Stable Isotopes as Tools for Evaluating Vitamin Contents and Bioactivity

Transcription

1 Research Center for utrition and Food Sciences Stable Isotopes as Tools for Evaluating Vitamin Contents and Bioactivity Michael Rychlik, BIAALYTIK Weihenstephan, Technische Universität München, Germany Sabine Mönch, TUM Michael etzel, CSIR, Australia Cornelia Witthöft, SLU Uppsala, Sweden Technische Universität München utline 1. Introduction: Stable isotopes, folates 2. Stable isotopes for accurate quantitations: : SIDA 3. SIDA in bioavailability studies 4. Double isotope studies: isotopes as tracers and as standards 5. Visions for stable isotope applications

2 The Atomar View: atural Stable Isotopes Element Hydrogen Carbon xygen itrogen Sulfur Isotope H-1 H-2 (Deuterium) C-12 C S-32 S-33 S-34 % Examples of isotopologically labelled compounds H H CH H H C H CH H H CH Analyte: Folic acid M r : 441 g/mol H D D CH H C CH D D CH Isotopologic Standard for Quantitation: [ 2 H 4 ]-Folic acid M r : 445 g/mol

3 Prinziple of the Stable Isotope Dilution Assay (SIDA) Isotopologic Standard RT: RT: MS Clean-up Derivatization Time (min) Analyte Folates: Introduction Folates involved in methylations of amino acids and nucleotides H CH H H C H CH CH H Average intake in Europe covers only 5 % of the recommended dose Evident / possible consequences of deficiency: - neural tube defects - elevated plasma level of homocysteine coronary heart disease - colon cancer?????? - alzheimer s disease

4 Structural Variety of Folates M CH H H C H CH C H CH CH possible variations: oxidation status of the pterin ring M linkage with c1 moieties n number of glutamate moieties Already known natural vitamers: about 5 compounds labile, occurring only in traces all vitamers have to be quantified C H n SIDA of Folates in Spinach 1..8 Intensity UV 28 nm Intensity R T: AA: H 4 folate SRM 446/299 Detection by LC-MS/MS Intensity R T: AA: [ 2 H 4 ]-H 4 folate SRM 45/33 R T: AA: SRM 46/ Methyl-H 4 folate Intensity 4 2 R T: 17.4 AA: [ 2 H 4 ]-5-Methyl-H 4 folate SRM 464/ Formyl-H 4 folate R T: AA: RT: A A: SRM 474/ [ 2 H 4 ]-5-Formyl-H 4 folate RT: AA: SRM 478/ Tim e (m in)

5 Foods Rich in Folates µg/1g Spinach SIDA wn data LC-FD Lit. data MA Lit. data Camembert cheese Wheat germs Brokkoli Mungo beans Whole-meal bread ot only contents, also bioavailability of food folates is important for dietary recommendations! Current assumptions: 5 % definition dietary folate equivalents to max 8 % for added folic acid to foods utline 1. Introduction: Stable isotopes, folates 2. Stable isotopes for accurate quantitations: : SIDA 3. SIDA in bioavailability studies 4. Double isotope studies: isotopes as tracers and as standards 5. Visions for stable isotope applications

6 Bioavailability: : Design of a first human study 26 healthy volunteers Saturated with folate by gavage of folic acid before the trial Design: random cross-over in ranges of 1 week: 11 blood samplings within 24 h - Meal A: 5 g spinach - Meal B: 5 g wheat germs - Meal C: 2 g Camembert cheese - Meal D: 4 µg folic acid in water Design of a first human study 5-Me-THF [ 2 H 4 ]-5-Me-THF (IS) [ 2 H 4 ]-Folate (IS) Endogenous Folates

7 Determination of Bioavailability: : AUC nmol/l spinach AUC plasma folate AUC: area under the curve nmol/l folic acid AUC base line Zeit in h Results: Mean Plasma Folate Curves after subtracting the base level at t= (nmol/l) 25 Camembert Wheat germs Spinach 2 Folic acid Time of sampling (h)

