201703040 Final edited MS [4 December, 2017]

Usefulness of Martin’s Method for Estimation of Low-Density Lipoprotein

Cholesterol in Coronary Atherosclerosis

Soie Chung

Department of Laboratory Medicine, Seoul National University College of Medicine and Seoul
National University Hospital Healthcare System Gangnam Center, Seoul, Korea

Address all correspondence to:

Soie Chung
Department of Laboratory Medicine
Seoul National University Hospital Healthcare System Gangnam Center
39 th FL. GFC, Teheran-ro 152, Gangnam-gu,
Seoul, 06236,
Korea.

Phone: +82-2-2112-5686;
Fax: +82-2-2112-5794
E-mail: soiec@naver.com

Short title: Estimation of Low-Density Lipoprotein Cholesterol in Coronary Atherosclerosis

Keywords: low-density lipoprotein cholesterol, coronary atherosclerosis, Friedewald equation

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Significance of the study

This study used a homogeneous assay to demonstrate that low-density lipoprotein cholesterol

estimated by Martin’s method showed better concordance compared to Friedewald’s equation in

coronary atherosclerosis. Hence, this Martin’s method could be used instead of Friedewald’s equation

in medical practice, because it is influenced less by triglyceride levels than Friedewald’s equation.

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Abstract

Objective: This study was conducted to validate Martin’s method in coronary atherosclerosis in

comparison with Friedewald’s equation. Subjects and Methods: A total of 299 participants with a

coronary artery calcium score (CACS) ≥ 300 and a serum triglyceride (TG) level < 400 mg/dL at

Seoul National University Hospital Healthcare System Gangnam Center, Seoul, Korea were enrolled

in this study. The Low-density lipoprotein cholesterol (LDL-C) was directly measured with a

homogeneous assay (DLDL) and estimated by both Friedewald’s equation (FLDL) and Martin’s

method (MLDL). Overall concordances between DLDL and LDL-C estimates were calculated as the

percent agreement. The McNemar test was used to compare the rate of reclassification of participants

with FLDL and MLDL and determine which differed significantly from each other. Results: Overall

concordance between DLDL and MLDL was slightly higher than that between DLDL and FLDL

(73.2 vs. 70.9%, p <0.001). The FLDL showed poor performance when the TG level was ≥ 200

mg/dL, mostly by underestimation, which was 64.7% discordance with DLDL. The reclassification

rate by MLDL, however, did not exceed 35.3% in all of the TG groups. Conclusions: Martin’s

method to estimate LDL-C using the strata-specific TG: VLDL ratio showed 2-fold better

concordance with LDL-C measured with a direct homogeneous assay in coronary atherosclerosis

compared to Friedewald’s equation, when the TG level was ≥ 200 mg/dL. This finding suggests

that MLDL could be a better alternative for estimating LDL-C compared to FLDL when the TG level

is ≥ 200 mg/dL in coronary atherosclerosis.

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Introduction

Cardiovascular diseases (CVDs) are the number one cause of death worldwide [1]. An

estimated 17.5 million people died from CVDs in 2012, which was 31% of all global deaths [1]. Of

these deaths, an estimated 7.4 million were due to coronary heart disease (CHD), and 6.7 million

were due to stroke [1].

Radiographically detectable coronary-artery calcium is a marker of subclinical CHD and

predicts coronary events [2]. In a previous study, coronary artery calcium scores (CACSs)

demonstrated incremental values for the prediction of coronary heart disease over that of the standard

coronary risk factors, and the prediction value was greatest when the CACS was over 300 [3].

Prevention of CVD is important, since it may lead to serious clinical sequelae [4]. Changing

lifestyle behaviors and reducing cardiovascular risk factors are well known approach for prevention

of CVD [4]. Low-density lipoprotein cholesterol (LDL-C) is a major modifiable cardiovascular risk

factor [5, 6]. Therefore, accurate assessment of LDL-C level is important for making decisions about

therapeutic plans. In clinical practice, LDL-C estimated by Friedewald’s equation is generally

utilized when direct measurement is unavailable or costly [7].

