Liquid chromatographic determination of hydroxyproline in tissue samples

doi:10.1016/S1570-0232(03)00248-4

Journal of Chromatography B

Volume 791, Issues 1–2, 5 July 2003, Pages 427-430

https://doi.org/10.1016/S1570-0232(03)00248-4 Get rights and content

Abstract

We describe a reversed-phase assay of hydroxyproline in rat lung tissue using sarcosine for the internal standard and pre-injection reaction with both o-phthalaldehyde (OPA) and 9-fluorenylmethylchloroformate (FMOC). Intra-assay variability in the concentration range of 25–500 μM hydroxyproline was less than 1%. Normal rat (left) lung was found to have a hydroxyproline content of 1.08±0.18 mg/lung. This ability to measure minute amounts of hydroxyproline is being applied to the measure of collagen and pathological fibrosis.

Introduction

This method was developed to satisfy a need to measure small changes in the amount of fibrosis in rat lung tissue used as a model for human asthma. Hydroxyproline is one of three predominant amino acids constituting collagen. We sought to develop an assay that could quantify changes in pulmonary fibrosis by measuring increases in pulmonary hydroxyproline content.

The use of fluorescent derivatives is common for the measurement of amino acids. An initial reaction with o-phthalaldehyde (OPA) is currently used to react with the primary amines, leaving secondary amines such as proline and hydroxyproline to react with other fluors [1]. 7-Chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) [2], phenylisothiocyanate (PITC) [3], and 4-dimethylaminoazobenzene-4′-sulfonyl chloride (dabsyl chloride, DABS-Cl) [4] have all been used in the secondary pre-column derivatization of hydroxyproline. HPLC has been tried in the measurement of the hydroxyproline-NBD-Cl derivative but yielded suboptimal sensitivity in the aqueous mobile phases [5]. Monboisse et al. have demonstrated the specificity and sensitivity of an HPLC assay combining sequential precolumn derivatization with OPA and 9-fluorenylmethylchloroformate (FMOC) [6].

Einarsson and separately Mazzi et al. reported the application of this OPA/FMOC process to the HPLC analysis of the hydroxyproline in seawater, urine, and serum [7], [8]. The FMOC derivative was measured by fluorescence detection and yielded an extremely sensitive assay for hydroxyproline. Our method reported below adapts these prior works to provide a technique suitable for measuring hydroxyproline in rat lung tissue.

Section snippets

Materials

Hydroxyproline, sarcosine, sodium acetate, sodium monophosphate, o-phthalaldehyde, iodoacetamide, and FMOC were all reagent grade and were purchased from Aldrich (Milwaukee, WI, USA). Chemical grade β-mercaptoethanol, ethyl ether, boric acid, and sodium hydroxide were obtained from Fisher Scientific, as were the HPLC-grade acetonitrile and glacial acetic acid. Reverse osmosis purified water was passed through a Barnstead water filtering system (Barnstead International, Dubuque, IA, USA). The

Results

The derivatized hydroxyproline peak eluted at 4.7 min, with the sarcosine standard, proline, and FMOC eluting at 11.7, 16.5, and 23 min, respectively (Fig. 1). The assay provided excellent reproducibility in the range of 25–500 μM hydroxyproline, and the coefficients of variation at all concentrations of the standards used for the curve were under 0.93%. Linear regression yielded an equation of HYP (μM)=32.29×(Peak Area)−9.5 (r²= 0.999).

The average amount of hydroxyproline in five normal rat

Discussion

Our initial attempts to apply the derivatization reported by Mazzi and Einarsson to hydrolyzed tissue were promising. One change from the previously published methods was the addition of a slightly supra-stoichiometric amount of NaOH to the acid hydrolysate to decrease the volume of borate buffer required to establish a pH of 9.5 for the derivatization steps. Another modification was the use of three ether washes to minimize the amount of unreacted FMOC in the HPLC sample. Although the washes

Acknowledgments

We are indebted to the enthusiasm and technical support of Ms. Jennifer Brown and Mr. Tabarius Smith towards the development of this methodology.

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Citation Excerpt :
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Short communicationLiquid chromatographic determination of hydroxyproline in tissue samples

Abstract

Introduction

Section snippets

Materials

Results

Discussion

Acknowledgments

J. Chromatogr.

Collagen Rel. Res.

Anal. Biochem.

J. Chromatogr.

Short communication
Liquid chromatographic determination of hydroxyproline in tissue samples