8 Results Bioavailability relative to folic acid = 1 % Lower limit of 9 % confidence interval (AUC) Spinach 523 µg 43.4 % Wheat germs 236 µg 19.5 % Camembert 254 µg 11.4 % Mean (AUC) 73. % 33. % 19.9 % Upper limit of 9 % confidence interval (AUC) % 56. % 34.7 % Summary of the First Study Bioavailability of certain food folates: Spinach 7-8 %, wheat germs appr. 3%, camembert 2% High inter individual differences (appr. ±1 %) Definition of dietary folate equivalents (5 % bioavailability of food folates) is still justified

9 First Study: pen Questions 5-Me-THF? [ 2 H 4 ]-5-Me-THF (IS) [ 2 H 4 ]-Folate (IS) Endogene Folate? Further isotopologically labelled folates H H H CH H C H CH H H Analyte: Folic acid M r : 441 g/mol CH H D D CH H C CH D D CH Isotopologic Standard for Quantitation: [ 2 H 4 ]-Folic acid M r : 445 g/mol H 13 CH H C 13 CH 13 CH Isotopologic Standard for Tracing [ 13 C 5 ]-Folic acid M r : 446 g/mol

10 utline 1. Introduction: Stable isotopes, folates 2. Stable isotopes for accurate quantitations: : SIDA 3. SIDA in bioavailability studies 4. Double isotope studies: isotopes as tracers and as standards 5. Visions for stable isotope applications ext Study: Stable Isotope as Tracer 5-Me-THF [ 13 C 5 ]-5-Me-THF (m/z 465) End. Folates [ 13 C 5 ]- Folates in cooperation with C. Witthöft, Uppsala

11 ext Study: Double Isotope Model 5-Me-THF [ 13 C 5 ]-5-Me-THF (m/z 465) [ 2 H 4 ]-5-Me-THF (IS, m/z 464) End. Folates [ 13 C 5 ]- Folates [ 2 H 4 ]-Folates (IS) in cooperation with C. Witthöft, Uppsala Differentiation of Isotopologues in TripleQuadMS Multiple Reaction Monitoring A H CH 3 CH H C CH + MS/MS H CH 3 H C + H CH m/z 46 m/z 313 H B H CH 3 D D CH H C CH + MS/MS H CH 3 H D D C + D D D D H CH m/z 464 m/z 317 H C H CH 3 H C 13 CH 13 CH + MS/MS H CH 3 H C H 13 CH m/z 465 m/z 313 H

12 Plasmafolates after Dosage of [ 13 C 5 ]-Folic acid Intensity, cps Intensity, cps Intensity, cps Intensity, cps XIC of +MRM (14 pairs): 1.e4 5.. MRM: 46./ MRM: 464./ e4 5.. MRM: 465./ Time, min Max. 35. cps. Max. 35. cps. 5-MethylTHF m/z Max. 1.e4 cps. [ 2 H 4 ]-MethylTHF m/z Max cps. [ 13 C 5 ]-5-MethylTHF m/z Aims of the Second Bioavailability Study Bioavailability of different folates in sub-saturated humans Behaviour of folic acid vs. 5-methyltetrahydrofolate in fortified foods Bioavailability of added folates to different matrices

13 Hazard from Fortification with Folic Acid? USA Canada mandatory fortification mandatory fortification Colorectal Cancer incidence pre- and post-mandatory fortification Source: Mason et al., Cancer Epidemiology, Biomarkers & Prevention (27) 16: 1325 Visions for the Use of Isotopes in Vitamin Research Tracing vitamins from fortified foods Bioavailability (fortification efficiency) Metabolism (benefit or hazard) Multivitamer assays eed for different labels, differentiation possible? Intrinsic labelling for tracing of endogenous vitamins for production of complex vitamins

14 Acknowledgement PhD students Sabine Mönch, Barbara Büttner und Veronica Öhrvik Cooperation partners Prof. Cornelia Witthöft, SLU Uppsala Dr. Michael and Gabi etzel, CSIR Brisban Dr. Thomas Frank, Bad Soden Funding by 8th IFDC rganizers Swedish Research Council FRMAS German Research Association