However, there are several limitations in Friedewald’s equation. First, a fixed ratio of

triglyceride (TG) levels to very low-density lipoprotein cholesterol (TG/VLDL-C) of 5:1, as used in

Friedewald’s equation, does not provide an accurate estimate of VLDL-C. Indeed, the Lipid Research

Clinics Prevalence Study showed that the mean TG: VLDL-C ratio ranged from 5.2 to 8.9 [8].

Furthermore, there are several clinical conditions that may alter TG/VLDL-C ratio and lead to

inaccurate assessment of LDL-C when using Friedewald’s equation. In patients with Type III

hyperlipoproteinemia, VLDL is abnormally high with cholesterol relative to TG that results in

underestimation of VLDL-C and an overestimation of LDL-C [9]. In diabetic patients, Friedewald

equation underestimated, on average 8% serum LDL-C and even more than 10% in patients with TG

levels between 200 and 400mg/dL [10]. In patients with alcoholic liver cirrhosis, Friedewald equation

significantly underestimated, on average 15%, LDL-C compared to direct measurement using

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ultracentrifugation method [11].

Second, Friedewald’s equation requires fasting serum to estimate LDL-C, since

chylomicronemia in non-fasting status cause overestimation of VLDL-C and underestimation of

LDL-C [9, 12]. Similarly, the Friedewald equation tends to become less reliable for estimating LDL-

C when plasma triglyceride concentrations increase, because the ratio of VLDL-C to serum

triglyceride gradually changes as serum triglyceride concentrations increase [9, 13]. Thus the error

in estimated LDL-C gradually increases as fasting serum triglyceride concentrations increase [9, 13].

Although the formula’s inaccuracies at TG levels ≥ 400mg/dL were well recognized by Friedewald

et.al [14], but when TG levels are under 400mg/dL, the LDL-C estimated by Friedewald equation

underestimate LDL-C and thus misclassify CVD risk [7, 15, 16]. Especially if TG levels are ≥ 150

mg/dl, Friedewald’s estimation commonly classifies LDL-C as < 70 mg/dl, despite directly measured

levels that were ≥ 70 mg/dl [15].

Third, in the era when the Friedewald equation was proposed, an LDL-C < 70 mg/dL was not

yet established as an ideal secondary prevention target for treatment of high-risk patients [17,18]. In

fact, an LDL-C level in this range was not included in original training data set used in deriving the

Friedewald equation [14]. Therefore, Martin et al. [18] proposed a novel method of applying an

adjustable factor for the TG/VLDL-C ratio based on triglycerides and non-high density lipoprotein

cholesterol (HDL-C) concentrations.

Several studies applying this novel method had been reported [19, 20]. However, Martin’s

equation has not been validated in coronary atherosclerosis. Hence, in this study, Martin’s method

was compared to the traditional Friedewald equation with a direct homogeneous assay for estimating

LDL-C in coronary atherosclerosis predicted using coronary artery calcium scores.

Subjects and Methods

Study population

The medical records of 2773 persons who underwent a general health check-up and

completed an examination of total cholesterol, HDL-C, TG, DLDL, and CTCA at the Seoul National

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University Hospital Healthcare System Gangnam Center from September 2012 to March 2014 were

reviewed. The study protocol was reviewed and approved by the institutional review board of Seoul

National University Hospital (IRB No. H-1612-001-809). Since the current study was performed as

a retrospective study using the database and medical records, informed consent was waived by the

board. Exclusion criteria were TG levels ≥ 400 mg/dL and CACS < 300. Based on exclusion

criteria, of the 2773 patients, 299 participants were enrolled in this study.

Demographic characteristics, anthropometric data, and laboratory findings

Demographic characteristics and anthropometric data were acquired using medical

questionnaires, nurse interviews, and health examinations. Data of total cholesterol, triglyceride (TG),

low-density lipoprotein (LDL) cholesterol (DLDL), and high-density lipoprotein (HDL) cholesterol

were obtained from the medical record.

