Aging markers in human urine: A comprehensive, non-targeted LC-MS study

Abstract

Age markers in urine, non-targeted metabolomics, human aging, LC-MS, highly 23 correlated metabolites, leucine, pseudouridine, 24 25 Short title: Aging markers in human urine 26 27 Summary 28Metabolites in human biofluids document individual physiological status. We 29 conducted comprehensive, non-targeted, non-invasive metabolomic analysis of urine 30 from 27 healthy human subjects, comprising 13 youths (30±3 yr) and 14 seniors 31 (76±4 yr). Quantitative analysis of 99 metabolites revealed 55 that were linked to 32 aging, displaying significant differences in abundance between the two groups. 33These include 13 standard amino acids, 5 methylated, 4 acetylated, and 9 other 34 amino acids, 6 nucleosides, nucleobases, and derivatives, 4 sugar derivatives, 5 35 sugar phosphates, 4 carnitines, 2 hydroxybutyrates, 1 choline, and 1 ethanolamine 36 derivative, and glutathione disulfide. Abundances of 53 compounds decreased, while 37 2 increased in elderly people. Many age-linked markers were highly correlated; 42 of 38 55 compounds, showed Pearson's correlation coefficients larger than 0.70. As 39 metabolite profiles of urine and blood are quite different, age-related information 40 in urine components offer yet more valuable insights into aging mechanisms of 41 endocrine system and related organ systems. 42 43Results 71 Collection of urine samples 72Samples of morning urine, immediately after awaking, were collected 73 from healthy volunteer subjects (elderly, 75.8±3.9 yr, and young, 30.6±3.2 yr; 74 Supplementary Table s1 shows gender and BMI) in Onna Village, Okinawa, 75Japan. Precautions taken for sample collection are described in the Materials 76 and Methods. Basic data analytical procedures were similar to those previously 77 described (Chaleckis et al., 2016) (Teruya et al., 2019) (Kameda et al., 2020). 78 79 99 urine metabolites identified 80 Ninety-nine urinary metabolites, about half of which are amino acids and 81 their derivatives, were identified and quantified using liquid chromatography -mass 82 spectrometry (LC -MS) and MZmine 2 (Pluskal et al., 2010a) (Teruya et al., 2019) 83 (Supplemental Table s2). These compounds were subdivided into 12 groups, 84 containing 17 standard amino acids, 12 methylated amino acids, 6 acetylated amino 85 acids and 15 other amino acids, 12 nucleosides, nucleobases, and derivatives, 4 sugar 86 derivatives, 6 sugar phosphates, 3 vitamins and coenzymes (pantothenate, 4-87 aminobenzoate, nicotinamide), 4 choline and ethanolamine derivatives, 8 carnitines, 88 11 organic acids, and 1 antioxidant (oxidized form of glutathione, GSSG). Small amino 89 acids, such as glycine and alanine, were not detected in our analysis due to the 90 mass cutoff (100 m/z) used. Levels of individual compounds, categorized by 91 abundance as H (high, >10 8 ), M (medium, 10 7~1 0 8 ) or L (low, <10 7 ), were estimated 92 based upon mass spectroscopic peak area (Chaleckis et al., 2014) (Chaleckis et al., 93 6 2016). Some compounds varied widely from one individual to the next and are 94 denoted as H-L, H-...