A simple quantitative method for collagen typing in tissue samples: its application to human liver with schistosomiasis.

Abstract

Collagen chains separated on 5% SDS-acrylamide gels were stained with a 0.1% Sirius F3BA solution in saturated aqueous picric acid. After destaining the gels with methanol: acetic acid: water (30: 7 : 63), they were scanned at 540 nm and the area under each peak was determined. After that, the bands were sliced and the slices incubated overnight with trypsin at 37 degrees C. The color of the slices was eluted completely when the denatured collagen was ingested with trypsin. The absorbance of the color eluted was determined at 540 nm. The results obtained demonstrated that both procedures are reproducible and linear from 10 to at least 120 micrograms of protein. The correlation coefficient between both procedures was greater than 95%. The color is stable and the same end-point is obtained after destaining. In order to test the usefulness of the procedure in the typing of collagens from parenchymatous tissues, liver biomatrix was prepared from normal and cirrhotic human specimens obtained at autopsy. Over 95% of the collagen originally present in each liver was recovered in the corresponding biomatrix . We also showed that over 80-85% of biomatrix collagen could be solubilized with pepsin. These extracts were neutralized to pH 7.0 to inactivate pepsin and were applied onto the acrylamide gels. After electrophoresis and staining with Sirius red the types of collagen were determined by densitometric analysis. Our findings confirmed the results obtained by others using more complex and time consuming methodologies, mainly that type I and type III collagens are present in the normal liver in equal concentrations and that the ratio of I/III is 1. In all the cirrhotic livers investigated, the ratio of type I/type III collagen was greater than 1 due to an increase in type I collagen.