Qualitative and quantitative studies of heparin and chondroitin sulfates in normal human plasma.
Heparin was extracted and purified from normal human plasma, and full characterization of its structure and physico-chemical properties was achieved for the first time. Plasma was submitted to exhaustive proteolytic treatment with papain, trypsin, chymotrypsin, collagenase and pepsin, anion-exchange chromatography and precipitation with organic solvents. By this procedure, we recovered heparin (about 0.7 mg/100 ml of plasma) and chondroitin sulfate (about 0.1 mg/100 ml of plasma). Chondroitin sulfate has a peak molecular mass of about 15,630, and it is composed of about 60% nonsulfated disaccharide, 3.5% disaccharide 6-monosulfate and about 40% disaccharide 4-monosulfate, with a sulfate-to-carboxyl ratio of 0.41. Heparin, identified by agarose-gel electrophoresis, is constituted by about 40% slow-moving component and about 60% fast-moving species. This glycosaminoglycan had a peak molecular mass of about 7000, and was identified as 'typical' heparin by its constituent disaccharide composition. About 70% of disaccharides were identified as trisulfated disaccharide, and about 18% as disulfated disaccharides, 3% as monosulfated disaccharides and 10% as nonsulfated disaccharide. Heparin extracted from normal human plasma has a high sulfate-to-carboxyl ratio (2.47) and in vitro anticoagulant activity of about 70 I.U. A more quantitative and statistical analysis performed on 10 ml of plasma obtained from 10 human healthy volunteers revealed a heparin level of 0.54 +/- 0.17 mg/100 ml plasma (mean +/- standard deviation) with a coefficient of variation of about +/- 32%. These findings demonstrate for the first time the presence of heparin molecules in normal human plasma and confirm the importance of adequate extraction processes to purify a molecule that strongly interacts with plasma protein components. This is discussed in light of other authors that described a polysaccharide molecule named heparan sulfate in human plasma.
Volpi
N
N
Department of 'Biologia Animale', University of Modena, Italy.
Cusmano
M
M
Venturelli
T
T
D002240:Carbohydrate Sequence; D002808:Chondroitin Lyases / Q000378:metabolism; D002809:Chondroitin Sulfates / Q000097:blood* / Q000302:isolation & purification / Q000378:metabolism; D004187:Disaccharides / Q000032:analysis; D004587:Electrophoresis, Agar Gel; D006025:Glycosaminoglycans / Q000097:blood / Q000302:isolation & purification / Q000378:metabolism; D006493:Heparin / Q000097:blood* / Q000302:isolation & purification / Q000378:metabolism; D019763:Heparin Lyase; D006801:Humans; D008969:Molecular Sequence Data; D011133:Polysaccharide-Lyases / Q000378:metabolism
D004187:Disaccharides; D006025:Glycosaminoglycans; D006493:Heparin; D002809:Chondroitin Sulfates; D002808:Chondroitin Lyases; D011133:Polysaccharide-Lyases; D019763:Heparin Lyase