containing lipoproteins. Using this assay, we determined the serum Lp(a)-C concentrations and characterized the relationship between serum Lp(a)-C concentrations and the Lp(a) mass.
2. Subjects and methods 2.1. Subjects We enrolled outpatients (men, n = 33; women, n = 22) visiting the Toranomon Hospital (Tokyo, Japan) who agreed to our evaluation of their serum lipid profiles (mean age, 63 ± 13 y; age range, 23–84 y). In our earlier experiences [8] the distribution of the serum Lp(a) concentrations showed skewing toward lower values in the Japanese population, with 90% of the population having values b30 mg/dl and this distribution was consistent with the serum concentration profile reported for whites [1]. However, since the aim of the current study was to determine the cholesterol content of Lp(a), we intentionally selected subjects with relatively high serum Lp(a) concentrations. The study was conducted with the approval of the institutional review board of Toranomon Hospital.
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The 55 participants were classified into 3 groups according to their lipid profiles and underlying conditions: group 1: normal serum lipid profile, defined as serum total cholesterol b240 mg/dl and serum triglyceride b150 mg/dl (n= 17); group 2: hyperlipidemia, defined as serum cholesterol N240 mg/dl, serum triglyceride N150 mg/dl, or treatment with lipid-lowering agents (n = 26); group 3: type 2 diabetes mellitus, diagnosed based on the criteria established by the World Health Organization [15] (n= 12). In group 2, hyperlipidemia could be further divided into hypercholesterolemia (n = 16), including familial hypercholesterolemia (n= 6), diagnosed based on the characteristic features (i.e., LDL cholesterol N200 mg/dl, thickening of the Achilles tendon [N9 mm], and history of marked hypercholesterolemia or CHD in first-degree relatives), mixed hyperlipidemia (n= 6), and hypertriglyceridemia (n= 4). In group 3, 7 participants with diabetes mellitus also had hyperlipidemia.
LDL-C” (after subtracting Lp(a)-C) were evaluated by the paired t-test. Values of p b 0.05 were considered significant. 3. Results 3.1. Assay conditions To determine the effect of the incubation time on the adsorption of Lp(a), we incubated each serum specimen (150 μl) with the immunoaffinity gel (715 μl aliquots containing 0.5 mg IgG protein) for various
A
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Adsorption rate (%)
2.2. Determination of the serum Lp(a) and lipid concentrations Serum Lp(a) concentrations were determined by a latex immunosorbent assay using polyclonal anti-Lp(a) goat serum [Inotec Lp(a), Sanwa, Tokyo] [16]. Serum concentrations of total cholesterol, triglyceride and high-density lipoprotein cholesterol were determined using routine laboratory methods.
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2.3. Determination of the serum concentration of Lp(a)-C by immunoaffinity gel
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2.3.1. Preparation of the immunoaffinity gel IgG (10 mg) prepared from a polyclonal anti-Lp(a) goat antiserum (SHIMA Laboratories Co. LTD) was coupled to cyanogen bromideactivated Sepharose 4B (1.67 g; Pharmacia Fine Chemicals, Uppsala, Sweden), according to the manufacturer's instructions. The gel was suspended in 0.01 mol/l Tris–HCl buffer (final volume, 10 ml) containing 0.15 mol/l NaCl (pH 8.0), and then 715-μl aliquots (0.5 mg protein) were divided into tubes and stored in a refrigerator until the Lp(a)-C measurement. It was confirmed in a preliminary study that the gel can be stored at 4 °C for at least 2 months.
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2.4. Statistical analysis Results were expressed as mean ± standard deviation (SD) or range. Linear regression analysis was performed to evaluate the association between the serum concentrations of Lp(a)-C and Lp(a). Comparisons between groups were performed by two-tailed Student's t-test. The differences between the serum LDL-C as calculated by the Friedewald formula (“calculated LDL-C”) and the “corrected
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Incubation time (min)
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Lp(a)-C concentrations (mg/dl)
2.3.2. Measurement of serum Lp(a)-C To determine the optimal conditions for measurement of the serum Lp(a)-C, each serum specimen (0–250 μl) was mixed with immunoaffinity gel (715 μl) and incubated for 0–180 min under gentle shaking at room temperature. The mixture was then centrifuged at 3000 rpm for 15 min, and the Lp(a) concentration in the supernatant was determined. The supernatant Lp(a) concentration was corrected for serum dilution during incubation with the gel by simultaneous measurement of the serum protein as the internal reference. Serum Lp(a)-C adsorption was calculated as ([Serum Lp(a)supernatant Lp(a)] × 100)/Serum Lp(a). To determine the serum Lp(a)-C concentrations in the study participants, each serum specimen (150 μl) was mixed with immunoaffinity gel (715 μl) and incubated under gentle shaking for 120 min at room temperature. The mixture was then centrifuged at 3000 rpm for 15 min, and the cholesterol concentration in the supernatant was determined after correction for the serum dilution. Serum Lp(a)-C concentration was determined by subtracting the supernatant cholesterol concentration from the serum total cholesterol.
