EXTRAVASCULAR DISTRIBUTION OF FLUORESCENT ALBUMIN, GLOBULIN AND FIBRINOGEN IN CONNECTIVE TISSUE STRUCTURES

Abstract

Adult albino rats were injected intravenously with labelled homologous albumin, globulins and fibrinogen. Labelling was done with acid rhodamine B (C.I. No. 45100) and fluorescein isothiocyanate. The amount of fluorescent dye bound to proteins was measured spectrophotometrically. Paper electrophoretic mobility and clotting time of fibrinogen were also checked after labelling. As a control, free fluorescent dye solutions were injected into another lot of rats. Animals were killed at intervals from 10 minutes to 12 days. Blood samples were taken to determine spectrophotometrically the concentration of labelled proteins in the circulation. Paraffin or unfixed fresh frozen sections from all tissues were examined with the fluorescence microscope. Histoimmunological studies were also made using fluorescent antibody against the different rat serum fractions. Coons's technique was applied to tissue sections from normal rats or from animals previously injected with unlabelled serum fractions and killed between 3 and 12 hours later. The following principal observations were made: 1) Globulins bind a larger amount of dye than albumin, while fibrinogen bound the least. 2) Labelling does not change grossly the electrophoretic mobility of albumin and globulins but slightly modified the clotting time of fibrinogen. 3) Time-concentration curves of albumin and globulins in the circulation are very much alike and reveal a fast and a slow component with 2.6 day time half-life for the former and 3.1 half-life for the latter. 4) Histologically labelled albumin and globulins are seen first in the lumen and on the walls of all blood vessels, and up to 8 hours they appeared to accumulate widely in connective tissue structures as well as in basement membranes of dermis and the mucosae of digestive, respiratory and urogenital tracts; fluorescence does not cross the basement membranes in most organs, nor does it pass the perichondrium, periosteum, or blood capillaries in the nervous system into surrounding parenchyma. Finally, labelled proteins began to disappear from all these sites after 12 hours, but persist longer in the macrophages of liver, lymph nodes, spleen and bone marrow. Fibrinogen was not seen diffusing out from vessels to the surrounding connective tissue. 5) That in animals previously injected with unlabelled serum fractions, histoimmunological techniques also showed the highest specific fluorescence in sections of connective tissue, stained with labelled antialbumin and antiglobulins; labelled antifibrinogen gave only a weak fluorescence in the same structutres. 6) These results confirm and extend our previous studies on the role of connective tissue as a transient depot for the extravascular pool of serum proteins.