A simplified method for the analysis of hydroxyproline in biological tissues

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Reddy, G. Kesava¹; Enwemeka, Chukuka S.¹

A simplified method for the analysis of hydroxyproline in biological tissues

Clin Biochem. June 1996;29(3):225-229

©1996 The Canadian Society of Clinical Chemists

Affiliations

¹Department of Physical Therapy, University of Kansas Medical Center, 3901 Rainbow Blvd., Kansas City, KS 66160
Abstract and keywords

A critical study of the different steps involved in previous procedure for hydroxyproline assay allows the direct measurement of collagen content in tissue homogenates without losing the advantages of the method. The procedure is based on alkaline hydrolysis of the tissue homogenate and subsequent determination of the free hydroxyproline in hydrolyzates. Chloramine-T was used to oxidize the free hydroxyproline for the production of a pyrrole. The addition of Ehrlich's reagent resulted in the formation of a chromophore that can be measured at 550 nm. Optimal assay conditions were determined using tissue homogenate and purified acid soluble collagen along with standard hydroxyproline. Critical parameters such as the amount of chloramine-T, sodium hydroxide, p-dimethylaminobenzaidehyde, pH of the reaction buffer, and length of oxidation time were examined to obtain satisfactory results. The method has been applied to samples of tissue homogenate and purified acid soluble collagen, with recovery of added hydroxyproline of 101 ± 6.5 and 104 ± 6.0 (SD) percent, respectively. The method is highly sensitive and reproducible when used to measure the imino acid in tissue homogenates. The modified hydroxyproline assay presented in this communication will be useful for routine measurement of collagen content in extracts of various tissue specimens. In addition, the modified method can be used for batch processing of column fractions to monitor the collagen concentrations during purification.

Keywords: hydroxyproline; tissue hydrolyzate; collagen; Ehrlich's reagent
Links

DOI: 10.1016/0009-9120(96)00003-6

PubMed: 8740508

WoS: A1996UJ73000005
History

Received: 1995-09-8

Accepted: 1995-12-11

Cited Works (2)

Year	Entry
1967	Stegemann H, Stalder K. Determination of hydroxyproline. Clin Chim Acta. November 1, 1967;18(2):267.
1950	Neuman RE, Logan MA. The determination of hydroxyproline. J Biol Chem. 1950;184(1):299-306.

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