Short communicationLiquid chromatographic determination of hydroxyproline in tissue samples
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.
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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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Simple determination of L-hydroxyproline in idiopathic pulmonary fibrosis lung tissues of rats using non-extractive high-performance liquid chromatography coupled with fluorescence detection after pre-column derivatization with novel synthetic 9-acetylimidazol-carbazole
2017, Journal of Pharmaceutical and Biomedical AnalysisCitation Excerpt :L-Hyp also plays a key role in the synthesis and stability of collagen [3]. Hence, L-Hyp is a characteristic amino acid and an important marker that can be used to directly measure the collagen content in IPF tissues [4–6]. In recent years, L-Hyp has been attracting increasing attention as an important biomarker of IPF.