Determination of l-glutamic acid and γ-aminobutyric acid in mouse brain tissue utilizing GC-MS/MS.

A rapid and selective method for the quantitation of neurotransmitters, l-Glutamic acid (GA) and γ-Aminobutyric acid (GABA), was developed and validated using gas chromatography-tandem mass spectrometry (GC-MS/MS). The novel method utilized a rapid online hot GC inlet gas phase sample derivatization and fast GC low thermal mass technology. The method calibration was linear from 0.5 to 100µg/mL, with limits of detections of 100ng/mL and 250ng/mL for GA and GABA, respectively. The method was used to investigate the effects of deletion of organic anion transporter 1 (Oat1) or Oat3 on murine CNS levels of GA and GABA at 3 and 18 mo of age, as compared to age matched wild-type (WT) animals. Whole brain concentrations of GA were comparable between WT, Oat1-/-, and Oat3-/- 18 mo at both 3 and 18 mo of age. Similarly, whole brain concentrations of GABA were not significantly altered in either knockout mouse strain at 3 or 18 mo of age, as compared to WT. These results indicate that the developed GC-MS/MS method provides sufficient sensitivity and selectivity for the quantitation of these neurotransmitters in mouse brain tissue. Furthermore, these results suggest that loss of Oat1 or Oat3 function in isolation does not result in significant alterations in brain tissue levels of GA or GABA.

Inosine and hypoxanthine as novel biomarkers for cardiac ischemia: from bench to point-of-care.

Cardiac ischemia associated with acute coronary syndrome and myocardial infarction is a leading cause of mortality and morbidity in the world. A rapid detection of the ischemic events is critically important for achieving timely diagnosis, treatment and improving the patient's survival and functional recovery. This minireview provides an overview on the current biomarker research for detection of acute cardiac ischemia. We primarily focus on inosine and hypoxanthine, two by-products of ATP catabolism. Based on our published findings of elevated plasma concentrations of inosine/hypoxanthine in animal laboratory and clinical settings, since 2006 we have originally proposed that these two purine molecules can be used as rapid and sensitive biomarkers for acute cardiac ischemia at its very early onset (within 15 min), hours prior to the release of heart tissue necrosis biomarkers such as cardiac troponins. We further developed a chemiluminescence technology, one of the most affordable and sensitive analytical techniques, and we were able to reproducibly quantify and differentiate total hypoxanthine concentrations in the plasma samples from healthy individuals versus patients suffering from ischemic heart disease. Additional rigorous clinical studies are needed to validate the plasma inosine/hypoxanthine concentrations, in conjunction with other current cardiac biomarkers, for a better revelation of their diagnostic potentials for early detection of acute cardiac ischemia.

Phase I safety trial of intravenous ascorbic acid in patients with severe sepsis.

BACKGROUND: Parenterally administered ascorbic acid modulates sepsis-induced inflammation and coagulation in experimental animal models. The objective of this randomized, double-blind, placebo-controlled, phase I trial was to determine the safety of intravenously infused ascorbic acid in patients with severe sepsis. METHODS: Twenty-four patients with severe sepsis in the medical intensive care unit were randomized 1:1:1 to receive intravenous infusions every six hours for four days of ascorbic acid: Lo-AscA (50 mg/kg/24 h, n = 8), or Hi-AscA (200 mg/kg/24 h, n = 8), or Placebo (5% dextrose/water, n = 8). The primary end points were ascorbic acid safety and tolerability, assessed as treatment-related adverse-event frequency and severity. Patients were monitored for worsened arterial hypotension, tachycardia, hypernatremia, and nausea or vomiting. In addition Sequential Organ Failure Assessment (SOFA) scores and plasma levels of ascorbic acid, C-reactive protein, procalcitonin, and thrombomodulin were monitored. RESULTS: Mean plasma ascorbic acid levels at entry for the entire cohort were 17.9 ± 2.4 µM (normal range 50-70 µM). Ascorbic acid infusion rapidly and significantly increased plasma ascorbic acid levels. No adverse safety events were observed in ascorbic acid-infused patients. Patients receiving ascorbic acid exhibited prompt reductions in SOFA scores while placebo patients exhibited no such reduction. Ascorbic acid significantly reduced the proinflammatory biomarkers C-reactive protein and procalcitonin. Unlike placebo patients, thrombomodulin in ascorbic acid infused patients exhibited no significant rise, suggesting attenuation of vascular endothelial injury. CONCLUSIONS: Intravenous ascorbic acid infusion was safe and well tolerated in this study and may positively impact the extent of multiple organ failure and biomarkers of inflammation and endothelial injury. TRIAL REGISTRATION: ClinicalTrials.gov identifier NCT01434121.

