Biomedical subjects

Hilmi Orhan

Publications and source records attributed to Hilmi Orhan.

8 recordsLinked to original sources

PubMed2006

Misincorporation of free m-tyrosine into cellular proteins: a potential cytotoxic mechanism for oxidized amino acids.

In vitro studies demonstrate that the hydroxyl radical converts L-phenylalanine into m-tyrosine, an unnatural isomer of L-tyrosine. Quantification of m-tyrosine has been widely used as an index of oxidative damage in tissue proteins. However, the possibility that m-tyrosine might be generated oxidatively from free L-phenylalanine that could subsequently be incorporated into proteins as an L-tyrosine analogue has received little attention. In the present study, we demonstrate that free m-tyrosine is toxic to cultured CHO (Chinese-hamster ovary) cells. We readily detected radiolabelled material in proteins isolated from CHO cells that had been incubated with m-[14C]tyrosine, suggesting that the oxygenated amino acid was taken up and incorporated into cellular proteins. m-Tyrosine was detected by co-elution with authentic material on HPLC and by tandem mass spectrometric analysis in acid hydrolysates of proteins isolated from CHO cells exposed to m-tyrosine, indicating that free m-tyrosine was incorporated intact rather than being metabolized to other products that were subsequently incorporated into proteins. Incorporation of m-tyrosine into cellular proteins was sensitive to inhibition by cycloheximide, suggesting that protein synthesis was involved. Protein synthesis using a cell-free transcription/translation system showed that m-tyrosine was incorporated into proteins in vitro by a mechanism that may involve L-phenylalanine-tRNA synthetase. Collectively, these observations indicate that m-tyrosine is toxic to cells by a pathway that may involve incorporation of the oxidized amino acid into proteins. Thus misincorporation of free oxidized amino acids during protein synthesis may represent an alternative mechanism for oxidative stress and tissue injury during aging and disease.

Animals↗

PubMed2006

Comparative dural closure techniques: a safety study in rats.

BACKGROUND: Some neurosurgical procedures have high morbidity and mortality rates due to cerebrospinal fluid (CSF) fistula development, particularly when dural defects are in relatively inaccessible areas or surrounded by friable dura. We used a rat model to test 4 different dural closure techniques to determine which one was significantly superior for achieving a watertight dural closure with minimal harm to brain tissue. METHODS: The rats were randomly divided into 2 groups. The first group (group A, n = 40) was used to test the strength of the adhesivity for CSF leakage. Histopathologic studies were used to evaluate the granulation tissue between the dura and dural graft. Effects on the brain tissue were studied in the second group (group B, n = 40) where lipid peroxidation was determined. These 2 groups consisted of 5 subgroups: control, methyl metacrylate, n-butyl cyanoacrylate, fibrin glue, and CO(2) laser. RESULTS: Methyl metacrylate and CO(2) laser techniques were inadequate for stopping dural leakage and had harmful effects on brain tissue. Cerebrospinal fluid leak was observed only in 1 rat in the n-butyl cyanoacrylate subgroup and this result was statistically significant (P = .0005), but lipid peroxidation levels for this material showed that it was not safe for dural closure in case it leaked through the dural defect. The lipid peroxidation levels of the fibrin glue subgroup were not statistically significantly different from the control group (P = .440). CONCLUSIONS: Fibrin glue was the safest material with a CSF leakage risk that was higher than n-butyl cyanoacrylate (25% vs 12.5%) but acceptable. This study showed no relationship between the CSF leak and histopathologic findings for sealant properties of the tissue adhesives.

Animals↗

PubMed2006

Synthesis and evaluation of in vitro antioxidant capacities of some benzimidazole derivatives.

New, except 1d, melatonin analogue benzimidazole derivatives were synthesized and characterized in the present study. The potential role of melatonin as an antioxidant by scavenging and detoxifying ROS raised the possibility that compounds that are analogous to melatonin can also be used for their antioxidant properties. Therefore the antioxidant effects of the newly synthesized compounds were investigated in vitro by means of their inhibitory effect on hydrogen peroxide-induced erythrocyte membrane lipid peroxidation (EMLP) and on various erythrocyte antioxidant enzymes viz. superoxide dismutase (SOD), catalase (CAT) and glucose-6-phosphate dehydrogenase (G6PD). The synthesized benzimidazole derivatives showed remarkable antioxidant activity in vitro in the H2O2-induced EMLP system. Furthermore their effects on various antioxidant enzymes are discussed and evaluated from the perspective of structure- activity relationships.

Antioxidants↗

PubMed2005

Quantification of urinary o,o'-dityrosine, a biomarker for oxidative damage to proteins, by high performance liquid chromatography with triple quadrupole tandem mass spectrometry. A comparison with ion-trap tandem mass spectrometry.

