In a retem report a urinary hydroxyproline-containing peptide was preliminarily suggested as a possible useful biomarker of bone resorption, with a perspective to replace hydroxyproline assay in the diagnostice of osteopathy in clinical laboratories. The authors of the study developed a Simple and rapid way for screening the peptide in morning non-hydrolysed urine samples using HPLC with fluorometric detection. The procedure employed successive addtition of two reagents common in HPLC, i.e. OPA and FMOC, finto the sample. The former reagent reacted with primary amino groups ohly, leaving thus the latter to attack the secondary oves. The FMOC-treated imino acids, i.e. the peptide, proline, hydroxyproline, and also 3,4-dehydroproline as the interval standard, are intensively fluorescent products detected at correspondingly adjusted wavelengths, whereas the OPA-treated forms pase the detector unrecorded. Hundreds of randomly selected individuals were subjected to the peptide assay in non-hydrolysed morning urine samples and to hydroxyproline assay after urine hydrolyees. The values, corrected to creatinine excretion, afforded a high degree of correlation (r = 0.972), supporting thus an assumption tkat the so easily screened peptide might replace the determination of urinary hydroxyproline. In this report we developed an alternative procedure for the peptide based on capillary GC. Such an approach requires a preceding chemical modification of the compounds of interest to create volatile derivatives amenable to GC. This can be done smoothly with, e.g., ethyl chloroformate (ECF) in water containing media, tkat resembles the action of FMOC in HPLC, bot in contrast to fit also carboxylic groups are esterifled. Prior to the derivatization, the urine samples were subjected to amino acid uptake on a small bed of cation exchanger placed in a fllter tip and eluted afterwards with reaction médium of the subsequent chemical treatment. The total time required for pretreatment, derivation and GC analysis came to about 15 min, being thus equal to tkat of the HPLC procedure. Both methods were found correlated dosely (r = 0.944). Moreover, the peptide was identifled as dipeptide of proline with 4-hydroxyproline (PHP).

        Key words: PHP-dipeptide, bone resorption marker, GC assaay, GC vs. HPLC assay