**3. Methods for measurement and lipids, lipoproteins with special attention to LDL-cholesterol measurement**

### **3.1 Basic lipid assays and the short history of lipid and lipoprotein measurement**

The first cumbersome but at that time reliable colorimetric methods for cholesterol and triglyceride assays were developed in the first half of the last century. From today's point of view obsolete methods were crucial for understanding the association between lipid metabolism and cardiovascular disease. Later there were replaced with enzymatic assays and adjusted for the use in automatic analyzers. Isolation of lipoproteins by ultracentrifugation giving basis for the today lipoprotein terminology was introduced in the 40's of the last century. Analytical and preparative ultracentrifugation is also today the gold standard method for quantification, separation and research on lipoproteins. About the same time paper electrophoresis (later replaced by agarose and polyacrylamide gel) gave the rise not only to an alternative nomenclature (alpha, pre-beta and beta lipoproteins) but also to the epoch-making phenotypic classification of dyslipoproteinemias by Fredrickson & Lees in 1965 and a better understanding of the pathobiochemistry of atherosclerosis. This methodology is currently reserved only for specialized applications. (For an excellent review on this topic see Mcnamara et al., 2006).

Triglyceride and total cholesterol assays are the starting points of lipid status assessment also today but their information value is different. Strictly speaking both are "artefacts" but whereas total triglycerides provide an important information about the presence of TRLs, total cholesterol itself has a limited diagnostic value.

### **3.2 Estimation of LDL-cholesterol**

Lipoproteins are prone to differential precipitation in arteficial conditions (e.g. heparinmanganese or dextran-magnesium solutions) and this allowed to develop methods to separate them without ultracentrifugation or electrophoresis. Based on this procedure in 70's first the direct measurement of HDL-C was developed and introduced into clinical chemistry. On the other side direct measurement of LDL-C was not possible until the end of the century. In the 25 year long interim period there was only one possibility to estimate the

Lipid and Lipoprotein Abnormalities in Chronic Renal Insufficiency: Review 357

**4.1 Metaanalysis of results achieved with Friedewald equation compared to reference** 

Our metaanalysis contains data from all available published papers dealing (entirely or as a part of broader study) with analytical accuracy of LDL-C assessment according to the Friedewald equation as compared with the reference UC method in CRI patients. The methodology of the metanalysis is described in our previous paper (Gaško et Sánchez-Meca, 2009). It includes studies from 1990 until the end of year 2010. Four studies were found but two of them were further divided according to the details of the study. The basic data of the

**Study, Year Probands Location % Weight Inclusion criteria**  Nauck & al 1, 1996 887 Germany 0.0% healthy (not included) Nauck & al 2, 1996 136 Germany 23.2% HD, TG < 4.56 mmol/l Nauck & al 3, 1996 171 Germany 29.2% CAPD, TG < 4.56 mmol/l Pedro-Botet & al, 1996 101 Spain 17.2% HD, age 20 - 80 years Bairaktari & al 1, 2001 54 Greece 9.2% HDs, TG < 2.26 mmol/l Bairaktari & al 2, 2001 38 Greece 6.5% HD, TG 2,26 – 4.52 mmol/l Bairaktari & al, 2004 86 Greece 14.7% HDs TG < 4.5 mmol/l

Table 10. Studies included in metaanalysis, divided into subgroups according to inclusion

**Model Study name Statistics for each study Difference in means and 95% CI Difference Lower Upper in means limit limit**

Fig. 3. Difference in means LDL-C with 95% CI in 4 studies included with 6 subgroups of patients with severe nephropathy on different forms of dialysis, with fixed and random effects model summary difference. Forest plot. Units: mmol/l. Patients included 586.

For patients in different dialysis regimes the summary differences of means of LDL-C is 0.234 mmol/l, what presents a bias of -4.9%. The information value of this result is much higher when compared with results on healthy probands od with results of patients with

**-1,00 -0,50 0,00 0,50 1,00 Favours FF Favours BQ**

**4. Analytical accuracy of LDL-C in kidney disease and hemodialysis** 

studies are summarized in Table 10 and the results of metaanalysis in Fig 3.

