Quantitative assessment of betainized compounds and associations with dietary and metabolic biomarkers in the randomized study of the healthy Nordic diet (SYSDIET).

BACKGROUND: Recently, a group of betainized compounds have been suggested to play a role in health effects in relation to a whole-grain-rich diet. OBJECTIVES: The aims of this study were to develop a quantitative mass spectrometric method for selected betainized compounds in human plasma, and to investigate their association with nutrient intake and measures of metabolic health in participants of the SYSDIET study. METHODS: The SYSDIET study was a controlled randomized intervention including individuals with metabolic syndrome, where the healthy Nordic diet (HND) group increased intakes of whole grains, canola oil, berries, and fish, whereas the control diet (CD) group consumed low-fiber cereal products, milk fat, and restricted amounts of fish and berries. A quantitative LC combined with triple quadrupole MS method for betainized compounds was developed and applied to fasting plasma samples from baseline (week 0) and the end of the intervention (week 18 or 24). Concentrations of betainized compounds were correlated with intakes of selected nutrients and fiber and measures of metabolic health. RESULTS: Pipecolic acid betaine (PAB) concentrations were significantly higher in the HND group than in the CD group (P = 0.00032) at the end of the intervention and correlated directly (P < 0.0001) with intakes of dietary fiber (r = 0.376) and a biomarker related to whole-grain rye intake, namely the ratio of alkylresorcinol C17:0 to C21:0 (r = 0.442). PAB was associated inversely with fasting plasma insulin consistently at the beginning and at the end of the intervention (P < 0.001, r = -0.300; P < 0.01, r = -0.250, respectively), as well as IL-1 receptor antagonist (P < 0.01, r = -0.232 at the beginning; P < 0.01, r = -0.236 at the end) and serum LDL/HDL cholesterol (P < 0.01, r = -0.239 at the beginning; P < 0.01, r = -0.241 at the end). CONCLUSIONS: Among adults with the metabolic syndrome, PAB plasma concentrations were associated with fasting insulin, inflammation, and lipids and were significantly increased with adoption of the HND. Further studies are needed to clarify the biological functions of betainized compounds. This trial was registered at clinicaltrials.gov as NCT00992641.

Simple, direct, and informative method for the assessment of CYP2C19 enzyme inactivation kinetics.

Many clinically relevant drug interactions involving cytochrome P450 inhibition are mediated by mechanism-based inactivation (MBI). Time-dependent inhibition is one of the major features distinguishing between reversible inhibition and MBI. It thus provides a useful screening approach for early drug interaction risk assessment. Accordingly, we developed an easy and informative fluorometric method for the assessment of CYP2C19 enzyme inactivation kinetics. Dibenzylfluorescein (DBF) is widely used as a profluorescent probe substrate for P450 activity and inhibition assays, but its use has been considered to be limited to traditional endpoint assays. We monitored CYP2C19-catalyzed metabolism of DBF using synthesized fluorescein benzyl ester and fluorescein benzyl ether along with commercially available fluorescein as intermediate standards. Furthermore, we demonstrated the use of DBF in a kinetic assay as a progress curve analysis for straightforward determination of whether a compound is a time-dependent inactivator of CYP2C19. The recombinant human CYP2C19 inactivation kinetics of isoniazid, ticlopidine, and tranylcypromine were evaluated, and their key kinetic parameters were measured from the same experiment. The known mechanism-based inactivators, isoniazid and ticlopidine, exhibited clear time-dependent inactivation with K(I) and k(inact) values of 250.5 ± 34 µM and 0.137 ± 0.006 min(-1) and 1.96 ± 0.5 µM and 0.135 ± 0.009 min(-1), respectively. Tranylcypromine did not display any time-dependent inhibition, which is consistent with its reported mechanism of competitive inhibition. In summary, DBF is suitable for use in the progress curve analysis approach and can be used as an initial screen to identify compounds that require more detailed investigations in drug interaction optimization.