Quantification of hydroxyproline in small amounts of skin tissue using isocratic high performance liquid chromatography with NBD-F as fluorogenic reagent.
In order to determine the collagen content of small amounts of skin tissue, we developed a new, simple and highly sensitive method of measuring the quantity of hydroxyproline (Hyp) using isocratic high performance liquid chromatography (HPLC) with a fluorogenic agent, 7-fluoro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-F). The recovery rate of Hyp and reproducibility of the assay were high, and the test was sensitive enough to detect Hyp in less than 1 mg of skin tissue. This method is clinically useful for ensuring accurate diagnosis and for monitoring specific skin conditions using small human skin samples collected in biopsies.
Kakinuma
Miwa
M
Department of Dermatology, Tokyo Medical University, 6-7-1 Nishi-Shinjuku, Shinjuku-ku, Tokyo 160-0023, Japan. kakimiwa@aol.com
Watanabe
Yasuo
Y
Hori
Yusuke
Y
Oh-I
Tsunao
T
Tsuboi
Ryoji
R
D009327:4-Chloro-7-nitrobenzofurazan / Q000031:analogs & derivatives / Q000737:chemistry; D000596:Amino Acids / Q000032:analysis / Q000302:isolation & purification; D000818:Animals; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D003964:Diaphragm / Q000737:chemistry; D005456:Fluorescent Dyes / Q000737:chemistry; D006909:Hydroxyproline / Q000032:analysis*; D007668:Kidney / Q000737:chemistry; D008099:Liver / Q000737:chemistry; D008297:Male; D009132:Muscles / Q000737:chemistry; D009206:Myocardium / Q000737:chemistry; D051381:Rats; D017207:Rats, Sprague-Dawley; D015203:Reproducibility of Results; D012867:Skin / Q000737:chemistry*
D000596:Amino Acids; D005456:Fluorescent Dyes; C032930:7-fluoro-4-nitrobenzo-2-oxa-1,3-diazole; D009327:4-Chloro-7-nitrobenzofurazan; D006909:Hydroxyproline