Amir Kiani

Determination of oseltamivir carboxylic acid in human serum by solid phase extraction and high performance liquid chromatography with UV detection

This study was aimed at developing a fast and sensitive method for determination of oseltamivir carboxylic acid (OCA), the active moiety of anti-influenza agent, oseltamivir phosphate, in human serum by high performance liquid chromatography and UV detection. The analyte and an internal standard (vanillin) were extracted from human serum by a solid phase extraction (SPE) procedure. Chromatographic separation was achieved using a reverse phase C18 column with a mobile phase consisting of 0.05M phosphate buffer containing triethylamine (1mL/L; pH 3.0) and acetonitrile (70:30, v/v). The detection wavelength was set at 215nm. The average recoveries of the drug and internal standard were 98 and 85%, respectively. The calibration curve was linear over a concentration range of 15-6400ng/mL of OCA in human serum. The lower limits of detection and quantification were 5 and 15ng/mL, respectively. The coefficient variation values of both inter- and intra-day analysis were less than 12% whereas the percentage error was less than 4.5. The stability of the drug at the serum samples maintained at -40 degrees C for 60 days was found to be 100% from the initial value and no interferences were found from either endogenous components in serum or commonly co-administrated antiviral drugs. The validated method was applied to a randomized cross-over bioequivalence study of two different oseltamivir phosphate preparations in 24 healthy volunteers.

In vitro and in vivo anti-angiogenesis effect of shallot (Allium ascalonicum): a heat-stable and flavonoid-rich fraction of shallot extract potently inhibits angiogenesis.

This study has been undertaken to elucidate the anti-angiogenic properties of shallot extract in vitro and in vivo and also to define the responsible fraction and its stability. After preparation of the extract of shallot bulbs with 50% ethanol, the extract was successively fractionated into n-hexane, ethyl acetate, n-butanol and aqueous fractions. The ethyl acetate fraction was further fractionated to three subfractions using thin layer chromatography. Anti-angiogenic activity of fractions and subfractions were examined on human umbilical vein endothelial cells (HUVECs) in collagen matrix and chicken chorioallantoic membrane (CAM) models. Among the fractions, ethyl acetate fraction and one of its subfractions potently inhibited angiogenesis in vitro and in vivo. Furthermore, ethyl acetate fraction sustained its inhibitory effect significantly even after treatment in high thermal and low pH conditions. These findings provided a useful basis for further investigations on shallot as a useful herb with therapeutic or preventive activity against angiogenesis related disorders.

Matrix metalloproteinase-2 functional promoter polymorphism G1575A is associated with elevated circulatory MMP-2 levels and increased risk of cardiovascular disease in systemic lupus erythematosus patients

Matrix metalloproteinase-2 (MMP-2) is a zinc dependent endonuclease that degrades type IV collagen, the major structural component of basement membranes. MMP-2 functional promoter polymorphism G1575A affects circulating level of MMP-2 and may be considered an important genetic determinant of cardiovascular disease (CVD) in systemic lupus erythematosus (SLE) patients. In this study, association between MMP-2 1575A allele with serum MMP-2, neopterin and lipid-lipoprotein levels and with SLE and developing CVD was investigated. The present case–control study consisted of 109 SLE patients with and without CVD (mean age, 35.6 years) and 101 gender- and age-matched, unrelated, healthy controls (mean age, 37.1 years) from the population in the west of Iran. MMP-2 1575G/A polymorphism was detected by polymerase chain reaction (restriction fragment length polymorphism) PCR-RFLP, serum MMP-2, neopterin and lipid levels were determined by enzyme-linked immunosorbent assay (ELISA), high-performance liquid chromatography (HPLC) and enzyme assay, respectively. The presence of MMP-2 G1575A allele was found to be associated with SLE and developed CVD (OR = 1.78, p = 0.029 and OR = 3.43, p = 0.025, respectively). The SLE patients with MMP-2 A (G/A + A/A) allele had higher MMP-2 activity (301 ± 166 vs. 194 ± 35.5, p = 0.002), neopterin (29.4 ± 39.4 vs. 7.3 ± 4.6, p = 0.005), LDL-C (120 ± 25.7 vs. 87 ± 39.3, p = 0.045) and lower HDL-C (39.6 ± 11 vs. 45.9 ± 11.8, p = 0.031) levels than the control subjects. There was a significantly positive correlation between MMP-2 level with neopterin, total cholesterol and TG levels and negative correlation with HDL-C level in SLE patients with CVD. MMP-2 G1575A allele may be a risk factor for SLE. The carriers of this allele have high levels of MMP-2, neopterin, total cholesterol and TG and lower levels of HDL, thus, they are more likely to develop heart disease.

