Urinary Proteomic Biomarkers of Trabecular Bone Volume Change during Army Basic Combat Training

Abstract

ABSTRACT Changes in bone microarchitecture resulting from chronic physical stress such as that experienced during U.S. Army Basic Combat Training (BCT) underscore its adaptive potential. There is a need for noninvasive biomarkers of adaptive bone formation that can be obtained at scale in real-world settings. Differential mass spectrometry (dMS), a label-free quantitative proteomics technique, may provide useful information about bone adaptation based on the proteomic features of urine. Purpose The purpose of this study is to optimize a dMS-based urinary proteomic technique and evaluate the relationship between urinary proteome content and adaptive changes in bone microarchitecture during BCT. Methods Urinary proteomes were analyzed with an optimized dMS technique in two groups of 13 recruits (N = 26) at the beginning (Pre) and end (Post) of BCT. Matched by age (21 ± 4 yr), sex (16 W), and baseline tibial trabecular bone volume fractions (Tb.BV/TV), these groups were distinguished by the most substantial (High) and minimal (Low) improvements in Tb.BV/TV. Differential protein expression was analyzed with mixed permutation ANOVA and false discovery proportion-based adjustment for multiple comparisons. Results Tibial Tb.BV/TV increased from pre- to post-BCT in High (3.30 ± 1.64%, P < 0.0001) but not Low (−0.35 ± 1.25%, P = 0.4707). The optimized dMS technique identified 10,431 peptides from 1368 protein groups that represented 165 integrative biological processes. Seventy-four urinary proteins changed from pre- to post-BCT (P = 0.0019), and neutrophil-mediated immunity was the most prominent ontology. Two proteins (immunoglobulin heavy constant gamma 4 and C-type lectin domain family 4 member G) differed from pre- to post-BCT in High and Low (P = 0.0006). Conclusions The dMS technique can identify more than 1000 urinary proteins. At least 74 proteins are responsive to BCT, and other principally immune system–related proteins show differential expression patterns that coincide with adaptive bone formation.