Markers of cholesterol synthesis and absorption and sphingolipid profile in preeclampsia

[T. D. Antonić Krejović]

139 str.

Medicinske nauke – Farmacija - Medicinska biohemija / Medical Sciences – Pharmacy - Medical Biochemistry

Uprkos napretku u razjašnjavanju patofizioloških mehanizama uključenih u razvoj preeklampsije, jedina efikasna terapija je porođaj, a rizik za razvoj preeklampsije se najčešće procjenjuje na osnovu demografskih karakteristika i faktora rizika prisutnih kod majke, koji imaju niske pozitivne prediktivne vrijednosti. Kako kardiovaskularne bolesti i preeklampsija dijele niz zajedničkih faktora rizika, a poremećaji u metabolizmu lipida i lipoproteina se smatraju ključnim faktorima u patogenezi ateroskleroze i razvoju kardiovaskularnih bolesti, nameće se potreba za detaljnijim ispitivanjem promjena u metabolizmu lipida i njihovog značaja u predikciji rizika za razvoj preeklampsije. Cilj ovog istraživanja bio je longitudinalno praćenje specifičnih komponenti lipidoma kod žena sa visokorizičnom trudnoćom, analiza razlika u specifičnim elementima lipidoma između trudnica sa i bez preeklampsije, i procjena dijagnostičke tačnosti ispitivanih parametara. U studiju je uključeno 90 trudnica sa visokim rizikom sa razvoj preeklampsije, a koje su se javile radi praćenja trudnoće u Ginekološko-akušersku kliniku „Narodni Front“ u Beogradu. Na osnovu primarnog ishoda trudnoće, ispitanice su klasifikovane u grupu trudnica sa visokorizičnom trudnoćom (RG), koja nije razvila preeklampsiju, i grupu trudnica sa preeklampsijom (PG). U prvoj fazi istraživanja određeni su osnovni klinički i biohemijski parametri i osnovni parametri lipidnog profila tokom prvog, drugog, trećeg trimestra i pred porođaj. Koncentracije neholesterolskih sterola (NHS) u serumu i lipoproteinskim česticama visoke gustine (HDL) su određene metodama tečne hromatografije sa tandem masenom detekcijom (HPLC-MS/MS), prethodno implementiranim i validiranim u laboratoriji Katedre za medicinsku biohemiju, a izabrani sfingolipidi HPLC-MS/MS metodom razvijenom za potrebe ove studije, takođe, u četiri tačke tokom gestacije. Masena koncentracija apolipoproteina M (apoM) je određivana tehnikom enzimskog imunoeseja. Aktivnosti lecitin-holesterol aciltransferaze (LCAT) i holesterol-estar transfernog proteina (CETP) su procijenjene na osnovu brzine stvaranja i prenosa holesterol estara. Aktivnost paraoksonaze 1 (PON1) mjerena je kinetički korišćenjem paraoksona kao supstrata. U okviru studije uspješno je optimizovana i validirana HPLC-MS/MS metoda za kvantifikaciju sfingozina, sfinganina, sfingozin-1-fosfata (S1P), sfinganin-1-fosfata, ceramida C16:0 (Cer C16:0) i C24:0 (Cer C24:0), i sfingomijelina C16:0 (SM C16:0). Zbog izraženog efekta matriksa, prilikom konstrukcije kalibracionih krivih kao matriks je korišten pool humane plazme. Varijacije unutar serija su bile 3,8-18,8%, a između serija 4,1-19,4%. Tačnost se kretala u opsegu od ± 15% od nominalne vrijednosti. Koncentracije triglicerida su bile značajno više u PG u odnosu na RG (p <0,05 u svim ispitivanim tačkama), dok je koncentracija HDL holesterola bila značajno niža u PG u 2. trimestru u poređenju sa RG (p <0,05). Povećanje koncentracije holesterola u RG je bilo praćeno značajnim povećanjem koncentracija holesterolskih prekursora dezmosterola (p <0,001), 7-dehidroholesterola (p <0,05) i latosterola (p <0,001). S druge strane, u PG uprkos povećanju koncentracije ukupnog holesterola, nisu uočene značajne promjene u koncentracijama dezmosterola (p = 0,427) i 7- dehidroholesterola (p =0,368), već samo u koncentraciji latosterola (p <0,05). Porast koncentracije holesterolskh prekursora krajem trudnoće u RG bio je praćen značajnim padom koncentracija markera apsorpcije kampesterola (p <0,05) i β-sitosterola (p <0,05), dok u PG uprkos porastu koncentracije latosterola nije bilo značajnih promjena u koncentracijama biljnih sterola (p =0,364 za kampesterol i p =0,426 za β-sitosterol), što upućuje na djelimično izmjenjenu homeostazu holesterola u PG. Uočeni su različiti obrasci u sfingolipidnim profilima između ispitivanih grupa. Koncentracije S1P u RG su se povećavale od 1. trimestra do porođaja (p <0,001)...

