Ispitivanje biomarkera homeostaze holesterola i metabolizma vitamina D kod pacijenata sa kolorektalnim karcinomom

Abstract

Kolorektalni karcinom (CRC) je maligno oboljenje sa visokom prevalencijom u razvijenim zemljama. Holesterol poseduje mnogobrojne funkcije, te se pretpostavlja da ima ulogu i u procesima maligne transformacije i proliferacije. Procena efikasnosti procesa sinteze i apsorpcije holesterola, određivanjem cirkulišućih nivoa neholesterolskih sterola (NHS), može pružiti značajne podatke o metabolizmu holesterola, kao i njegovom uticaju na status drugih biomolekula (vitamin D, steroidni hormoni). Ciljevi ovog istraživanja bili su uvođenje i validacija metoda tečne hromatografije sa tandem masenom detekcijom (HPLC-MS/MS) za kvantifikaciju markera sinteze (dezmosterol i latosterol) i apsorpcije holesterola (kampesterol, β-sitosterol) u serumu i izolovanoj serumskoj frakciji lipoproteina visoke gustine (HDL), kao i razvoj i validacija metode za kvantifikaciju prekursora (7-dehidroholesterol, 7-DHC) i metabolita vitamina D (25(OH)D3, 24,25(OH)2D3) u serumu. Osim toga, ispitivali smo i status markera sinteze i apsorpcije holesterola, kao i metabolizma vitamina D kod osoba sa CRC, i nastojali da utvrdimo povezanost između metabolizma ovih jedinjenja, kao i da procenimo njihov prediktivni potencijal za CRC. U istraživanju je učestvovao 101 pacijent sa dijagnozom CRC i 114 zdravih ispitanika. Pacijenti su podeljeni prema patološkim karakteristikama na sledeće grupe: karcinom kolona (CA kolona) i karcinom rektuma (CA rektuma); gradus I i objedinjeni gradusi II i III; stadijumi A i B, te stadijumi C i D. Koncentracije NHS u serumu i HDL frakciji, kao i koncentracije parametara statusa vitamina D u serumu, određivani su HPLCMS/ MS metodama. Na osnovu koncentracija NHS u serumu i HDL frakciji, izračunati su različiti indeksi homeostaze holesterola, dok je odnos metabolita vitamina D (VDMR) izračunat na osnovu koncentracija 25(OH)D3 i 24,25(OH)2D3 u serumu. Rezultati studije su pokazali da je holesterol glavni interferent prilikom određivanja NHS, pa je u tom smislu izvršena priprema odgovarajućeg matriksa za kvantitativnu analizu. Varijacije unutar serije su bile 4.7-10.3% za NHS u serumu i 3.6-13.6% za NHS u HDL frakciji, a između serija 4.6-9.5% za NHS u serumu i 2.5–9.8% za NHS u HDL frakciji. Studije prinosa analita su pokazale zadovoljavajuće rezultate: 89.8–113.1% za NHS u serumu i 85.3–95.8% za NHS i HDL frakciji. Pacijenti su imali značajno niže koncentracije NHS u serumu (p<0,001, za sve) i u HDL frakciji (p<0,001 za dezmosterol i β-sitosterol, p<0,005 za kampesterol, p<0,05 za latosterol)...

Colorectal cancer (CRC) is a malignancy highly prevalent in developed countries. Cholesterol is a biomolecule with multiple functions and suspected important role in malignant transformation and proliferation. Assessing the efficiency of cholesterol synthesis and absorption processes, by determining circulating levels of non-cholesterol sterols (NCS), can provide significant data on cholesterol metabolism as well as its effect on other biomolecules (vitamin D, steroid hormones). The objectives of this study were to introduce and validate liquid chromatography - tandem mass detection (HPLC-MS/MS) methods for quantification of cholesterol synthesis (desmosterol and lathosterol) and cholesterol absorption (campesterol, β-sitosterol) markers in serum and serum high density lipoprotein fraction (HDL), as well as the method for quantification of vitamin D status parameters (7-dehydrocholesterol (7-DHC), 25(OH)D3, 24,25(OH)2D3). In addition, we examined the status of cholesterol synthesis and absorption markers, as well as vitamin D metabolites in individuals with CRC, and explored the association between these two metabolic pathways, as well as evaluated their predictive potential for CRC. The study involved 101 patients with CRC diagnosis and 114 healthy subjects. Patients were divided according to pathological characteristics into the following groups: colon cancer (colon CA) and rectal cancer (rectal CA); grade I, and grade II and III combined; stages A and B and stages C and D. NCS concentrations in serum and HDL fraction, as well as concentrations of serum vitamin D status parameters, were determined by HPLC-MS/MS. Based on NCS concentrations in serum and HDL fraction, different cholesterol homeostasis indices were calculated. Vitamin D metabolite ratio (VDMR) was calculated based on serum 25(OH)D3 and 24,25(OH)2D3 concentrations. The study results showed that cholesterol was the main interferent in the determination of NCS, and in this respect, the appropriate matrix for quantitative analysis was prepared. Within-series variations were 4.7-10.3% for serum NHS and 3.6-13.6% for NCS in HDL fraction, whereas between series variations were 4.6-9.5% for serum NHS and 2.5-9.8% for NCS in HDL fraction. Recovery study has shown satisfactory results: 89.8–113.1% for serum NCS and 85.3–95.8% for NHS and HDL fraction. Patients had significantly lower NCS concentrations (p<0.001, for all) in serum and in the HDL fraction (p<0.001 for desmosterol and β-sitosterol, p<0.005 for campesterol, p<0.05 for latosterol)...