Pretreatment procedure for the microdetermination of chondroitin sulfate in plasma and urine.
A new, simple, and rapid pretreatment method for the determination of chondroitin sulfate, dermatan sulfate, and hyaluronan from urine and blood plasma samples has been developed. Plasma proteins were first converted into small peptides by digestion using a nonspecific protease, actinase E, and the resulting small peptides were removed by centrifugal filtration. The retained, residual crude glycosaminoglycans, including chondroitin/dermatan sulfates and hyaluronan, were converted into unsaturated disaccharides through the action of chondroitin sulfate lyses. Next, these disaccharides were recovered and purified using centrifugal filtration together with DeltaDi-UA2S, added as an internal standard. The filtered disaccharide mixture was analyzed by HPLC with fluorometric postcolumn derivatization using 2-cyanoacetamide as a fluorogenic reagent. This method was applied to a pharmacokinetic study of chondroitin sulfate administered intravenously to mice. The half-life of the administered chondroitin sulfates, having molecular masses from 6 to 50 kDa, varied depending on their molecular sizes. This new method should be useful for studies on the metabolic fate of exogenously administered glycosaminoglycans in small experimental animals.
Sakai
Shinobu
S
Graduate School of Pharmaceutical Sciences, Chiba University, 1-33, Yayoi-cho, Inage-ku, Chiba 263-8522, Japan.
Onose
Jun-ichi
J
Nakamura
Haruka
H
Toyoda
Hidenao
H
Toida
Toshihiko
T
Imanari
Toshio
T
Linhardt
Robert J
RJ
D000818:Animals; D002808:Chondroitin Lyases / Q000378:metabolism*; D002809:Chondroitin Sulfates / Q000097:blood* / Q000493:pharmacokinetics / Q000652:urine; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D003871:Dermatan Sulfate / Q000097:blood* / Q000652:urine; D004187:Disaccharides / Q000032:analysis* / Q000378:metabolism; D005374:Filtration / Q000379:methods; D006025:Glycosaminoglycans / Q000097:blood / Q000652:urine; D006207:Half-Life; D006820:Hyaluronic Acid / Q000097:blood* / Q000652:urine; D007275:Injections, Intravenous / Q000379:methods; D008297:Male; D051379:Mice
D004187:Disaccharides; D006025:Glycosaminoglycans; C020341:glucosaminoglycans; D003871:Dermatan Sulfate; D006820:Hyaluronic Acid; D002809:Chondroitin Sulfates; D002808:Chondroitin Lyases