1. © Copyright 2009 by the American Association for Clinical Chemistry Agreement Between Fasting and Postprandial LDL Cholesterol Measured with 3 Methods in Patients with Type 2 Diabetes Mellitus S.S. Lund, M. Petersen, M. Frandsen, U.M. Smidt, H.- H. Parving, A.A. Vaag, and T. Jensen February 2011 http://www.clinchem.org/cgi/content/full/57/2/298 © Copyright 2011 by the American Association for Clinical Chemistry Journal Club

2. © Copyright 2009 by the American Association for Clinical Chemistry Introduction:  The use of fasting or nonfasting lipid measurements is debated. LDL cholesterol (LDL-C) is considered the primary target of lipid lowering therapy. (Adult Treatment Panel III (ATP-III), Circulation 2002). International guidelines recommend using fasting samples to assess LDL-C (e.g., ATP III). However, the use of nonfasting (postprandial) samples is more convenient.

3. © Copyright 2009 by the American Association for Clinical Chemistry Introduction (cont.):  The relation between fasting and nonfasting LDL-C: At the population level, nonfasting LDL-C is typically lower than fasting LDL-C as measured by the mean nonfasting-fasting difference. However, at the individual level (i.e., for patient management), the extent of agreement between nonfasting and fasting LDL-C is probably a more relevant measure.

4. © Copyright 2009 by the American Association for Clinical Chemistry Introduction (cont.):  Assessment of LDL-C. The LDL-C reference method,  -quantification (BQ), is cumbersome – it involves ultracentrifugation. In daily practice, LDL-C is usually calculated by the Friedewald equation (FE), or measured by a direct assay (DA). Unlike DA, the use of FE is recommended only in fasting samples and if triglycerides (TG) are <4.52 mM (400 mg/dL). Contrary to recommendations, many routine clinics and research studies use FE in nonfasting samples.

5. © Copyright 2009 by the American Association for Clinical Chemistry Introduction (cont.):  Patients with type-2 diabetes (T2DM): In the postprandial state, T2DM patients are often characterized by elevations of TG. The elevated postprandial TG could interact with or influence the measurement of LDL-C.  Study aim: To evaluate the agreement between fasting and postprandial LDL-C measured with 3 methods in patients with T2DM.

6. © Copyright 2009 by the American Association for Clinical Chemistry Questions:  How can fasting and postprandial concentrations of LDL-C be investigated?  Which LDL-C methods should be included in the investigation?

7. © Copyright 2009 by the American Association for Clinical Chemistry Material and methods:  Study protocol: After an overnight fast of minimum 10 h, 74 T2DM patients were served a standard fat-rich breakfast meal. The meal consisted of typical western foods: Bread, butter, cheese, milk, jam, and sausage (3515 kJ, 54% fat (50 g total fat),13% protein, 33% carbohydrates). Any medications were taken with the meal. Only drinking water was allowed postprandially. Plasma was sampled at time 0 (fasting) as well as 1.5, 3.0, 4.5 and 6 h (postprandially). Data are presented for 66 patients with complete measurements.

8. © Copyright 2009 by the American Association for Clinical Chemistry Material and methods:  Study protocol (cont.): Measurements: Total cholesterol (TC) and TG: enzymatic methods (Roche). HDL cholesterol (HDL-C): Homogeneous assay (HDL-C Plus, Roche). LDL-C: 1)Modified BQ (MBQ): Ultracentrifugation for 18 h at 105.000g. The bottom fraction (d>1.006 g/mL) containing the LDL and HDL particles was brought to volume and analyzed for TC and HDL-C. LDL-C was calculated as bottom fraction TC – HDL-C. 2)FE: LDL-C (mM) = TC – HDL-C – TG/2.2 (use TG/5 if in mg/dL) in samples with TG <4.52 mM (400 mg/dL). 3)DA: Homogeneous assay (LDL-C Plus, Roche), attempts to inhibit the cholesterol reactivity in non-LDL particles. This enables enzymatic measurement of LDL-C.

9. © Copyright 2009 by the American Association for Clinical Chemistry Questions:  How should agreement between fasting and postprandial LDL-C be evaluated?  What is a clinically relevant difference in LDL-C?

10. © Copyright 2009 by the American Association for Clinical Chemistry Statistics:  Agreement was evaluated according to the Bland-Altman method (Lancet 1986): The ‘limits of agreement’ (LOA) given by the mean  1.96 x standard deviation (i.e., mean and 95% LOA) of the postprandial-fasting differences was calculated and plotted. Acceptable agreement was concluded if the mean and 95% LOA of the postprandial-fasting difference in LDL-C was within  0.20 mM (  7.7 mg/dL). This was considered to be a clinically relevant difference. (Law, BMJ 2003; Linsel-Nitschke, PlosOne 2008).

