Assessment of biomarkers of cholesterol homeostasis and vitamin D metabolism in patients with colorectal cancer

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)..

Interleukin-6: uloga u laboratorijskoj dijagnostici inflamacije i ograničenja pri određivanju

Uvod i cilj. Određivanje koncentracije interleukina-6 (IL-6) od velikog je značaja za pra- ćenje toka bolesti i oporavka pacijenata od COVID-19 infekcije. Cilj ovog ispitivanja je da se objasni uticaj koncentracije biotina kao interferencije na određivanje koncentracije IL-6 nekompetitivnom ECLIA metodom. Metode. Korišćene su ECLIA i hemiluminiscentne metode za određivanje koncentracije IL-6 (Roche e411 - ECLIA metoda i IMMULITE 1000 - hemiluminiscencija). Za procenu interfe- rencije od značaja su bili anamnestički, kao i podaci o terapiji koju pacijent koristi. Rezultati. Dobijene su značajne razlike u koncentracijama IL-6 prilikom određivanja sa ove dve različite metode. Koncentracije IL-6 su bile višestruko niže kod ECLIA metode (<1.5 pg/mL za Roche e411-ECLIA i 8.5 pg/mL za IMMULITE 1000). Interferencija usled prisustva egzogenog biotina u ispitivanom uzorku može uzrokovati „lažno“ negativne rezultate prilikom određivanja ovog parametra ECLIA metodom. Zaključak. Određivanje IL-6 imunohemijskim testovima je ograničeno mogućim interferen- cijama. Egzogeni biotin u visokim koncenracijama značajno utiče na nivo IL-6 u serumu kada se određivanje vrši ECLIA metodom. Preporuka je informisati pacijente o adekvatnoj pripremi za imunohemijske analize ukoliko primenjuju oralne preparate koji sadrže biotin i time svesti mogućnost greške pri izdavanju laboratorijskih rezultata na najmanju moguću mer

Prediction of in vivo Bioavailibility by in vitro Characterization of Ethylenediamine Dipropanoic Acid Derivatives with Cytotoxic Activity

O,O'-diethyl-(S, S)-ethylenediamine-N, N'-di-2-(3-cyclohexyl) propanoate (DE-EDCP) is novel substance with cytotoxic activity in human leukemic cells. The aim of this study has been to predict in vivo bioavailability of the DE-EDCP and its potential metabolite (S, S)-ethylenediamine-N, N'-di-2-(3-cyclohexyl) propanoic acid (EDCP) by in vitro characterization which includes determination of lipophilicity and passive membrane permeability. There has also been evaluated inter-laboratory reproducibility of the bio-analytical method which was previously developed and validated for non-clinical study of the DE-EDCP and EDCP. Distribution coefficient n-octanol/water was 1.68 and 0.03, and apparent permeability coefficient was 4 x 10(-4) cm/s and 20 x 10(-4) cm/s, for the DE-EDCP and EDCP, respectively. Observed results have shown that the DE-EDCP is more lipophilic with better membrane retention, but the EDCP has better pass through the membrane. Also, there has been demonstrated a reproducibility and robustness of the proposed bio-analytical method

Izazovi primene hromatografskih tehnika u postavljanju dijagnoze SARS-CoV-2 infekcije

