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

Abstract

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.

Toxic Effect of a Marine Bacterium on Aquatic Organisms and Its Algicidal Substances against Phaeocystis globosa

“…Hypoxanthine is widely used in food industry as an index for evaluating meat or fish freshness [ 36 ]. Hypoxanthine is also commonly used in medical and clinical conditions [ 37 , 38 ]. However, few studies about hypoxanthine on the algicidal activity against harmful algae have been reported.…”

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

“…We have developed a chemiluminescence method using a microplate luminometer to address the issues of equipment size, ease of use, speed of analysis, specificity, sensitivity and reproducibility. 30 Our simple method utilizes a small quantity of plasma and employs enzymes to convert inosine and hypoxanthine to their metabolic products. As these substrate-specific enzymes turn over, free radicals are generated and excite a sensitive luminescent material, which generates a quantifiable blue-green light.…”

“…In particular, our published results using chemiluminescence technology, one of the most affordable and sensitive analytical techniques currently available, demonstrated a rapid, sensitive, and highly reproducible method for measuring plasma total hypoxanthine concentrations in healthy individuals versus cardiac patients with confirmed cTnT elevation. 30 Therefore, our proposed candidate biomarkers (inosine/hypoxanthine) may follow the similar path as IMA biomarker, which also has low specificity and high sensitivity and was approved by FDA for the use in conjunction with other FDA-approved cardiac tests (e.g. ECG and cTn) to improve patient diagnostic accuracy for cardiac ischemia.…”

Inosine as a Tool to Understand and Treat Central Nervous System Disorders: A Neglected Actor?

“…Inosine is also a potential biomarker in plasma from patients experiencing non-traumatic chest pain or unstable angina due to potential acute cardiac ischemia/myocardial infarction (Farthing et al, 2007;Ameta et al, 2016). High levels of this nucleoside have already been reported in mouse hearts (Farthing et al, 2006) and in observational studies in humans (Al-Shamiri et al, 2009;Farthing et al, 2011). However, early evidence has shown that adenosine, but not inosine, may cause chest pain in healthy individuals.…”