Inhibitory effect of bikunin on calcium oxalate crystallization in vitro and urinary bikunin decrease in renal stone formers

Two proteins of 17 and 24 kDa, respectively, which were immunologically rel ated to bikunin, were purified from urine of healthy men, using in the last step a trypsin CNBr-sepharose affinity column. These pro teins strongly in hibited calcium oxalate (CaOx) crystallization in two in vitro models. In t he first model, the presence of 8 mu g/ml protein in a medium containing 0. 76 mM CaCl2 (with Ca-45) and 0.76 mM ammonium oxalate inhibited the crystal lization process by 80%, as estimated by supernatant radioactivity after 60 min of incubation. A similar inhibition was observed in the second turbidi metric model, where the CaOx crystallization kinetics were followed for 10 min at 620 nm in a medium containing 4 mM CaCl2 and 0.5 mM Na(2)Ox. These p roteins were used as standard protein for the development of an enzyme-link ed immunosorbent assay (ELISA) in urine. Mean (+/- SEM) urinary bikunin con centration in 18 healthy subjects was 5.01 +/- 0.91 mu g/ml. This was a con centration range of strong inhibitory activity in vitro. Bikunin values wer e nearly 50% lower (2.54 +/- 0.42 mu g/ml, P = 0.007) in 31 CaOx renal ston e formers (having weddelite crystals in their first morning urine) than in the healthy volunteers. A correlation was found between urinary bikunin and alpha-1 microglobulin concentrations in the control group (y = 0.73x + 1.0 9, r(2) = 0.8) while no such correlation existed in the lithiasis group. In conclusion, bikunin exerts a strong inhibitory action of CaOx crystallizat ion in vitro. Its involvement in urinary CaOx crystallization of stone form ers is highly probable, based on the significant decrease in its urinary co ncentration in the majority of stone formers studied.