Preventivne mere, rano otkrivanje i potvrda novih slučajeva infekcije, predstavljaju osnov u sprečavanju širenja i suzbijanju ininfektivne bolesti COVID-19. Primena visoko osetljive, spe- cifične real-time RT-PCR metode predstavlja zlatni standard u detekciji SARS-CoV-2. Brza i pouzdana dijanoza su neophodni za efikasno praćenje bolesti, ali veliki broj lažno negativnih slučajeva omogućio je nekontrolisanu transmisiju infekcije. Nove metode za detekciju SARS-CoV-2 iz nazofaringealnog brisa zasnovane na principu tečne hromatografije sa masenom spektrometrijom (HPLC-MS/MS) isključivo se koriste u istraživačke svrhe. Hromatografski testovi omogućavaju istovremenu detekciju više različitih, specifičnih peptidnih markera za identifikaciju SARS-CoV-2. Na ovaj način, moguće mutacije u genskoj sekvenci virusa, lako mogu biti prevaziđene. Upotreba gasne hromatografije sa spektrometrijom pokretljivosti jona (GC-IMS) za detekciju odabranih molekula u izdahnutom vazduhu pacijenata sa COVID-19 može omogućiti neinvazivnu, brzu i tačnu, „point of care” potvrdu dijagnoze bolesti. Uprkos superiornim analitičkim performansama hromatografskih tehnika, njihova primena u rutinskoj laboratorijskoj praksi je retka. Pored opreme, njihova primena zahteva obučeno osoblje i „in house” procedure validacije i verifikacije metoda. Protokoli validacije hromato- grafskih metoda se oslanjaju na preporuke date u naučnim publikacijama i različitim smer- nicama, te su istraživački orijentisani. Najčešće korišćene su EMA, FDA i CLSI smernice za postupke i procedure validacije metoda. Međutim, ove smernice dozvoljavaju različita tuma- čenja i ostavljaju analitičaru da odluči koji od parametara validacije su neophodni. Različiti preanalitički i analitički aspekti hromatografskih metoda diktiraju složenost kriterijuma vali- dacije. Zbog toga je neophodno izdvojiti najvažnije postupke validacije hromatografskih teh- nika (određivanje linearnosti, LOQ i LOD vrednosti, tačnosti i preciznosti metode) i primeniti dostupnu opremu i naučna saznanja

LncRNAs as Regulators of Atherosclerotic Plaque Stability

Current clinical data show that, despite constant efforts to develop novel therapies and clinical approaches, atherosclerotic cardiovascular diseases (ASCVD) are still one of the leading causes of death worldwide. Advanced and unstable atherosclerotic plaques most often trigger acute coronary events that can lead to fatal outcomes. However, despite the fact that different plaque phenotypes may require different treatments, current approaches to prognosis, diagnosis, and classification of acute coronary syndrome do not consider the diversity of plaque phenotypes. Long non-coding RNAs (lncRNAs) represent an important class of molecules that are implicated in epigenetic control of numerous cellular processes. Here we review the latest knowledge about lncRNAs’ influence on plaque development and stability through regulation of immune response, lipid metabolism, extracellular matrix remodelling, endothelial cell function, and vascular smooth muscle function, with special emphasis on pro-atherogenic and anti-atherogenic lncRNA functions. In addition, we present current challenges in the research of lncRNAs’ role in atherosclerosis and translation of the findings from animal models to humans. Finally, we present the directions for future lncRNA-oriented research, which may ultimately result in patient-oriented therapeutic strategies for ASCVD

Cholesterol homeostasis is dysregulated in women with preeclampsia

Introduction The link between preeclampsia and dyslipidemia has been established. Even though lipid profile parameters have been intensively investigated in the pathology of preeclampsia, their accurate molecular mechanisms of action have not been fully decoded. Objectives We aimed to identify the specifics of cholesterol metabolism in women affected by late‑onset preeclampsia and single out potential biomarkers associated with late‑onset syndrome. Patient s and methods A total of 90 pregnant women with a priori risk for preeclampsia were monitored at 4 time points during gestation and, based on the outcome of pregnancy, they were classified into the high‑risk group (70 women) and the preeclampsia group (20 women). Cholesterol metabolic profiling was done using liquid chromatography‑tandem mass spectrometry. Result s The only significant change in the preeclampsia group was an increase in the lathosterol level (P = 0.001). The first‑trimester lathosterol level was higher in the preeclampsia group compared with the high‑risk group (P = 0.02). Further, in the preeclampsia group, positive correlations were found between desmosterol and β‑sitosterol (ρ = 0.474; P = 0.03) in the third trimester, desmosterol and campesterol changes between the second and the first (ρ = 0.546; P = 0.02), and the third and first trimesters (ρ = 0.754; P <0.001), as well as between the desmosterol and β‑sitosterol differences between the third and first trimesters (ρ = 0.568; P = 0.01). No similar correlations were found in the high‑risk group. Conclusions Late‑onset preeclampsia could be associated with an altered lipid profile. By studying the quantitative metabolic signatures of cholesterol, we might assume that both cholesterol synthesis and absorption are increased, that is, there is an imbalance in the cholesterol homeostasis regulation in women affected by the disease