Expression and function of organic cation and anion transporters (SLC22 family) in the CNS.

A major function of the blood brain barrier (BBB) and blood cerebrospinal fluid barrier (BCSFB) is to exert selective control over the flux of organic cations and anions into and out of the CNS compartment. These barriers are dynamic tissues that accomplish this task by expressing dozens of transporter proteins representing numerous transporter families. One such family, belonging to the Solute Carrier (SLC) superfamily, is the organic cation/anion/zwitterion (SLC22) family of transporters, which includes the organic cation transporters (OCTs/OCTNs) and organic anion transporters (OATs). SLC22 transporters interact with a broad range of compounds that include drugs of abuse, environmental toxins/toxicants, opioid analgesics, antidepressant and anxiolytic agents and neurotransmitters and their metabolites. Defining the transport mechanisms controlling the CNS penetration, disposition and clearance of such compounds is fundamental to advancing our understanding of the underlying mechanisms that regulate CNS homeostasis and impact neuronal health. Such information might help direct efforts to improve the efficacy and clinical outcomes of current and future therapeutic agents used in the treatment of CNS disorders. This review focuses on highlighting the identification of the SLC22 transporter family, current knowledge of OCT and OAT expression within the CNS (including brain capillaries, choroid plexus and brain regions relevant to monoaminergic neuronal signaling), and recent data regarding behavioral changes related to mood and anxiety disorders and altered responses to stimulants and antidepressants in SLC22 loss of functions models (knockout/knockdown). In vitro and in vivo evidence of SLC22 localization and transport characteristics within the CNS compartment are summarized.

A rapid and simple chemiluminescence method for screening levels of inosine and hypoxanthine in non-traumatic chest pain patients.

A rapid and simple chemiluminescence method was developed for detection of inosine and hypoxanthine in human plasma. The method utilized a microplate luminometer with direct injectors to automatically dispense reagents during sample analysis. Enzymatic conversions of inosine to hypoxanthine, followed by hypoxanthine to xanthine to uric acid, generated superoxide anion radicals as a useful metabolic by-product. The free radicals react with Pholasin(®) , a sensitive photoprotein used for chemiluminescence detection, to produce measurable blue-green light. The use of Pholasin(®) and a chemiluminescence signal enhancer, Adjuvant-K™, eliminated the need for plasma clean-up steps prior to analysis. The method used 20 µL of heparinized plasma, with complete analysis of total hypoxanthine levels (inosine is metabolized to hypoxanthine using purine nucleoside phosphorylase) in approximately 3.7 min. The rapid chemiluminescence method demonstrated the capability of differentiating total hypoxanthine levels between healthy individuals, and patients presenting with non-traumatic chest pain and potential acute cardiac ischemia. The results support the potential use of chemiluminescence methodology as a diagnostic tool to rapidly screen for elevated levels of inosine and hypoxanthine in human plasma, potential biomarkers of acute cardiac ischemia.

A simple and sensitive HPLC fluorescence method for determination of tadalafil in mouse plasma.