The LDL-C estimation by Friedewald equation (FLDL) and Martin’s method (MLDL) were

calculated as previously described [14, 20]. The FLDL was calculated using fixed ratio of 5 for

VLDL-C: TG. Thus, FLDL was expressed as total cholesterol minus HDL-C) minus TG/5 in

milligrams per deciliter. The MLDL was calculated using median of the ratio of TG to VLDL-C by

non-HDL-C and TG strata acquired from 900,605 people (table.1) [18]. Thus, MLDL was expressed

as total cholesterol minus HDL-C minus TG/ (Strata specific median VLDL-C: TG ratio).

Coronary computed tomography (CT) angiography (CTCA) was performed using a 256-slice

multidetector CT scanner (Brilliance iCT 256; Philips Medical Systems, Cleveland, OH, USA). The

CACS was calculated onsite on a dedicated workstation with the analysis software Wizard VB10B

(Somaris/5 VB10B-W, Syngo; Siemens, Erlangen, Germany), and quantitative CACS was calculated

according to the method described by Agatston et al. [21].

Statistical analysis

All statistical analyses were performed using Statistical Package for the Social Sciences

(SPSS) 22.0 for Windows (SPSS, Chicago, IL, USA) and MedCalc for Windows version 16.8.4.0

(MedCalc Software, Mariakerke, Belgium). All statistical outcomes were based on two-sided tests,

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and p- values < 0.05 were considered to be significant.

For continuous variables, data are expressed as median and interquartile ranges, since their

distribution was not normal by the Kolmogorov-Smirnov test (p <0.05). For categorical variables,

data are expressed as percentages. Bland-Altman plots were created to compare DLDL and FLDL or

DLDL and MLDL. A paired t-test was performed to compare the mean difference between (DLDL-

FLDL) and (DLDL- MLDL). Linear regression was performed between DLDL and FLDL or DLDL

and MLDL. Concordance in classification between FLDL and DLDL was examined through cross-

tabulations by DLDL categories according to the National Cholesterol Education Program Adult

Treatment Panel III (NCEP-ATP III) guideline [5]. The McNemar test was used to compare overall

concordance between LDL-cholesterol estimates and whether the rate of reclassification of

participants with FLDL or MLDL significantly differed from each other by DLDL categories or TG

groups. Pearson’s Chi-square test was used to compare the reclassification rate of FLDL or MLDL

among the four TG groups (TG <100 mg/dL; TG 100-149 mg/dL; TG 150-199 mg/dL; and TG 200-

399 mg/dL). Comparisons of (DLDL-FLDL) or (DLDL-MLDL) values according to TG groups were

analyzed using Kruskal-Wallis test.

Results

The demographic characteristics and lipid profile or overall concordance of FLDL and MLDL

compared to DLDL of the participants in this study are shown in table 2. The median and interquartile

ranges of DLDL, FLDL, and MLDL in our study were as follows: DLDL, 107 (89-130); FLDL, 99

(77-128); and MLDL 101 (83-128) mg/dL. The median CACS and interquartile range were 607 and

423-1017, respectively.

The distribution of TG: VLDL-C ratios is shown in fig.1. The median TG: VLDL-C ratio was

5.1 (4.5-5.8) (table 2). Most of estimates using both FLDL and MLDL were within 2 SD compared

with DLDL depicted by Bland-Altman plot (fig.2). The mean difference between DLDL and MLDL

was slightly lower than that of DLDL and FLDL (7.4(-7.8, 22.7) vs. 5.3 (-7.4, 18.1)) (p<0.001). Also,

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linear regression analysis revealed a good correlation between both FLDL and MLDL with DLDL

(fig. 3). However, MLDL showed a better fit with DLDL (MLDL, r=0.98, p< 0.001 vs. FLDL, r=0.97,

p<0.001) compared to FLDL. Overall concordance between DLDL and MLDL as an agreement

with LDL-C treatment classes recommended by NCEP-ATP III was slightly higher than that between

DLDL and FLDL (73.2 vs. 70.9%, p<0.001).

