Folic acid. Erik Konings Nestlé Research Center Switzerland

Transcription

1 Folic acid Erik Konings Nestlé Research Center Switzerland

2 Outline Background - Terminology/Chemical Structure - History/Occurrence - Metabolism/RDA - Benefits/Risks Current analytical techniques - Microbiology - LC - Biospecific methods Regulatory information - Infant formulae and follow-up formulae (IF, FUF) - Foods for Special Medical Purposes (FSMP)

3 Terminology Folic acid: - synthetic form (supplements, fortified food) - a monoglutamate - no attachments to basic molecule Folates - natural forms (plant and animal tissues) - polyglutamates - usually have a one-carbon attachement

4 Chemical structure

5 Chemical structure

6 History 1931: Lucy Wills et al. demonstrated that an autolysed yeast preparation (Marmite) was effective against nutritional megaloblastic anaemia in pregnant women. 1941: Mitchell et al. processed 4 tons of spinach to obtain a purified substance with acidic properties which was an active growth factor for rats and L. casei. They named the factor folic acid. 1946: Angier et al. accomplished synthesis and determination of structure of folic acid.

7 Occurrence Folates present in majority of foods; liver, yeast extracts, green leafy vegetables, legumes, and some fruits are especially rich sources. They contain 30 (orange) 1000 (Marmite) µg folates/100g. Low-fat milk 5 µg/100 ml

8 Metabolism EFSA meeting summary report, January 2009

9 Dietary Folate Equivalents: Application of Folate Bioavailability µg DFE = µg food folate + (1.7 x µg synthetic folic acid) Assumes: - 50% bioavailability of natural dietary folate (Sauberlich et al 1987). - 85% bioavailability of added folic acid (Pfeiffer et al. 1997). - Thus, synthetic is 85/50 more available. Institute of Medicine, 1998

10 Recommended Daily Allowances (RDA) Europe: µg/d (adults), µg/d (children), 400 µg/d (pregnant women) US: 400 µg DFE/d, 400 µg/d (pregnant women) Safe Upper Level: 1000 µg folic acid/day (SCF, IOM)

11 Benefits and risks of folic acid/folate Benefits Risks Certain Anaemia and Deficiency Neural Tube defects Mask Pernicious Anaemia Probable Stroke Accelerates existing Tumour Possible Heart disease Cancer occurrence Cognetive decline Alzheimer s Disease Other Birth Defects Accelerates cognitive decline Impairs immune function Interferes with Methotroxate Therapy EFSA meeting summary report, January 2009

12 Current Analytical Techniques Codex Type II methods: AOAC ; EN Microbiology Analysis: Turbidimetric methods based on absolute requirement of certain microorganisms for folic acid/folate. Organism multiplies in proportion to concentration of limiting folic acid/folate Extent of growth is ascertained by measuring turbidity produced Lactobacillus casei

13 Disadvantages MBA Technique lengthy, manipulative Compromised by variable vitamer response Susceptible to interference from growthstimulating artifacts Poor precision

14 Koontz et al JAOAC Macaroni, pizza: folic acid fortified CV: 9-11%, others >45%

15 Current method for routine folate analysis by HPLC Affinity chromatography purification HPLC separation and electrochemical detection Diaz de la Garza et al. Proc Natl Acad Sci USA 2004;101: Modified from Bagley and Selhub, Clin Chem 2000;46: Research setting Jesse F. Gregory, University Florida, First International Vitamin Conference, 2010, Denmark.

16 thf 16.7m 5 formyl-thf 26.1m dhf 27.1m 10 formyl-dhf 21.7m Response (µa) 5 methyl-thf 19.5m 5,10 methenyl-thf 23.8m folic acid 28.8m Research setting Typical Food Folate Analysis (Monoglutamyl folate standards) Rev. Phase HPLC w/ Electrochemical Detection Retention time (minutes) Jesse F. Gregory, University Florida, First International Vitamin Conference, 2010, Denmark.

17 Analytical methods must include calibration and measurement of biologically active vitamers All natural and added vitamers. Bioactive vitamers that can be oxidation products in food or formed during analysis (e.g. 10-formyldihydrofolate). Do analytical results reflect actual pattern of vitamers in food? Jesse F. Gregory, University Florida, First International Vitamin Conference, 2010, Denmark.

18 Mixed vegetables CRM 485 Pig Liver CRM 487 Milk Powder CRM 421 Wholemeal Flour CRM 121 Total folate MBA µg/100g 315± ±70 142±7 50±4 Total folate LC µg/100g 195± ±58 107±2 37±3 Konings et al. 1999, 2001: LC values 25% lower compared to MBA Karilioto et al. (University Helsinki) confirmed this recently for cereal products, at the First International Vitamin Conference in Copenhagen, 2010.

