Objective: Measurement of the immunoreactive urinary albumin (ir-uAlb) concentration by immunological methods was found to be an effective method to identify disease activity in Crohn's disease (CD). Recently a size-exclusion (SE) high performance liquid chromatography (HPLC) method was developed to measure both ir-uAlb and non-immunoreactive urinary albumin (total, t-uAlb). We aimed to follow-up one of our CD patients with frequent remissions and exacerbation phases comparing the changes of disease activity parameters and the concentration of ir-uAlb and t-uAlb. The surprising results led us to perform measurements in greater depth.
Material and methods: Concentration of ir-uAlb was measured by immunoturbidimetry (IT) and t-uAlb by SE-HPLC. Albumin peak of SE-HPLC was collected and applied to a reversed-phase (RP) HPLC and to gel-electrophoresis. Eluted peaks of RP-HPLC and identified bands of gel-electrophoresis were analyzed using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS).
Results: The concentration of t-uAlb was 15 times higher than that of the ir-uAlb during active state. The RP-HPLC and the gel-electrophoresis separation proved that albumin peak by size-exclusion consists of three different peaks. MALDI-TOF/MS measurements identified α1-acid-glycoprotein and Zn-α2-glycoprotein as major, and albumin as minor protein.
Conclusions: Peak of albumin of SE-HPLC contains a significant amount of glycoprotein during the active phase of CD, which could not be detected in remission. Urinary α1-acid-glycoprotein and/or Zn-α2-glycoprotein could be an ideal disease activity biomarker of CD.