Carbonic anhydrase II was detected in urine of a patient with osteosarcoma during high-dose methotrexate and leucovorin rescue therapy

Abstract

Using a 5-20% (linear gradient) polyacrylamide slab gel electrophoresis, we studied the urinary protein collected from a 14-year-old girl with osteosarcoma during high-dose methotrexate (MTX) and leucovorin rescue therapy. A distinct protein band of approximately 30 kDa (P-30 protein) was observed in addition to albumin. We have never before encountered a 30 kDa urinary protein. Therefore, we first studied the P-30 protein by peptide mass fingerprinting and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). We obtained the amino acid sequence "YDPSLKPLS-VSYDQATSLR", which is the same as that of amino acids (40-58) of human carbonic anhydrase II (CAII). Then, we confirmed this protein by Western blotting using anti-human CAII antibody. This enzyme distributes widely in the human bodies, in erythrocytes, kidney, and pancreas. We considered that this CAII originated from the kidney in association with high-dose MTX and leucovorin rescue therapy. Key words: carbonic anhydrase II, high-dose methotrexate and leucovorin rescue therapy, urinary protein, osteosarcoma, mass spectrometry. 10-8-1.5×10-7 M, at 24 h, 48 h and 72 h after a 5-h MTX (100 mg/kg body weight) infusion, respectively 5). These levels of blood MTX were within safe intervals 6) , i.e., lower than 1×10-5 M, 1×10-6 M and 1×10-7 M, at 24 h, 48 h and 72 h after start of the infusion, respectively 6). We also studied 1) liver function tests (serum AST, ALT, LDH, ALP and total bilirubin) and 2) renal function tests (serum Na, K, Cl, creatinine and BUN). However, no difference was seen in the data before and during this therapy 5). In these cases, no side effects were seen except for slight mucositis. Peyriere reported that during high-dose MTX therapy for a 14-year-old boy with osteosarcoma, severe intoxication was observed together with high MTX plasma concentration, acute renal failure, and hepatitis, followed by