ORIGINAL ARTICLE

Does Friedewald Formula Underestimate the Risk of IschemicHeart Disease?

Ekta Bansal • Navpreet Kaur

Received: 3 June 2013 / Accepted: 30 September 2013

� Association of Clinical Biochemists of India 2013

Abstract Traditionally Friedewald formula has been used

to calculate low density lipoprotein cholesterol (LDL-C)

concentration though now direct homogenous methods for its

measurement are also available. Clinical guidelines recom-

mend the use of calculated LDL-C to guide therapy because

the evidence base for cholesterol management is derived

almost exclusively from trials that use calculated LDL, with

direct measurement of LDL-C being reserved for those

patients who are non fasting or with significant hypertriglyc-

eridemia. In this study our aim was to compare calculated and

direct LDL and their variation at different cholesterol and

triglyceride levels. Fasting lipid profile estimation was done

on 503 outpatients in a tertiary hospital. Both direct and cal-

culated LDL were then compared. Mean fasting direct LDL

was found to be higher than calculated LDL in 87.1 % of

subjects by 8.64 ± 8.35 mg/dl. This difference was seen a all

levels of cholesterol and triglyceride. Using 130 mg/dl LDL

cholesterol as cut off fewer subjects were classified as high

risk by calculated LDL than direct LDL. In conclusion, direct

LDL is higher than calculated LDL. Compared with direct

measurement, the Friedewald calculation underestimates the

risk for ischemic heart disease.

Keywords Friedewald formula � Direct LDL �Calculated LDL

Introduction

The concentration of low-density lipoprotein cholesterol

(LDL-C) is one of the strongest markers of atherosclerosis

and predictor for assessing coronary heart disease (CHD)

risk. Strong positive association between increased LDL-C

and CHD has been well documented [1–3]. The National

Cholesterol Education Programme’s (NCEP) Adult Treat-

ment Panel III (ATP III) deemed that LDL-C concentration

was the primary basis for treatment and appropriate patients’

classification in risk categories [4]. The serum LDL-C con-

centration used to classify adults for high risk of heart disease

is: desirable\130 mg/dl, borderline high risk 130–159 mg/

dl, high risk[160 mg/dl. The goal for subjects with two or

more risk factors like diabetes, family history, hypertension,

smoking, low high density lipoprotein cholesterol (HDL) is

to achieve LDL-C concentration of 100 mg/dl [4]. Therefore

accurate and precise measurements of patients’ LDL-C

concentration is necessary to appropriately identify indi-

viduals with hypercholesterolemia and to monitor the

response to diet and drug treatments.

The reference method for LDL-C concentration mea-

surement, which combines ultracentrifugation-polyanion

precipitation, is not readily available and also impractical

in the routine laboratory [5]. A new generation of direct

homogenous assays [6, 7] for LDL-C determination in

serum has been developed with a satisfactory degree of

accuracy but at the same time these assays are expensive.

Friedewald’s formula [8] is still the most commonly

employed procedure in clinical laboratories for the esti-

mation of LDL-C concentration and it has been considered

acceptable for patients’ classification [4]. It is often used in

developing countries due to its simplicity, convenience and

low cost. Although convenient, the Friedewald calculation

suffers from several well-established limitations, which led

an Expert Panel convened by the NCEP to recommend

development of accurate direct LDL-C estimation methods

[5]. Earlier direct methods had limitations for general use.

Recently, a new generation of homogeneous methods

E. Bansal (&) � N. Kaur

Department of Biochemistry, DMC&H, Ludhiana 141001,

Punjab, India

e-mail: dr_ekta_bansal@dmch.edu

123

Ind J Clin Biochem

DOI 10.1007/s12291-013-0392-2

capable of full automation has been introduced that uses

specific reagents of various types to selectively expose and

directly measure the cholesterol associated with LDL.

As already reported by other authors the homogeneous

methods and Friedewald’s formula are not capable of

providing identical results [9, 10].

The present study was aimed to assess the difference in

direct LDL and calculated LDL as per Friedewald’s for-

mula in population from Punjab, North India.

Materials and Methods

503 outpatients who underwent lipid profile estimation were

selected for the study. Samples were collected after over-

night fasting. All estimations were done on Cobas c system

on the same day the sample was collected. Total cholesterol

(TC) and triglyceride (TG) levels were measured enzymat-

ically by CHOD-PAP and GPO-PAP methods (Roche

Diagnostics GmbH, Mannheim, Germany), respectively

according to the manufacturer’s specifications. All patients

who had TGs more than 400 mg/dl were excluded from the

study. High-density lipoprotein cholesterol (HDL-C) was

measured using a homogeneous assay without precipitation

(Roche Diagnostics GmbH, Mannheim, Germany) [11].

