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Reliability of Calculated Low-Density Lipoprotein Cholesterol

      Aggressive low-density lipoprotein cholesterol (LDL-C)–lowering strategies are recommended for prevention of cardiovascular events in high-risk populations. Guidelines recommend a 30% to 50% reduction in at-risk patients even when LDL-C concentrations are between 70 and 130 mg/dl (1.8 to 3.4 mmol/L). However, calculation of LDL-C by the Friedewald equation is the primary laboratory method for routine LDL-C measurement. We compared the accuracy and reproducibility of calculated LDL-C <130 mg/dl (3.4 mmol/L) to LDL-C measured by β quantification (considered the gold standard method) in 15,917 patients with fasting triglyceride concentrations <400 mg/dl (4.5 mmol/L). Both variation and bias of calculated LDL-C increased at lower values of measured LDL-C. The 95% confidence intervals for a calculated LDL-C of 70 mg/dl (1.8 mmol/L) and 30 mg/dl (0.8 mmol/L) were 60 to 86 mg/dl (1.6 to 2.2 mmol/L) and 24 to 60 mg/dl (0.6 to 1.6 mmol/L), respectively. Previous recommendations have emphasized the requirement for a fasting sample with triglycerides <400 mg/dl (4.5 mmol/L) to calculate LDL-C by the Friedewald equation. However, no recommendations have addressed the appropriate lower reportable limit for calculated LDL-C. In conclusion, calculated LDL-C <30 mg/dl (0.8 mmol/L) should not be reported because of significant deviation from the gold standard measured LDL-C results, and caution is advised when using calculated LDL-CF values <70 mg/dl (1.8 mmol/L) to make treatment decisions.
      Calculation of low-density lipoprotein cholesterol (LDL-C) by the Friedewald equation (total cholesterol – low-density lipoprotein cholesterol [HDL-C] – [triglycerides/5], referred to as LDL-CF) has been the standard of practice for decades and remains the primary laboratory method for routine LDL-C orders. Surprisingly, the analytical performance of the Friedewald equation at low LDL-C concentrations has not been previously defined in a large cross-sectional outpatient population. Here we report on the accuracy and reproducibility of calculated LDL-CF <130 mg/dl (3.4 mmol/L) compared with LDL-C measured by β quantification (LDL-CßQ), which is considered the gold standard LDL-C method and is the foundation on which the Friedewald equation was based.

      Methods

      Patient data were accessed in compliance with the Mayo Clinic Institutional Review Board. The present study included 15,917 patients with clinically ordered LDL-CßQ. Serum LDL-CßQ measurement included quantitation of HDL-C and triglycerides that were subsequently used to calculate LDL-CF. Inclusion criteria were total serum triglycerides <400 mg/dl (4.5 mmol/L) and LDL-CF <130 mg/dl (3.4 mmol/L). The mean patient age was 50 ± 19 years; 46% were women (Table 1). Patients were directed to fast for a minimum of 8 hours before sample collection. Cholesterol (Cholesterol Generation 2; Roche Diagnostics, Indianapolis, Indiana) and triglycerides (Roche Diagnostics) were measured using a Cobas c501 (Roche Diagnostics). To determine LDL-CßQ, 1.0 ml serum was centrifuged for 15 hours at 86,000g (Beckman LE-80K ultracentrifuge, Type 25 rotor; Beckman Coulter, Inc., Brea, California). The very low–density layer was removed according to volumetric guide (275 μl). The remaining fraction was weighed (required density 1.00 to 1.07 g/ml) and then reconstituted to 1.0 ml using distilled water. Cholesterol was measured before and after precipitation of LDL using a dextran sulfate solution (Pointe Scientific, Canton, Michigan). LDL-CF was calculated as described earlier. The Mayo Clinic Cardiovascular Medicine clinical testing laboratory is certified by the Centers for Disease Control and Prevention Lipid Standardization Program for quantitation of cholesterol, HDL-C, triglycerides, and LDL-CßQ. LDL-C precision was determined by daily repeat analysis (n = 37) of pooled serum (LDL-CßQ mean 109 mg/dl, SD 4.2 mg/dl, %CV 3.9%; LDL-CF mean 107 mg/dl, SD 3.2 mg/dl, %CV 3.1%). Graphical regression and statistical analyses were performed using JMP software (SAS Inc., Cary, North Carolina).
      Table 1Demographic and lipid characteristics for patients with calculated LDL-CF in clinically relevant categories
      LDL-CF Category< 30 mg/dL30-69 mg/dL70-99 mg/dL100-129 mg/dLOverall
      N1873,0496,3066,37515,917
      Female, N (%)66 (35%)1,145 (38%)2,800 (44%)3,202 (50%)7,213 (46%)
      Age, mean±SD48±2353±2250±2150±1950±19
      Lipids, mg/dL; median (IQR)
       Measured LDL-CßQ33 (27 - 39)64 (57 - 71)90 (82 - 98)116 (108 - 124)97 (79 - 113)
       Calculated LDL-CF22 (15 - 27)58 (49 - 64)86 (78.2 - 93)113 (106 - 121)93 (74 - 110)
       Total Cholesterol86 (70 - 102)125 (112 - 139)158 (146 - 171)190 (177 - 203)167 (144 - 188)
       HDL Cholesterol32 (22 - 43)39 (31 - 48)43 (36 - 53)46 (38 - 55)43 (35 - 53)
       Triglycerides155 (67 - 255)120 (81 - 189)121 (83 - 183)127 (89 - 189)123 (85 - 187)