A simple and sensitive high-performance liquid chromatographic (HPLC) method utilizing fluorescence detection was developed for the determination of the phosphodiesterase type 5 inhibitor tadalafil in mouse plasma. This method utilizes a simple sample preparation (protein precipitation) with high recovery of tadalafil (∼98%), which eliminates the need for an internal standard. For constituent separation, the method utilized a monolithic C(18) column and a flow rate of 1.0mL/min with a mobile phase gradient consisting of aqueous trifluoroacetic acid (0.1% TFA in deionized water pH 2.2, v/v) and acetonitrile. The method calibration was linear for tadalafil in mouse plasma from 100 to 2000ng/mL (r>0.999) with a detection limit of approximately 40ng/mL. Component fluorescence detection was achieved using an excitation wavelength of 275nm with monitoring of the emission wavelength at 335nm. The intra-day and inter-day precision (relative standard deviation, RSD) values for tadalafil in mouse plasma were less than 14%, and the accuracy (percent error) was within -14% of the nominal concentration. The method was utilized on mouse plasma samples from research evaluating the potential cardioprotective effects of tadalafil on mouse heart tissue exposed to doxorubicin, a chemotherapeutic drug with reported cardiotoxic effects.

An in vivo-in vitro study of cefepime and cefazolin dialytic clearance during high-flux hemodialysis.

STUDY OBJECTIVES: To assess the influence of in vitro and in vivo hemodialysis with a new high-flux dialyzer on the clearance of cefazolin and cefepime; to assess the correlation of in vivo dialytic clearance of these antibiotics with blood flow rate; and to assess the correlation between in vitro and in vivo dialytic clearances of these antibiotics. DESIGN: Prospective, open-label, dialysis clearance study. SETTING: A tertiary-care, university health science center. PATIENTS: Five adults who received high-flux hemodialysis 3 times/week. Intervention. For the in vivo experiment, patients received a single intravenous infusion of cefazolin 1 g and cefepime 1 g before dialysis and then underwent a modified hemodialysis session. For the in vitro experiment, a buffered simulated plasma water (SPW) solution containing cefazolin and cefepime was used. Hemodialysis for both experiments was performed with use of a new high-flux polysulfone dialyzer. MEASUREMENTS AND MAIN RESULTS: Cefazolin and cefepime dialytic clearances were determined at blood and/or SPW flow rates of 100, 200, 300, and 400 ml/minute after a 15-minute equilibration period. The degree of correlation of in vitro and in vivo clearances with blood flow rate was determined. Cefepime dialytic clearance increased proportionally with blood flow rate (p<0.01), reaching a maximum mean +/- SD value of 178.9 +/- 24.3 ml/minute at a blood flow rate of 400 ml/minute. Cefazolin dialytic clearance ranged from a mean +/- SD of 42.3 +/- 7.7 to 52.7 +/- 16 ml/minute; no significant correlation was noted between blood flow rate and dialytic clearance. In vitro cefazolin and cefepime dialytic clearances increased proportionally with SPW flow rate (p<0.05). After adjusting the in vitro cefazolin and cefepime dialytic clearances based on their degrees of protein binding, the correlation between the in vitro and in vivo cefepime dialytic clearances was significant (r(2)=0.91, p=0.04), but no significant correlation was noted between the in vitro and in vivo cefazolin clearances (r(2)=0.61, p=0.22). CONCLUSION: The in vivo hemodialysis clearances of cefepime and cefazolin with the new high-flux polysulfone dialyzer used in this study are markedly higher than values reported with conventional dialyzers but similar to values observed with other high-flux hemodialyzers. The in vivo dialytic clearance of cefazolin was significantly lower than the in vitro values, most likely due to cefazolin's high degree of protein binding. These results highlight the limitation of directly applying in vitro data to clinical situations.

Effects of salicylic acid on post-ischaemic ventricular function and purine efflux in isolated mouse hearts.

