Şükrü Beydemir

In Vitro Inhibition of Human Carbonic Anhydrase I and II Isozymes with Natural Phenolic Compounds

Inhibition of two human cytosolic carbonic anhydrase (hCA, EC 4.2.1.1) isozymes I and II with some natural phenolic derivatives was investigated using the esterase assay with 4‐nitrophenyl acetate as substrate. Resveratrol, catechin, silymarin, dobutamin, and curcumin showed KI values in the range of 4.47–9.47 mm for hCA I and of 2.86–7.44 μm against hCA II, respectively. These natural product phenols were generally competitive inhibitors with 4‐nitrophenylacetate as substrate. Some natural phenols investigated here showed effective hCA II inhibitory effects, in the same range as the clinically used sulfonamide acetazolamide, and might be used as leads for generating enzyme inhibitors possibly targeting other CA isoforms that have not been yet assayed for their interactions with such agents.

The effect of ethanol on erythrocyte carbonic anhydrase isoenzymes activity: An in vitro and in vivo study

The ethanol is a widely consumed as sedative-hypnotic drug throughout the world. In this study, the effects of ethanol were investigated on carbonic anhydrase (CA) enzyme activities both in vitro in human erythrocyte and in vivo in Sprague-Dawley rat erythrocyte. For in vitro study, the human carbonic anhydrase-I (HCA-I) and -II (HCA-II) are purified by Sepharose 4B–L-tyrosine-sulphanilamide affinity chromatography. In vivo CA enzyme activity was determined colorimetrically by using CO2-hydration method of Wilbur and Anderson. Rat blood samples were taken from each rat before and after the ethanol administration at different times (1 h, 3 h, and 5 h). Rat erythrocyte CA activity was significantly inhibited by pharmacological dosage of the ethanol (2 mL.kg− 1) for up to 3 h (p < 0.001) following intraperitoneally administration. The ethanol showed in vitro inhibitory effects on HCA-I and HCA-II hydratase activity, determined by colorimetrically using the CO2-hydratase method. The inhibitor concentrations causing up to 50% inhibition (IC50) were 2.09 M for HCA-I (r2:0.9273) and 1.83 M for HCA-II (r2:9749). In conclusion, it was demonstrated that carbonic anhydrase enzyme in erythrocytes was significantly inhibited by the ethanol both in in vitro and in vivo.

A Study on the In Vitro Antioxidant Activity of Juniper (Juniperus communis L.) Fruit Extracts

Abstract This study aimed at evaluating the in vitro antioxidant activity of water and ethanol extracts of juniper (Juniperus communis L., Family Cupressaceae) fruit. The antioxidant properties of both Juniper extracts were studied using different antioxidant assays, including reducing power, free radical scavenging, superoxide anion radical scavenging, hydrogen peroxide scavenging, and metal chelating activities. Both the water and the ethanol extracts exhibited strong total antioxidant activity. The concentrations of 20, 40, and 60 µg/mL of water and ethanol extracts of juniper fruit showed 75%, 88%, 93%, 73%, 84%, and 92% inhibition on peroxidation of linoleic acid emulsion, respectively. On the other hand, 60 µg/mL of standard antioxidant such as butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), and α‐tocopherol exhibited 96, 96, and 61 inhibitions on peroxidation of linoleic acid emulsion, respectively. However, both extracts of juniper had effective reducing power, free radical scavenging, superoxide anion radical scavenging, hydrogen peroxide scavenging, and metal chelating activities at these same concentrations (20, 40, and 60 µg/mL). Those various antioxidant activities were compared to BHA, BHT, and α‐tocopherol as standard antioxidants. In addition, total phenolic compounds in both aqueous and ethanolic juniper extracts were determined as gallic acid equivalents. Accordingly, these results indicate that juniper has in vitro antioxidant properties and these may be major reasons for the inhibition of lipid peroxidation properties.

