Primena LC‐MS/MS metoda u ispitivanju parametara oksidativnog stresa

Oxidative stress is a phenomenon that occurs due to the disturbance in the balance between the production of reactive oxygen species and the ability of biological systems to remove the resulting compounds. Oxidative stress is involved in the pathogenesis of many disorders in the human organism. This indicates the importance of quantification of oxidative stress parameters in biological samples. Traditionally, these parameters are determined by biochemical tests. Although these tests are routinely performed, they have many drawbacks. To determine the exact concentration of selected compounds, more sensitive analytical methods are becoming more important. In the modern scientific literature, liquid chromatography-tandem mass spectrometry (LC-MS/MS) is increasingly mentioned (1). This method, with adequate selection of stationary and mobile phases, enables quantification of very low concentrations of selected parameters. In addition, these methods can simultaneously determine the concentration of many selected components. However, it is necessary to take into account that LC-MS/MS methods require a very purified biological sample from which the proteins have been maximally removed. In this work, we will present the results of determination of cysteine, cystine, reduced and oxidized glutathione in patients with microcellular lung cancer. The use of LC-MS/MS methods is becoming increasingly common for the analysis of oxidative stress markers in biological fluids. In the future, we are expected to move to even more modern, fully automated methods, which simultaneously purify and analyze samples.Oksidativni stres je pojava koja nastaje usled narušavanja ravnoteže između proizvodnje i nagomilavanja reaktivnih jedinjenja kiseonika u organizmu i sposobnosti bioloških sistema da ukloni nastala jedinjenja. Oksidativni stres učestvuje u patogenezi mnogih poremećaja kao što su kardiovaskularne bolesti, dijabetes i bolesti bubrega. Ovo ukazuje na značaj određivanja odabranih parametara oksidativnog stresa u uzorcima biološkog materijala. Tradicionalno, najveći broj parametara se određuje biohemijskim testovima. Iako se ovi testovi rutinski izvode, oni imaju mnoge nedostatke. Da bi se odredila tačna koncentracija izabranih parametara, koji se u uzorcima nalaze u veoma niskim koncentracijama, sve veću prednost imaju osetljivije analitičke metode. U savremenoj naučnoj literaturi se u te svrhe sve više spominje tečna hromatografija spregnuta sa masenim detektorom (LC-MS/MS) (1). Ova metoda, uz adekvatan izbor stacionarne i mobilne faze, omogućava kvantifikaciju veoma niskih koncentracija odabranih jedinjenja. Sem toga, ovim metodama se istovremeno, u jednom uzorku, može odrediti koncentracija većeg broja odabranih komponenti. Ipak, neophodno je voditi računa o tome da tečna hromatografija spregnuta sa masenim detektorom zahteva dobro prečišćen uzorak biološkog materijala iz kog su maksimalno uklonjeni proteini. U ovom radu biće prikazani rezultati određivanja cisteina, cistina, redukovanog i oksidovanog glutationa kod pacijenata sa mikrocelularnim karcinomom pluća. Upotreba LC-MS/MS metoda je sve uobičajenija za analizu markera oksidativnog stresa u biološkim tečnostima zbog svoje osetljivosti. U budućnosti se očekuje razvoj ka još savremenijim, potpuno automatizovanim metodama, kojima se istovremeno uzorci prečišćavaju i analiziraju.VIII Kongres farmaceuta Srbije sa međunarodnim učešćem, 12-15.10.2022. Beogra

Analysis of the Prognostic Potential of Schlafen 11, Programmed Death Ligand 1, and Redox Status in Colorectal Cancer Patients