Acetyl salicylic acid (aspirin) is one of the most widely used drugs in the world. Various plasma concentrations of aspirin and its predominant metabolite, salicylic acid, are required for its antiarthritic (1.5-2.5 mM), anti-inflammatory (0.5-5.0 mM) or antiplatelet (0.18-0.36 mM) actions. A recent study demonstrated the inhibitory effects of both aspirin and salicylic acid on oxidative phosphorylation and ATP synthesis in isolated rat cardiac mitochondria in a dose-dependent manner (0-10 mM concentration range). In this context, the present study was conducted to determine the effects of salicylic acid on inosine efflux (a potential biomarker of acute cardiac ischaemia) as well as cardiac contractile function in the isolated mouse heart following 20 min of zero-flow global ischaemia. Inosine efflux was found at significantly higher concentrations in ischaemic hearts perfused with Krebs buffer fortified with 1.0 mM salicylic acid compared with those without salicylic acid (12575+/-3319 vs. 1437+/-348 ng ml(-1) min(-1), mean+/-SEM, n=6 per group, p<0.01). These results indicate that 1.0 mM salicylic acid potentiates 8.8-fold ATP nucleotide purine catabolism into its metabolites (e.g. inosine, hypoxanthine). Salicylic acid (0.1 or 1.0 mM) did not appreciably inhibit purine nucleoside phosphorylase (the enzyme converts inosine to hypoxanthine) suggesting the augmented inosine efflux was due to the salicylic acid effect on upstream elements of cellular respiration. Whereas post-ischaemic cardiac function was further depressed by 1.0 mM salicylic acid, perfusion with 0.1 mM salicylic acid led to a remarkable functional improvement despite moderately increased inosine efflux (2.7-fold). We conclude that inosine is a sensitive biomarker for detecting cardiac ischaemia and salicylic acid-induced effects on cellular respiration. However, the inosine efflux level appears to be a poor predictor of the individual post-ischaemic cardiac functional recovery in this ex vivo model.

An HPLC method for determination of inosine and hypoxanthine in human plasma from healthy volunteers and patients presenting with potential acute cardiac ischemia.

A simple and sensitive high-performance liquid chromatography (HPLC) method utilizing ultraviolet (UV) detection was developed for the determination of inosine and hypoxanthine in human plasma. For component separation, a monolithic C(18) column at a flow rate of 1.0 mL/min with an aqueous mobile phase of trifluoroacetic acid (0.1% TFA in deionized water pH 2.2, v/v) and methanol gradient was used. The method employed a one-step sample preparation utilizing centrifugal filtration with high component recoveries (approximately 98%) from plasma, which eliminated the need of an internal standard. The method demonstrated excellent linearity (0.25-5 microg/mL, R>0.9990) for both inosine and hypoxanthine with detection limits of 100 ng/mL. This simple and cost effective method was utilized to evaluate potential endogenous plasma biomarker(s), which may aid hospital emergency personnel in the early detection of acute cardiac ischemia in patients presenting with non-traumatic chest pain.

Simple HPLC-UV method for determination of iohexol, iothalamate, p-aminohippuric acid and n-acetyl-p-aminohippuric acid in human plasma and urine with ERPF, GFR and ERPF/GFR ratio determination using colorimetric analysis.

A simple high-performance liquid chromatographic (HPLC) method was developed for the simultaneous determination of iohexol, iothalamate, p-aminohippuric acid (PAH) and n-acetyl-p-aminohippuric acid (n-acetyl-PAH) in human plasma and urine. A C(18) column at a flow rate of 1 ml/min with an aqueous mobile phase of trifluoroacetic acid (0.1% TFA in deionized water (pH 2.2), v/v) and methanol gradient was used for component separation. The plasma and urine assay demonstrated linearity from 10 to 50 microg/ml for iohexol and iothalamate, 5 to 40 microg/ml for PAH and 2.5 to 40 microg/ml for n-acetyl-PAH. The HPLC plasma and urine results obtained for PAH were used to calculate the subject kidney effective renal plasma flow (ERPF) and the iohexol results were used to calculate the subject kidney glomerular filtration rate (GFR). The HPLC results for PAH were then compared to an alternative colorimetric method for analyzing PAH to determine if subject metabolism (acetylation) of PAH affected the ERPF results obtained using the colorimetric method, the subsequent ERPF/GFR ratio and clinical impression of subject patient kidney function. The method was utilized in several different clinical studies evaluating the effect of kidney function from medications (phase IV evaluations) marketed for patients with cardiovascular disease.