Automated Turbidimetric Benzalkonium Chloride Method for Measurement of Protein in Urine and Cerebrospinal Fluid

Various methods have been used to measure protein in urine and cerebrospinal fluid (CSF). Urinary protein can be determined by turbidimetric assays using trichloroacetic acid (1), sulfosalicylic acid (2), or benzethonium chloride (BTC) (3)(4); the biuret assay (5)(6); or protein dye-binding assays using Coomassie Brilliant Blue (CBB) (7)(8)(9)(10)(11)(12)(13), Pyrogallol Red (PYR)-molybdate (14)(15), or Ponceau S (16). The BTC, CBB, and PYR methods can be automated. Shephard and Whiting (17) used benzalkonium chloride (BC) as a precipitating agent for the determination of protein in urine and CSF by nephelometry. In this study we developed an automated turbidimetric method using BC and compared it with conventional automated methods. Albumin fraction V (bovine), disodium molybdate · 2 H2O, NaOH, sodium azide, sodium benzoate, potassium oxalate, and succinic acid were obtained from Merck; BTC, Coomassie Brilliant Blue G 250, EDTA, and PYR were from Sigma; BC was from Serva; and phosphoric acid was from Riedel de Haen. A commercially available PYR reagent (M-TP) and protein calibrators were obtained from Beckman Coulter. A globulin solution containing mostly IgG and IgM and no albumin was obtained from the Refik Saydam Center of Hygiene, Turkey. We developed a new method that uses the same principle as the method of Iwata and Nishikaze (3) but with BC instead of BTC. BC is a detergent and causes protein denaturation in an alkaline medium. The NaOH/EDTA reagent (reagent 1) contained 33 mmol/L EDTA and 0.5 mol/L NaOH. The BC solution (5 g/L; reagent 2) was prepared from a 50 g/L aqueous stock solution of BC. Calibrators containing 50, 100, 200, 500, and 1000 mg/L protein were …