Alpha-1-microglobulin: inhibitory effect on calcium oxalate crystallization in vitro and decreased urinary concentration in calcium oxalate stone formers.
In the past few years, alpha-1-microglobulin (alpha1m) has been copurified from human urine with bikunin, a potent inhibitor of calcium oxalate (CaOx) crystallization in vitro. In this study, we have purified alpha1m without bikunin contamination and investigated its possible role in CaOx crystallization by in vitro and in vivo studies. Alpha-1m was purified with an anti-alpha1m antibodies CNBr-activated sepharose column. Two molecular species of alpha1m of respectively 30 and 60 kDa were purified. For each protein, two blots of 30 and 60 kDa cross-reacted with anti-alpha1m antibodies, suggesting that these two forms were derived one from the other. Both protein species inhibited CaOx crystallization in a dose-dependent manner in two in vitro tests. In the first test, the presence of alpha1m of 30 kDa (8 microg/ml) in a medium containing 0. 76 mM CaCl(2) (with (45)Ca) and 0.76 mM Ox(NH(4))(2) inhibited CaOx crystallization by 38% as estimated by supernatant radioactivity after 1 h of agitation. In the second test, CaOx kinetics were examined for 3 to 10 min in a turbidimetric model at 620 nm. The presence of alpha1m of 30 kDa in a medium containing 4 mM CaCl(2) and 0.5 mM Na(2)Ox inhibited CaOx crystallization by 41.5%, as estimated by the slope modification of turbidimetric curve. Alpha-1m can be considered as another inhibitor of urinary CaOx crystal formation, as shown by the present in vitro studies. Using an ELISA assay, we found that urinary alpha1m concentration was significantly lower in 31 CaOx stone formers than in 18 healthy subjects (2.95 +/- 0.29 vs 5.34 +/- 1.08 mg/l respectively, P = 0.01). The decreased concentration of alpha1m in CaOx stone formers could be responsible in these patients, at least in part, for an increased risk of CaOx crystalluria.
Tardivel
S
S
Laboratoire du Métabolisme Minéral des Mammiféres, EPHE, Physiologie, Faculté de Pharmacie, F-92296 Châtenay-Malabry Cedex, France.
Médétognon
J
J
Randoux
C
C
Kébédé
M
M
Drüeke
T
T
Daudon
M
M
Hennequin
C
C
Lacour
B
B
D000293:Adolescent; D000328:Adult; D000368:Aged; D000818:Animals; D015153:Blotting, Western; D002118:Calcium / Q000652:urine; D002129:Calcium Oxalate / Q000037:antagonists & inhibitors* / Q000652:urine; D002846:Chromatography, Affinity; D003460:Crystallization; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D006023:Glycoproteins / Q000276:immunology / Q000302:isolation & purification / Q000494:pharmacology / Q000652:urine*; D006801:Humans; D008297:Male; D008562:Membrane Glycoproteins; D008875:Middle Aged; D008970:Molecular Weight; D019815:Oxalic Acid / Q000652:urine; D011817:Rabbits; D014360:Trypsin Inhibitor, Kunitz Soybean; D014545:Urinary Calculi / Q000652:urine*
D006023:Glycoproteins; D008562:Membrane Glycoproteins; C109148:SPINT2 protein, human; D002129:Calcium Oxalate; D014360:Trypsin Inhibitor, Kunitz Soybean; D019815:Oxalic Acid; D002118:Calcium