Reclassification rates of FLDL or MLDL by individual DLDL NCEP-ATP III treatment

classes and TG level groups are shown in table 3. There was no significant difference in

reclassification rates between FLDL and MLDL estimated DLDL by NCEP-ATP III treatment

classes. Analysis by TG groups showed that in the TG <100 mg/dL group, the reclassification rate

of FLDL was significantly lower than that of MLDL (24.8 vs 33.3%, p=0.012); in the TG 200-399

mg/dL group, the reclassification rate of MLDL was significantly lower than that of FLDL as

analyzed by the McNemar test (35.3 vs. 64.7, p=0.031) (table 3). The TG levels of 200-399 mg/dL

showed the greatest reclassification rate, especially by FLDL among all of the TG groups based on

Pearson’s Chi-square test (64.7%, p<0.001) (table 3). Underestimation of FLDL accounted for this

reclassification (fig. 4). In contrast, the reclassification rates by MLDL were 13.1-35.3% in all of the

TG groups analyzed by Pearson’s Chi-square test (p=0.006). The median and interquartile ranges of

differences between DLDL and FLDL gradually increased as TG levels increased (TG <100 mg/dL,

5 (1-9); TG 100-149 mg/dL, 8 (4-12); TG 150-199 mg/dL, 11 (7-15); and TG 200-399 mg/dL, 17

(13-22); p<0.001). However, the differences between DLDL and MLDL across TG levels were

similar to each other (TG <100 mg/dL, 7 (2-11); TG 100-149 mg/dL, 5 (2-10); TG 150-199 mg/dL,

4 (-3-10); and TG 200-399 mg/dL, 4 (-3-10); p=0.023).

Discussion

This study showed that LDL-cholesterol calculated by Martin’s approach had better overall

concordance with directly measured LDL-cholesterol compared to that obtained by Friedewald’s

method in coronary atherosclerosis. The discordance between FLDL and DLDL was mostly due to

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underestimation, which was mostly observed when the TG level was ≥ 200 mg/dL, reaching 64.7%,

thereby indicating that Friedwald’s method is an inappropriate tool for actual assessment of LDL-

cholesterol levels.

In this study, the overall concordance of FLDL or MLDL compared with DLDL was lower than

that from previous studies [18, 20, 23]. The difference could be due to patient groupings, the direct

LDL-cholesterol measurement method, or race. In this study, the patients were confined to those

with coronary atherosclerosis but the patients of previous studies [18, 20, 23] were conducted in the

general population. The median age of the patient group in this study was older, and the proportion

of the male patients was considerably higher, compared to those of previous studies [18, 20, 23].

The older patients in this study could have led to low non-HDL-C, which probably affected FLDL

and MLDL differences from DLDL compared to previous studies [18, 20, 23]. The second,

Martin’s or Meeusens’ studies had used β-quantification as the reference LDL-cholesterol

measurement method. Although the direct homogeneous assay showed good performance in a

healthy population, all of the direct homogeneous assays on the market have shown unacceptable

total error for the diseased group, such as people with cardiovascular disease or conditions that might

be expected to affect lipoprotein methods [22]. Third, racial differences and related differences in

dietary patterns might have affected the TG: VLDL ratio [20] and probably led to low concordance

between FLDL and DLDL or MLDL and DLDL in our population compared to that of previous

studies of Martin et al. [18] or Meeusen et al. [23]

Two of the 3 previous studies concluded that MLDL showed better overall concordance [18, 23].