      Results

      LDL-CF results were significantly lower than LDL-CßQ across the entire cohort. LDL-CF values ranged from −16 to 129 mg/dl (−0.4 to 3.3 mmol/L), whereas LDL-CßQ ranged from 7 to 164 mg/dl (0.2 to 4.2 mmol/L). Among patients with LDL-CF between 70 and 99 mg/dl (1.8 to 2.6 mmol/L), the median difference between LDL-CF and LDL-CßQ was −4 mg/dl with an IQR of −9 to 0 mg/dl (0.1 mmol/L; −0.2 to 0 mmol/L). Underestimation of LDL-CF was exacerbated at lower LDL-CF ranges, with a median difference of −11 mg/dl (IQR −21 to −5 mg/dl [−0.3 mmol/L, IQR −0.5 to −0.1 mmol/L]) for patients with LDL-CF <30 mg/dl (<0.8 mmol/L).
      Overall, the median triglyceride concentration was 123 mg/dl (1.4 mmol/L). LDL-CF underestimation worsened with increasing triglycerides; however, there was no significant correlation between LDL-CF and triglycerides. Serum triglycerides were <200 mg/dl (2.26 mmol/L) in 12,528 patients (79%) and in 2,485 patients (77%) with LDL-CF <70 mg/dl (1.8 mmol/L).
      Increased discordance between LDL-CF and LDL-CßQ at low concentrations was because of both increased variation and increased bias in the calculated LDL-CF results (Figure 1). The 95% confidence interval for an LDL-CF of 70 mg/dl (1.8 mmol/L) was 60 to 86 mg/dl (1.6 to 2.2 mmol/L), and the 95% confidence interval for an LDL-CF of 30 mg/dl (0.8 mmol/L) increased to 24 to 60 mg/dl (0.6 to 1.6 mmol/L), further demonstrating the increased variability with lower LDL-CF.
      Figure thumbnail gr1
      Figure 1Calculated LDL-C deviates from β-quantification–measured LDL cholesterol at low concentrations. (A) LDL-CF plotted as a function of LDL-CßQ for 15,917 patients with triglycerides <400 mg/dl (4.5 mmol/L) and LDL-CF <130 mg/dl (3.4 mmol/L). Dotted line represents line of identity. (B) Bias of LDL-CF ([calculated – measured]/measured) plotted as a function of LDL-CßQ. Patients were binned according to LDL-CßQ, and each point represents 100 patients.