The Schlafen 11 (SLFN11) protein has recently emerged as pivotal in DNA damage conditions, with predictive potential for tumor response to cytotoxic chemotherapies. Recent discoveries also showed that the programmed death ligand 1 (PD-L1) protein can be found on malignant cells, providing an immune evasion mechanism exploited by different tumors. Additionally, excessive generation of free radicals, redox imbalance, and consequential DNA damage can affect intestinal cell homeostasis and lead to neoplastic transformation. Therefore, our study aimed to investigate the significance of SLFN11 and PD-L1 proteins and redox status parameters as prognostic biomarkers in CRC patients. This study included a total of 155 CRC patients. SLFN11 and PD-L1 serum levels were measured with ELISA and evaluated based on redox status parameters, sociodemographic and clinical characteristics, and survival. The following redox status parameters were investigated: spectrophotometrically measured superoxide dismutase (SOD), sulfhydryl (SH) groups, advanced oxidation protein products (AOPP), malondialdehyde (MDA), pro-oxidant–antioxidant balance (PAB), and superoxide anion (O2•–). The prooxidative score, antioxidative score, and OXY-SCORE were also calculated. The results showed significantly shorter survival in patients with higher OXY-SCOREs and higher levels of serum SLFN11, while only histopathology-analysis-related factors showed significant prognostic value. OXY-SCORE and SLFN11 levels may harbor prognostic potential in CRC patients

Oksidativni stres i dužina telomera kod COVID‐19 pacijenata

The pathogenesis of the COVID-19 infection caused by the SARS-CoV-2 coronavirus is the subject of active research worldwide. As a complex disease, it is suspected that oxidative stress plays a role in its pathogenesis and contributes to the progressive shortening of telomeres. Patients aged 25–84 years (n=31) were monitored at the time of diagnosis, as well as at control on the 14th and 21st days. Redox status parameters were determined: total oxidant status (TOS), superoxide anion radical (O2.-), prooxidant-antioxidant balance (PAB), advanced protein oxidation products (AOPP), malondialdehyde (MDA), and ischemia- modified albumin (IMA) as prooxidants and total antioxidant status (TAS), superoxide- dismutase (SOD) and paraoxonase 1 (PON1) enzyme activity, total antioxidant and total oxidant status ratio (TAS/TOS), and total sulfhydryl groups (SHG) as antioxidants. Patients have the shortest telomeres at diagnosis and their length increases during recovery. A statistically significant difference in rTL was shown in patients at the time of diagnosis and after 21 days (p=0.008). rTL has a statistically significant negative correlation with SHG in patients at the time of diagnosis (ρ=-0.386, p=0.038), as well as in patients after 14 days (ρ =- 0.389, p=0.037). After 21 days, a statistically significant positive correlation was shown between rTL and SOD (ρ =0.381, p=0.046). The obtained results indicate progressive shortening of telomeres caused by COVID-19 infection, impaired redox status, as well as the change of these parameters during recovery, which confirms the link between oxidative stress and rTL, as well as the participation of these processes in the pathogenesis of COVID- 19.Patogeneza COVID-19 infekcije izazvane koronavirusom SARS-CoV-2 je predmet aktivnog istraživanja širom sveta. Kako je u pitanju složena bolest, smatra se da i u njenoj patogenezi leži oksidativni stres, koji doprinosi progesivnom skraćivanju telomera. Pacijenti starosti 25-84 godina (n=31) praćeni su u trenutku postavljanja dijagnoze, kao i na kontroli 14. i 21. dana. Parametri redoks statusa određivani su iz seruma ili plazme odgovarajućim spektrofotometrijskim metodama, dok je iz leukocita periferne krvi merena relativna dužina telomera (rTL) pomoću qPCR metode. Od parametara oksidativnog statusa određivani su: totalni oksidantni status (TOS), superoksidni anjon radikal (O2.- ), prooksidantno- antioksidantni balans (PAB), uznapredovali produkti oksidacije proteina (AOPP), malondialdehid (MDA), ishemijom modifikovan albumin (IMA). Kao parametri antioksidativne zaštite određivani su: totalni antioksidantni status (TAS), aktivnost enzima superoksid-dismutaze (SOD) i paraoksonaze 1 (PON1), odnos totalnog antioksidantnog i totalnog oksidantnog statusa (TAS/TOS), kao i ukupne sulfhidrilne grupe (SHG). Pacijenti u trenutku postavljanja dijagnoze imaju najkraće telomere i njihova dužina se povećava tokom oporavka. Pokazana je statistički značajna razlika u rTL kod pacijenata u trenutku postavljanja dijagnoze i nakon 21 dan (p=0,008). rTL je u statistički značajnoj negativnoj korelaciji sa SHG kod pacijenata u trenutku postavljanja dijagnoze (ρ=-0,386, p=0,038), kao i kod pacijenata nakon 14 dana oporavka (ρ=-0,389, p=0,037). Nakon 21 dan, pokazana je statistički značajna pozitivna korelacija između rTL i SOD (ρ=0,381, p=0,046). Dobijeni rezultati ukazuju na progresivno skraćenje telomera izazvano COVID-19 infekcijom, narušen redoks status, kao i na promenu ovih parametara tokom oporavka, koja potvrđuje vezu između oksidativnog stresa i rTL, kao i učešće ovih procesa u patogenezi COVID-19.VIII Kongres farmaceuta Srbije sa međunarodnim učešćem, 12-15.10.2022. Beogra