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20 Total folate (MBA) in CRM421 Milk Powder HKMA 03 HKMA 04 HKMA 05 HKMA 06 HKMA 07 HKMA 10 HKMA 11 HKMA 12 HKMA 13 HKMA 04 PAMA 05 HPMA 06 HPMA 12 CPMA 13 CPMA LC The certification of the mass fractions of vitamins in four reference materials. P.M. Finglas, K.J. Scott, C.M. Witthöft, H. van den Berg, I. de Froidmont-Görtz. Commission of the European Communities, 1998.

21 UPLC/MS/MS procedure 13 C-Folic Acid (internal std) Protease Homogenize Buffer & Heat Amylase T. Gbatu, J. Szpylka, A. Gheney, AOAC Annual meeting 2008.

22 Deconjugation with Rat Plasma Conjugase 37 deg C for 22 hr 100 deg C for 5 min Monoglutamates Polyglutamates (n = 1-8) T. Gbatu, J. Szpylka, A. Gheney, AOAC Annual meeting 2008.

23 Average µg/100g SD µg/100g RSD % Value µg/100g Infant formula Baby food Protein powder RTE cereal Yogurt drin Soymilk Onion Powder Blackeye peas Eggs Banana Spinach Chicken liver T. Gbatu, J. Szpylka, A. Gheney, AOAC Annual meeting 2008.

24 Biospecific methods Labelled (radioisotope or enzyme) and non-labelled (optical biosensor) Biospecific protein binding and immuno assay (may detect natural folates but not accurately quantified) Optical biosensor promissing; commercial kit for folic acid using an anti-folic acid monoclonal AB: RSD for Infant Formulas: 3.6% (8-250 µg) (Fonterra) AB currently changed for FBP to determine total folates

25 Regulatory information CODEX STAN (Revision 2007) for infant formula and formulas for special medical purposes intended for infants CODEX STAN for follow-up formula EU: 2006/141/EC on infant formulae and follow-on formulae EU: 1999/21/EC on dietary foods for special medical purposes In addition regulations for Australia/New Zealand (FSANZ: Standard Infant Formula Products), US (FD&C Act, chapter IV, section 412), China (GB Infant Formula and Follow-on Formula) were consulted

26 Compositional criteria are set for specific populations and product categories Infant formulae and follow-up formulae for infants (0-6 months) and young children (6-12 months) respectively. Foods for Special Medical Purposes (FSMP) for infants (<12 months) and adults.

27 Infant formulae and follow-up formulae Origine Product Target pop Min Max Unit Codex Infant formula 0-6 months (GUL) µg/100 kcal Follow-up formula 6-12 months 4 NS µg/100 kcal EU Infant formula 0-6 months µg/100 kcal Follow-on formula 6-12 months µg/100 kcal Powdered formulae: ± 100 kcal/20 g µg/100g Liquid formulae: ± 70 kcal/100 ml µg/100 ml

28 Foods for Special Medical Purposes (FSMP) Origine Product Target pop Min Max Unit Codex FSMP <1y (GUL) µg/100 kcal EU FSMP <1y 4 25 µg/100 kcal >1y µg/100 kcal FSMP as sole nutrition adults: kcal/day; products 1-2 kcal/ml µg/100ml In US for FSMP at least DRI has to be provided

29 Further requirements in regulations. Advisory list of nutrient compounds for use in foods for special dietary uses intended for infants and young children (Codex, CAC GL (revised 2008)) - N-Pteroyl-L-glutamic acid - calcium-l-methyl-folate (Formulas for Special Medical Purposes) Essential composition expressed for formulae when reconstituted as instructed. (2006/141/EC; Codex STAN )

30 Further requirements in regulations. When establishing minimum and maximum amounts, both naturally occuring in ingredients and added nutrients are taken into account (Codex STAN ) However, minimum and maximum levels in regulations are expressed as folic acid rather than folic acid/folates

31 Tolerances Country Double-sided tolerance One-sided tolerance No tolerance Belgium Italy Germany Austria France UK Denmark Finland Ireland USA Thailand Singapore Philippines Nigeria Nepal Korea Indonesia India Egypt Canada Chili UAE Saudi Aribia Africa except Egypt Codex /80-130/150/200 70/80-130/50 a a /120 n 70/80-200/ / A /80 N x x x x

32 C A method is said to be fit-for-purpose if C m > 1.6 (two-sided specifications) USL LSL 6 SD USL LSL SD( ir 6 k m ) method LSL Specification spread (USL-LSL) USL SD method is the method variability SD(iR) is the standard deviation of intermediate reproducibility of the method k is the number of replicates defined in the method protocol for routine analyses (generally 1 or 2) Method spread (6*SD method )

33 Example C m = x SD(iR) /1 (K =1) SD(iR) = (120-90)/(1.6 * 6) = 3%

34 Conclusion MBA still seen as the reference method Due to disadvantages, look out for other technique LC methods in unfortified foods tend to give 20-30% lower values compared to MBA For testing compliance in fortified foods, UPLC/MSMS for at least folic acid and 5MTHF seems option for future reference method