Cholesterol Metabolic Profiling of HDL in Women with Late-Onset Preeclampsia

A specific feature of dyslipidemia in pregnancy is increased high-density lipoprotein (HDL) cholesterol concentration, which is probably associated with maternal endothelium protection. However, preeclampsia is most often associated with low HDL cholesterol, and the mechanisms behind this change are scarcely explored. We aimed to investigate changes in HDL metabolism in risky pregnancies and those complicated by late-onset preeclampsia. We analyze cholesterol synthesis (cholesterol precursors: desmosterol, 7-dehydrocholesterol, and lathosterol) and absorption markers (phytosterols: campesterol and β-sitosterol) within HDL particles (NCSHDL), the activities of principal modulators of HDL cholesterol’s content, and major HDL functional proteins levels in mid and late pregnancy. On the basis of the pregnancy outcome, participants were classified into the risk group (RG) (70 women) and the preeclampsia group (PG) (20 women). HDL cholesterol was lower in PG in the second trimester compared to RG (p < 0.05) and followed by lower levels of cholesterol absorption markers (p < 0.001 for campesterolHDL and p < 0.05 for β-sitosterolHDL). Lowering of HDL cholesterol between trimesters in RG (p < 0.05) was accompanied by a decrease in HDL phytosterol content (p < 0.001), apolipoprotein A-I (apoA-I) concentration (p < 0.05), and paraoxonase 1 (PON1) (p < 0.001), lecithin–cholesterol acyltransferase (LCAT) (p < 0.05), and cholesterol ester transfer protein (CETP) activities (p < 0.05). These longitudinal changes were absent in PG. Development of late-onset preeclampsia is preceded by the appearance of lower HDL cholesterol and NCSHDL in the second trimester. We propose that reduced capacity for intestinal HDL synthesis, decreased LCAT activity, and impaired capacity for HDL-mediated cholesterol efflux could be the contributing mechanisms resulting in lower HDL cholesterol

Effects of Gestational Diabetes Mellitus on Cholesterol Metabolism in Women with High-Risk Pregnancies: Possible Implications for Neonatal Outcome

Metabolic disorders in pregnancy, particularly gestational diabetes mellitus (GDM), are associated with an increased risk for adverse pregnancy outcome and long-term cardiometabolic health of mother and child. This study analyzed changes of serum cholesterol synthesis and absorption markers during the course of high-risk pregnancies, with respect to the development of GDM. Possible associations of maternal lipid biomarkers with neonatal characteristics were also investigated. The study included 63 women with high risk for development of pregnancy complications. Size and proportions of small low-density (LDL) and high-density lipoprotein (HDL) particles were assessed across trimesters (T1–T3), as well as concentrations of cholesterol synthesis (lathosterol, desmosterol) and absorption markers (campesterol, β-sitosterol). During the study, 15 women developed GDM, while 48 had no complications (non-GDM). As compared to the non-GDM group, women with GDM had significantly higher triglycerides in each trimester, while having a lower HDL-C level in T3. In addition, they had significantly lower levels of β-sitosterol in T3 (p < 0.05). Cholesterol synthesis markers increased across trimesters in both groups. A decrease in serum β-sitosterol levels during the course of pregnancies affected by GDM was observed. The prevalence of small-sized HDL decreased in non-GDM, while in the GDM group remained unchanged across trimesters. Newborn’s size in the non-GDM group was significantly higher (p < 0.01) and inversely associated with proportions of both small, dense LDL and HDL particles (p < 0.05) in maternal plasma in T1. In conclusion, high-risk pregnancies affected by GDM are characterized by altered cholesterol absorption and HDL maturation. Advanced lipid testing may indicate disturbed lipid homeostasis in GDM