Oxidative Stress and Inflammatory Biomarkers in Patients with Diabetic Foot

Background and Objectives: Diabetic foot (DF) development is driven by complex interactions of hyperglycemia, inflammation, and oxidative stress (OS). We aimed to investigate OS and inflammatory biomarkers in patients with DF and their potential to improve early diagnosis and management of DF. Materials and Methods: The prooxidant–antioxidant balance (PAB), superoxide dismutase (SOD), total oxidative status (TOS), total sulfhydryl groups (SHG), routine biochemical parameters, and complete blood count were determined in 42 patients with type-2 DM, of which 23 patients had DF, while 19 patients were without DF complications. The neutrophils-to-lymphocyte ratio (NLR) was evaluated as a biomarker of inflammation. Results: Patients with DF had significantly higher (p < 0.05) PAB levels (170 ± 33.9 U/L) compared to those without DF complications (142 ± 31.3 U/L). In addition, patients with DF had significantly reduced SOD activities (p < 0.01). NLR values were significantly higher in the DF group (median: 2.8; interquartile range: 2.0–4.3) than in the group without DF (median: 1.4; interquartile range: 1.4–2.1; p < 0.01). A positive correlation was found between the PAB and NLR index (r = 0.449; p < 0.05). The diagnostic accuracy of both PAB (AUC = 0.741; p < 0.01) and NLR (AUC = 0.760; p < 0.01) was estimated as acceptable. Conclusions: In conclusion, the development of DF is associated with enhanced OS and inflammation processes. PAB and NLR could be useful non-invasive biomarkers of DF development

