Journal
TISSUE ENGINEERING PART C-METHODS
Volume 23, Issue 4, Pages 243-250Publisher
MARY ANN LIEBERT, INC
DOI: 10.1089/ten.tec.2017.0018
Keywords
hydroxyproline; 4-dimethyl(amino)benzaldehyde; perchloric acid; quantitative biochemistry
Categories
Funding
- National Institutes of Health (NIH) [R01AR067821, R01DE015038]
- NIH T32 grant [OD011147]
- National Science Foundation Graduate Research Fellowship [1650042]
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Collagen quantification has long been relevant to biomedical research and clinical practice to characterize tissues and determine disease states. The hydroxyproline assay, while a broadly employed method of quantifying collagen, uses perchloric acid to dissolve Ehrlich's reagent. Since perchloric acid poses occupational safety hazards and high costs, in this study, a new hydroxyproline assay was developed that replaces perchloric acid with a relatively safer and cheaper alternative, hydrochloric acid (HCl). To validate this biochemical technique, first, using either acid to dissolve Ehrlich's reagent, the assays were completed for native and engineered collagenous tissues. No statistical differences were identified between the assays (p = 0.32). Subsequently, both biochemical techniques were compared to amino acid analysis, considered a proteomics gold standard. Interestingly, utilizingHCl in lieu of perchloric acid yielded greater concordance with amino acid analysis (rho(c) = 0.980) than did the traditional assay (rho(c) = 0.947); that is, the HCl-based assay more closely estimates hydroxyproline content, and, consequently, true collagen content. Thus, using Ehrlich's reagent containing HCl in the hydroxyproline assay represents an advance in both mitigating laboratory safety hazards and improving biochemical collagen quantification.
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