A microtitre format assay for proline in human serum or plasma.

Abstract Background : Recent studies have suggested that low serum proline concentration may be associated with low bone mineral density. However, further investigation of this association has been hampered by the lack of a relatively high throughput assay for proline in biological fluids. Here we report a sensitive and specific microtitre plate format assay for proline which exploits the chemical interaction between proline and isatin. Methods : Human serum or plasma is deproteinised by incubation with sodium citrate buffer pH4.1 at 95 °C, and the supernatant is reacted with isatin at 95 °C for 3 h. The resultant blue coloured product is quantitated sprectrophometrically. Results : This assay yields a linear standard curve in the range 15 μmol/l to 1 mmol/l ( r =0.998±0.002; n =8 determinations) with a sensitivity of 31±11 μmol/l. None of the other proteogenic amino acids are detected ( Conclusions : This method provides the first reliable micro-titre format assay for proline in human serum.