Despite advancements in elucidating the pathophysiological mechanisms involved in the development of preeclampsia, delivery remains the only effective therapy. The risk of developing preeclampsia is most commonly assessed based on demographic characteristics and maternal risk factors, which have low positive predictive values. Since cardiovascular diseases and preeclampsia share numerous risk factors, and lipid and lipoprotein metabolism disorders are considered key factors in the pathogenesis of atherosclerosis and the development of cardiovascular diseases, there is a need for a more detailed investigation of lipid metabolism changes and their significance in predicting the risk of preeclampsia. The aim of this study was to longitudinally monitor specific components of the lipidome in women with high-risk pregnancies, analyze differences in specific lipidome elements between women with and without preeclampsia, and evaluate the diagnostic accuracy of the examined parameters. The study included 90 pregnant women at high risk for developing preeclampsia, who were monitored at the Gynecology and Obstetrics Clinic "Narodni Front" in Belgrade. Based on the primary pregnancy outcome, participants were classified into a high-risk pregnancy group (RG) that did not develop preeclampsia and a preeclampsia group (PG). In the first phase of the study, basic clinical and biochemical parameters, as well as lipid profile parameters, were determined during the first, second, and third trimesters, and before delivery. Concentrations of non-cholesterol sterols (NCS) in serum and high-density lipoprotein particles (HDL) fraction were measured using liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) methods previously implemented and validated in the laboratory of the Department of Medical Biochemistry. Selected sphingolipids were measured using an HPLCMS/ MS method developed specifically for this study, also at four gestational points. The mass concentration of apolipoprotein M (apoM) was determined using an enzyme immunoassay technique. The activities of lecithin-cholesterol acyltransferase (LCAT) and cholesterol ester transfer protein (CETP) were assessed based on the rates of cholesterol ester formation and transfer. Paraoxonase 1 (PON1) activity was measured kinetically using paraoxon as a substrate. In this study, an HPLC-MS/MS method was successfully optimized and validated for quantifying sphingosine, sphinganine, sphingosine-1-phosphate (S1P), sphinganine-1-phosphate, ceramide C16:0 (Cer C16:0), ceramide C24:0 (Cer C24:0), and sphingomyelin C16:0 (SM C16:0). Due to a pronounced matrix effect, a pool of human plasma was used as the matrix for constructing calibration curves. Intraassay variations ranged from 3.8% to 18.8%, while inter-assay variations were between 4.1% and 19.4%, with accuracy within ±15% of the nominal values. Triglyceride concentrations were significantly higher in the PG compared to the RG (p < 0.05 at all points), while HDL cholesterol levels were significantly lower in PG during the 2nd trimester compared to RG (p < 0.05). In RG, an increase in cholesterol concentrations was accompanied by significant increases in cholesterol precursors desmosterol (p < 0.001), 7-dehydrocholesterol (p < 0.05), and lathosterol (p < 0.001). In PG, despite rising total cholesterol levels, no significant changes were observed for desmosterol (p = 0.427) or 7-dehydrocholesterol (p = 0.368), with only lathosterol showing a significant increase (p < 0.05). Late pregnancy increases in cholesterol precursors in RG were associated with significant decreases in absorption markers campesterol (p < 0.05) and β-sitosterol (p < 0.05). In contrast, PG showed no significant changes in phytosterols’ concentrations despite lathosterol increases (p = 0.364 for campesterol and p = 0.426 for β-sitosterol), indicating altered cholesterol homeostasis in PG. Distinct sphingolipid profiles were observed between the groups. S1P concentrations in RG increased from the 1st trimester to delivery (p < 0.001)...

preeklampsija, lipidni status, neholesterolski steroli, prekursori holesterola, fitosteroli, HDL, sfingolipidi, sfingozin-1-fosfat, ceramidi, sfingomijelin

preeclampsia, lipid profile, non-cholesterol sterols, cholesterol precursors, phytosterols, HDL, sphingolipids, sphingosine-1-phosphate, ceramides, sphingomyelin

615:618.3(043.3)

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