1. A broad spectrum of proteins has been detected within calcium stones. A newcomer to the field of urolithiasis is the blood protein inter-alpha-inhibitor. Inter-alpha-inhibitor comprises three protein chains linked by chondroitin sulphate: two heavy chains, H1 (65 kDa) and H2 (70 kDa) and a light chain (approx. 30 kDa) most commonly known as bikunin. The physiological function of the two heavy chains is unknown; nor has their presence been reported in urine. However, bikunin has been implicated in various renal diseases, including urolithiasis. 2. This study was undertaken to determine which chains of inter-alpha-inhibitor are actually present in calcium kidney stones. Organic extracts were obtained from 10 calcium stones and analysed by SDS/PAGE and Western blotting. The H1 and H2 chains of inter-alpha-inhibitor were detected in 9 of the 10 stones, but only one stone contained a protein with a molecular mass close to that of bikunin (30-35 kDa). 3. These results demonstrate for the first time that H1 and H2 are present in stones and show that the bikunin chain of inter-alpha-inhibitor may not be the only part of the molecule implicated in stone formation.
The bikunin peptide chain of the protease inhibitor inter-alpha-inhibitor (IalphaI) has been reported to be an inhibitor of calcium oxalate (CaOx) crystallization, and hence has been proposed as having a role in CaOx kidney stone formation. However, further experimental evidence is required to assess if fragments of IalphaI other than bikunin may play a role in the regulation of crystallization events in stone formation. The aim of the present study was to assess the effects of IalphaI and several of its derivatives on CaOx crystallization in a seeded inorganic system and to compare these effects with those of a known inhibitor of crystallization, prothrombin. IalphaI was purified from a preparation of human plasma and fragmented by alkaline hydrolysis, and two of its peptide chains, bikunin and heavy chain 1 (H1), were purified further by HPLC. Their purity was confirmed by SDS/PAGE. Using Coulter counter and [(14)C]oxalate analysis and scanning electron microscopy, IalphaI, its H1 chain and bikunin from urine and from plasma were shown to be relatively weak inhibitors of CaOx crystallization in vitro at expected physiological concentrations. It was concluded that members of the IalphaI family may not be as important in kidney stone formation as has been generally proposed, although further studies are required before a possible role for IalphaI and its fragments in stone formation can be unambiguously discounted.
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