A microtitre format assay for proline in human serum or plasma.
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 pH 4.1 at 95 degrees C, and the supernatant is reacted with isatin at 95 degrees C for 3 h. The resultant blue coloured product is quantitated sprectrophometrically.


RESULTS
This assay yields a linear standard curve in the range 15 micromol/l to 1 mmol/l (r=0.998+/-0.002; n=8 determinations) with a sensitivity of 31+/-11 micromol/l. None of the other proteogenic amino acids are detected (<0.3% detection at 10 mmol/l) and the closely related metabolite hydroxyproline is only very weakly detected (3% detection at 10 mmol/l). Using human serum, the assay has linear dilution characteristics and a mean spike recovery of 107+/-5%. Repeated re-measurement of the same serum sample yields an intra-assay coefficient of variation (CV) of 4.8% and an inter-assay CV of 6.1%.


CONCLUSIONS
This method provides the first reliable micro-titre format assay for proline in human serum.
Grainger
David J
DJ
Department of Medicine, University of Cambridge, Box 157, Addenbrooke's Hospital, Cambridge CB2 2QQ, UK. djg15@cam.ac.uk
Aitken
Sri
S
D002021:Buffers; D006801:Humans; D006909:Hydroxyproline / Q000032:analysis; D007510:Isatin / Q000097:blood / Q000737:chemistry; D007700:Kinetics; D011392:Proline / Q000097:blood* / Q000737:chemistry; D012015:Reference Standards; D012680:Sensitivity and Specificity
D002021:Buffers; D007510:Isatin; D011392:Proline; D006909:Hydroxyproline