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Technical Briefs |
Dept. of Clin. Biochem., Natl. Women's Hosp., Private Bag 92189, Auckland 3, New Zealand
1
Clin. Trials Res. Unit, Univ. of Auckland, Auckland;
2
Dept. of Nephrol., Christchurch Hosp., Christchurch, New Zealand;
a author for correspondence: fax 64-9-630-9996
Increased plasma cholesterol, particularly that portion associated with LDL, is an established risk factor for coronary heart disease (1)(2). As a consequence, it is widely recommended (3) that LDL-cholesterol be determined in individuals with increased total cholesterol. Because isolation of the LDL fraction requires ultracentrifugation, a technique not generally available in service laboratories, the concentration of LDL-cholesterol is usually calculated by the formula of Friedewald et al. (4).
The Friedewald formula provides an adequate estimate of LDL-cholesterol for most fasting specimens but is known to be less reliable as triglyceride concentration increases (5). Moreover, in patients with chronic renal failure, in whom an accumulation of remnant particles and increased concentrations of IDL-cholesterol have been found (6)(7), the estimate is possibly further compromised (8).
Here we report results on specimens obtained from patients on dialysis with chronic renal failure that show no greater disparity between measured and estimated LDL-cholesterol concentrations than those seen in other patient groups in which the Friedewald formula is frequently used. Additionally we have reexamined the effect of IDL and VLDL composition on the reliability of estimations made with the formula.
Two sets of blood specimens taken 6 months apart were obtained from 106 patients receiving hemodialysis or continual ambulatory peritoneal dialysis (9). Within this 6-month period, some patients received an inhibitor of hydroxymethylglutaryl-CoA reductase, some an inhibitor of angiotensin-converting enzyme, some both treatments, and some placebo only. Blood collected after an overnight fast and separated within 4 h provided serum specimens for analysis.
Total cholesterol and triglyceride were assayed enzymically with reagents from Boehringer Mannheim. HDL-cholesterol was assayed after precipitation of the apo B-containing lipoproteins with phosphotungstate and magnesium ions (10). The performance of these assays was regularly certified by reference to samples provided by the Centers for Disease Control and Prevention, Atlanta, GA. Ultracentrifugation was carried out as described by others (11) for 15 h at 120 000g with a TFT 80.4 fixed-angle rotor in a Centrikon T-2070 centrifuge (Kontron Instruments). Cholesterol and triglyceride contents of supernatant fractions obtained after the tubes were sliced (11) were measured; the fraction floating at a relative density (d) of 1.006 was taken to represent VLDL and that floating at d 1.019 obtained by adjustment of 20 volumes of serum with 1 volume of NaBr solution (d = 1.27, ~3.9 mol/L) was taken to represent VLDL plus IDL. The contents of IDL alone were calculated by subtraction. When in other experiments this technique was extended to permit sequential isolation of LDL and of HDL (11), the mean recovery of total cholesterol was 90% (range 86–95%) and of triglyceride was 92% (range 88–95%).
LDL-cholesterol was calculated as total cholesterol - HDL-cholesterol - VLDL-cholesterol; VLDL-cholesterol was either measured directly (after ultracentrifugation) or calculated as 0.456 x total triglyceride concentration expressed in mmol/L (Friedewald).
Analysis of 204 specimens with complete data from the 106 patients showed concentrations (mmol/L) of total cholesterol of 3.16–9.25 (median 6.00), triglyceride of 0.48–7.74 (median 2.01), VLDL-cholesterol of 0.09–3.78 (median 0.92), IDL-cholesterol of 0.15–1.58 (median 0.45), and HDL-cholesterol of 0.36–2.28 (median 0.93). LDL-cholesterol derived from ultracentrifugation data ranged from 1.13 to 7.27 (median 3.92) mmol/L.
Thirty-six specimens (18%) had a LDL-cholesterol calculated from the Friedewald formula that differed from the ultracentrifugation value by >10%. This proportion of specimens with significant error is similar to those proportions (15–19%) found in much larger series of patients of more diverse clinical conditions (12)(13). The proportion is substantially less than the 42% of 45 individuals with chronic renal failure reported by others (8), despite the lower lipid values in their series.
Recognition that the Friedewald formula is unreliable in the presence of chylomicronemia and hypertriglyceridemia has led to the use of various indices for the exclusion of such specimens. Total triglyceride concentration commonly is used (5), although a low total cholesterol:triglyceride ratio has been proposed as a more relevant criterion for rejection (14). Additionally, others have identified increased cholesterol:triglyceride ratios in VLDL and IDL and increased IDL-cholesterol alone as being associated with increased error in the formula (8).
