Measurement of changes in urinary neopterin and total neopterin in body builders using SCX HPLC

Abstract

Body building is a sport where ultrastructual damage to muscle fibres aids the development of dense muscle layers. Using a new strong cation exchange (SCX) based chromatography technique to measure neopterin and 7,8-dihydroneopterin, we investigated whether this muscle damage caused increased levels of inflammation. Urine samples were collected over eight consecutive mornings from 10 natural competitive body builders. Samples were analysed using SCX high performance liquid chromatography (HPLC) with urine volume corrected for creatinine and specific gravity (SG). The majority of subjects showed large changes in both neopterin and total neopterin (7,8-dihydroneopterin+neopterin) levels, though the mean data for the group showed no significant change over the week. There was no evidence of the high intensity resistance training causing an accumulation of inflammation as the values for all the body builders returned to close to the starting values after 2 days rest. The SCX analysis had an intra-specific viability of 3.04% and the inter-specific viability was 5.42%. Urine volume correction with SG was found to give the same values as using creatinine. Creatinine and specific gravity are both reliable methods for correcting for urine volume while SCX HPLC provides a new means of measuring urinary neopterin and total neopterin.

Realising the Potential of Urine and Saliva as Diagnostic Tools in Sport and Exercise Medicine

“…Improvement in diagnostic methodologies, however, has led to the development of specific and sensitive immunoassays [25] and chromatographic [26] and spectrometric assays [27] capable of providing an overview of an individual's molecular profile through saliva and urine. Haematological parameters [28][29][30] that provide a comprehensive overview of stress relative to acute and chronic exposure have been investigated extensively in exercise research.…”

“…Non-invasive alternatives such as urine and saliva collection offer reliable indicators that do not restrict the ability to quantify ''stress''. Certain urinary biomarkers have been shown to repeatedly provide a precise quantification and analysis of exercise-induced stress and disease or illness that does not require the use of an invasive procedure [26,[31][32][33][34][35][36][37]. For example, providing a measurement of the total free (un-bound) hormone concentration in the case of saliva more accurately reflects the active form in the body [38].…”

“…Modern quantification methods include the reaction with o-nitrobenzaldehyde [234], reverse phase [235][236][237] and cation-exchange high-performance liquid chromatography (HPLC) [26], cation-pairing HPLC [238], mass spectrometry, and gas chromatography [239]. The advantage of chromatography is the simultaneous determination of creatinine with the analyte that requires creatinine correction.…”

“…The advantage of chromatography is the simultaneous determination of creatinine with the analyte that requires creatinine correction. Usually analysed at 234 nm by ultra-violet detection, the method has been used effectively in the quantification correction of neopterin [26,240,241], desmosine [242], catecholamines, and metanephrines [243].…”

“…As a result of the known variations in creatinine excretion, SG has been sought after as a viable alternative and used in recent exercise research [20]. The minimal studies that have compared creatinine and SG have shown SG performs as well as creatinine in correcting urinary analyte concentrations [26,229,[258][259][260] and assessing patient and pre-exercise hydration status [261][262][263]. Its limited use is a result of finite research which has not elucidated reference ranges that are critical for diagnosis.…”