An automated scintillation counting system with high efficiency for continuous analysis: Cross-links of [3H]NaBH4-reduced collagen
✍ Scribed by Gerald L. Mechanic
- Publisher
- Elsevier Science
- Year
- 1974
- Tongue
- English
- Weight
- 366 KB
- Volume
- 61
- Category
- Article
- ISSN
- 0003-2697
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✦ Synopsis
A completely mobile apparatus is described for automatic column monitoring and analysis of radioactive-labeled compounds by the scintillation counting of aqueous effluents from chromatographic columns. The system is extremely versatile, is highly efficient for tritium-labeled substances, and uses only approximately 10% of the sample, saving 90% for further work. The system may be easily assembled from readily available items. In physio-logi& studies of the analysis of cross-links from ['HlNaBHa-reduced collagen, the output of work was increased by ninefold.
The macromolecular structure, organization, and packing of collagen molecules into support matrices is intimately related to the intermolecular covalent cross-links stabilizing the matrices. A commonly used method for the stabilization, to hydrolysis and subsequent detection of labile aldehydic cross-link precursors and their labile Schiff base cross-links is the reduction with [3H] NaBH,, producing tritium-labeled primary alcohols from the aldehydes and secondary amines from the Schiff bases.
The more usual analyt.ical methods (l-3) involve chromatography of the reduced collagen hydrolyzate on a single column using gradient elution, and collection ,of 200400 fractions. Procedures then involve aliquoting each fraction into a scintillation vial using a Lang-Levy constriction pipet, adding 10 ml of scintillation fluid to each vial, stirring, and counting in a scintillation counter. This manual process of analysis usually takes %3 days for each sample, and is laborious, tedious, time consuming and expensive, considering man hours, scintillation vials, and fluid. In comparative studies involving qualitative and quantitative analyses of cross-links and their precursors, the complexity of the elution profiles of the various collagenous tissues (4-6) makes analysis especially difficult, since each analysis must be repeated two to three times to ascertain the positions and quantities of the cross-links in the elution profiles (1-3).
In this laboratory we have screened patients with possible connective 349