Alireza Arabshahi

Liquid chromatography tandem mass spectrometry identification of proanthocyanidins in rat plasma after oral administration of grape seed extract.

Proanthocyanidin rich plant extracts derived from grape seed extract (GSE), hawthorn and cranberry are on markets for their preventive effects against cardiovascular diseases and uroinfections in woman. However, the importance of these health beneficial effects of these botanicals remains elusive due to incomplete understanding of uptake, metabolism and bioavailability of proanthocyanidins in vivo. In the present study rats were given GSE orally (300 mg/kg, twice a day) and blood and urine were collected over a 24 h period. Monomeric catechins and their methylated metabolites, and proanthocyanidins up to trimers were detected in blood samples treated with GSE using LC-MS/MS operating in the multiple reaction monitoring (MRM) mode. A new tetramethylated metabolite of dimeric proanthocyanidin (m/z 633) in GSE-treated urine was tentatively identified. Using LC-MS/MS, (+)-catechin and (-)-epicatechin were identified in the brain conclusively. These data suggested that GSE catechins cross the blood brain barrier and may be responsible for the neuroprotective effects of GSE.

Simultaneous determination of 11 phytoestrogens in human serum using a 2 min liquid chromatography/tandem mass spectrometry method

A rapid 2 min liquid chromatography-tandem mass spectrometry (LC-MS/MS) method operating in multiple reaction ion monitoring mode was developed and validated that allows for the characterization and simultaneous quantification of 11 phytoestrogen metabolites with mass transitions m/z 241/119 (equol), 253/132 (daidzein), 255/149 (dihydrodaidzein), 257/108 (O-desmethylangolesin), 269/133 (genistein), 283/184 (glycitein), 267/191 (formononetin), 289/109 (biochanin A), 267/91 (coumestrol), enterodiol (301/253), and enterolactone (297/253). The method was demonstrated to be specific and sensitive, and a linear response for each phytoestrogen was observed over a range of 1-5000 ng/mL in human serum with the exception of dihydrodaidzein, whose lower limit of quantification was 2 ng/mL. The separation was carried out on a Synergi Polar-RP 2.5 micron (50 mm x 2.0 mm i.d.) column at 50 degrees C with water and acetonitrile (both containing 10 mM ammonium acetate) as the mobile phase under gradient conditions at a flow rate of 0.75 mL/min. This LC-MS/MS method is very useful for high-throughput analysis of phytoestrogens and proved to be simple, sensitive, reproducible, and reliable.

Tissue distribution of puerarin and its conjugated metabolites in rats assessed by liquid chromatography tandem mass spectrometry

Puerarin (an isoflavone C-glucoside from kudzu root) has been the focus of several studies investigating its potential effects on health benefits. In this study, we determined single dose tissue distribution of puerarin and its metabolites in order to examine whether they undergo selective uptake by specific organs. Puerarin was administered orally (50 mg/kg) to rats and the concentration of puerarin in tissue compartments was determined using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Puerarin was widely distributed in rat tissues with highest concentrations in lungs (799+/-411.6 ng/g wet tissues). In addition, we examined the excretion of puerarin into the bile. LC-MS/MS analysis of bile samples collected after infusing puerarin directly into the portal vein indicated that puerarin was excreted into the bile predominantly in the form of unconjugated puerarin. This report identifying puerarin in several organs including kidney and pancreas may explain its beneficial effects in diabetes.

Chronic dietary Kudzu Isoflavones Improve Components of Metabolic Syndrome in Stroke-prone Spontaneously Hypertensive Rats

The present study tested the long-term effects of dietary kudzu root extract supplementation on the regulation of arterial pressure, plasma glucose, and circulating cholesterol in stroke-prone spontaneously hypertensive rats (SP-SHR). Female SP-SHR were maintained for 2 months on a polyphenol-free diet, with or without the addition of 0.2% kudzu root extract. Half of the rats in each diet group were ovariectomized, whereas the other half remained intact. Following 2 months on the diets, the 0.2% kudzu root extract supplementation (compared to control diet) significantly lowered arterial pressure (11-15 mmHg), plasma cholesterol, fasting blood glucose (20-30%), and fasting plasma insulin in both the ovariectomized and intact SP-SHR. These results indicate that long-term dietary kudzu root extract supplementation can improve glucose, lipid, and blood pressure control in intact and ovariectomized SP-SHR.

