We describe a fully automated particle-enhanced turbidimetric assay for cystatin C in undiluted serum and EDTA-plasma. The throughput is 90 samples per hour and urgent samples can be analyzed in 7 min. The assay range (0.4-14.1 mg/L) covers the concentration range in health and disease. The within- and between-run imprecision is 0.9% and 2.2%, respectively. Analytical recovery of additions of recombinant cystatin C averaged 98%. Rheumatoid factors (< or = 323,000 IU/L), bilirubin (< or = 150 mumol/L), hemoglobin (< or = 1.2 g/L), and triglycerides (< or = 8.5 mmol/L) do not interfere in the assay. In view of the superior (by ROC analysis) diagnostic accuracy of serum concentrations of cystatin C for reduced glomerular filtration rate (GFR) in comparison with creatinine, cystatin C seems an attractive alternative to creatinine for estimation of GFR.
Aims: Resistance mechanisms are important limiting factors in the treatment of solid malignancies with cis-diamminedichloroplatinum(II) (cisplatin). To gain further understanding of the effects of acquired cisplatin-resistance, we compared a human malignant pleural mesothelioma cell line (P31) to a sub-line (P31res1.2) with acquired cisplatin-resistance. Methods and Results: The role of Na+,K+,2Cl--cotransport (NKCC1) activity in cisplatin-induced morphological changes and acquired cisplatin-resistance was investigated in a time-resolved manner. Acquisition of cisplatin-resistance resulted in markedly reduced NKCC1 activity, absence of cisplatin-induced early membrane blebbing, and increased basal caspase-3 activity. At equitoxic cisplatin concentrations, P31res1.2 cells had a faster activation of caspase-3 than P31 cells, but the end-stage cytotoxicity and number of cells with DNA fragmentation was similar. Bumetanide inhibition of NKCC1 activity in P31 cells repressed cisplatin-induced early-phase membrane blebbing but did not increase P31 cell resistance to cisplatin. Conclusions: Together, these results suggest that active NKCC1 was necessary for cisplatin-induced early membrane blebbing of P31 cells, but not for cisplatin-resistance. Thus, acquisition of cisplatin-resistance can affect mechanisms that have profound effects on cisplatin-induced morphological changes but are not necessary for the subsequent progression to apoptosis.
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