Determination of cholesterol in erythrocyte membrane

Abstract

Quantification of cholesterol in biological membranes is an important step toward understanding of metabolism of intracellular cholesterol, composition of cell membrane and plasma lipid profile. The aim of our study was to optimize the method for determination of cholesterol in erythrocyte membrane and then to use this method in the determination of cholesterol concentration in erythrocyte membrane in the studied group of blood samples. Cholesterol in erythrocyte membrane was determined in dry lipid extract of erythrocyte membrane by the enzymatic manual CHOD-PAP method. Cholesterol in erythrocyte membrane and plasma lipid parameters (total cholesterol HDL-cholesterol, LDL-cholesterol and triglycerides) were determined in blood samples of 58 females, obtained by routine health control. Lipid parameters were determined by standard biochemical methods. We examined the relationship between cholesterol in erythrocyte membrane and other plasma lipid parameters as well as other atherogenic risk factors (BMI, blood pressure). Optimization of the method for determination of cholesterol in erythrocyte membrane was based on the observation that primary cholesterol standards prepared by dissolving crystal cholesterol cannot be used due to the interference of the dissolved dry extract in organic solvents with the enzymatic reagent. Commercial standard solutions of cholesterol were used for calibration because they contain detergents for solubilisation of the dry extract in enzymatic reagent. The obtained mean value for cholesterol in erythrocyte membrane, as mmol/L erythrocyte is 4.44 ± 1.019; median 4.65 5-th percentile is 2.70, and 95-th percentile is 6.26. In the examined female group we tested cholesterol concentration in erythrocyte membrane according to age. Two groups were formed (females below and above 50 years) using nonparametric t-test no statistically significant difference was found between these two age groups (p>0.05), while plasma lipid parameters of total cholesterol, triglycerides and LDL-cholesterol were different in the examined groups (p lt 0.05). By Spearmen nonparametric correlation method we found no statistically significant correlation between cholesterol in erythrocyte membrane and other atherogenic risk factors.

Određivanje membranskog holesterola je važan stepen u utvrđivanju povezanosti intracelularnog metabolizma holesterola, sastava ćelijske membrane i plazmatskog lipidnog profila. Prvi cilj ovog rada je bio optimizacija metode za određivanje membranskog holesterola što je podrazumevalo izbor standarda i primenu enzimskog testa. Holesterol u membrani eritrocita određivan je ručno u suvom lipidnom ekstraktu membrane, enzimskom CHOD-PAP metodom firme Randox. Optimizirana metoda je primenjena za određivanje koncentracija holesterola u membrani eritrocita kod 58 žena kojima je krv uzeta na rutinskom sistematskom pregledu. U ispitivanim uzorcima određen je i serumski lipidni profil standardnim biohemijskim metodama. Ispitivane žene su podeljene u dve starosne grupe: na mlađe od 50 godina i na starije od 50 godina. Ispitan je i stepen povezanosti između membranskog holesterola sa jedne strane i plazmatskih lipida i drugih faktora rizika za aterosklerozu (starosti, BMI, sistolni i dijastolni pritisak) sa druge strane. Optimizacijom metode za određivanje membranskog holesterola došlo se do zaključka da se ne mogu koristiti primarni standardni rastvori holesterola dobijeni rastvaranjem kristalnog holesterola u organskom rastvaraču zbog slabe rastvorljivosti suvog ekstrakta u enzimskom reagensu i uticaja pojedinih rastvarača na bojenu reakciju. Za kalibraciju treba koristiti komercijalne standardne rastvore holesterola jer sadrže deterdžente koji omogućavaju rastvorljivost suvog ekstrakta. U ispitivanoj populaciji dobijena srednja vrednosti za holesterol u membrani eritrocita (izražene kao mmol/L eritrocita) iznosi 4,44 ± 1,019, mediana 4,65, 5-ti procenat iznosi 2,70, a 95-ti procenat je 6,26. Poređenjem ispitivanih parametara između dve starosne grupe za holesterol u eritrocitnoj membrani nije bilo značajne razlike (p>0,05) dok je za ukupan holesterol, LDL-holesterol i trigliceride ova razlika bila statistički značajna (p lt 0,05). Primenom Spearmanove neparametarske korelacije nije nađena statistički značajna korelacija između membranskog holesterola i drugih faktora rizika za aterosklerozu.