Simultaneous quantification of F2-isoprostanes and prostaglandins in human urine by liquid chromatography tandem-mass spectrometry

A specific and sensitive LC-MS/MS method for analysis of F(2)-isoprostanes (F(2)-IsoPs) and prostaglandins (PGs) in urine was developed and validated to examine the levels of F(2)-IsoPs and prostaglandin F(2α) (PGF(2α)), in human urine in patients undergoing cardiac surgery. The rapid extraction for F(2)-IsoPs and PGs from urine was achieved using a polymeric weak anion solid phase extraction cartridge. The base-line separation of 8-iso-PGF(2α), 8-iso-15(R)-PGF(2α), PGF(2α), and 15(R)-PGF(2α) was carried out on a Hydro-RP column (250mm×2.0mm i.d., Phenomenex, CA) using a linear gradient of methanol:acetonitrile (1:1, v/v) in 0.1% formic acid at a flow rate of 0.2mL/min. The method was proved to be accurate and precise for simultaneous quantification of each analyte over a linear dynamic range of 0.05-50ng/mL with correlation coefficients greater than 0.99. The intra-day and inter-day assay precision at the lowest quality control (0.07ng/mL) level were less than 17%. The mean extraction recoveries of F(2)-IsoPs and PGs were in a range of 79-100%. In applications of this method to patients undergoing cardiac surgery, post-surgery urinary concentrations of 8-iso-PGF(2α) increased significantly in patients (n=14) who did not develop acute kidney (AKI) (pre-surgery 0.344±0.039 vs. post-surgery 0.682±0.094ng/mg creatinine, p<0.01), whereas there was no significant change in this isoprostane in the patients (n=4) who developed AKI (pre-surgery 0.298±0.062 vs. post-surgery 0.383±0.117ng/mg creatinine, NS). Therefore, the method is suitable for the analysis of individual F(2)-IsoPs and PGF(2α)s in both clinical and research studies.

Hemodynamic characteristics of midazolam, propofol, and dexmedetomidine in healthy volunteers

STUDY OBJECTIVE To study the effect of intravenous (IV) sedation on blood pressure (BP), heart rate (HR), and respiratory rates (RR) to determine if IV sedatives differ with respect to their effect on BP, HR, and RR. DESIGN Prospective, randomized, single-blinded, placebo-controlled study. SETTING Monitored patient care room at a clinical research center. SUBJECTS 60 healthy ASA physical status 1 volunteers. INTERVENTIONS Subjects were randomized to receive, in increasing doses, one of three IV sedatives: propofol, midazolam, or dexmedetomidine; or saline control. MEASUREMENTS Blood pressure (systolic, diastolic), HR, and RR were recorded. MAIN RESULTS A significant dose-dependent BP reduction occurred with dexmedetomidine and, to a lesser degree, with propofol; and there was good agreement of predicted versus measured drug concentrations for all sedatives. Blood pressure and HR of participants who received midazolam did not change. CONCLUSIONS When administered in sedative doses, dexmedetomidine and, to a lesser extent, midazolam, reduces BP in a dose-dependent fashion. Dexmedetomidine also reduces HR. Midazolam does not affect BP or HR.

Supplemental Dietary Racemic Equol Has Modest Benefits to Bone but Has Mild Uterotropic Activity in Ovariectomized Rats

Soy isoflavones and their metabolites, with estrogenic activity, have been considered candidates for reducing postmenopausal bone loss. In this study, we examined the effect of dietary equol, a bioactive metabolite of the soy isoflavone daidzein, on equol tissue distribution, bone parameters, and reproductive tissue activity using an adult ovariectomized (OVX) rat model. An 8-wk feeding study was conducted to compare 4 dietary treatments of equol (0, 50, 100, 200 mg/kg diet) in 6-mo-old OVX female Sprague-Dawley rats. A dose response increase in tissue equol concentrations was observed for serum, liver, kidney, and heart, and a plateau occurred at 100 mg equol/kg diet for intestine. In OVX rats receiving 200 mg equol/kg diet, femoral calcium concentration was greater than those receiving lower doses but was still less than SHAM (P < 0.05), and other bone measures were not improved. Tibia calcium concentrations were lower in OVX rats receiving 100 and 200 mg equol/kg diet compared with the OVX control rats. Trabecular bone mineral density of tibia was also lower in equol-fed OVX rats. At this dietary equol intake, uterine weight was higher (P < 0.05) than in other OVX groups but lower than the SHAM-operated intact rats. The 200 mg/kg diet dose of dietary equol significantly increased proliferative index in the uterine epithelium. Dietary equol had no stimulatory effect on mammary gland epithelium. We conclude that in OVX rats, a dietary equol dose that had modest effect on bone also exerts mild uterotropic effects.

