Dinitrophenyl derivatization of imino acids, spectral characteristics and HPLC analysis: application in urinary peptide-derived hydroxyproline and proline assay.
BACKGROUND
Assay of urinary imino acids, in particular peptide derived, is of immense utility in diagnosis of collagen-related disorders. The often-used methods for hydrolysis of urinary peptides need a long time and are cumbersome, hence the need for relatively simpler, but effective methods.


METHODS
The method described, based on alkaline hydrolysis by autoclaving for 60 min followed by pre-column dinitrophenyl (DNP) derivatization and high-performance liquid chromatography (HPLC) analysis, demonstrates the complete hydrolysis and stability of urinary peptide derived imino acids.


RESULTS
DNP derivatives of both imino acids had identical lambda max (380 nm) with molar epsilon of 28.224 x 10(3) and 17.036 x 10(3), respectively, for hydroxyproline (Hyp) and proline (Pro). HPLC run, extending up to 18 min, resolved major components of collagen products, namely Hyp, Hyl, Gly, Pro and Lys, with retention times of 6.5, 9.8, 10.5, 11.2 and 12.55 min, respectively. The assay method conformed to linear response for individual amino acid concentrations of 0.5-4.0 nmol per injection, with goodness of fit (r(2) value) 0.99 for both Hyp and Pro, and detection limit of 0.05-4.0 nmol of DNP derivatives. The recovery of Pro and Hyp, when spiked with urine prior to hydrolysis, were found to be 95% and 92%, respectively.


CONCLUSIONS
Alkaline hydrolysis by autoclaving and DNP derivatization of imino acids followed by HPLC provides a method for the analysis of peptide-derived Hyp and Pro in urine. Hence, it is of utility to study collagen disorders.
Badadani
Mallikarjun
M
Department of Neurochemistry, National Institute of Mental Health and Neuro Sciences, Hosur Road, Bangalore 560029, Karnataka, India.
Babu
S V Suresh
SV
Shetty
K Taranath
KT
D002364:Caseins / Q000737:chemistry; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004136:Dinitrobenzenes / Q000737:chemistry*; D005780:Gelatin / Q000737:chemistry; D006801:Humans; D006868:Hydrolysis; D006909:Hydroxyproline / Q000737:chemistry / Q000652:urine; D007098:Imino Acids / Q000032:analysis* / Q000737:chemistry; D010455:Peptides / Q000737:chemistry / Q000652:urine; D011392:Proline / Q000737:chemistry / Q000652:urine; D011506:Proteins / Q000737:chemistry; D015203:Reproducibility of Results
D002364:Caseins; D004136:Dinitrobenzenes; D007098:Imino Acids; D010455:Peptides; D011506:Proteins; D005780:Gelatin; D011392:Proline; D006909:Hydroxyproline