PT - JOURNAL ARTICLE AU - Vinicius da Eira Silva AU - Vitor de Salles Painelli AU - Samuel Katsuyuki Shinjo AU - Eduardo Maffud Cilli AU - Craig Sale AU - Bruno Gualano AU - Maria Concepción Otaduy AU - Guilherme Giannini Artioli TI - Comparison between proton magnetic resonance spectroscopy and high-performance liquid chromatography to quantify muscle carnosine in humans AID - 10.1101/568923 DP - 2019 Jan 01 TA - bioRxiv PG - 568923 4099 - http://biorxiv.org/content/early/2019/03/07/568923.short 4100 - http://biorxiv.org/content/early/2019/03/07/568923.full AB - Proton magnetic resonance spectroscopy (1H-MRS) has been used as a non-invasive alternative to quantify carnosine in human muscle. It is unclear whether 1H-MRS is a valid and reliable method. 1H-MRS validity and reliability was examined in a series of in vitro and in vivo studies. In the in vitro study, phantoms containing different concentrations of carnosine, imidazole, histidine and bovine serum albumin (BSA) were submitted to 1H-MRS to verify: 1) signal linearity; 2) whether other sources of imidazole could contribute to carnosine signal. In the in vivo study, carnosine was determined in the m. gastrocnemius by 1H-MRS and by high-performance liquid chromatography (HPLC, a reference method) in muscle biopsy samples from 16 young men. Test-retest reliability was determined with (n=10) and without (n=5) voxel repositioning and re-shimming. Convergent validity (n=16) was determined by comparing carnosine values obtained with 1H-MRS vs. HPLC. Discriminant validity (n=14) was determined by measuring carnosine before and after 4 weeks of β-alanine supplementation. In vitro carnosine signal showed excellent linearity (Pearson correlation: r=0.999). Histidine and imidazole, but not BSA, emitted quantifiable signals in the same chemical shift of carnosine. A clear loss in signal quality was shown in the signal obtained in vivo. 1H-MRS coefficient of variation without repositioning voxel was 6.6% and increased to 16.9% with voxel repositioning. 1H-MRS was able to detect a significant increase in muscle carnosine after β-alanine supplementation, both a substantial disagreement with HPLC was shown. 1H-MRS showed adequate discriminant validity, but limited reliability and poor agreement with the reference method.Key points summaryAlthough proton magnetic resonance spectroscopy (1H-MRS) was developed to quantify carnosine in human muscle as a non-invasive alternative method to high-performance liquid chromatography (HPLC) in extracts from human muscle biopsy, a thorough assessment of 1H-MRS validity is lacking. Thus, we examined signal linearity in vitro, matrix effect in vivo, as well as reliability, convergent validity and discriminant validity of in vivo 1H-MRS for the determination of carnosine in human muscle using in vitro and in vivo experiments.An excellent 1H-MRS in vitro signal linearity was shown for carnosine across the physiological range, although broadening and signal losses were observed when 1H-MRS was performed in vivo.Free histidine and imidazole also emitted quantifiable signals at the same chemical shift of carnosine, which could constitute a source of error in carnosine quantification. Large protein (e.g., bovine serum albumin) did not emitted signal, thereby indicating they do not constitute a source of error.1H-MRS can detect and quantify muscle carnosine in vivo, and it is sensitive to detect increases in muscle carnosine brought about by β-alanine supplementation.Despite being sensitivity, 1H-MRS showed poor test-retest reliability, especially due to voxel repositioning and re-shimming.A poor agreement was shown for muscle carnosine determination between 1H-MRS and HPLC performed in muscle biopsies taken at the closest possible site (m. gastrocnemius).Caution should be exercised when interpreting muscle carnosine data obtained with 1H-MRS.