Hemoglobin-associated oxidative stress in the pericardial compartment of postoperative cardiac surgery patients

Atherosclerosis and valvular heart disease often require treatment with corrective surgery to prevent future myocardial infarction, ischemic heart disease, and heart failure. Mechanisms underlying the development of the associated complications of surgery are multifactorial and have been linked to inflammation and oxidative stress, classically as measured in the blood or plasma of patients. Postoperative pericardial fluid (PO-PCF) has not been investigated in depth with respect to the potential to induce oxidative stress. This is important because cardiac surgery disrupts the integrity of the pericardial membrane surrounding the heart and causes significant alterations in the composition of the pericardial fluid (PCF). This includes contamination with hemolyzed blood and high concentrations of oxidized hemoglobin, which suggests that cardiac surgery results in oxidative stress within the pericardial space. Accordingly, we tested the hypothesis that PO-PCF is highly pro-oxidant and that the potential interaction between inflammatory cell-derived hydrogen peroxide with hemoglobin is associated with oxidative stress. Blood and PCF were collected from 31 patients at the time of surgery and postoperatively from 4 to 48 h after coronary artery bypass grafting, valve replacement, or valve repair (mitral or aortic). PO-PCF contained high concentrations of neutrophils and monocytes, which are capable of generating elevated amounts of superoxide and hydrogen peroxide through the oxidative burst. In addition, PO-PCF primed naive neutrophils resulting in an enhanced oxidative burst upon stimulation. The PO-PCF also contained increased concentrations of cell-free oxidized hemoglobin that was associated with elevated levels of F2α isoprostanes and prostaglandins, consistent with both oxidative stress and activation of cyclooxygenase. Lastly, protein analysis of the PO-PCF revealed evidence of protein thiol oxidation and protein carbonylation. We conclude that PO-PCF is highly pro-oxidant and speculate that it may contribute to the risk of postoperative complications.

Lipoxin A4, a 5‐lipoxygenase pathway metabolite, modulates immune response during acute respiratory tularemia

Respiratory infection with Francisella tularensis (Ft) is characterized by a muted, acute host response, followed by sepsis‐like syndrome that results in death. Infection with Ft establishes a principally anti‐inflammatory environment that subverts host‐cell death programs to facilitate pathogen replication. Although the role of cytokines has been explored extensively, the role of eicosanoids in tularemia pathogenesis is not fully understood. Given that lipoxin A4 (LXA4) has anti‐inflammatory properties, we investigated whether this lipid mediator affects host responses manifested early during infection. The addition of exogenous LXA4 inhibits PGE2 release by Ft‐infected murine monocytes in vitro and diminishes apoptotic cell death. Tularemia pathogenesis was characterized in 5‐lipoxygenase‐deficient (Alox5−/−) mice that are incapable of generating LXA4. Increased release of proinflammatory cytokines and chemokines, as well as increased apoptosis, was observed in Alox5−/− mice as compared with their wild‐type counterparts. Alox5−/− mice also exhibited elevated recruitment of neutrophils during the early phase of infection and increased resistance to lethal challenge. Conversely, administration of exogenous LXA4 to Alox5−/− mice made them more susceptible to infection thus mimicking wild‐type animals. Taken together, our results suggest that 5‐LO activity is a critical regulator of immunopathology observed during the acute phase of respiratory tularemia, regulating bacterial burden and neutrophil recruitment and production of proinflammatory modulators and increasing morbidity and mortality. These studies identify a detrimental role for the 5‐LO–derived lipid mediator LXA4 in Ft‐induced immunopathology. Targeting this pathway may have therapeutic benefit as an adjunct to treatment with antibiotics and conventional antimicrobial peptides, which often have limited efficacy against intracellular bacteria.

Isoflavone metabolism in domestic cats (Felis catus): Comparison of plasma metabolites detected after ingestion of two different dietary forms of genistein and daidzein1

Some felid diets contain isoflavones but the metabolic capacity of cats toward isoflavones is relatively unknown, despite the understanding that isoflavones have divergent biological potential according to their metabolite end products. The objective of this study was to determine the plasma metabolites detectable in domestic cats after exposure to 2 different dietary forms of isoflavones, either as a soy extract tablet (n = 6) or as part of a dietary matrix (n = 4). Serial blood samples were collected after isoflavone exposure to identify the plasma metabolites of each cat. Genistein was detected in its unconjugated form or as a monosulfate. Daidzein was detected as both a mono- and disulfate as well as in its unconjugated form. Other daidzein metabolites detected included equol mono- and disulfate, dihydrodaidzein, and O-desmethylangolensin. No β-glucuronide metabolites of either isoflavone were detected. Equol was produced in markedly fewer cats after ingestion of a soy extract tablet as a single oral bolus compared with cats consuming an isoflavone-containing diet. The detectable metabolites of the isoflavones, genistein and daidzein, in domestic cat plasma after dietary ingestion has been described in the present study for the first time. The metabolic capacity for isoflavones by domestic cats appears to be efficient, with only minimal proportions of the ingested amount detected in their unconjugated forms. This has implications for the potential of isoflavones to exert physiological activity in the domestic cat when consumed at concentrations representative of typical dietary intake.