Christine Farthing

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.

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.

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 patients 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.

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.

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

Abstract 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.

A Simple High-Performance Liquid Chromatographic Method for the Simultaneous Determination of Monoamine Neurotransmitters and Relative Metabolites with Application in Mouse Brain Tissue

As a means to evaluate the role of organic solute carriers in CNS homeostasis, a simple and rapid high-performance liquid chromatographic method utilizing ultraviolet and electrochemical detectors was developed and validated for the simultaneous determination of the neurotransmitters dopamine, norepinephrine, and serotonin and their metabolites in mouse brain homogenate. For analyte separation, the method utilized a C18 column with a mobile phase of 75 mM sodium dihydrogen phosphate (monohydrate), 1.7 mM 1-octanesulfonic acid sodium salt, 25 µM EDTA, 10% acetonitrile, and 1% triethylamine with a final pH of 3.0 adjusted using phosphoric acid. The method was linear for dopamine, norepinephrine, serotonin, 3,4-dihydroxyphenylacetic acid, homovanillic acid, and 5-hydroxyindoleacetic acid from 20–1000 ng/mL (r ≥ 0.993) with detection limits of 5 ng/mL for dopamine, norepinephrine, 3,4-dihydroxyphenylacetic acid, and homovanillic acid, and of 20 ng/mL for serotonin and 5-hydroxyindoleacetic acid. The method was linear over the range of 10–200 µg/ml for quinolinic acid, xanthurenic acid, and nicotinic acid (r ≥ 0.993) with detection limits of 1 µg/mL for all components. At a flow rate of 1 mL/min, the analytical run time was less than 10 min. The method was successfully applied to the quantitation of these compounds in mouse whole brain homogenates.

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.