Sildenafil is a strong activator of mammalian carbonic anhydrase isoforms I–XIV

Sildenafil citrate, a phosphodiesterase-5 (PDE5) inhibitor widely used for the treatment of erectile dysfunction was investigated for its interaction with the zinc-enzyme carbonic anhydrase (CA, EC 4.2.1.1), as it has in its molecule a piperazine moiety also found in some CA activators (CAAs). Sildenafil was a potent, low micromolar activator of several CA isozymes, such as CA I, VA and VI (K(A)s in the range of 1.08-6.54microM), and activated slightly less the isoforms CA III, IV and VA (K(A)s of 13.4-16.8microM). CA isozymes II, IX, XIII and XIV showed activation constants in the range of 27.5-34.0microM, whereas the least activated isoforms were CA VII and XII (K(A)s of 72.9-73.0microM). Sildenafil citrate was also given orally to Sprague-Dawley rats at 1mg/kg body weight. Red blood cell CA activity was inhibited in the treated animals at 3-5h post-administration (in the range of 60-85%), probably due to NO/nitrite formed by PDE5 inhibition or by another, unknown mechanism. Whether CA activation by sildenafil has clinical consequences in humans is beyond the scope of the present work and warrants further studies.

Effects of low molecular weight plasma inhibitors of rainbow trout (Oncorhynchus mykiss) on human erythrocyte carbonic anhydrase-II isozyme activity in vitro and rat erythrocytes in vivo.

The effects of low molecular weight plasma inhibitors from rainbow trout (Oncorhynchus mykiss) (RT) were investigated on the carbonic anhydrase enzyme (CA) activities in in vitro human and in in vivo Sprague–Dawley rat erythrocytes. The RT blood was used as extracellular fluid (plasma) source and plasma inhibitors were obtained by dialysis of the plasma. For the in vitro study, human carbonic anhydrase-II (HCA-II) isozyme was obtained by Sepharose 4B-l-tyrosine-sulfanylamide affinity chromatography with an overall purification of about 646-fold. The enzyme (specific activity of 7750 EU/mg protein) was obtained with a yield of 71.1% and SDS-PAGE showed a single band. From in vitro studies, the I50 value for RT plasma inhibitors obtained was 0.37 mg/ml. From in vivo studies on rat erythrocytes, CA activity was significantly inhibited by the inhibitors from the extracellular fluid of RT for up to 3 h (p<0.05) following intraperitoneal administration.

In vitro inhibitory effects of some heavy metals on human erythrocyte carbonic anhydrases

The inhibition of two human carbonic anhydrase (HCA, EC 4.2.1.1) isozymes, the cytosolic HCA I and II, with heavy metal salts of Pb(II), Co(II) and Hg(II)has been investigated. Human erythrocyte CA-I isozyme was purified with a specific activity of 920 EUmg− 1 and a yield of 30% and CA-II isozyme was purified with a specific activity of 8000 EUmg− 1 and a yield of 40% using Sepharose-4B-L tyrosine-sulfanilamide affinity gel chromatography. The overall purification was approximately 104-fold for HCA-I and 900-fold for HCA-II. The inhibitory effects of different heavy metals (lead, cobalt and mercury) on CA activity were determined at low concentrations using the esterase method under in vitro conditions. Ki values for these metals were calculated from Lineweaver-Burk graphs as 1.0, 3.22 and 1.45 mM for HCA-I and 0.059, 1.382 and 0.32 mM for HCA-II respectively. Lead was a noncompetitive inhibitor for HCA-I and competitive for HCA-II, cobalt was competitive for HCA-I and noncompetitive for HCA-II and mercury was uncompetitive for both HCA-I and HCA-II. Lead was the best inhibitor for both HCA-I and HCA-II.

Intravenous anesthetics inhibit human paraoxonase-1 (PON1) activity in vitro and in vivo.

OBJECTIVES Here we evaluated the in vitro and in vivo effects of the intravenous anesthetics, etomidate, propofol, and ketamine, on the activity of human serum paraoxonase (hPON1). DESIGN AND METHODS hPON1 was purified from human serum using simple chromatographic methods, including DEAE-Sephadex anion exchange and Sephadex G-200 gel filtration chromatography. RESULTS The three anesthetics dose-dependently decreased in vitro hPON1 activity. Inhibition mechanisms are: etomidate was noncompetitive, propofol was competitive, and ketamine was uncompetitive. In vivo studies were performed on five patients for each drug. PON1 was significantly inhibited by 0.3 mg/kg etomidate (p<0.05), 2 mg/kg propofol (p<0.001), and 1 mg/kg ketamine (p<0.05) for up to 5 min following intravenous administration. CONCLUSIONS Our results showed that anesthetics significantly inhibit hPON1 activity, both in vitro and in vivo, with rank order etomidate>propofol>ketamine in vitro, and propofol>etomidate>ketamine in vivo.