Paraoxonase 1 (PON1) 55 polymorphism, lipid profiles and psoriasis

Background  Paraoxonase 1 (PON1) is a serum high‐density lipoprotein‐bound enzyme with antioxidant function. It hydrolyses lipid peroxides, protecting low‐density lipoproteins from oxidative modifications. Patients with psoriasis are at greater risk of oxidative stress, which is associated with abnormal plasma lipid metabolism.

Butyrylcholinesterase K variants increase the risk of coronary artery disease in the population of western Iran

The conflicting results of several studies suggest that there is an association between the butyrylcholinesterase‐K variant (BCHE‐K, G1615A/Ala539Thr) and the risk of developing coronary artery disease (CAD) in diabetes and non‐diabetic subjects. The objective of this study was to determine whether the presence of the BCHE‐K variant exacerbates the risk of CAD in patients from western Iran with and without type 2 diabetes mellitus (T2DM). This case‐control study comprised 464 subjects undergoing their first coronary angiography. They were matched and randomly assigned into four groups: CAD+T2DM+ (CAD/T2DM), CAD+DM− (CAD/ND), CAD−DM+ (T2DM/NCAD) and CAD−DM−(control). The BCHE‐K variant was detected by PCR‐RFLP. The BCHE‐K allele frequency in CAD patients with and without T2DM [total CAD (TCAD)] and separately for each group (CAD/T2DM and CAD/ND) was significantly higher than in the control group (21.1 % versus 13.3 % (p = 0.001), 22.4 % versus 13.3 % (p = 0.001) and 19.7 % versus 13.3 % (p = 0.015), respectively). The odds ratios (ORs) for the BCHE‐K heterozygous and homozygous variants in TCAD subjects were 1.65 (95 % CI 1.17–2.3; p = 0.004) and 4.3 (1.05–19.4; p = 0.048); for CAD/T2DM individuals 1.76 (1.2–2.6; p = 0.004) and 4.73 (0.96–23.3; p = 0.052); and for CAD/ND patients 1.53 (1.05–2.3; p = 0.029) and 3.88 (0.8–19.7; p = 0.7), respectively. The OR of the BCHE‐K allele was found to be 1.74 (1.1–2.4; p = 0.001) in TCAD subjects, 1.87 (1.12–1.48; p = 0.001) in the CAD/T2DM group and 1.59 (1.04–1.4; p = 0.016) in CAD/ND subjects. These data suggest that the BCHE‐K allele increases the risk of CAD in the population (with and without DM) in western parts of Iran, and its presence intensifies the risk of CAD in T2DM. The fact that the BCHE‐K allele, even in the heterozygous form, exacerbates the risk of CAD in this population, suggests that a specific therapeutic intervention should be considered for this particular group of patients.

Association between butyrylcholinesterase activity and phenotypes, paraoxonase192 rs662 gene polymorphism and their enzymatic activity with severity of rheumatoid arthritis: correlation with systemic inflammatory markers and oxidative stress, preliminary report.