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Sample volume ( µ I) Fig. 1. (A) Relationship between serum Lp(a) adsorption by the gel and the incubation period. Each serum specimen (150 μl) was mixed with immunoaffinity gel (715 μl) and incubated under gentle shaking for 0–180 min at room temperature. The mixture was then centrifuged at 3000 rpm for 15 min, and the Lp(a) concentration in the supernatant was determined; the supernatant Lp(a) concentration was corrected for serum dilution during incubation with the gel by simultaneous measurement of serum protein as the internal reference. Serum Lp(a) adsorption was calculated as ([Serum Lp(a)-supernatant Lp(a)] × 100)/Serum Lp(a). Results are expressed as mean ± SD of 3 samples: Sample 1, 63.4 mg/dl Lp(a); Sample 2, 86.6 mg/dl Lp(a); Sample 3, 90.3 mg/dl Lp(a). (B) Relationship between serum volume and serum Lp(a)-C concentration. This was evaluated using 2 serum samples [○, Lp(a) 63.4 mg/dl; ●, Lp(a) 86.6 mg/dl] run in parallel. Increasing volumes of serum were mixed with immunoaffinity gel (715 μl) and incubated under gentle shaking for 120 min at room temperature. The mixtures were then centrifuged at 3000 rpm for 15 min, and the cholesterol concentrations in the supernatant were determined; the supernatant cholesterol concentration was corrected for serum dilution during incubation with the gel by simultaneous measurement of serum protein as the internal reference. Serum Lp(a)-C concentration was calculated as (Serum cholesterol–Supernatant cholesterol).
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3.2. Validation of the Lp(a)-C assay 3.2.1. Specificity Recovery of non-Lp(a)-associated cholesterol from the gel was determined by incubating serum specimens containing a very small amount of Lp(a) (1.4 mg/dl) with the gel. We found that 99.7% of the total cholesterol (mean of five replicate experiments) could be recovered after incubation with the gel, indicating the lack of crossreactivity with non-Lp(a)-associated cholesterol. After incubation of the serum specimens with the gel and centrifugation, the supernatant Lp(a)-C concentrations were below the detection sensitivity of 1 mg/ dl in all the samples.
40
Lp(a)- C concentration (mg/dl)
durations. We found that the serum Lp(a)-C adsorption was greater than 80% at 30 min and reached a plateau at approximately 2 h (Fig.1, A). Therefore, in subsequent experiments, incubation was carried out for 2 h. To determine the effect of serum volume on the adsorption of the serum Lp(a)-C, increasing volumes of serum were incubated with the immunoaffinity gel, and we found that the adsorption of Lp(a) increased in a linear serum-volume-dependent manner. The serum concentration of Lp(a)-C could be measured up to N50 mg/dl (Fig. 1, B).
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y = 0.273x + 0.666 r = 0.923 n = 55
35 30 25 20 15 10 5 0
0
20
40
60
80
100
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Lp(a) concentration(mg/dl) Fig. 2. Relationship between serum Lp(a) and Lp(a)-C concentrations.
3.4. Serum Lp(a), Lp(a)-C, Lp(a)-C/Lp(a), and Lp(a)-C/TC ratio in the three subject groups 3.2.2. Reproducibility Within-assay reproducibility was estimated by using 2 serum specimens (each specimen, 5 replicate experiments), one with moderate elevation of the Lp(a) concentration (63.4 mg/dl) and one with marked elevation of the Lp(a) concentration (129.8 mg/dl). The serum Lp(a)-C concentrations of these specimens were 22.0 ± 1.4 and 36.6 ± 1.1 mg/dl (CV 6.4% and 3.1%), respectively. Inter-day assay error was evaluated by comparing the assay results of 2 serum samples (Lp(a) concentrations, 67.7 and 89.7 mg/dl) on 7 different days. The serum Lp(a)-C concentrations in these samples were 22.1 ± 1.0 and 28.6 ± 0.85 mg/dl (CV 5.0% and 3.0%), respectively.