11. © Copyright 2009 by the American Association for Clinical Chemistry Statistics (cont.):  Interpretation of the Bland-Altman method: The LOA is not a 95% confidence interval (CI) for the mean difference. It is a prediction interval within which 95% of the individual data points, e.g., postprandial- fasting LDL-C differences, can be expected to lie. About half of data points, differences, will typically lie between the mean and either of the lower or higher 95% LOA, respectively. The 95% LOA can also be interpreted as the estimated ‘maximum’ disagreement between two methods of measurement, e.g., fasting and postprandial LDL-C. The Bland-Altman method provides optimal clinical information, that for the population, the mean difference, and that for the individual, the LOA.

12. © Copyright 2009 by the American Association for Clinical Chemistry Results: Table 1. Patient characteristics. Data are median (range) or numbers (%). *Calculated by FE. To convert mM to mg/dL multiply by 38.67 for cholesterol and by 88.57 for TG. Gender (men/women)55/11 Age (years)61.7 (44.4; 81.2) Known duration of diabetes (years)5 (0; 29) Body mass index (kg/m 2 )24.6 (18.8; 28.2) HemoglobinA 1c (%)7.7 (5.2; 10.7) Glucose-lowering therapy, (Diet only/oral agents/insulin) 25/41/0 Fasting TC (mM)4.70 (2.80; 6.60) Fasting LDL-C (mM)*2.85 (1.50; 4.90) Fasting HDL-C (mM)1.14 (0.56; 1.93) Fasting TG (mM)1.27 (0.40; 3.40) Ongoing statin treatment17 (26) Known cardiovascular disease15 (23)

13. © Copyright 2009 by the American Association for Clinical Chemistry Figure 1. Fasting and postprandial LDL-C concentrations in T2DM patients measured by a modified  quantification, the Friedewald equation and a direct assay. Data are mean  SE. For all methods, mean LDL-C concentrations at all postprandial times were significantly lower, typically by about 0.2 to 0.4 mM, than time 0 (fasting), p<0.005 versus fasting for all. Data are also shown for TC, HDL-C and TG (mean  SE, or geomean) (*p<0.05 versus fasting for these).

14. © Copyright 2009 by the American Association for Clinical Chemistry Figure 2. Bland-Altman plots of postprandial and fasting LDL-C measured by 3 methods in T2DM patients. Difference: Postprandial minus fasting LDL-C; Average: Mean of postprandial and fasting LDL-C. Solid line: The identity line. Dashed black lines: Mean difference with 95% LOA. Dashed grey lines: 95% CI. Grey area: The window of acceptable agreement of  0.2 mM. Acceptable agreement was unmet for all LDL-C methods at all times (e.g., LOA: –0.52 to –0.89 mM at 4.5 h; data not shown at 3.0 or 6.0h).

15. © Copyright 2009 by the American Association for Clinical Chemistry Misclassification according to LDL-C <2.6 mM (ATP-III) using postprandial LDL-C: Table 2. Data are percentages and number of patients. For each method, the reference is the fasting LDL-C for that method. All: The 66 patient-group. Statin users: The 17 statin users. Misclassified patients: Those with fasting LDL-C >= 2.6 mM and at least one postprandial LDL-C <2.6. A: Misclassified patients (n 1 ) among patients with fasting LDL-C >= 2.6 mM (n 2 ). B: Misclassified patients (n 1 ) among patients with at least one postprandial LDL-C <2.6 mM (n 2 ). In patients with fasting LDL-C >= 2.6 mM (columns A), 10-38%, and up to 63% of statin users, were misclassified into lower ATP-III risk categories using postprandial instead of fasting LDL-C. In patients with postprandial LDL-C <2.6 mM (columns B), 24-50%, and up to 36% of statin users, were similarly misclassified.

16. © Copyright 2009 by the American Association for Clinical Chemistry Question:  Do statin treatment or high postprandial TG influence the difference between postprandial and fasting LDL-C?

17. © Copyright 2009 by the American Association for Clinical Chemistry Figure 3. Fasting and postprandial LDL-C in T2DM patients partitioned by statin treatment or TG. Data are mean  SE. TG partition was based on at least 1 postprandial TG concentration >2.08 mM (the median postprandial TG concentration). †p 2.08 mM meant greater postprandial decrease in LDL-C for all methods, most pronounced for FE.

18. © Copyright 2009 by the American Association for Clinical Chemistry Summary:  For 3 LDL-C methods the mean LDL-C decreased by 0.2-0.4 mM postprandially in T2DM patients.  Postprandial and fasting LDL-C disagreed for all methods with 95% LOA (estimated ‘maximum’ disagreement) ranging from –0.52 to –0.89 mM at 4.5 h. That is, for FE, about half of patients may disagree of from ~0.4 mM (the mean) to ~0.9 mM (the LOA) lower postprandial than fasting LDL-C.  Using postprandial LDL-C, FE misclassified 38% and two-thirds of statin users into lower ATP-III risk categories.  Unlike statin use, postprandial TG >2.08 mM had greater disagreement between fasting and postprandial LDL-C for all methods.

19. © Copyright 2009 by the American Association for Clinical Chemistry Authors’ conclusions: In T2DM patients, postprandial LDL-C concentrations might differ substantially from fasting concentrations, with postprandial LDL-C concentrations usually being lower. The findings support that, irrespective of the method, postprandial LDL-C should not be used for assessing CVD risk.