A microtitre format assay for proline in human serum or plasma

Area of Science:

• Biochemistry
• Analytical Chemistry
• Clinical Chemistry

Background:

• Low serum proline concentration is potentially linked to reduced bone mineral density.
• Previous research was limited by the absence of a high-throughput proline assay.
• A novel assay for proline in biological fluids is presented.

Purpose of the Study:

• To develop a sensitive and specific microtitre plate assay for quantifying proline in human serum.
• To facilitate further investigation into the association between proline levels and bone mineral density.

Main Methods:

• A microtitre plate assay utilizing the chemical reaction between proline and isatin.
• Serum or plasma deproteinization followed by reaction with isatin at 95°C.
• Quantification of the resulting blue color product spectrophotometrically.

Main Results:

• The assay demonstrates linearity from 15 µmol/l to 1 mmol/l (r=0.998) with a sensitivity of 31±11 µmol/l.
• High specificity with negligible detection of other amino acids and weak detection of hydroxyproline.
• Consistent results with linear dilution, mean spike recovery of 107±5%, and intra-assay/inter-assay coefficients of variation of 4.8% and 6.1%, respectively.

Conclusions:

• This study presents the first reliable microtitre format assay for proline in human serum.
• The developed assay is suitable for high-throughput analysis.
• Enables further research into the clinical significance of serum proline levels.