We plotted the number of errors detected against each index arranged in
centiles (Fig. 1
). The median (range) for each index was: total triglyceride
concentration, 2.01 (0.48–7.74) mmol/L; total cholesterol:triglyceride
ratio, 2.92 (0.86–7.86); cholesterol:triglyceride ratio in VLDL, 0.75
(0.31–1.28); IDL-cholesterol 0.45 (0.15–1.58) mmol/L;
cholesterol:triglyceride ratio in IDL, 2.69 (1.11–6.80). With total
triglyceride concentration and total cholesterol:triglyceride
ratio (by inverse ranking so that increased incidence of error was
positively associated with the index), error detection was similar with
the number of errors initially small but rising steeply from the 60th
centile. By contrast, with the ratio of cholesterol:triglyceride in
VLDL and in IDL and with IDL-cholesterol concentration, the errors were
more evenly distributed among the centiles. Quantitatively, 18 errors
(50%) were included when triglyceride concentration was 3.51 mmol/L
(86th centile), total cholesterol:triglyceride ratio was 1.91 (81st
centile), cholesterol:triglyceride ratio in VLDL was 0.82 (70th
centile), IDL-cholesterol was 0.53 mmol/L (65th centile), and
cholesterol:triglyceride ratio in IDL was 2.51 (38th centile).
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The ranking by total triglyceride concentration and total cholesterol:triglyceride ratio demonstrates that these values are far better suited as criteria for deciding whether it is valid to calculate LDL-cholesterol. Moreover, the results confirm that total triglyceride concentration alone seems to be the most simple and appropriate criterion to use, with total cholesterol:triglyceride ratio offering no improvement in discrimination.
In our study, 24 of 41 specimens (59%) with triglyceride concentration
>3.2 mmol/L had an unreliable estimate of LDL-cholesterol from the
Friedewald formula, with all but two being an overestimate. While such
overestimation is typical of abnormal VLDL composition as seen in Type
III hyperlipoproteinemia (5), there appeared to be no
clear association between cholesterol:triglyceride ratio in VLDL and
the incidence of error (Fig. 1
). It seems improbable that the disparity
between our results and those of others (8) could be
explained by effects of drug treatment. Indeed, the conclusion was
unchanged when the 50 specimens from patients on an
hydroxymethylglutaryl-CoA reductase inhibitor were removed from
analysis.
In summary, use of the Friedewald formula in a group of patients expected to give aberrant results provided reliable data in the majority of cases. Moreover, triglyceride concentration was a suitable index of reliability. However, restricting use of the formula to those specimens having a triglyceride concentration of <4.5 mmol (4 g)/L (13) included a group above the 80th centile in which the error rate exceeded 50%. Limiting use of the formula to those below the 80th centile and having a triglyceride concentration of <3.2 mmol (2.8 g)/L excluded the majority of errors and left 151 of 163 (93%) with a valid estimate. By contrast with the results of others (8)(14), we found little influence of VLDL or IDL on the reliability of the Friedewald formula and no advantage with total cholesterol:triglyceride ratio as a discriminant.
Acknowledgments
The study of the effects of simvastatin and enalapril on serum lipoprotein concentrations in patients on dialysis, from which this work derives, was supported by a grant from Merck, Sharp and Dohme.
References
The following articles in journals at HighWire Press have cited this article:
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  A. C. Alibegovic, L. Hojbjerre, M. P. Sonne, G. van Hall, B. Stallknecht, F. Dela, and A. Vaag Impact of 9 Days of Bed Rest on Hepatic and Peripheral Insulin Action, Insulin Secretion, and Whole-Body Lipolysis in Healthy Young Male Offspring of Patients With Type 2 Diabetes Diabetes, December 1, 2009; 58(12): 2749 - 2756. [Abstract] [Full Text] [PDF]
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 E. T. Bairaktari, K. I. Seferiadis, and M. S. Elisaf Evaluation of Methods for the Measurement of Low-Density Lipoprotein Cholesterol Journal of Cardiovascular Pharmacology and Therapeutics, January 1, 2005; 10(1): 45 - 54. [Abstract] [PDF]
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 M. Nauck, G. R. Warnick, and N. Rifai Methods for Measurement of LDL-Cholesterol: A Critical Assessment of Direct Measurement by Homogeneous Assays versus Calculation Clin. Chem., February 1, 2002; 48(2): 236 - 254. [Abstract] [Full Text] [PDF]
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