      Discussion

      This study highlights serious limitations to calculated LDL-CF at concentrations <70 mg/dl (1.8 mmol/L). In an illustrative example, a 37-year-old man had a total cholesterol of 135 mg/dl (3.5 mmol/L), HDL-C of 47 mg/dl (1.2 mmol/L), and triglycerides of 173 mg/dl (2.0 mmol/L). The calculated LDL-CF was 53 mg/dl (1.4 mmol/L), whereas the measured LDL-CßQ was much higher at 84 mg/dl (2.2 mmol/L). Given sufficient risk factors, the measured value may indicate an adjustment to lipid-lowering therapies. However, if the calculated LDL-CF of 53 mg/dl was the only result available, then the statin dosage would likely not be titrated and may even be lowered.
      Previous studies in small patient populations have demonstrated greater bias in LDL-CF in children with type 1 diabetes,
      • Sibal L.
      • Neely R.D.
      • Jones A.
      • Home P.D.
      Friedewald equation underestimates low-density lipoprotein cholesterol at low concentrations in young people with and without type 1 diabetes.
      patients taking anacetrapib or a statin/ezetimibe combinations,
      • Davidson M.
      • Liu S.X.
      • Barter P.
      • Brinton E.A.
      • Cannon C.P.
      • Gotto Jr., A.M.
      • Leary E.T.
      • Shah S.
      • Stepanavage M.
      • Mitchel Y.
      • Dansky H.M.
      Measurement of LDL-C after treatment with the CETP inhibitor anacetrapib.
      • Miller W.G.
      • Myers G.L.
      • Sakurabayashi I.
      • Bachmann L.M.
      • Caudill S.P.
      • Dziekonski A.
      • Edwards S.
      • Kimberly M.M.
      • Korzun W.J.
      • Leary E.T.
      • Nakajima K.
      • Nakamura M.
      • Nilsson G.
      • Shamburek R.D.
      • Vetrovec G.W.
      • Warnick G.R.
      • Remaley A.T.
      Seven direct methods for measuring HDL and LDL cholesterol compared with ultracentrifugation reference measurement procedures.
      and patients who undergo LDL apheresis.
      • Scharnagl H.
      • Nauck M.
      • Wieland H.
      • Marz W.
      The Friedewald formula underestimates LDL cholesterol at low concentrations.
      Additional studies have established and confirmed the variability of HDL-C methods, particularly at low cholesterol and high triglycerides concentrations.
      • Miida T.
      • Nishimura K.
      • Okamura T.
      • Hirayama S.
      • Ohmura H.
      • Yoshida H.
      • Miyashita Y.
      • Ai M.
      • Tanaka A.
      • Sumino H.
      • Murakami M.
      • Inoue I.
      • Kayamori Y.
      • Nakamura M.
      • Nobori T.
      • Miyazawa Y.
      • Teramoto T.
      • Yokoyama S.
      Validation of homogeneous assays for HDL-cholesterol using fresh samples from healthy and diseased subjects.
      • Nakamura M.
      • Kayamori Y.
      • Iso H.
      • Kitamura A.
      • Kiyama M.
      • Koyama I.
      • Nishimura K.
      • Nakai M.
      • Noda H.
      • Dasti M.
      • Vesper H.W.
      • Miyamoto Y.
      LDL cholesterol performance of beta quantification reference measurement procedure.
      However, there have been no studies that have systematically evaluated LDL-CF in a cross-sectional outpatient population. Furthermore, there are no recommendations that address the appropriate lower reportable limit for LDL-CF.
      Clinicians need to be aware of the LDL-C method used in their practice and know the limitations associated with the method. Calculated LDL-CF has several limitations clearly defined in the inaugural work by Friedewald and colleagues that include a requirement for the patient to fast and a triglyceride limit of 400 mg/dl.
      • Friedewald W.T.
      • Levy R.I.
      • Fredrickson D.S.
      Estimation of the concentration of low-density lipoprotein cholesterol in plasma, without use of the preparative ultracentrifuge.
      Despite pressures to report an LDL-C in the medical record for all at-risk patients, the Friedewald equation should not be used if triglycerides are elevated. Routine use of measured LDL would significantly increase the cost of testing. In our institution, we routinely calculate and use non-HDL-C
      • Lopez-Jimenez F.
      • Simha V.
      • Thomas R.J.
      • Allison T.G.
      • Basu A.
      • Fernandes R.
      • Hurst R.T.
      • Kopecky S.L.
      • Kullo I.J.
      • Mulvagh S.L.
      • Thompson W.G.
      • Trejo-Gutierrez J.F.
      • Wright R.S.
      A summary and critical assessment of the 2013 ACC/AHA guideline on the treatment of blood cholesterol to reduce atherosclerotic cardiovascular disease risk in adults: filling the gaps.
      per guideline recommendations.
      • Jacobson T.A.
      • Ito M.K.
      • Maki K.C.
      • Orringer C.E.
      • Bays H.E.
      • Jones P.H.
      • McKenney J.M.
      • Grundy S.M.
      • Gill E.A.
      • Wild R.A.
      • Wilson D.P.
      • Brown W.V.
      National Lipid Association recommendations for patient-centered management of dyslipidemia: part 1-executive summary.
      Expert Panel on Detection, Evaluation, and Treatment of High Blood Cholesterol in Adults
      Executive Summary of The Third Report of The National Cholesterol Education Program (NCEP) Expert Panel on Detection, Evaluation, and treatment of high blood cholesterol in adults (Adult treatment Panel III).
      Limitations in the analytical performance are of particular importance when evaluating changes in LDL-C over time. The reference change value (RCV) is defined as the minimum change required to be certain that serial values differ beyond analytical and biologic variations (RCV=2.77CVa2+CVi2). The normal biologic variability (CVi) for LDL-C is 8% to 10%
      • Alexander K.S.
      • Kazmierczak S.C.
      • Snyder C.K.
      • Oberdorf J.A.
      • Farrell D.H.
      Prognostic utility of biochemical markers of cardiovascular risk: impact of biological variability.
      ; thus, the RCV for LDL-C is ∼25%. However, the confidence intervals for LDL-CF values <70 mg/dl (1.8 mmol/L) span 30 to 40 mg/dl (0.8 to 1.0 mmol/L), much greater than the 25% RCV. Therefore, reporting a calculated LDL-CF in patients with a true LDL-C <70 mg/dl (1.8 mmol/L) may mask clinically significant changes because of the variability inherent in the performance of the Friedewald equation below this threshold. For these reasons, our institution no longer reports LDL-CF <30 mg/dl (0.8 mmol/L) because of the marked deviation from the gold standard LDL-CßQ results. Additionally, a comment is included which states that the calculated LDL-CF deviates significantly from measured LDL-CßQ when LDL-CF is <70 mg/dl (1.8 mmol/L).

      Disclosures

      This work was not supported by any grants, contracts, or other forms of financial support or any relations with industry. The authors have no conflicts of interest to disclose.

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