We recently described an isotope dilution reversed-phase liquid chromatography-atmospheric pressure chemical ionization-ion-trap-tandem mass spectrometry (HPLC-APCI-MS/MS) method for the quantitative determination of oxidized amino acids in human urine, including o,o'-dityrosine, a specific marker of protein oxidation. In the present study, we investigated the possibility to use a triple quadrupole instrument for the analysis of this biomarker in urine. The two instruments were compared in terms of sensitivity, specificity and reproducibility. Results showed that the triple quadrupole instrument reaches 2.5-fold higher sensitivity (LOD=0.01 microM) compared to the previously used ion-trap instrument. Precision of the present assay is as follows: in-day variation is 4.6% and inter-day variation is 17%. The currently developed method was applied to a group of smoker urine samples. The mean urinary o,o'-dityrosine concentration was 0.08+/-0.01 microM. Expressed per urinary creatinine concentration, this corresponds to 10.1+/-0.4 micromol/mol creatinine. This is comparable to the previously reported values of 5.8+/-0.3 micromol/mol creatinine in non-smokers night-time urines, and 12.3+/-5 micromol/mol creatinine in day-time urines measured by the ion-trap instrument.

Biomarkers↗

PubMed2005

Erythrocyte antioxidant defense response against cigarette smoking in humans--the glutathione S-transferase vulnerability.

Cigarette smoking leads to uptake of a multitude of reactive chemicals including many electrophiles and may also give rise to oxidative stress. Human red blood cells are important targets for electrophilic and oxidant foreign compounds. We investigated the oxidative stress in erythrocytes upon cigarette smoking, and the response of antioxidant defense system against it. With this aim, simultaneous determination of erythrocyte superoxide dismutase (SOD), selenium dependent glutathione peroxidase (Se-GPx), catalase (CAT), glutathione S-transferase (GST) activities and plasma levels of thiobarbituric acid reactive substances (TBARS), and the degree of erythrocyte membrane lipid peroxidation (EMLP) were carried out in blood samples of smokers and their controls. Plasma TBARS levels and EMLP in smokers were significantly higher than the control levels (p < 0.01 and p < 0.005, respectively). SOD activity was diminished in smokers compared to nonsmoker controls (p < 0.005). Erythrocyte Se-GPx activity was also found significantly diminished in smokers (p < 0.005), while plasma Se-GPx activity was not changed. We observed that erythrocyte CAT activity was not different in smokers compared to nonsmoker controls. We found that the erythrocyte GST activity is significantly lower in young adult smokers (3.03 +/- 0.18 U/mg protein; mean +/- SEM; n = 46) than in nonsmoking contemporaries (3.98 +/- 0.26 U/mg protein; mean +/- SEM; n = 41). Together with previously reported data, it can be concluded that the decrease in GST activity leads to extra GST synthesis during erythrocyte proliferation. The same data were also analyzed for the sex differences. The statistically significant differences remained the same between nonsmoker and smoker females. Only EMLP degree and SOD activity were significantly different between nonsmoker and smoker males; however, when compared the parameters between male and female nonsmokers, GST activity was found to be significantly higher in females than that of males.

Adult↗

PubMed2004

Simultaneous determination of tyrosine, phenylalanine and deoxyguanosine oxidation products by liquid chromatography-tandem mass spectrometry as non-invasive biomarkers for oxidative damage.

We developed an isotope dilution HPLC-atmospheric pressure chemical ionization-tandem mass spectrometry (HPLC-APCI-MS/MS) method for the simultaneous determination of p-tyrosine, phenylalanine, o,o'-dityrosine, m-tyrosine, o-tyrosine, 3-chlorotyrosine and 3-nitrotyrosine and 8-hydroxy-2'-deoxyguanosine (8-OHdG) that requires no extensive sample pre-treatment. p-[(2)H(4)]Tyrosine and o,o'-[(2)H(6)]dityrosine were used as internal standards. Calibration curves of the method were linear (r(2)=0.990-0.999) over a concentration range of 0.03-10 microM for o-tyrosine; 0.04-10 microM for 3-nitrotyrosine and 3-chlorotyrosine; 0.05-10 microM for o,o'-dityrosine; and for m-tyrosine; 1.0-100 microM for p-tyrosine and for phenylalanine; and 0.01-10 microM for 8-OHdG. The detection limits were from 0.025 to 0.05 microM for the tyrosine derivatives; 0.01 microM for 8-OHdG; and 0.5 microM for p-tyrosine and for phenylalanine, respectively. Within-day coefficients of variation (CV) for spiked human urine samples ranged from 2.7 to 7.0%, except for 8-OHdG (13.7%). Between-day variations ranged from 7.9 to 13.0%, except for o-tyrosine (CV = 18.2%), and for 8-OHdG (CV = 24.7%). The background levels of p-tyrosine, phenylalanine, o,o'-dityrosine, and o-tyrosine in morning urine of eight healthy volunteers were 3890+/-590, 3420+/-730, 5.8+/-0.3, and 9.2+/-1.5 micromol/mol creatinine, respectively. Using the present HPLC-APCI-MS/MS method, the urinary background levels of m-tyrosine, 3-chlorotyrosine, 3-nitrotyrosine and 8-OHdG were below the limit of detection.

Calibration↗

PubMed2004

Evaluation of a multi-parameter biomarker set for oxidative damage in man: increased urinary excretion of lipid, protein and DNA oxidation products after one hour of exercise.