The in vitro and in vivo inhibitory effects of some sulfonamide derivatives on rainbow trout (Oncorhynchus mykiss) erythrocyte carbonic anhydrase activity.

The in vitro and in vivo inhibitory effects of 5-(3α, 12α-dihydroxy-5-β-cholanamido)-1,3,4-thiadiazole-2-sulfonamide (1), 5-(3α, 7α, 12α-trihydroxy-5-β-cholanamido)-1,3,4-thiadiazole-2-sulfonamide (2), 5-(3α, 7α, 12α-triacetoxy-5-β-cholanamido)-1,3,4-thiadiazole-2-sulfonamide (3) and acetazolamide on rainbow trout (Oncorhynchus mykiss) (RT) erythrocyte carbonic anhydrase (CA) were investigated. The RT erythrocyte CA was obtained by affinity chromatography with a yield of 20.9%, a specific activity of 422.5 EU/mg protein and a purification of 222.4-fold. The purity of the enzyme was confirmed by SDS-PAGE. Inhibitory effects of the sulfonamides and acetazolamide on the RT erythrocyte CA were determined using the CO2-Hydratase method in vitro and in vivo studies. From in vitro studies, it was found that all the compounds inhibited CA. The obtained I50 value for the sulfonamides (1), (2) and (3) and acetazolamide were 0.83, 0.049, 0.82 and 0.052 μM, respectively. From in vivo studies, it was observed that CA was inhibited by the sulfonamides (1), (2) and (3) and acetazolamide.

Protective role of l-carnitine supplementation against exhaustive exercise induced oxidative stress in rats

The objective of this study was to investigate temperature dependent effects of oral l-carnitine supplementation on exhaustive exercise induced oxidative damage in rats. 42 male Spraque Dawley rats were randomly divided into seven experimental groups. These groups were formed as three non-carnitine exercise groups, three carnitine-exercise groups and a sedentary group. l-carnitine was given intraperitoneally to the carnitine-exercise groups 1h before the exercise in 100mg/kg. Blood was collected to measure paraoxonase-1 (PON1) activity, plasma malondialdehyde (MDA), low-density lipoprotein (LDL) and cholesterol concentrations. These biomarkers were measured in venous blood samples collected before and after the rats swam in pools at different water temperatures (18°C, 28°C and 38°C). In the non-carnitine group, exercise caused a significant decrease in PON1 activity and a significant elevation in MDA concentration at 28°C compared to the sedentary group. No significant alterations were evidenced in LDL and cholesterol concentrations upon exercise. The decrease in PON1 activity became higher with increasing temperature whereas the elevation in MDA levels increased at 18°C. In the l-carnitine supplementation group, recovery in PON1 activity was observed significant at 28°C and very significant at 38°C. MDA concentration was almost the same with that of the non-carnitine group at 18 and 38°C, but it significantly decreased at 28°C. Considering the recovery in PON1 and MDA levels at 28°C, which is the temperature of the sedentary group; our results suggest that l-carnitine supplementation has a protective role on exhaustive exercise-induced oxidative stress. Findings of this study also demonstrate influences of thermal stress on these parameters during exhaustive exercise.

Evaluation of the impacts of antibiotic drugs on PON 1; a major bioscavenger against cardiovascular diseases

Paraoxonase 1 (PON1) is an antiatherogenic enzyme which is also an organophosphate hydrolyzer. It has crucial roles in detoxification of highly toxic substances and protecting LDL against oxidation. Decrease in the levels of this enzyme is a great risk for the patients with cardiovascular diseases, diabetes mellitus, chronic renal failure, rheumatoid arthritis, hyperthyroidism, and age-related macular degeneration. Therefore, inhibitors and activators of PON1 must be well-characterized, and drug studies would be a good starting point in this regard. Moreover, purification of PON1 has been a challenge for scientists due to its tight association with HDL. Here we report the purification of human serum PON1 using very simple methods and investigation of the interactions between the enzyme and some commonly used antibiotics. We purified PON1 from human serum with a high specific activity, and used the pure enzyme for inhibition studies. We observed that some antibiotics inhibit the enzyme at very low doses while some are efficient at higher doses. The antibiotics exhibited different inhibition mechanisms. We concluded that usage of these antibiotics would be very dangerous in some cases.