The Use of Liquid Chromatography-Mass Spectrometry for the Identification and Quantification of Urinary Iminodipeptides in Prolidase Deficiency

Abstract

It has been reported that the urine of patients with prolidase deficiency contains various iminodipeptides with a carboxyl-terminal proline (hydroxyproline). These iminodipeptides have hitherto been detected indirectly by acid hydrolysis or enzymatic digestion, followed by amino acid analysis. In the present study, it was shown that X-Pro could be distinguished from Pro-X when the iminodipeptides were analysed directly by liquid chromatography coupled with atmospheric pressure ionization mass Spectrometry (LC/API-MS), with scanning of the protonated molecule ions ([M + H]+). The same procedure also successfully quantified urinary iminodipeptides from patients with prolidase deficiency. A quantitative investigation of two siblings with prolidase deficiency revealed that the patient with severe clinical symptoms excreted more iminodipeptides than the other who did not have serious symptoms. LC/API-MS also revealed iminodipeptides (Gly-Hyp and Pro^Hyp) in the urine of the mother of the patients and in normal volunteers. Patients excreted much more Pro-Hyp than normal volunteers, whereas no quantitative differences were found between the mother and controls. In patients, the excretion of large quantities of X-Pro is due to their very low prolidase activity towards this type of substrate. In the erythrocytes of patients, prolidase activity towards X-Hyp was extremely low; even in the mother and normal volunteers, it was remarkably low in comparison with the activity against X-Pro.

Measurement of Iminodipeptides in the Serum of Patients with Prolidase Deficiency Using Liquid Chromatography-Mass Spectrometry

“…Prolidase activities against X-Pro and X-Hyp were determined in erythrocytes from patients, their mother and normal subjects, using the method described previously (11). The prolidase activities of patients towards GlyPro and Pro-Pro were very low compared with contröls, and the mother had approximately half äs much activity äs normal subjects, which agreed with a previous report (12).…”

“…After 10 min at 90 °C with Chinard's reagent (10), which consisted of ninhydrin, phosphoric acid and acetic acid, liberated proline was estimated by spectrophotometry (wavelength 515 nm). The activity towards X-Hyp was assayed according to the method reported previously (11).…”

The role of emerging techniques in the investigation of prolidase deficiency: From diagnosis to the development of a possible therapeutical approach

“…These obstacles have been partially overcome through the development of techniques with increased precision of analysis. The application of HPLC interfaced with atmospheric pressure ionization mass spectrometry (API-MS), for instance, allowed Kodama et al [36] and Sugahara et al [37] to observe and quantify in the urine of patients with prolidase deficiency the quasi-molecular ions [M + H] + of various imidodipeptides containing proline or hydroxyproline at the Cterminus. Although it was never used for diagnostic purposes on urine of PD patients, this method was further improved by Sugahara et al [38] to measure simultaneously both imidodipeptides containing N-or C-terminal proline.…”