The serum concentrations of lipids, Lp(a), Lp(a)-C, Lp(a)-C/ Lp(a), and Lp(a)-C/TC ratio are shown in Table 1. The serum Lp(a) and Lp(a)-C concentrations were similar in the three groups of subjects. In the present study, 20 of the 55 subjects (36%) were taking lipid-lowering agents, typically a statin. The Lp(a)-C/Lp(a) ratio in the subjects under treatment with statins (25.8 ± 8.4%, n = 16) was similar to that in the subjects not taking statins (31.3 ± 8.6%, n = 35; p = 0.086). The mean contributions of the serum Lp(a)-C to the serum TC and non-HDL-C were 6.3 and 8.6%, respectively, and, at serum Lp(a) concentrations of N50 mg/dl, these values increased to 10.2 ± 3.8 and 14.2 ± 5.9%, respectively.
3.3. Cholesterol content of Lp(a)
3.5. Contribution of Lp(a)-C to conventional “calculated LDL-C”
To determine the serum Lp(a) concentrations, we carried out immunoaffinity gel analysis by incubating 150 μl serum of each study subject for 2 h; the mean Lp(a) concentration in the 55 subjects was determined to be 45 ± 28 mg/dl (range, 9–129 mg/dl) (Table 1). As shown in Fig. 2, the serum Lp(a)-C concentration and serum Lp(a) mass were correlated (r = 0.923, p b 0.001). The mean cholesterol content of Lp(a), determined as Lp(a)-C/Lp(a), was 29.5 ± 10.4%. The cholesterol contents of Lp(a) according to the serum Lp(a) concentrations were 30.3 ± 14.1 (0–30 mg/dl Lp(a); n = 23), 29.8 ± 8.3 (3060 mg/dl Lp(a); n = 15), and 28.3 ± 5.6% (≥60 mg/dl Lp(a); n = 17). Thus, the Lp(a)-C/Lp(a) ratio was not affected by the serum Lp(a) concentration, remaining constant at least up to a serum Lp(a) concentration of 129 mg/dl.
The differences between the “calculated LDL-C” and “corrected LDLC” [after subtracting Lp(a)-C from the “calculated LDL”] increased as the serum Lp(a) concentrations increased, and reached the mean value of 21 mg/dl at serum Lp(a) concentrations of N50 mg/dl (Table 2). Further, when the value of the [(“calculated LDL-C”-“corrected LDL-C)/“corrected LDL-C”] × 100 was calculated, the “calculated LDL-C” was 20.4% higher than the “corrected LDL-C”, indicating that the “calculated LDL” overestimated the serum LDL-C by 20.4%. 4. Discussion We demonstrated that cholesterol constitutes approximately 30% of each Lp(a) molecule and that the serum Lp(a)-C concentrations
Table 1 Serum concentrations of lipids, Lp(a) and Lp(a)-cholesterol in the study subjects.
Total NL HL DM
n (M/F)
Age (yr)
TC (mg/dl)
TG (mg/dl)
HDL-C (mg/dl)
Lp(a) (mg/dl)
Lp(a)-C (mg/dl)
Lp(a)-C/Lp(a) (%)
Lp(a)-C/TC (%)
55(33/22) 17(12/5) 26(15/11) 12(6/6)
63 ± 13 58 ± 14 66 ± 13⁎ 64 ± 10
210 ± 39 189 ± 37 223 ± 35⁎⁎ 209 ± 39
121 ± 57 98 ± 32 128 ± 67 137 ± 58⁎
53 ± 15 51 ± 14 57 ± 18 48 ± 8
45 ± 28 49 ± 32 47 ± 29 33 ± 17
13 ± 8 13 ± 9 14 ± 9 10 ± 5
29.5 ± 10.4 27.4 ± 8.9 30.8 ± 7.5 30.0 ± 14.8
6.3 ± 4.1 6.9 ± 4.4 6.5 ± 4.4 4.7 ± 2.5
Data represent means ± SD. NL, normolipidemia; HL, Hyperlipidemia; DM, diabetes mellitus; TC, total cholesterol; TG, triglyceride; HDL-C, high density lipoprotein cholesterol; Lp(a), lipoprotein(a); and Lp(a)C, lipoprotein(a) cholesterol. ⁎ p b 0.05 (vs. NL). ⁎⁎ P b 0.01 (vs. NL).
