MSACL 2023 Abstract

Case Description:
Study cohort conducted with 100 inflammatory bowel disease (IBD) patients (20-60 years, 40 females) whom diagnosed according to standard clinical, radiological and pathological criteria and 100 healthy controls (20-60 years, 40 females). Of the patients 58/100 had Crohn`s Disease (CD), 42/100 had ulcerative colitis (UC) and 29/100 were in inflammatory state (serum high sensitivity C reactive protein (hsCRP) concentrations >5mg/L). Standard serum and citrate plasma samples were withdrawn between 8:00 - 10:00 hours in the clinic after overnight fasting and stored at -20 °C until the analysis. Complete blood count (CBC), high sensitivity C reactive protein (hsCRP), fecal calprotectin (fCal) were determined with standard methods in serum matrix. 3-Hydroxyisovalerylcarntine (3HIVc) levels were determined by LC-MS/MS citrate plasma matrix.

Background:
Studies on biotin's role in the immune response have emergently revealed that biotin is related to inflammation and that its deficiency leads to an increase in the level of proinflammatory cytokines. A biotin-deficient diet was recently shown to induce a colitis-like phenotype in mice, alleviated by biotin substitution. Mice with dextran sulphate sodium (DSS)-induced colitis showed biotin deficiency and significantly reduced biotin absorption. Oral biotin substitution reversed DSS colitis and induced remission. Diagnosis of biotin deficiency is rather challenging since based solely on measurement of circulating biotin levels has been shown to be insufficiently sensitive for clinical purposes. Increased urinary excretion of biotin metabolites, is suggested as a sensitive indicator of biotin deficiency. However, 24 h urine collection is time consuming, expensive, and the test relies on correct collection of samples by the patient, thus if this is not done properly the results may be inaccurate. 3HIVc increases in the case of biotin deficiency as a response to the decreased activity of biotin dependent enzyme methyl crotonyl-coenzyme. Analysis of circulating 3HIVc levels by LC-MS/MS has been found to be one of the most sensitive markers of biotin depletion.

MS Method and Results:
For measurement of 3HIVc in citrate plasma the commercial LC-MS/MS kit KM 3200 (Immundiagnostik AG, Germany) was used. This kit enables the measurement of 3HIVc after a fast precipitation reaction.
Sample preparation was achieved by adding the precipitation solution including an isotopically labelled internal standard in 96-well plates. After vortexing and centrifugation, supernatants were diluted and injected into an ACQUITY UPLC H Class (Waters) with an injection cycle of 3 mins. Transitions of 3HIVc were measured in positive mode by ESI-MS/MS on TQXS (Waters). For calibration, six levels (0.47-88.82 ng/mL) covering the complete physiological range were used. Results were verified by injections of three quality control (QC)-levels throughout the measurement. The assay was validated after FDA guidelines. Finally, the performance f the kit was evaluated was evaluated on a second LC-MS system (TSQ vantage, Thermo Scientific), providing robust application.

Patients with IBD were found to have significantly higher 3HIVc levels than controls (6.1±2.4 ng/mL vs 5.1±2.1 ng/mL respectively, p = 0.004). 3HIVc concentrations were compared according to different disease characteristics within the patient group: circulating 3HIVc levels were found to be similar in patients with CD and UC (5.2±2.1 ng/mL vs. 4.9±2.1, p = 0.451). Patients were also found to have similar circulating 3HIVc concentrations irrespective of whether or not inflammatory activity was present (5.1±2.0 ng/mL in inflammatory vs. 5.7±2.4 ng/mL in noninflammatory conditions, p = 0.131).

Discussion and Conclusion:
The commercial LC-MS/MS assay enabled sensitive, and accurate quantification of 3HIVc. The kit allows easy implementation into the clinical laboratory, on a wide range of mass spectrometers. The clinical findings of this study are in line with the existing preclinical evidence indicating that biotin deficiency is more common in patients with IBD in comparison to healthy controls in a clinical setting. However, surprisingly, the biotin status of patients with IBD was not found to differ according to inflammatory status. Therefore, there is still a room for more comprehensive look into the relationship between IBD related inflammation and biotin deficiency and 3HIVc measurement with LC-MS/MS provides sensitive results for clinics to define biotin repletion to achieve with this future perspective. Finally, the assay is on the way to IVDR certification in Europe.