However, Meeusen et al. [23] concluded that overall concordance between DLDL and the 2 LDL-C

estimates were similar to each other (76.9% [95% CI, 75.2–79.4] for FLDL vs. 77.7% [95% CI, 76.0–

79.6] for MLDL). In this study, although MLDL only increased 2.3% of overall concordance with

DLDL compared to that of FLDL (70.9% [95% CI, 65.9-75.9] for FLDL vs. 73.2% [95% CI, 68.2-

77.9] for MLDL), the reclassification rate of FLDL was twice as much of that of MLDL when TG

was ≥ 200 mg/dL (64.7 vs. 35.3%; p<0.031). Martin’s original derivation cohort and KNHANES

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data also showed a significance difference in concordance between DLDL and the 2 LDL-C estimates

when the TG level was ≥ 200 mg/dL (KNHANES, 62.0% [95% CI, 58.6-65.4] for FLDL vs. 74.6%

[95% CI,71.5-77.6] for MLDL; derivation cohort, 4483/11116 for FLDL vs. 3761/4476 for MLDL)

[18, 20]. This finding is important because physicians usually rely on FLDL when the TG level is

lower than 400 mg/dL [24]. However, in our study, when TG was ≥ 200 mg/dL, FLDL

underestimated 17 mg/dL at the median, indicating Friedewald’s equation is an inappropriate

assessment tool for estimating LDL-C in coronary atherosclerosis patients, especially with TG ≥

200 mg/dL.

At TG levels below 100 mg/dL, FLDL showed a lower reclassification rate compared to

MLDL, implying FLDL estimates LDL-C better than MLDL at low TG levels. This finding suggested

that the discordance of FLDL from DLDL resulted from high TG levels in the present study

population.

A limitation of this study was that concordance between each LDL-C estimate and DLDL

was not analyzed based on TG levels.

Conclusions

In this study, the LDL-cholesterol estimated by Martin’s method provided better concordance

with that measured by a direct homogeneous assay in coronary atherosclerosis. The reclassification

rate of FLDL was greatest when TG levels were ≥ 200 mg/dL; hence, the MLDL method could be

an economical method for estimating LDL-cholesterol in coronary atherosclerosis, especially when

the TG levels are ≥ 200 mg/dL.

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Table 1. Median for the ratio of triglycerides to very low-density lipoprotein cholesterol by non-high-
density lipoprotein cholesterol and triglyceride strata (180-cell)
Triglyceride Non-HDL-C, mg/dL
Levels, mg/dL <100 100-129 130-159 160-189 190-219 ≥220
7-49 3.5 3.4 3.3 3.3 3.2 3.1
50-56 4 3.9 3.7 3.6 3.6 3.4
57-61 4.3 4.1 4.0 3.9 3.8 3.6
62-66 4.5 4.3 4.1 4.0 3.9 3.9
67-71 4.7 4.4 4.3 4.2 4.1 3.9
72-75 4.8 4.6 4.4 4.2 4.2 4.1
76-79 4.9 4.6 4.5 4.3 4.3 4.2
80-83 5 4.8 4.6 4.4 4.3 4.2
84-87 5.1 4.8 4.6 4.5 4.4 4.3
88-92 5.2 4.9 4.7 4.6 4.4 4.3
93-96 5.3 5.0 4.8 4.7 4.5 4.4
97-100 5.4 5.1 4.8 4.7 4.5 4.3
101-105 5.5 5.2 5.0 4.7 4.6 4.5
106-110 5.6 5.3 5.0 4.8 4.6 4.5
111-115 5.7 5.4 5.1 4.9 4.7 4.5
116-120 5.8 5.5 5.2 5.0 4.8 4.6
121-126 6 5.5 5.3 5.0 4.8 4.6
127-132 6.1 5.7 5.3 5.1 4.9 4.7
133-138 6.2 5.8 5.4 5.2 5.0 4.7
139-146 6.3 5.9 5.6 5.3 5.0 4.8
147-154 6.5 6.0 5.7 5.4 5.1 4.8
155-163 6.7 6.2 5.8 5.4 5.2 4.9
164-173 6.8 6.3 5.9 5.5 5.3 5.0
174-185 7 6.5 6.0 5.7 5.4 5.1
186-201 7.3 6.7 6.2 5.8 5.5 5.2
202-220 7.6 6.9 6.4 6.0 5.6 5.3
221-247 8 7.2 6.6 6.2 5.9 5.4
248-292 8.5 7.6 7.0 6.5 6.1 5.6
293-399 9.5 8.3 7.5 7.0 6.5 5.9
400-13975 11.9 10.0 8.8 8.1 7.5 6.7

Note. HDL-C: High-density lipoprotein cholesterol. cited from reference #18.

