ABSTRACT
Inhibition of kynurenine 3-monooxygenase (KMO) protects against multiple organ dysfunction (MODS) in experimental acute pancreatitis (AP). We aimed to precisely define the kynurenine pathway activation in relation to AP and AP-MODS in humans, by carrying out a prospective observational study of all persons presenting with a potential diagnosis of AP for 90 days. We sampled peripheral venous blood at 0, 3, 6, 12, 24, 48, 72 and 168 hours post-recruitment. We measured tryptophan metabolite concentrations and analysed these in the context of clinical data and disease severity indices, cytokine profiles and C-reactive protein (CRP) concentrations. 79 individuals were recruited (median age: 59.6 years; 47 males, 59.5%). 57 met the revised Atlanta definition of AP: 25 had mild, 23 moderate, and 9 severe AP. Plasma 3-hydroxykynurenine concentrations correlated with contemporaneous APACHE II scores (R2 = 0.273; Spearman rho = 0.581; P < 0.001) and CRP (R2 = 0.132; Spearman rho = 0.455, P < 0.001). Temporal profiling showed early tryptophan depletion and contemporaneous 3-hydroxykynurenine elevation. Furthermore, plasma concentrations of 3-hydroxykynurenine paralleled systemic inflammation and AP severity. These findings support the rationale for investigating early intervention with a KMO inhibitor, with the aim of reducing the incidence and severity of AP-associated organ dysfunction.
ABSTRACT
A hydrophilic interaction chromatography (HILIC)/mass spectrometric assay was developed for the determination of zanamivir, a neuraminidase inhibitor used to treat influenza, in rat and monkey plasma. An organic solvent with hydrophilic properties, methanol, was used to precipitate proteins in plasma to assure the highly polar zanamivir of staying in solution. Chromatographic separation was obtained using a HILIC silica column with multiple reaction monitoring turboionspray positive ion detection. The stable label of zanamivir, [(13)C(1)(15)N(2)] GR121167C, was used as the internal standard. The assay was validated for the determination of zanamivir in rat and monkey plasma. The lower and upper limits of quantitation were 2 and 10000 ng/mL, using 0.05 mL plasma aliquot, respectively. The signal to noise ratio of a typical 2 ng/mL was approximately 5:1. The inter-day precision (relative standard deviation) and accuracy (relative error) in rat plasma, derived from the analysis of validation samples at 5 concentrations, ranged from 6 to 10% and -6.5 to 0.2%, respectively. The inter-day precision (relative standard deviation) and accuracy (relative error) in monkey plasma, derived from the analysis of validation samples at five concentrations, ranged from 2 to 8% and -2.3 to 2.1%, respectively. Zanamivir was found to be stable for at least 5 days at approximately -80 degrees C and at room temperature in plasma. This assay incorporates a simple protein precipitation with methanol and hydrophilic interaction chromatography which is sensitive, accurate, precise, and is being used to support oral formulation and toxicokinetic studies in rat and monkey, respectively.