586 100.0%

Nauck et al 2, 1996 0,020 -0,045 0,085 Nauck et al 3, 1996 -0,070 -0,117 -0,023 Pedro-Botet et al 1, 1996 -0,310 -0,398 -0,222 Bairaktari et al 1, 2001 -0,110 -0,164 -0,056 Bairaktari et al 2, 2001 -0,390 -0,521 -0,259 Bairaktari et al, 2004 -0,180 -0,250 -0,110 Fixed -0,115 -0,142 -0,089 Random -0,163 -0,259 -0,068

**ultracentrifugation method** 

criteria.

concentration of LDL-C in everyday practice. It was the calculation according to Friedewald formula (Friedewald et al, 1972). The formula was based on the analysis of a large LDL database created in research laboratories using ultracentrifugation. It was however clear that a calculation from three (TC, TG, HDL-C) measured values with their own analytical uncertainity is only a recourse from necessity.

The uncertainity of the currently employed 3rd generation methods is much lower than that of the Friedewald formula or the older methods but from strictly analytical point of view they are also not sufficient to obtain accurate results (Bairaktari et al., 2005). Another unsolved problem is that the assay methods from different providers are not yet harmonized at all (Fuentes-Arderiu et al, 2009, Miller et al, 2010).

### **3.3 Apoprotein measurements**

The discovery of immunochemical methodology in 70's made apoprotein assays possible. In the same time their function and their role in lipid metabolism was identified. According to recent view some of them belong to parameters providing important information when assessing cardiovascular disease risk (Ritz & Wanner, 2004, Batista et al, 2005) There are three apoproteins which should be measured in each patiens with elevated risk of cardiovascular disease: Apoprotein B100, apoprotein A-I and lipoprotein(a).

Only one molecule of ApoB100 is present in each LDL-type particle (Fig 2). ApoB100 is a big molecule (m.w. 550 kDa) crucial already in the synthesis of VLDL and playing an essential role in the removal of LDL from the circulation. Measurement of ApoB100 in routine clinical practice is possible and in contrast to dynamic and heterogenous entities as "HDLcholesterol" and "LDL-cholesterol" is an unambiguously defined analyte. The concentration of ApoB100 therefore provides the best information about the presence of LDL-type particles circulating in the blood (Olofsson et al, 2007). Elevated concentration apoB100 and normal or slightly elevated LDL-C or nonHDL-C is an indirect but valuable marker of small dense LDL particles

Apoprotein A-I is a small molecule (28 kDa) and HDL particles contain several of them. They are activators of LCAT, that means the concentration of Apo A-I is a marker of HDL particle function and the metabolic activity of the particles and not their size or number.

Fig. 2. Apoprotein B100 is a big ringlike flexible molecule holding the whole particle together in its different stages of metabolism and making its binding to LDL-receptor

concentration of LDL-C in everyday practice. It was the calculation according to Friedewald formula (Friedewald et al, 1972). The formula was based on the analysis of a large LDL database created in research laboratories using ultracentrifugation. It was however clear that a calculation from three (TC, TG, HDL-C) measured values with their own analytical

The uncertainity of the currently employed 3rd generation methods is much lower than that of the Friedewald formula or the older methods but from strictly analytical point of view they are also not sufficient to obtain accurate results (Bairaktari et al., 2005). Another unsolved problem is that the assay methods from different providers are not yet

The discovery of immunochemical methodology in 70's made apoprotein assays possible. In the same time their function and their role in lipid metabolism was identified. According to recent view some of them belong to parameters providing important information when assessing cardiovascular disease risk (Ritz & Wanner, 2004, Batista et al, 2005) There are three apoproteins which should be measured in each patiens with elevated risk of

Only one molecule of ApoB100 is present in each LDL-type particle (Fig 2). ApoB100 is a big molecule (m.w. 550 kDa) crucial already in the synthesis of VLDL and playing an essential role in the removal of LDL from the circulation. Measurement of ApoB100 in routine clinical practice is possible and in contrast to dynamic and heterogenous entities as "HDLcholesterol" and "LDL-cholesterol" is an unambiguously defined analyte. The concentration of ApoB100 therefore provides the best information about the presence of LDL-type particles circulating in the blood (Olofsson et al, 2007). Elevated concentration apoB100 and normal or slightly elevated LDL-C or nonHDL-C is an indirect but valuable marker of small

Apoprotein A-I is a small molecule (28 kDa) and HDL particles contain several of them. They are activators of LCAT, that means the concentration of Apo A-I is a marker of HDL particle function and the metabolic activity of the particles and not their size or number.

Fig. 2. Apoprotein B100 is a big ringlike flexible molecule holding the whole particle together in its different stages of metabolism and making its binding to LDL-receptor

uncertainity is only a recourse from necessity.

**3.3 Apoprotein measurements** 

dense LDL particles

harmonized at all (Fuentes-Arderiu et al, 2009, Miller et al, 2010).

cardiovascular disease: Apoprotein B100, apoprotein A-I and lipoprotein(a).
