Microspectrophotometric detection of heparin in mast cells and basophilic granulocytes stained metachromatically with Toluidine Blue O.

Abstract

A qualitative microspectrophotometric detection method for heparin in situ has been developed, using data obtained previously with a model system of polyacrylamide films containing pure glycosaminoglycans (Tas, 1975). This technique, based on the unique metachromatic properties of heparin with Toluidine Blue O in glycerol, has been worked out with rat peritoneal and mesenteric mast cells. After the smears containing the stained cells had been mounted in glycerol, a change with time of the recorded metachromatic peaks to lower wavelengths was found, leading to an equilibrium phase after some days. The metachromatic peaks recorded in this phase appeared to resemble closely the peak obtained for the heparin-Toluidine Blue O complex under similar conditions in the model experiments. With rat mast cells it was found that nucleic acids, basic proteins, histamine and lipids had no appreciable influence on the position of the final recorded peaks, nor did they influence the slope of the time course very much. This observed decrease with time in the wavelengths of the metachromatic peaks can be explained by the time necessary for equilibration of the cells in glycerol and by the possible influence of lower sulphated glycosaminoglycans on the peak of the heparin-Toluidine Blue O complex. It was found that the method can be used to detect unequivocally the presence of heparin in cells, even if they also contain up to 75% (mole/mole) of other, lower sulphated glycosaminoglycan. Only a limited number of cells is necessary with this method - in contrast to biochemical determinations. For the first time the presence of heparin in normal human basophilic granulocytes and mast cells has been proved directly. The experiments indicate the occurrence of virtually similar sulphated heparins in human mast cells and basophilic granulocytes, as well as in pig mast cells. A higher sulphated heparin, however, might be present in rat mast cells.