Uticaj različitih uslova čuvanja uzoraka i kontaminacije uzoraka bakterijama na koncentracije rutinskih biohemijskih parametara

Background: The pre-analytical (PA) phase is the most vulnerable phase of laboratory testing procedure, with critical procedures-collection, handling, sample transport, and time and temperature of sample storage. The aim of this study was to examine if different anticoagulants, storage conditions, and freeze-thaw cycles (FTCs) influence the concentrations of basic biochemical parameters. In parallel, the presence and the effect of sample microbiological contamination during routine laboratory work were examined. Methods: Two plasma pools (EDTA, and sodium-fluoride/potassium oxalate plasma (NaF)) were stored at +4C˚/-20˚C. Total cholesterol (TC), glucose, triglycerides (TG), urea, total protein (TP), and albumin concentrations were measured using Ilab 300+. Sample microbiological contamination was determined by 16S rRNA sequence analysis. The experiment encompassed a 5 day-period: Day 1–fresh sample, Day 2–1st FTC, Day 3–2nd FTC, Day 4–3rd FTC, Day 5–4th FTC. The appearance of bacteria in two consecutive samples was the experiment's endpoint. Results: During 4 FTCs there were no changes in plasma urea concentrations. Glucose was stable in EDTA+4˚C and NaF- 20˚C until the 3rd FTC (P=0.008, P=0.042, respectively). Changes in protein concentrations followed the zig-zag pattern. TG concentrations changed significantly in the EDTA-20˚C sample after 1st and 4th FTCs (P=0.022, P=0.010, respectively). In NaF samples no contamination was observed during 4 FTCs. Conclusions: Urea and glucose concentrations were robust. Changes in lipid and protein concentrations after FTCs follow complex patterns. Bacterial growth was not observed in NaF plasma samples. This can promote NaF use in analytical procedures in which microbiological contamination affects the quality of analysis.Uvod: Preanaliti~ka (PA) faza je slo`en proces koji ~ine: prikupljanje, rukovanje, transport i skladi{tenje uzoraka, i predstavlja najzna~ajniji izvor laboratorijskih gre{aka. Cilj ovog istra`ivanja je bio da se ispita stabilnost osnovnih biohemijskih parametara u zavisnosti od uslova skladi{tenja uzoraka i broja ciklusa zamrzavanja-odmrzavanja (FTC). Pored toga, ispitivano je prisustvo bakterijske kontaminacije uzoraka tokom rutinskog laboratorijskog rada. Metode: Dva »pool«-a plazme (etilendiaminotetrasir}etna kiselina (EDTA) i natrijum-fluorid/kalijum oksalat (NaF)) su skladi{tena na +4 ˚C/-20 ˚C. Koncentracije ukupnog holesterola (TC), glukoze, triglicerida (TG), uree i albumina su odre|ene kori{}enjem BioSystems reagenasa (holesterol oksidaza/peroksidaza, glukoza oksidaza/peroksidaza, glice rol fosfat oksidaza/peroksidaza, ureaza/salicilat, od- nosno bromkrezol zeleno metodama, sukcesivno) na Ilab 300+ analizatoru. Bakterijska kontaminacija uzoraka je potvr|ena 16S rRNA sekvencioniranjem. Eksperiment je sproveden tokom 5 uzastopnih dana: 1. dan – sve` uzorak, 2. dan – 1. FTC, 3. dan –2. FTC, 4. dan – 3. FTC, 5. dan – 4. FTC. Zavr{nu ta~ku eksperimenta predstavljala je pojava bakterija u dva uzastopna uzorka. Rezultati: Tokom 4 FTC koncentracije uree u plazmi se nisu zna~ajno razlikovale. Koncentracija glukoze je bila stabilna u EDTA +4 ˚C i NaF -20 ˚C do 3.FTC (P=0,008, P=0,042, redom). Koncentracije TG su se zna~ajno pro- menile u uzorku EDTA -20 ˚C nakon 1. i 4. FTC-a (P=0,022, P=0,010, redom). U uzorcima NaF plazme nije do{lo do bakterijske kontaminacije tokom 4. FTC. Zaklju~ak: Koncentracije uree i glukoze su bile stabilne tokom trajanja eksperimenta. Promene u koncentracijama lipida nakon FTC prate slo`ene obrasce. Rast bakterija nije prime}en u uzorcima NaF plazme, te upotreba ovog anti- koagulansa mo`e biti opravdana u analiti~kim proce - durama podlo`nim uticaju mikrobiolo{ke kontaminacije

