Ahmet Bayrak

Serum paraoxonase 1 activity, asymmetric dimethylarginine levels, and brachial artery flow-mediated dilatation in women with polycystic ovary syndrome

OBJECTIVE To evaluate endothelial function via serum asymmetric dimethylarginine (ADMA) levels, paraoxonase 1 (PON1) activity, and brachial artery flow-mediated dilatation (FMD) in women with polycystic ovary syndrome (PCOS). DESIGN Prospective case-control study. SETTING University hospital. PATIENT(S) Thirty patients with PCOS with a mean age of 24.33 ± 4.50 years and 30 healthy controls matched for body mass index (BMI) and age. INTERVENTION(S) Endothelial function was assessed biochemically with serum ADMA levels and serum PON1 activity and functionally with brachial artery FMD by ultrasonography. MAIN OUTCOME MEASURE(S) Serum ADMA levels, serum PON1 activity, brachial artery FMD, hormonal and biochemical parameters. RESULT(S) Patients with PCOS had higher levels of free testosterone and insulin, and higher waist-hip ratio and Ferriman Gallwey scores when compared with the controls. Fasting glucose and homeostasis model assessment of insulin resistance were not different between the groups. There was no statistically significant difference in ADMA levels between two groups. Serum PON1 activity and brachial artery FMD were statistically significantly lower in women with PCOS. There was negative correlation between ADMA and PON1 in patients with PCOS. CONCLUSION(S) Serum PON1 activity and brachial artery FMD, as markers of endothelial dysfunction and cardiovascular risk, were statistically significantly lower in women with PCOS compared with healthy controls matched for age and BMI. Endothelial dysfunction may be seen at earlier ages in patients with PCOS.

Differential hydrolysis of homocysteine thiolactone by purified human serum 192Q and 192R PON1 isoenzymes

Human serum paraoxonase 1 (PON1) is a HDL-associated enzyme that catalyzes the hydrolysis of a variety of aromatic carboxylic acid esters and several organophosphates. Recently it has been suggested that a physiological substrate of serum PON1 is homocysteine thiolactone which is a putative risk factor in atherosclerosis. In this study, human (192)Q and (192)R PON1 isoenzymes were purified from the respective phenotype human serum, using a protocol consisting of ammonium sulfate precipitation and four chromatography steps: gel filtration, ion-exchange, non-specific affinity, and a second ion-exchange. Using paraoxon as substrate, overall purification fold was found as 742 for (192)R PON1 and 590 for (192)Q PON1. The final purified enzymes were shown as single protein bands close to 45kDa on SDS-PAGE and confirmed by Western blot. Substrate kinetics were studied with phenyl acetate, paraoxon and homocysteine thiolactone. Both PON1 isoenzymes showed mixed type inhibition with phenyl acetate. K(m) values of (192)Q and (192)R PON1 for homocysteine thiolactone were 23.5mM and 22.6mM respectively. For (192)R PON1, the V(max) was 2.5-fold and k(cat)/K(m) was 2.6-fold higher than those for (192)Q PON1 when homocysteine thiolactone is used as substrate. The present data suggest that defining (192)Q and (192)R PON1 isoforms could be a good predictor and prognostic marker in the cardiovascular risk assessment.

Purification and kinetic properties of rabbit liver paraoxonase 1

Paraoxonase 1 (PON1) is synthesized in the liver and secreted into the blood, where it is associated exclusively with HDL. In this study, rabbit liver PON1 enzyme was purified to homogeneity using a new purification approach, and the kinetic properties of the enzyme were investigated using phenyl acetate and homocysteine thiolactone as substrates. Rabbit liver PON1 was purified through the preparation of liver microsomal fraction, Sephacryl S300 HR gel filtration chromatography, DEAE Trisacryl M ion-exchange chromatography and hydroxyapatite chromatography steps. Using this method, rabbit liver PON1 was purified 576 times with a specific activity of 2726 U/mg protein. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis revealed the obtained enzyme as a single protein band close to 40 kDa. The Km of the this enzyme was found as 0.55+/-0.024 mM for phenyl acetate and 17.31+/-1.2 mM for homocysteine thiolactone. In this study, a new approach was used to purify PON1 enzyme from rabbit liver and for the first time in the literature, its kinetics was studied with homocysteine thiolactone as substrate.