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Table 2 Comparison of “conventional calculated LDL-C” and “corrected LDL-C” values. Lp(a) values mg/dl)
n
Calculated LDL-C (mg/dl)
Corrected LDL-C (mg/dl)
Difference (range) (mg/dl)
Difference§ (%)
Total 0–b30 30–b50 50≤
55 24 11 20
132 ± 32 128 ± 38 140 ± 36 134 ± 25
120 ± 33 122 ± 38 128 ± 35 112 ± 25
12.8 ± 2.7⁎ (3–36) 6.2 ± 2.7⁎ (3–11) 11.7 ± 2.7⁎ (7–16) 21.0 ± 6.9⁎ (11–36)
11.9 ± 8.9 5.8 ± 3.5 9.7 ± 2.6 20.4 ± 9.0
Results are expressed as mean ± SD. §[(“calculated LDL-C” -“corrected LDL-C”)/“corrected LDL-C”] × 100. Differences between “calculated LDL-C” and “corrected LDL-C” were evaluated with paired t test t test. ⁎ p b 0.001.
were significantly correlated with the serum Lp(a) mass over the range of 9 to 129 mg/dl. Our results indicate that the contribution of Lp(a)-C to the serum cholesterol should be taken into account in clinical subjects, especially those with markedly increased serum Lp(a) concentrations. Lp(a) is known to be a cholesterol-rich lipoprotein [9–12]; however, Lp(a)-C is not typically assessed in clinical studies. Previously, we studied subjects with markedly increased serum Lp(a) concentrations (i.e., N100 mg/dl) [8] and patients with type 2 diabetes mellitus [17]. During an analysis conducted to identify the independent risk factors for CHD, we considered that in subjects with markedly increased serum Lp(a) concentrations, the serum concentrations of LDL-C may be overestimated when calculated using the Friedewald formula [18], because Lp(a)-C is also included in the calculation of the serum LDL-C in this formula. Further, Lp(a) and LDL-C cannot be evaluated as independent variables. Therefore, we attempted to determine the actual cholesterol content of Lp(a). Earlier studies that analyzed the chemical composition of isolated Lp(a) reported that cholesterol constitutes 25% to 35% of each Lp(a) molecule [9–12]. However, no convenient method was available to evaluate the serum Lp(a)-C in patients, because preparative ultracentrifugation was necessary to isolate the lipoproteins. Thus, few studies have evaluated serum Lp(a)-C, and its clinical significance remains unclear. Li et al. measured the serum Lp(a)-C concentrations by precipitating Lp(a) with polyethylene glycol 6000, and reported that serum LDL-C was overestimated in subjects with serum Lp(a) concentrations N30 mg/dl [13]. Seman et al. measured the serum Lp(a)-C concentrations in the Framingham study population using lectin, a substance shown to non-specifically trap Lp(a), and reported that increased Lp(a)-C concentrations were an independent risk factor for CHD [19]. However, the clinical usefulness of polyethylene glycol 6000 and lectin was not assessed further. In the present study, we developed an immunoaffinity gel assay which separates Lp(a) from other cholesterol-containing lipoproteins. The assay was found to be satisfactory in terms of the specificity and reproducibility, and, therefore, to be suitable for clinical application. Our findings suggest that the cholesterol content of Lp(a) molecules does not vary depending on the underlying disease or condition. This result is consistent with earlier reports suggesting that most lipidlowering agents, including statins (HMG-CoA reductase inhibitors), exert no effect on the serum Lp(a) concentrations [1]. Apo(a) is heterogeneous in size, and the size of apo(a) determines the serum Lp(a) concentrations [1,20]. In the present study, we did not perform apo(a) phenotyping and therefore did not determine the effect of Lp(a) size heterogeneity on the serum Lp(a)- C concentration. However, no differences in the serum Lp(a)C/Lp(a) ratio were detected among subjects grouped according to the serum Lp(a) concentrations. Our study reveals that in patients with markedly increased serum Lp(a) concentrations (N50 mg/dl), serum low-density lipoprotein cholesterol (LDL-C) value may be overestimated when calculated
