Spectrophotometric determination of hydroxyproline in connective tissue on the nanogram level

✍ Scribed by G. Bondjers; S. Björkerud

• Publisher: Elsevier Science
• Year: 1973
• Tongue: English
• Weight: 457 KB
• Volume: 52
• Category: Article
• ISSN: 0003-2697
• DOI: 10.1016/0003-2697(73)90054-7

✦ Synopsis

An ultramicromethod

for the determination of hydroxyproline in submicrogram tissue samples is presented. The procedure may be used for the determination of down to 6 ng of hydroxyproline, with a standard deviation of a series of replicates of 0.20 ng. The method has been applied to samples of aortic media, with a recovery of added hydroxyproline of 103 + 4.4% (SD).

In contrast to other mammalian proteins, collagen contains a high concentration of the imino acid, hydroxyproline. Several investigators have used hydroxyproline measurement for determining amount, of collagen in various structures. However, occurrence of structural heterogeneity may decrease the value of the information obtained by such extrapolations (I) due to the large tissue samples necessary for measurements of hydroxyproline by the available methods. In addition, the sensitivity of these methods does not allow t,he determination of hydroxyproline content of smaller connective-tissue-containing structures, as for example the tympanic membrane, or structurally homogeneous or defined samples of other tissues. The purpose of the present investigation was to develop a method for the determination of nanogram amounts of hydroxyproline in struct,urally defined tissue samples in the microgram range.

METHODS

Equipnzent

Acid hydrolysis of the samples was performed in 1 ml glass ampoules (Trident glass container, Johnsen and Jgrgensen Ltd., London, England), and 75 X 10 mm centrifuge tubes were used for the colour reaction. Volumes were measured with constriction pipets. Ampoules and tubes were prewashed with a strongly alkaline detergent, Extran (Merck AG) and carefully rinsed in distilled water. The glassware was then transferred to a combustion oven and heated to 500°C as described clsewhcrc 496