OBJECTIVES Evidences indicate that oxidative stress and inflammation are important processes in the development of destructive synovial tissue in rheumatoid arthritis (RA). The two major bioscavenger enzymes that are associated with inflammation and oxidative stress are human-butyrylcholinesterase (BuChE) and paraoxonase-1 (PON-1). Thus, the objective of this study was to determine the relation of BuChE phenotypes and PON-1 Q192R polymorphism with inflammatory markers such as anti-cytroline circulated peptide (CCP)-antibodies, CRP, neopterin, DAS28-CRP in RA patients. DESIGN AND METHODS In this study, we examined association of BuChE-phenotypes and activity, PON192rs662 (Q192R) polymorphism and its arylesterase activity (ARE) with systemic-inflammatory-markers and oxidative stress. The present case-control study consisted of 419-RA patients and 398 gender-age-matched unrelated healthy controls from west population of Iran. PON192rs662 polymorphism was detected by real-time-PCR. BuChE phenotype, TAC level, serum BuChE and ARE activities were determined spectrophotometrically. Anti-CCP-antibody and CRP were measured by ELISA and neopterin level was detected by HPLC. We used the EULAR activity criteria to measure DAS28-CRP. RESULTS We found that PON-1-Q192R was associated with severity of RA [remission-to-low and moderate-to-high in dominant Q/Q+Q/R vs. R/R: OR=2.27, p<0.001; codominant Q/Q vs. R/R: OR=1.65, p<0.001 and Q/R vs. R/R: OR=2.12, p=0.003; recessive Q/Q vs. R/R+Q/R: OR=1.79, p=0.032; and allele Q vs. R: OR=1.68, p<0.001] and presence of anti-CCP-antibody (codominant model Q/Q vs. R/R: OR=1.28, p=0.042). The carriers of Q/Q genotype PON-1-Q192R and BuChE non-UU-phenotype had higher ARE activity, serum levels of neopterin, anti-CCP antibody titer and number of tender-joint and lower activity of BuChE and serum level of TAC than that of R/R genotype and BuChE-UU-phenotype. CONCLUSIONS The current findings demonstrate for the first time that there is a link between systemic inflammatory markers, oxidative stress, the PON192rs662-Q allele and BuChE-non-UU-phenotype and their corresponding enzymatic activity which may be considered as a risk factor for the severity of RA for a population in Iran.

Matrix metalloproteinase 9 polymorphisms and systemic lupus erythematosus: correlation with systemic inflammatory markers and oxidative stress

Systemic lupus erythematosus (SLE) is an autoimmune disease that involves multiple organs and is characterized by persistent systemic inflammation. Among the effects of inflammatory mediators, the induction of matrix metalloproteinases-2 and -9 (MMP-2 and MMP-9) and oxidative stress has been demonstrated to be important in the development of SLE. In this study, the possible association between MMP-9 and MMP-2 functional promoter polymorphism, stress, and inflammatory markers with development of severe cardiovascular disease (CVD), high blood pressure (HBP), and lupus nephropathy (LN) in SLE patients was investigated. The present case-control study consisted of 109 SLE patients with and without CVD, HBP and LN and 101 gender- and age-matched unrelated healthy controls from a population in western Iran. MMP-2 –G1575A and MMP-9 –C1562T polymorphisms were detected by PCR-RFLP, serum MMP-2 and MMP-9, neopterin, malondialdehyde (MDA) and lipid levels were determined by ELISA, HPLC and enzyme assay, respectively. We found that MMP-9 –C1562 T and MMP-2 –G1575A alleles act synergistically to increase the risk of SLE by 2.98 times (p = 0.015). Findings of this study also demonstrated that there is a significant increase in the serum levels of MMP-2, neopterin and MDA and a significant decrease in serum level of MMP-9 in the presence of MMP-9-C1562 T and MMP-2 –G1575A alleles in SLE patients compared to controls. Further, SLE patients with MMP-9 (C/T + T/T) genotype had significantly higher serum concentrations of MMP-2, neopterin, MDA and LDL-C, but lower serum MMP-9 and HDL-C levels than corresponding members of the control group. MMP-9 (C/T + T/T) genotype increased risk of hypertension in SLE patients 2.71-fold. This study for the first time not only suggests that MMP-9 –C1562 T and MMP-2 –G1575A alleles synergistically increase the risk of SLE but also high serum levels of MDA, neopterin, and circulatory levels of MMP-2 and lower MMP-9 in SLE patients. This information may be important in the evaluation of SLE progression and in the elucidation of the mechanisms of the disease pathogenesis.

Synergistic effects of BuChE non-UU phenotype and paraoxonase (PON1) 55 M allele on the risk of systemic lupus erythematosus: influence on lipid and lipoprotein metabolism and oxidative stress, preliminary report