Novi biomarkeri u procjeni rizika za razvoj preeklampsije

Despite significant progress in improving pregnancy outcomes in recent decades, predicting the risk and treatment of preeclampsia are still major challenges in clinical practice (1). The aim of this study was to examine non-routine biomarkers in preeclampsia risk assessment. The study involved 90 women with high-risk pregnancies, 20 of whom developed preeclampsia by the end of pregnancy. Biochemical parameters were determined between the 12th and 13 th weeks of gestation. The results of the study showed that women who later developed preeclampsia had higher concentrations of lathosterol, cholesterol synthesis marker (p <0.05), inflammatory proteins - monocyte chemoattractant protein-1 (MCP-1), and resistin (p <0.01, both), as well as paraoxonase-1 (PON1) activity (p <0.05). Binary logistic regression analysis showed that higher concentrations of lathosterol, MCP-1, resistin, and PON-1 were associated with preeclampsia development. To determine whether the parameters significant in univariate analysis, are independent predictors of preeclampsia, we applied multivariate regression analysis. Clinical markers commonly used in risk assessment (maternal age and body mass index, mean arterial pressure, and uterine blood flow), lathosterol, MCP-1, resistin, and PON-1 were included in the model. MCP-1 and resistin stood out as significant independent predictors of preeclampsia. The diagnostic accuracy of the investigated model was excellent (AUC=0.859). The study results indicated the importance of a multi-marker approach in risk assessment for preeclampsia development.Uprkos značajnom napretku u poboljšanju ishoda trudnoće poslednjih decenija, predviđanje rizika i terapija preeklampsije su još uvijek veliki izazovi u kliničkoj praksi (1). Cilj ove studije je bio ispitivanje biomarkera koji se ne koriste u rutinskoj praksi u proceni rizika za razvoj preeklampsije. U studiji je učestvovalo 90 žena sa visokorizičnim trudnoćama, od kojih je 20 razvilo preeklampsiju do kraja trudnoće. Biohemijski parametri su određivani između 12. i 13. nedelje gestacije. Rezultati studije su pokazali da su žene koje su razvile preeklampsiju imale više koncentracije latosterola, markera sinteze holesterola (p <0,05), inflamatornih proteina - monocitnog hemoatraktantnog proteina-1 (MCP-1) i rezistina (p < 0,01, oba), kao i aktivnost enzima paraoksonaze-1 (PON1) (p <0,05). Binarna logistička regresiona analiza je pokazala da su više koncentracije latosterola, MCP-1, rezistina i PON-1 povezane sa razvojem preeklampsije. Da bi se utvrdilo da su parametri koji su se u univarijantnoj analizi pokazali značajnim, nezavisni prediktori preeklampsije, primjenili smo multivarijantnu regresionu analizu. U model su ušli klinički parametri koji se uobičajeno koriste u procjeni rizika (starost i indeks tjelesne mase majke, srednji arterijski pritisak i protok krvi kroz matericu), latosterol, MCP-1, rezistin i PON-1. MCP-1 i rezistin su se istakli kao značajni nezavisni prediktori preeklampsije. Pokazana je odlična dijagnostička tačnost ispitivanog modela (AUC=0,859). Rezultati ove studije su ukazali na značaj multimarkerskog pristupa u procjeni rizika za razvoj preeklampsije.VIII Kongres farmaceuta Srbije sa međunarodnim učešćem, 12-15.10.2022. Beogra