Markeri redoks statusa kod pacijenata sa nealkoholnom masnom bolešću jetre

Nonalcoholic fatty liver disease (NAFLD) is the most common chronic liver disease, present in up to 30% of the adult population worldwide (1). Triglycerides accumulation in hepatocytes (steatosis) represents the root cause of NAFLD and is associated with oxidative stress, which could further lead to fibrosis and cell death of hepatocytes (2). The aim of this research was to identify redox status markers for predicting the risk of developing steatosis. 158 participants were included. Steatosis was confirmed by ultrasound in 101 subjects, while the remaining 57 were in the control group. The following markers of redox status were determined in serum and plasma samples of all subjects: superoxide dismutase 1 (SOD1), paraoxonase (PON1), malondialdehyde (MDA) and superoxide anion (O2.-). For this purpose, spectrophotometric methods and enzyme immunosorbent assays were used. SOD1 was statistically significantly higher (P<0.001), while O2 .- was significantly lower in the patient group (P<0.001). SOD1 was significantly negatively correlated with O2.- (ρ= -0.494, P<0.001) and MDA (ρ= -0.242, P=0.002). Univariate binary logistic regression analysis showed a positive association between SOD1 and the presence of steatosis (OR=1.018, 95% CI 1.005-1.031; P=0.005), as well as a negative association between O2 .- and the presence of steatosis (OR=0.959, 95% CI 0.941-0.978; P<0.001). Multivariate analysis singled out SOD1 (OR=1.024, 95% CI 1.006-1.041; P=0.007) and O2.- (OR=0.965, 95% CI 0.942-0.989; P=0.004) as independent predictors for the presence of steatosis in our subjects. The redox status parameters, SOD1 and O2, respectively, showed a positive and negative prediction of the presence of statosis in our subjects.Nealkoholna masna bolest jetre (eng. nonalcoholic fatty liver disease, NAFLD) je najčešće hronično oboljenje jetre, prisutno i u do 30% adultne populacije širom sveta (1). Značajnu ulogu u nastanku NAFLD ima akumulacija triglicerida u hepatocitima – steatoza, koja je povezana sa oksidativnim stresom, a koji dalje vodi fibrozi i ćelijskoj smrti hepatocita (2). Cilj ovog istraživanja bio je identifikacija markera redoks statusa za predviđanje rizika za nastanak steatoze. Studija je obuhvatila 158 ispitanika iz Kliničko bolničkog centra Zemun. Steatoza je potvrđena ultrazvukom kod 101 ispitanika, dok je preostalih 57 činilo kontrolnu grupu. U uzorcima seruma i plazme svih ispitanika određeni su sledeći markeri redoks statusa: superoksid dismutaza 1 (SOD1), paraoksonaza (PON1), malondialdehid (MDA) i superoksidni anjon (O2.-). U tu svrhu korišćene su spektrofotometrijske metode i enzimski imunosorbentni testovi. SOD1 je bila statistički značajno viša (P<0,001), dok O 2.- značajno niži u grupi pacijenata (P<0,001), dok se PON1 i MDA nisu značajno razlikovali između grupa. SOD1 je bila u značajnoj negativnoj korelaciji sa O 2.- (ρ=-0,494, P<0,001) i MDA (ρ= - 0,242, P=0,002). Univarijatna binarna logistička regresiona analiza je pokazala pozitivnu asocijaciju između SOD1 i prisustva steatoze (OR=1,018, 95% CI 1,005-1,031; P=0,005), kao i negativnu asocijaciju između O 2.- i prisustva steatoze (OR=0,959, 95% CI 0,941-0,978; P<0,001). Multivarijantna analiza je izdvojila SOD1 (OR=1,024, 95% CI 1,006-1,041; P=0,007) i O2.- (OR=0,965, 95% CI 0,942-0,989; P=0,004) kao nezavisne prediktore za prisustvo steatoze kod naših ispitanika. Parametri redoks statusa, SOD1 i O 2.- redom, su pokazali pozitivnu, odnosno negativnu predikciju prisustva statoze kod naših ispitanika.VIII Kongres farmaceuta Srbije sa međunarodnim učešćem, 12-15.10.2022. Beogra

