Objective: To determine which chains of inter-alpha-inhibitor (I alpha I) are present in urine and whether they are also found in calcium oxalate (CaOx) crystals generated in human urine.
Materials and methods: Fresh urine specimens were collected from five women and five men with no previous history of stone disease. An aliquot of each urine was retained for analysis, the remainder treated with a standard load of oxalate and the CaOx crystals precipitated from each specimen demineralized with ethylenediamine tetracetic acid. The resulting organic extracts from crystals and their corresponding urine samples were subjected to sodium dodecyl sulphate gel electrophoresis analysis and Western blotting using a commercial polyclonal antibody to I alpha I.
Results: Heavy chain 1 (H1) and 2 (H2) of I alpha I were commonly found in every urine sample, and in the CaOx crystals precipitated from those urine samples. Several protein bands were visible in urine samples from both sexes in the molecular mass range 25-70 kDa, which may be bikunin or its fragments. As well as H1 and H2, the crystals from both sexes contained a protein band at approximately 33 kDa. In many cases there appeared to be no direct relationship between the proteins detected in the crystals and the urine samples from which they were derived, which probably reflects the well known instability of I alpha I and the occurrence of a range of bikunin fragments in urine.
Conclusion: These results show for the first time that H1 and H2 are present in human urine and urinary CaOx crystals, that the bikunin chain of I alpha I is not the only part of the molecule capable of participating in CaOx crystallization in urine, and in theory at least, in the regulation of crystallization events in stone formation. It is also apparent that significant fragmentation of I alpha I occurs both in vivo and in vitro, and this must be considered in any study attempting to elucidate the influence of this protein in the formation of CaOx stones.