Friedewald formula was used to obtain calculated LDL

cholesterol values (C-LDL = TC - HDL-C - TG/5).

Direct LDL cholesterol (D-LDL) was measured by a

homogenous enzymatic colorimetric assay offered by Ky-

owa Medex and distributed by Roche Diagnostics. The

principle of D-LDL determination is as follows: At pH 6.75

in the presence of magnesium ions and a sugar compound

the enzymatic reaction for cholesterol in very low-density

lipoprotein (VLDL) and chylomicrons is markedly

reduced. Selective micellary solublization of LDL choles-

terol by a non ionic detergent enables its measurement by a

conventional enzymatic reaction with cholesterol esterase,

cholesterol oxidase and peroxidase (Roche Diagnostics

GmbH, Mannheim, Germany) [6, 11]. This direct homo-

geneous assay has been shown to meet current NCEP cri-

teria for precision (CV \ 4 %), accuracy (bias \ 4 %)

versus reference method and for total analytical error

(\12 %) [4, 11]. Within run CVs for direct LDL-C were

0.9 % at 96.9 mg/dl and 1.3 % at 237 mg/dl and the total

CVs were 1.9 % at 56.8 mg/dl and 2.1 % at 153 mg/dl.

Paired t test, v2 test and Pearson’s correlation coefficient

was used to find the statistical significance.

Results

Mean age of the study group was 51.40 ± 12.39 years. 68 %

were males and 32 % were females. Mean level of cholesterol

was 184.01 ± 43.89 mg/dl and maximum number of patients

(38.8 %) had cholesterol level between 150 and 199 mg/dl.

Mean TG was 139.92 ± 65.83 mg/dl and 49.5 % patients had

TG level between 101 and 200 mg/dl. Mean level of direct

LDL was 118.10 ± 38.45 mg/dl whereas mean calculated

LDL level was 109.46 ± 36.30 mg/dl (Table 1). 87.1 % of

patients had direct LDL more than calculated while only

12.9 % had direct LDL equal to or less than calculated LDL.

Mean difference between the two was 8.64 ± 8.35 mg/dl

which was statistically significant (p = 0.000) (Table 2).

The difference between direct and calculated LDL was seen

at all levels of TG. It was 8.65 ± 6.1 mg/dl when TG was

between 101 and 200 mg/dl and increased up to 17.02 ±

7.73 mg/dl with TG between 201 and 300 mg/dl. Maximum

difference of 25.6 ± 5.70 mg/dl was seen when the level of

TGs was between 301 and 400 mg/dl (p = 0.000 and

r = 0.698). Similar result was seen when the difference was

compared at different levels of TC (p = 0.000 and r = 0.305)

except at low levels (50–99 mg/dl) (Table 3). Subjects were

divided into two categories taking NCEP criteria of 130 mg/dl

LDL cholesterol as cut off. It was seen that more number of

subjects were classified into higher risk category ([130 mg/dl)

by using direct LDL measurement than by calculated LDL

(Table 4). Figures 1 and 2 show a positive correlation

between the difference between direct and calculated LDL

and increasing levels of TG (r = 0.698) and cholesterol

(r = 0.305).

Discussion

Strategies for treatment of lipid abnormalities are primarily

based on LDL-C concentration. Therefore, LDL-C must be

Table 1 Mean baseline values

Age 51.40 ± 12.39 years

Total cholesterol 184.01 ± 43.89 mg/dl

Triglyceride 139.92 ± 65.82 mg/dl

HDL cholesterol 46.60 ± 12.28 mg/dl

Direct LDL cholesterol 118.10 ± 38.47 mg/dl

Calculated LDL cholesterol 109.46 ± 36.30 mg/dl

Table 2 Classification of subjects depending on the difference

between direct and calculated LDL

Difference between direct and calculated

LDL (mg/dl)