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Table 2. Median values of the baseline characteristics of patients (n=299)
Present study
Number of participants, n 299
Age, yrs. (median) 63 (58-70)
Male, n (%) 268 (89.6)
TC, mg/dL 180 (153-202)
TG, mg/dL 104 (73-147)
HDL-C, mg/dL 51 (43-59)
Non-HDL-C, mg/dL 125 (103-151)
TG:VLDL-C 5.1 (4.5-5.8)
DLDL, mg/dL 107 (89-130)
DLDL method Homogeneous assay
FLDL, mg/dL 99 (77-128)
MLDL, mg/dL 101 (83-128)
Overall concordance of FLDL, % (95% CI) 70.9 (65.9-75.9)
Overall concordance of MLDL, % (95% CI) 73.2 (68.2-77.9)

Values are presented as median (interquartile range) or a number (percentage).

Note. TC: Total cholesterol; TG: triglyceride; HDL-C: high-density lipoprotein cholesterol; Non-
HDL-C: Non-high-density lipoprotein cholesterol; VLDL-C: very-low density lipoprotein
cholesterol; DLDL: directly measured LDL-cholesterol; FLDL: LDL-cholesterol estimated using
Friedewald equation; MLDL: LDL-cholesterol estimated using Martin’s method:

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Table 3. Reclassification of directly measured low-density lipoprotein cholesterol (DLDL) by
estimation using Friedewald equation and Martin method in relation to DLDL levels or triglyceride
levels
DLDL, mg/dL (n) FLDL, n (%) MLDL, n (%) p value *
<70 (17) 0 (0.0) 1 (5.9) N/A

70-99 (100) 30 (30.0) 22 (22.0) 0.096

100-129 (106) 41 (38.7) 35 (33.0) 0.286

130-159 (55) 10 (18.2) 15 (17.3) 0.180

160-189 (16) 6 (37.5) 7 (43.7) 1.000

≥ 190 (5) 0 (0.0) 0 (0.0) N/A

TG levels, mg/dL (n)

<100 (141) 35 (24.8) 47 (33.3) 0.012

100-149 (84) 16 (19.0) 11 (13.1) 0.063

150-199 (40) 14 (35.0) 10 (25.0) 0.289

200-399 (34) 22 (64.7) 12 (35.3) 0.031

Values shown are n/N in group (%)

Note. DLDL: directly measured LDL-cholesterol; FLDL: LDL-cholesterol estimated using
Friedewald equation; MLDL: LDL-cholesterol estimated using Martin method; TG: Triglyceride.
*
p values were calculated with McNemar test.

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Figure Legends

Fig.1 Distribution of TG: VLDL-C ratio.

Note. TG: triglyceride; VLDL-C: very low-density lipoprotein cholesterol.

Fig.2 Bland-Altman plot for the comparison of directly measured LDL-cholesterol and LDL-

cholesterol estimated using Friedewald equation (A) and Martin method (B).

Note. DLDL: directly measured LDL-cholesterol; FLDL: LDL-cholesterol estimated using

Friedewald equation; MLDL: LDL-cholesterol estimated using Martin’s method.

Fig.3 Comparison of correlation between DLDL and FLDL (A) or MLDL (B).

Note. DLDL: directly measured LDL-cholesterol; FLDL: LDL-cholesterol estimated using

Friedewald equation; MLDL: LDL-cholesterol estimated using Martin’s method.

Fig.4 Overall discordance of estimated LDL-cholesterol using Friedewald equation and Martin’s

method according to triglyceride level by NCEP-ATP III guideline classification

Note. FLDL: LDL-cholesterol estimated using Friedewald equation; MLDL: LDL-cholesterol

estimated using Martin method.

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Fig.1

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Fig.2

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Fig.3

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Fig.4

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