Analysis of Tamm–Horsfall protein by high-performance liquid chromatography with native fluorescence

Tamm–Horsfall (TH) is a large glycoprotein which originates in the kidney and is very abundant in the urine. This protein has been measured mainly by immunoassays. Here we describe a different approach for its measurement based on high-performance liquid chromatography (HPLC) using a molecular exclusion column with native fluorescence detection in the ultraviolet range. This method in addition to measuring the level of the protein has the advantage of detecting changes in size or aggregation. Urine, 1 ml was mixed with 100 μl of 30% NaCl and left at 37 °C for 30 min. The urine was centrifuged at 12 000 rpm for 20 s. The precipitate was vortex-mixed and dissolved in a triethanolamine buffer. A 20 μl aliquot was injected on a Macrosphere GPC column which was eluted with phosphate buffer and the effluent was detected by a fluorometer set at 280 nm for excitation and 325 nm for emission. Since the protein has a very large molecular mass compared to other urinary and serum proteins we did not experience any interference. It elutes as the first peak (in ∼2.5 min on a 500 Å and 2.7 min on 1000 Å). The protein precipitates rapidly <60 min at 37 °C. The detection in the UV is sensitive for this protein down to 1 mg/l in absence of any concentration steps. The method was linear between 1 and 100 mg/l. The R.S.D. was 10.4% (mean 62, n=10). The mean level in 42 normal individuals was 31 mg/g creatinine and in 30 patients with proteinuria (different renal disorders) was 23 mg/g creatinine.