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 recommend 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 hypertriglyceridemia. 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 calculated 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 Treatment 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 concentration 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 individuals with hypercholesterolemia and to monitor the response to diet and drug treatments.
The reference method for LDL-C concentration measurement, 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 estimation 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 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 formula 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 overnight 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 enzymatically 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 Kyowa 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 cholesterol 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 homogeneous assay has been shown to meet current NCEP criteria 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, χ2 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).
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
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).
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
Fig. 2.
Relationship between total cholesterol and difference in directly measured and calculated LDL
Discussion
Strategies for treatment of lipid abnormalities are primarily based on LDL-C concentration. Therefore, LDL-C must be 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 levels >4.52 mmol/l (400 mg/dl), to non-fasting samples and to samples of patients with dysbetalipoproteinemia (Fredrickson 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 measurement 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 consistent 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 calculated 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 difference 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. 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 Vujovic 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 calculated 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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