Abstract

   Objective: To study the changes in urine metabolism in female water
   polo players before and after high-intensity training by using
   ultra-high performance liquid chromatography-mass spectrometry, and to
   explore the biometabolic characteristics of urine after training and
   competition. Methods: Twelve young female water polo players (except
   goalkeepers) from Shanxi Province were selected. A 4-week formal
   training was started after 1 week of acclimatization according to
   experimental requirements. Urine samples (5 mL) were collected before
   formal training, early morning after 4 weeks of training, and
   immediately after 4 weeks of training matches, and labeled as T1, T2,
   and T3, respectively. The samples were tested by LC-MS after
   pre-treatment. XCMS, SIMCA-P 14.1, and SPSS16.0 were used to process
   the data and identify differential metabolites. Results: On comparing
   the immediate post-competition period with the pre-training period (T3
   vs. T1), 24 differential metabolites involved in 16 metabolic pathways
   were identified, among which niacin and niacinamide metabolism and
   purine metabolism were potential post-competition urinary metabolic
   pathways in the untrained state of the athletes. On comparing the
   immediate post-competition period with the post-training period (T3 vs.
   T2), 10 metabolites involved in three metabolic pathways were
   identified, among which niacin and niacinamide metabolism was a
   potential target urinary metabolic pathway for the athletes after
   training. Niacinamide, 1-methylnicotinamide, 2-pyridone, L-Gln, AMP,
   and Hx were involved in two metabolic pathways before and after the
   training. Conclusion: Differential changes in urine after water polo
   games are due to changes in the metabolic pathways of niacin and
   niacinamide.

   Keywords: high-intensity training, LC-MS, urine, water polo

1. Introduction

   Since its first introduction by Nicholson [[34]1] in 1999, metabolomics
   has gradually developed and gained significance as a tool in systems
   biology. It can respond more directly to the physiological status of
   organisms and changes in the types and quantities of metabolites with
   molecular weights of 1000 Da or less. Mass spectrometry (MS) is used in
   metabolomics detection because it can detect multiple metabolites in a
   single experiment, given its high sensitivity and large dynamic range
   of monitoring. It can detect and track metabolite changes correlated
   with the target state in a global, non-targeted analysis
   [[35]2,[36]3,[37]4,[38]5].

   Among many other applications, metabolomics is applied in sports, for
   monitoring exercise intensity and body metabolism [[39]6], performance
   prediction [[40]7,[41]8], exercise in disease diagnosis and treatment
   [[42]9], and sports nutrition supplementation [[43]10]. In China,
   metabolomics is now being applied in choosing exercise and nutritional
   supplementation [[44]11], athlete selection [[45]12], and particularly
   in exploring the metabolic mechanisms of athletes [[46]13,[47]14] and
   the improvement in diseases by exercise [[48]15,[49]16].

   Water polo is a physical sport combining swimming, handball, and
   volleyball. It is a type of metabolic energy supply involving the
   phosphate source system, lactic energy system, and aerobic oxidation
   system [[50]17]. A human’s athletic ability largely depends on the
   body’s metabolism. Different exercise routines have differential
   changes in the metabolism. The changes in endogenous metabolites
   facilitating body transformation during exercise also have different
   effects on the metabolism, depending upon the type of exercise routine.
   Little has been reported about the effects of water polo training on
   the metabolic mechanisms of small molecules in the body This study
   analyzes the metabolic effects of water polo by identifying the changes
   in urinary metabolites in players before and after training, based on
   LC-MS technology. It also provides theoretical references for