Dužina telomera kao biomarker starenja i bolesti

As research related to healthspan and lifespan has become a hot topic, the necessity for a reliable and practical biomarker of aging (BoA), which can provide information about mortality and morbidity risk, along with remaining life expectancy, has increased. The chromosome terminus non-coding protective structure that prevents genomic instability is called a telomere. The continual shortening of telomeres, which affects their structure as well as function, is a hallmark of agedness. The aforementioned process is a potential cause of age-related diseases (ARDs), leading to a bad prognosis and a low survival rate, which compromise health and longevity. Hence, studies scrutinizing the BoAs often include telomere length (TL) as a prospective candidate. The results of these studies suggest that TL measurement can only provide an approximate appraisal of the aging rate, and its implementation into clinical practice and routine use as a BoA has many limitations and challenges. Nevertheless, measuring TL while determining other biomarkers can be used to assess biological age. This review focuses on the importance of telomeres in health, senescence, and diseases, as well as on summarizing the results and conclusions of previous studies evaluating TL as a potential BoA.Kako su istraživanja na temu starenja postala sve popularnija, javila se potreba za pouzdanim i praktičnim biomarkerom starenja, koji može pružiti informacije o riziku od mortaliteta i morbiditeta. Telomere su nekodirajući krajevi linearnih hromozoma koji održavaju njihovu stabilnost i integritet. Ćelijsko starenje i starenje organizma karakteriše progresivno skraćivanje telomera, što ugrožava njihovu strukturu i funkciju. Skraćivanje telomera je u vezi sa povećanom incidencom bolesti povezanih sa starenjem i lošom stopom preživljavanja, što ugrožava zdravlje i skraćuje životni vek. Stoga je dužina telomera dugo vremena prepoznata kao jedan od najboljih biomarkera starenja. Međutim, nedavna istraživanja ukazuju na to da dužina telomera može da pruži samo približnu procenu brzine starenja, pa je implementacija ovog biomarkera u kliničku praksu i rutinsku primenu praćena mnogim ograničenjima i izazovima. Uprkos tome, merenje dužine telomera, uz istovremeno određivanje drugih biomarkera, može poslužiti za procenu biološke starosti. Fokus ovog rada je na značaju telomera u ljudskom zdravlju, starenju i bolestima, kao i na sumiranju rezultata i zaključaka dosadašnjih studija koje su se bavile ispitivanjem dužine telomera kao potencijalnog kandidata za biomarker starenja

Correlation of Systemic Inflammation Parameters and Serum SLFN11 in Small Cell Lung Cancer—A Prospective Pilot Study

Background and objectives: The objective of this research was to analyze the correlation of the neutrophil-to-lymphocyte ratio (NLR), C-reactive protein (CRP), soluble programmed cell death ligand 1 (sPD-L1), and Schlafen 11 (SLFN11) with the response to first-line chemotherapy in a cohort of small cell lung cancer (SCLC) patients, and to determine their potential as predictive serum biomarkers. Materials and Methods: A total of 60 SCLC patients were included. Blood samples were taken to determine CRP, sPD-L1, and SLFN11 levels. The first sampling was performed before the start of chemotherapy, the second after two cycles, and the third after four cycles of chemotherapy. Results: The patients who died earlier during the study had NLR and SLFN11 concentrations significantly higher compared to the survivor group. In the group of survivors, after two cycles of chemotherapy, the NLR ratio decreased significantly (p < 0.01), but after four cycles, the NLR ratio increased (p < 0.05). Their serum SLFN11 concentration increased significantly (p < 0.001) after two cycles of chemotherapy, but after four cycles, the level of SLFN11 fell significantly (p < 0.01). CRP, NLR, and SLFN11 were significant predictors of patient survival according to Kaplan–Meier analysis. The combination of inflammatory parameters and SLFN11 with a cutoff value above the 75th percentile of the predicted probability was associated with significantly lower overall survival in SCLC patients (average survival of 3.6 months vs. 4.8 months). Conclusion: The combination of inflammatory markers and the levels of two specific proteins (sPD-L1, SLFN11) could potentially serve as a non-invasive biomarker for predicting responses to DNA-damaging therapeutic agents in SCLC

The influence of Klotho protein and prooxidant–antioxidant balance combination on the mortality of HD patients