Evaluation of predictive effect of PAF-AH on the prognosis of intensive care unit patients / Yoğun bakım hastalarında PAF-AH’ın prognoz üzerindeki prediktif etkisinin değerlendirilmesi

Abstract Objective: Determination of the factors associated with the intensive care unit (ICU) prognosis and mortality has important role in the clinical follow-up of the patients. Definition of novel biomarkers, beside older biomarkers available for evaluation of the outcome of these patients has been proposed. Platelet-activating factor acetylhydrolase (PAF-AH) is an enzyme that inactivates the platelet-activating factor. A reduction in the level of the PAF-AH has been demonstrated during systemic inflammation and multiple organ failure. This research aims to determine whether measurement of PAF-AH enzyme activity in ICUs can be used as a prognostic indicator like conventional biomarkers. Methods: Eighty five adult patients have been included. Following data have been recorded: preliminary C-reactive protein (CRP), lactate, albumin and PAF-AH values, APACHE II scores and discharge forms from ICU. Patients were divided in two groups with respect to APACHE II values: Group 1 (1-19) and Group 2 (≥20). Results: Observed mortality was 51.2%. In the APACHE II Group 2 patients, the values of CRP (p=0.001) and lactate (p=0.040) were significanty high, and the values of PAF-AH (p=0.008) and albumin (p=0.001) were significantly low. A statistically significant difference was found between PAF-AH values of exitus and alive patients (p=0.001). According to ROC analysis, the sensitivity and specificity of predicting mortality was 70.5% and 70.7% for CRP, 63.6% and 70.7% for lactate, 90.2% and 61.4% for albumin and 63.6% and 70% PAF-AH, respectively. Conclusion: Our study demonstrated that, in predicting the ICU mortality risk, sensitivity of the PAF-AH is similar to the sensitivity of the lactate, and specificity of the PAF-AH is better than that of the albumin. According to our results, PAF-AH can be included in the novel biomarkers. Özet Amaç: Yoğun Bakım unitelerinde mortalite ve prognozu etkileyen faktorlerin belirlenmesi, hastanın klinik durumunun takibi icin faydalıdır. Bu amacla rutin kullanılan diagnostik belirteclerin yanı sıra yeni belirteclerin kullanımı da gundemdedir. Platelet aktive edici faktor asetil hidrolaz (PAF-AH), PAF’ı inaktive eden enzimdir. Sistemik enflamasyonda ve multipl organ yetmezliklerinde dolaşımdaki PAF-AH duzeylerinde azalma gosterilmiştir. Bu araştırmada PAF-AH enzim aktivitesi olcumunun Yoğun Bakım unitelerinde prognoz gostergesi olarak kullanılıp kullanılamayacağını araştırdık. Metod: Seksen beş erişkin hasta calışmaya dahil edildi. Hastaların Yoğun Bakım’a kabuldeki C-reaktif protein (CRP), laktat, albumin ve PAF-AH değerleri ile APACHE II skorları ve yoğun bakımdan cıkış bicimleri kaydedildi. Hastalar APACHE II değerlerine gore iki gruba ayrıldılar; Grup 1 (1-19) ve Grup 2 (≥20). Bulgular: Yoğun Bakım Unitemizde gozlenen mortalite oranı %51.2 idi. APACHE II 2. grupta CRP (p=0.001) ve laktat (p=0.040) seviyeleri anlamlı olarak yuksek iken, PAF-AH (p=0.008) ve albumin (p=0.001) değerleri duşuktu. PAF-AH duzeyleri acısından olen hastalarla yaşayan gruplar arasında anlamlı bir farklılık tespit edildi (P=0.001). Yapılan ROC analizine gore, CRP’nin mortaliteyi gostermedeki duyarlılığı %70.5, ozgulluğu %70.7, laktatın duyarlılığı %63.6, ozgulluğu %70.7, albuminin duyarlılığı %90.2, ozgulluğu %61.4, PAF-AH’ın duyarlılık ve ozgulluğu ise sırasıyla %63.6 ve %70 idi. Sonuç: Calışmamızda PAF-AH’ın Yoğun Bakım mortalite riskini belirlemede laktat ile eşit duyarlılıkta olduğu, ozgulluğunun ise albuminden daha yuksek olduğu tespit edilmiştir. PAF-AH, Yoğun Bakım hastalarında prognozun değerlendirilmesi amacıyla kullanılabilecek yeni bir biyobelirtectir.