The objective of the present study was to evaluate a comprehensive set of urinary biomarkers for oxidative damage to lipids, proteins and DNA, in man. Eighteen moderately trained males (mean age 24.6+/-0.7) exercised 60min at 70% of maximal O2 uptake on a cycle ergometer. Urine fractions for 12 h were collected 1 day before, and for 3 consecutive days after exercise. As biomarkers of lipid peroxidation, 8 aldehydes (i.e. propanal, butanal, pentanal, hexanal, heptanal, octanal, nonanal and malondialdehyde-MDA)and acetone were analyzed in urines by gas chromatography with electron capture detection (GC-ECD). As a biomarker of protein oxidation, o,o'-dityrosine was analyzed in urine samples by a recently developed isotope dilution HPLC-atmospheric pressure chemical ionization (APCI)-tandem-mass spectrometry (HPLC-APCI-MS/MS) methodology. As a biomarker of oxidative DNA damage, urinary excretion of 8-hydroxy-2'-deoxyguanosine (8-OHdG) was measured by an ELISA method. On the day of exercise, significant increases were observed in urinary excretions of acetone (p < 0.025, n = 18) and butanal (p < 0.01, n = 18) in the 12h daytime fractions compared to the daytime fraction before exercise. The urinary acetone excretion was also significantly (p < 0.05) increased on the 1st day after exercise. Octanal and nonanal were increased in the daytime urine fraction on the 2nd day after exercise. However, these increases were of borderline significance (p = 0.09 and p = 0.07, respectively). Significantly elevated urinary o,o'-dityrosine amounts were observed in the daytime fraction on the day of exercise (p < 0.025) and on the 1st day after exercise (p = 0.07) compared to the before exercise daytime fraction. Excretion of urinary 8-OHdG was statistically significantly increased in the daytime fractions on the day of exercise (p = 0.07) and on the 1st day after exercise (p < 0.025) compared to before exercise daytime fraction. Increases in urinary excretions of acetone, propanal, pentanal, MDA and 8-OHdG significantly correlated with training status (hours of exercise/week) of the volunteers, while o,o'-dityrosine did not. To our knowledge, the present study is the first to evaluate a multi-parameter non-invasive biomarker set for damage to three main cellular targets of ROS. It shows that 1 h of exercise may already induce oxidative damage in moderately trained individuals and that the chosen urinary biomarkers are sensitive enough to monitor such damage.

8-Hydroxy-2'-Deoxyguanosine↗

PubMed2002

Circulating biomarkers of oxidative stress in complicated pregnancies.

Increased lipid peroxidation (LPO) and reduced antioxidant activity may contribute to the development of complications in pregnancy. The present study discusses the possibility of LPO and antioxidant activity in both maternal and umbilical cord blood as an indicator of oxygen radical activity. For this aim, pregnancies with hypertension and pre-eclampsia, diabetes mellitus (insulin dependent diabetes mellitus and gestational diabetes mellitus), oligohydramnios and abruptio placentae, as well as a healthy control group, were subjected in the present study. Simultaneous determination of glutathione S-transferase (GST), selenium dependent glutathione peroxidase (Se-GPx), catalase (CAT) activities and thiobarbituric acid reactive-substances (TBARs) levels were carried out in maternal erythrocyte and plasma in the antenatal period (in the third trimester) and immediately after the delivery. The same oxidative stress-related parameters were determined in umbilical cord blood as well. Erythrocyte GST activity was significantly increased in insulin-dependent diabetic pregnancy (IDDP) when compared to the control (P<0.05). Erythrocyte Se-GPx activity was found to be significantly increased in hypertensive preeclamptic pregnancy (HPP) (P<0.05) and in IDDP (P<0.05). Alterations in enzyme activities were accompanied by a simultaneous significant increase in the levels of TBARs in plasma samples of HPP (P<0.05), and IDDP (P<0.05). Enzyme activities were found to be significantly lower in cord blood samples than the maternal values, except GST. This enzyme represents about two- to threefold higher activity than those of the maternal activity in uncomplicated and complicated groups. Cord blood erythrocyte and plasma Se-GPx and CAT activities were decreased significantly in the HPP group when compared to the maternal value (P<0.05). Cord blood erythrocyte CAT activity was significantly decreased in the HPP group compared to the control (P<0.05). Cord blood TBARs levels were significantly lower than the before deliveries maternal value in the HPP group (P<0.05). No difference was detected between umbilical cord blood and maternal blood TBARs levels after delivery. The results of the present study suggest that oxidative stress and subsequent lipid peroxidation accompany the complications of hypertension, preeclampsia and diabetes mellitus in pregnancy. Maternal erythrocyte GST activity seems to be a sensitive indicator of oxidative stress in IDDP before delivery. The same enzyme can be used in cord blood as a biomarker of oxidative stress upon a sudden increase in oxygenation during delivery. These multiparameter biomarkers can also be used in monitoring the efficiency of antioxidant supplementation in complicated pregnant women, as has recently been suggested for diabetic and preeclamptic pregnancies.

Adult↗