There is some evidence indicating lipid peroxidation can affect progression of atherosclerosis, cardiovascular diseases (CVDs) and glomerulonephritis in systemic lupus erythematosus (SLE) patients. Human butyrylcholinesterase (BuChE) and paraoxonase-1 (PON1) are two major bioscavenger enzymes that are associated with inflammation, oxidative stress and lipid metabolism. Hyperlipidemia, increase in lipid oxidation reactions and defects in antioxidant status may lead to increased oxidative stress and high frequency of CVDs in SLE. It has also been suggested that deficiency in the function of the antioxidant system and an increase in reactive oxygen release (ROS) may play an important role in the pathogenesis of SLE. This study is the first investigation to examine the association of BuChE phenotypes, PON1 (L55M; PON-55-M) polymorphism, the levels of malondialdehyde (MDA), neopterin, lipid-lipoprotein and activities of BuChE and arylesterase activity (ARE) of PON with severity of SLE. The present case-control study consisted of 109 SLE patients and 101 gender- and age-matched, unrelated healthy control subjects from the population of west Iran. We found that the PON-55-M allele and BuChE non-UU act synergistically to increase the risk of SLE by 2.5 times (1.03–6.7, p = 0.044). There was a significant negative correlation between severity of SLE with serum BuChE activity (R = −0.31, p < 0.001) and positive correlation with serum neopterin level. The SLE patients with the PON-55-M (M/L + M/M) allele or with BuChE non-UU phenotype had significantly lower serum ARE and BuChE activities than those with PON-55-L/L or BuChE-UU phenotypes, respectively. In addition, their serum levels of MDA, neopterin and LDL-C were significantly elevated, suggesting that these individuals are more susceptible to CVD. However, further studies are needed to shed more light on the contribution of the M allele of PON1 and non-UU phenotypes of BuChE in the development of SLE in different ethnicities.

Plasma level of neopterin as a marker of disease activity in treated rheumatoid arthritis patients: Association with gender, disease activity and anti-CCP antibody

Immune system activation is known to be involved in the progression of rheumatoid arthritis (RA). The pro-inflammatory cytokine interferon-γ in various cells, including monocytes, induces neopterin production. Plasma level of neopterin has been measured in many autoimmune diseases and can be used as a marker of cellular immunity activation. In this study we measured the plasma level of neopterin in 418 treated RA patients and 398 age and sex matched healthy people by high pressure liquid chromatography (HPLC) method. Disease activity score was calculated in all patients by DAS-CRP method. Plasma level of neopterin was compared between RA and control groups. We also determined the association between neopterin level with gender and disease activity score in RA patients. Significantly higher level of neopterin was observed in RA patients compared to healthy controls. Moreover, there was higher neopterin level in male RA patients versus female patients. Plasma neopterin level was increased in patients with active disease and also was correlated with disease activity parameters. There was a significant correlation of plasma level of neopterin with age in both RA and control group and also age of onset and disease duration in RA patients. Anti-CCP positive patients had higher level of neopterin in comparison to anti-CCP negative patients and there was a significant correlation between neopterin level and anti-CCP titer. Our results indicated that neopterin is a sensitive marker for assaying background inflammation and disease activity score in RA patients and may be used as a marker for evaluation of therapy efficacy.

Apolipoprotein E Genotypes, Lipid Peroxidation, and Antioxidant Status among Mild and Severe Preeclamptic Women from Western Iran: Protective Role of Apolipoprotein ε2 Allele in Severe Preeclampsia

Objective. The aim of this study was to examine the association of apolipoprotein E (APOE) genotypes and oxidative stress with the risk of mild and severe preeclampsia. Methods. In a case–control study, 198 women with preeclampsia including 128 women with mild and 70 women with severe preeclampsia and 101 control pregnant women from Western Iran were studied. The APOE genotypes were identified using polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP) method. The serum level of malondialdehyde (MDA) and total antioxidant capacity (TAC) were determined using high-performance liquid chromatography and commercial kits, respectively. Results. The frequency of APOE ϵ2 allele in severe preeclamptic women (2.1%) was significantly (p = 0.008) lower than that in controls (9.4%). The presence of APOE ϵ2 allele was associated with around five times decreased risk of severe preeclampsia [OR = 0.21 (95% CI = 0.6–0.73, p = 0.014)]. A significantly higher serum level of MDA was observed in women with severe (10.87 ± 4.61 μM) and mild (9.81 ± 3.67 μM) preeclampsia compared with that in controls. A trend toward decrease serum level of MDA was observed according to the APOE alleles as ϵ2 < ϵ3 < ϵ4 (9.23, 10.22, and 10.43 μM, respectively). In preeclamptic women, an inverse correlation was detected between serum levels of MDA and HDL-C (r = −0.16, p = 0.029). However, there was a direct correlation between serum level of MDA with diastolic blood pressure (r = 0.15, p = 0.037). Conclusion. Our study in a population with Kurdish ethnic background indicates a protective role for APOE ϵ2 allele against severe preeclampsia that might be through high antioxidant capacity of this allele.