Stabilizer free hsa

The quantification of caprylate was performed with a free fatty acid quantification assay kit (abcam, Cambridge, UK). This kit provides a sensitive, enzyme-based method for colorimetric detection of long- and middle chain free fatty acids in biological samples. Concentrations of free fatty acids in the samples were calculated from standards which are included in the kit. To obtain a signal in the standard range (0 to 200 μM), the samples were diluted 1:100 with dilution reagent which was provided with the kit. The amount of caprylate was calculated in percent of the untreated 20% HSA-solution (Kedrion Biopharmaceuticals, Italy), and as negative control the stabilizer-free HSA (Sigma Aldrich, USA) was used.

The quantification of N-acetyltryptophanate was performed with a high performance liquid chromatography method. Samples were precipitated by adding 50 μl HSA sample to 250 μl methanol, vortexed, and cooled at -80°C for 20 minutes. After centrifugation (14000 g, 5 min) 20 μl of the supernatant was injected into the HPLC column (150 x 4.6 mm Nucleosil 100–5 C18 column combined with a 4 x 3 mm Nucleosil 100–5 C18 guard column; Macherey-Nagel, Düren, Germany). The temperature of the column oven was set to 35°C. The elution consisted of a linear gradient program from 30 to 50% methanol in water over 6.5 minutes, maintained at 50% methanol for 1 minute and returned to 30% methanol for 1 minute. The flow rate was 0.5 ml/min and absorbance detection at 280 nm was performed for NAT quantification. The amount of N-acetyltryptophanate was calculated in percent of the untreated 20% HSA-solution (Kedrion Biopharmaceuticals, Italy), and as negative control the stabilizer-free HSA (Sigma Aldrich, USA) was analyzed. The recovery of NTA using this HPLC procedure is 72.3 ± 0.5% (S1 Fig). It was determined by comparing the peak areas between NTA diluted in methanol and NTA spiked in stabilizer-free albumin solution.