Purpose: End-stage renal disease patients on chronic hemodialysis (HD) have a shortened life expectancy compared to the general population. The aim of this study was to evaluate a possible link between three new and emerging factors in renal pathophysiology: Klotho protein, telomere length in peripheral blood mononuclear cells (TL) and redox status parameters before HD (bHD) and after HD (aHD), and to test mortality prediction capability of these emerging parameters in a population of HD patients. Methods: The study included 130 adult patients with average age 66 (54–72), on HD (3 times per week; 4–5 h per session). Klotho level, TL, routine laboratory parameters, dialysis adequacy and redox status parameters: advanced oxidation protein products (AOPP), prooxidant–antioxidant balance (PAB), superoxide anion (O2.−), malondialdehyde (MDA), ischemia-modified albumin (IMA), total sulfhydryl group content (SHG), and superoxide dismutase (SOD) were determined. Results: Klotho concentration was significantly higher aHD; 68.2 (22.6–152.9) vs. bHD 64.2 (25.5–119.8) (p = 0.027). The observed increase in TL was not statistically significant. AOPP, PAB, SHG, and SOD activity were significantly increased aHD (p > 0.001). The patients with the highest mortality risk score (MRS) had significantly higher PAB bHD (p = 0.002). Significantly lower O2.− (p < 0.001), SHG content (p = 0.072), and IMA (p = 0.002) aHD were found in patients with the lowest MRS values. Principal component analysis revealed redox balance-Klotho factor as a significant predictor of high mortality risk (p = 0.014). Conclusion: Decreased Klotho and TL attrition as well as redox status disturbance could be connected with higher mortality rate in HD patients

Childhood obesity accelerates biological ageing: is oxidative stress a link?

Obesity is multifactorial pathophysiological condition with an imbalance in biochemical, immunochemical, redox status and genetic parameters values. We aimed estimate connection between relative leukocyte telomere lengths (rLRL) - biomarker of cellular aging with metabolic and redox status biomarkers values in a group of obese and lean children. The study includes 110 obese and 42 lean children and adolescents, both genders. The results suggested that rLTL are significantly shorter in obese, compared to lean group (p30kg/m2 according to logistic regression analysis was PCA derived antioxidant factor's score (OR: 1.66, 95th Cl: 1.05-2.6, p=0.029). PCA analysis confirmed oxidative stress importance in biological ageing caused by obesity and its multiple consequences related to prooxidants augmentation and antioxidants exhaustion and gave us clear signs of disturbed cellular homeostasis deepness, even before any overt disease occurrence.This is peer-reviewed version of the following article: Subošić, B., Zdravković, V., Ješić, M., Munjas, J., Kovačević, S., Guzonjić, A., Mitrović, J., Saso, L., Đuričić, I.,& Kotur-Stevuljević, J.. (2024). Childhood obesity accelerates biological ageing: is oxidative stress a link?. in British Journal of Nutrition Cambridge University Press.. [https://doi.org/10.1017/S0007114524000898

Correlation of Short Leukocyte Telomeres and Oxidative Stress with the Presence and Severity of Lung Cancer Explored by Principal Component Analysis

Lung cancer (LC) is the second most common malignancy and leading cause of cancer death. The potential "culprit" for local and systemic telomere shortening in LC patients is oxidative stress. We investigated the correlation between the peripheral blood leukocyte (PBL) telomere length (TL) and the presence/severity of LC and oxidative stress, and its usefulness as LC diagnostic marker. PBL TL was measured in 89 LC patients and 83 healthy subjects using the modified Cawthon RTq-PCR method. The relative PBL TL, found to be a potential diagnostic marker for LC with very good accuracy (P < 0.001), was significantly shorter in patients compared to the control group (CG) (P < 0.001). Significantly shorter telomeres were found in patients with LC TNM stage IV than in patients with stages I-III (P = 0.014), in patients without therapy compared to those on therapy (P = 0.008), and in patients with partial response and stable/progressive disease compared to those with complete response (P = 0.039). The total oxidant status (TOS), advanced oxidation protein products (AOPP), prooxidant-antioxidant balance (PAB) and C-reactive protein (CRP) were significantly higher in patients compared to CG (P < 0.001) and correlated negatively with TL in both patients and CG (P < 0.001). PCA showed a relation between PAB and TL, and between the EGFR status and TL. Oxidative stress and PBL telomere shortening are probably associated with LC development and progression