Abstract
BACKGROUND: N(τ)-methylhistidine released endogenously from muscle into urine is an index of muscle protein breakdown, and part of N(τ)-methylhistidine is excreted as the acetylated derivative, N-acetyl-N(τ)-methylhistidine. OBJECTIVES: The aim of this study was to measure the urinary concentrations of N(τ)-methylhistidine and N-acetyl-N(τ)-methylhistidine. A quantitative method was established using liquid chromatography-tandem mass spectrometry (LC-MS/MS) operated in Q1 single-ion monitoring (SIM) mode and multiple reaction monitoring (MRM) mode with stable-isotope dilution analysis. METHODS: We synthesized N-acetyl-N(τ)-methylhistidine and its isotopic analog for stable-isotope dilution analysis. Then, we validated a method for quantifying urinary concentrations of intact N(τ)-methylhistidine, N-acetyl-N(τ)-methylhistidine, and creatinine to precisely determine their concentrations in rats, humans, cattle, and dogs. Creatinine correction was applied to normalize for urine volume. RESULTS: For both SIM and MRM analysis, the acceptable linear ranges of detection were 5 pmol/mL to 10 nmol/mL with r(2) = 1.000 in SIM mode and MRM mode. The LC-MS/MS methods operated in both SIM and MRM transitions detected changes in the urinary excretion levels of N(τ)-methylhistidine and N-acetyl-N(τ)-methylhistidine in response to starvation and refeeding of rats. The methods also detected urinary concentrations of N(τ)-methylhistidine and N-acetyl-N(τ)-methylhistidine in cattle, humans, and dogs. CONCLUSIONS: These results suggest that the LC-MS/MS method operated in SIM mode and MRM mode can be used to measure urinary concentrations of N(τ)-methylhistidine and N-acetyl-N(τ)-methylhistidine.