using the Friedewald formula, because the “calculated” LDL-C includes significant amounts of Lp(a)-C as well. Accurate measurement of the serum LDL-C is important in order to establish appropriate diagnostic and therapeutic targets for the serum LDL-C. Clinical studies typically do not evaluate serum Lp(a)-C and “calculated LDL-C” as independent risk factors, because Lp(a)-C is considered as a part of the calculated LDL-C. However, our results indicate that the contribution of Lp(a)-C to serum cholesterol should be taken into account, especially in cases with marked elevation of the serum Lp(a) concentrations. The distribution of the serum Lp(a) concentrations in the population is known to show skewing towards lower values, with 90% of the population having values less than 30 mg/dl [1,8], and subjects with marked elevation of serum Lp(a) concentrations being thought to be rather rare. Thus, the cholesterol contents of Lp(a) have not attracted any clinical attention so far. However, subjects whose serum Lp(a) concentrations N50 mg/dl seem to be not so rare, especially in some specific patient subgroups. In our experience, the percentage of subjects with serum Lp(a) concentrations of over 50 mg/dl was 8.8% in patients with type 2 diabetes mellitus (31/352 cases)(17) and 15.6% (20/128) in patients with familial hypercholesterolemia. This may strengthen the importance of this study. In summary, we determined the cholesterol content of Lp(a) using an immunoaffinity gel assay developed by us, and demonstrated that Lp(a) contains approximately 30% cholesterol in each molecule. The serum Lp(a)-C values were found to be highly correlated with the serum Lp(a) mass. Most previous studies have overlooked Lp(a)-C when analyzing serum cholesterol, however, our results indicate that the contribution of Lp(a)-C to the serum cholesterol should be taken into account in clinical subjects, especially in subjects with markedly increased serum Lp(a) concentrations. References [1] Utermann G. Lipoprotein(a). In: Scriber CR, Beaudet AL, Aly WS, Vallo O, editors. The metabolic and molecular bases of inherited diseases. 8nd edn. NewYork: McGraw-Hill; 2001. p. 2753–89. [2] Danesh J, Collins R, Peto R. Lipoprotein(a) and coronary heart disease: meta-analysis of prospective studies. Circulation 2000;102:1082–5. [3] Kamstrup PR, Tybjærg-Hansen, Steffensen R, Nordestgaard BG. Genetically elevated lipoprotein(a) and increased risk of myocardial infarction. JAMA 2009;301:2331–9. [4] Erqou S, Kaptoge, Perry PL, et al. Lipoprotein(a) concentration and the risk of coronary heart disease, stroke, and nonvascular mortality. JAMA 2009;302: 412–23. [5] Nordestgaard BG, Chapman MJ, Ray K, et al. Lipoprotein(a) as a cardiovascular risk factor: current status. Eur Heart J 2010;31:2844–53. [6] Berg K. A new serum type system in man: the Lp(a) system. Acta Pathol Microbiol Scand 1963;59:362–82. [7] Scanu AM, Fless GM. Lipoprotein(a). Heterogeneity and biological relevance. J Clin Invest 1990;85:1709–15. [8] Murase T, Okubo M, Amemiya-Kudo M, et al. Impact of markedly elevated serum lipoprotein(a) concentrations (≥ 100mg/dl) on the risk of coronary heart disease. Metabolism 2007;56:1187–91. [9] Gaubatz JW, Heideman C, Gotto Jr A, Morrisett JD, Dahlen GH. Human plasma lipoprotein[a]: structural properties. J Biol Chem 1983;256:4582–9. [10] Fless GM, Rolih CA, Scanu AM. Heterogeneity of human plasma lipoprotein (a): isolation and characterization of the lipoprotein subspecies and their apoproteins. J Biol Chem 1984;259:11470–6. [11] Kostner GM, Ibovnik A, Holzer H, Grillhofer H. Preparation of a stable fresh frozen primary lipoprotein[a] (Lp[a]) standard. J Lipid Res 1999;40:2255–63. [12] Marcovina SM, Albers JJ, Scanu AM, et al. Use of a reference material proposed by the International Federation of Clinical and Laboratory Medicine to evaluate analytical methods for the determination of plasma lipoprotein(a). Clin Chem 2000;46:1956–67. [13] Li KM, Wilcken DEL, Dudman NPB. Effect of serum lipoprotein(a) on estimation of low-density lipoprotein cholesterol by the Friedewald formula. Clin Chem 1994;40:571–3. [14] Seman LJ, Jenner JL, McNamara JR, Schaefer EJ. Quantification of lipoprotein(a) in plasma by assaying cholesterol in lecithin-bound plasma fraction. Clin Chem 1994;40:400–3. [15] World Health Organization. Report of a WHO consultation, Part 1: diagnosis and classification of diabetes mellitus and itsu complication. Geneva: World Health Organization; 1999. [16] Hiraga T, Shimada M, Okubo M, Nakanishi K, Kobayashi T, Murase T. Lipoprotein (a) is an independent risk factor for multiple cerebral infarction. Atherosclerosis 1996;122:29–32.
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