Number Percentage

\10 242 55.2

10–20 144 32.9

[20 52 11.9

Mean difference between direct and calculated cholesterol was

8.64 ± 8.35 mg/dl. p value = 0.000

Ind J Clin Biochem

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accurately determined to establish a personal CHD risk

profile so as to initiate dietary adjustments, drug therapy

and to monitor their effects [4]. Friedewald’s formula has

been shown to be relatively reliable and recommended by

the NCEP as a routine method [5] for estimation of LDL-C

despite it having several well-established constraints. It

cannot be applied to samples containing TG lev-

els [4.52 mmol/l (400 mg/dl), to non-fasting samples and

to samples of patients with dysbetalipoproteinemia (Fred-

rickson Type III) [8, 11]. Some authors have demonstrated

that the formula should not be used in certain groups of

patients with diabetes, liver and renal dysfunction even

with TG levels \4.52 mmol/l [12–14]. The formula relies

on the accuracy of the TC, TG and HDL-C assays and also

on an additional mathematical term that is used to estimate

the VLDL-C concentration. It assumes a fixed relationship

between TC, TG, and HDL-C in fasting serum and is based

on the assumption that TG is only present as VLDL. Even

in the presence of small amounts of chylomicrons or

abnormal lipoproteins, the formula gives rise to falsely low

LDL cholesterol values. As well, the homogenous Roche

method we have used has some limitations [6] although it

has been reviewed by Nauck et al. [11] to be precise and

acceptably accurate. It gives an improvement in the mea-

surement of LDL-C in samples with high TG and may

assist better in classification of patients at risk categories

for cardiovascular diseases than Friedewald’s equation.

Our findings as regards Friedewald’s formula are con-

sistent with other published studies [9, 15, 16]. Just like in

these studies we have found that mean baseline direct LDL

obtained is higher than calculated LDL by 8.85 mg/dl. In a

previous study Jun and co-workers [15] revealed that cal-

culated or Friedewald-LDL-C differed significantly from

D-LDL-C over the concentration ranges of both TC and

TG. They found that the mean difference between C-LDL-

C and D-LDL-C was -9.1 % and assumed that this dif-

ference was critical for the evaluation of patients with

hyperlipidemia. Their study demonstrated that higher TG

resulted in a greater difference and increased TC was

associated with decreased difference. In our study we have

also got similar results which show that calculated LDL is

highly dependent on TG levels. With increasing TG levels

the difference between direct and calculated LDL also

increases with maximum difference seen when TG

is [300 mg/dl. Baruch et al. [17] in their study found that

in 60 % of subjects, the difference between D-LDL-C and

C-LDL-C was more than 5 mg/dl. One-third had greater

than a 15 mg/dl difference between D-LDL-C and C-LDL-

C, whereas 25 % had a greater than 20 mg/dl difference.

Table 3 Mean difference between direct and calculated LDL at

different cholesterol levels

Cholesterol range (mg/dl) Number Mean difference ± SD

50–99 7 -0.32 ± 8.23

100–149 118 4.78 ± 6.13

150–199 195 8.3 ± 7.9

200–249 148 11.7 ± 8.8

[250 35 12.42 ± 8.20

r value = 0.305 and p = 0.000

Table 4 Classification of subjects taking 130 mg/dl LDL-C as cut-

off level as per NCEP criteria

LDL cholesterol

(mg/dl)

Direct (number %) Calculated (number %)

\130 309 (61.4 %) 363 (72.2 %)

[130 194 (38.6 %) 140 (27.8 %)

p value = 0.005

Fig. 1 Relationship between

triglycerides and difference in

directly measured and

calculated LDL

Ind J Clin Biochem

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Similarly in our study nearly 45 % of patients have

[10 mg/dl difference between direct and calculated LDL

out of which 12 % had a difference of more than 20 mg/dl.

Our results are also supported by a Serbian study by Vu-

jovic et al. [18] who found a significantly higher direct

LDL as compared to LDL obtained by Friedwald formula.

They found that 82 % of patients had higher direct LDL

comparable to 87 % in our study.

There are few studies which have reported opposite

results. An Indian study by Anandaraja et al. [19] found

calculated or Friedewald LDL higher than direct LDL.

Similar results were reported by Mora et al. [20] who found

direct LDL to be lower by 5.8 mg/dl than calculated LDL.

Taking NCEP criteria into account,194 patients

(38.6 %) were found to have direct LDL [130 mg/dl

whereas 140 (27.8 %) had calculated LDL [130 mg/dl.

This means that 54 patients (10 %) were classified as low

risk by Friedewald estimation. This means that Friedewald

calculation underestimates the risk for CHD in around

10 % of the subjects who could have been instructed to

undergo a therapeutic lifestyle change.

In conclusion, compared with direct measurement, the

Friedewald calculation underestimates the risk for CHD.

Both TC and TG are significant variables affecting the

difference between the directly measured and the calcu-

lated LDL-C, over the entire range of TC and TG values.

Thus, for evaluating patients with hyperlipidemia, the

direct method of determining the LDL-C appears to be

more useful than the Friedewald calculation.

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