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Case Study: Folate Bioavailability Jess Gregory Food Science & Human Nutrition Dept. University of Florida.

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Presentation on theme: "Case Study: Folate Bioavailability Jess Gregory Food Science & Human Nutrition Dept. University of Florida."— Presentation transcript:

1 Case Study: Folate Bioavailability Jess Gregory Food Science & Human Nutrition Dept. University of Florida

2 Folate Bioavailability Outline:  Overview of folate absorption and physiology; past understanding of folate bioavailability.  Approaches to assessment of bioavailability (focus on recent methods & human relevance).  Recent advances in views of folate bioavailability.  Research needs.

3 5 or 10 Substituent (R) (methyl) (formyl) (formimino) (methylene) (methenyl) and 10 Position Polyglutamyl Tetrahydrofolates Folic (Pteroyl-L-Glutamic) Acid OH CH 2 N C O C H COOH CH 2 CH 2 C N NN N H 2 N N HO N H n C COOH H CH 2 CH 2 COOH H H H H 5 10 CH 3 CHO CH=NH CH 2 CH R OH CH 2 N C O C H COOH CH 2 CH 2 COOH H N NN N H 2 N N H

4 (plasma?) Pancreatic Juice PG 1 Dietary Folate PG n Urine Feces Pool A ol B (tissues) Bile (folates & catabolites)

5  Deconjugation by jejunal brush border pteroylpolyglutamate hydrolase, some additional role of pancreatic PPH.  Most absorption of food folate by saturable transport (Km ~1-2 µM)  At high intraluminal [folate] (> 5-10 µM), absorption by passive diffusion predominates. Folate Absorption

6  Variation among forms of monoglutamyl folate reported in humans, not in rats.  Polyglutamyl folates often exhibit ~75% bioavailability relative to mono. (range ~50-100%)  Variables: intestinal deconjugation, intestinal stability, transport, tissue uptake and retention, catabolism enterohepatic circulation, and renal excretion. Factors Affecting Bioavailability - Form of Folate:

7  Wide variation reported among foods for humans and rats (Tamura & Stokstad 1973; Clifford et al. 1990,'91).  Inconsistent reports for some foods.  Total folate in mixed diet, relative to formula diet, exhibited no more than 50% bioavailability (Sauberlich et al. 1987).  Increased dietary folate from foods had little impact on folate status of women (Cuskelly et al. 1996). Bioavailability of Folate in Food

8 Factors Affecting Bioavailability: Drug Effects  Antacids may impair folate absorption.  Many nonsteroidal anti-inflammatory drugs are antifolates.  Chronic alcohol abuse impairs folate deconjugation and absorption. It may also enhance catabolism.

9 Bioavailability Assessment Methods  Rat and chick bioassays.  Relevance to humans not established.  Short-term human studies.  Single dose of test material (food or supplement) compared to equivalent reference dose.  Based on area under curve of plasma folate response. Not suitable for assessment of low doses (< ~300 µg).

10 Bioavailability Assessment Methods  Long-term human studies.  Chronic administration of test and reference diets (~3-6 wk)  Measure plasma and RBC folate and/or plasma homocysteine.  Examples: Cuskelly et al. 1996, Brouwer et al

11 Short-Term Kinetic Approaches

12 Pharmacokinetic Study of Levoleucovorin (15 mg doses) DeVito et al., Clin. Pharmacy Time (h) Plasma 5-Methyl-THF (ng/mL above basal) oral iv

13 AUC Study of Spinach Folate Prinz-Langenohl et al., J. Nutr g Spinach 300g Spinach 0.4 mg FA folate-free Time, h Plasma Folate, nmol/L

14  Major advantage is specificity.  Several approaches to stable isotope labeling of folates. Multiple labeled forms are available for use. GCMS analysis is well established.  Convenient stable isotope protocols in humans using urinary folate (24-48 h) or short-term plasma folate. Isotopic Methods

15  Intrinsic/extrinsic labeling questions. Added tracers probably don’t fully mix with endogenous folates in all types of foods.  However, well designed studies do allow investigation of many aspects of folate bioavailability.  Radiolabeled folates. Convenient to use and analyze in animal studies. [ 14 C]Folates may be applicable in some human studies (accelerator MS – Clifford et al. 1999). Isotopic Methods

16 OH CH 2 N C O C COOH H N NN N H 2 N N H H H H CD 2 CD 2 COOH H H H 2 [Glu-H 4 ]Folic Acid OH CH 2 N C O C H COOH H N NN N H 2 N N H H H HD D CH 2 CH 2 COOH [3',5'- 2 H 2 ]Folic i d OH CH 2 N C O H OOH H N NN N H 2 N N H H H H H 2 H 2 OOH H H C CCCC 13 [Glu-C 5 ]Folic id

17 Time (h) Molar Ratio of Plasma Folates H 13 C 5 Rogers et al., J. Nutr Short-Term Plasma Kinetics of Oral and IV Doses (100 µg iv [ 2 H 2 ]FA) (400 µg oral [ 13 C 5 ]FA) Mean ± SEM, n=4.

18 Can In Vitro Methods Predict Folate Bioavailability In Vivo???  We originally hypothesized that bioavailability is governed via inhibition of intestinal deconjugation of polyglutamyl folates by components of foods.  If true, then an in vitro screen should predict bioavailability of naturally food folate.

19 Many Foods Contain Inhibitors of Brush Border Conjugase (PPH)  Extracts of many foods inhibit PPH – many act as competitive inhibitors.  Major inhibitors are organic acids (citric, malic, ascorbic, etc.). Bhandari and Gregory, AJCN 1990 Wei and Gregory, J. Agric. Food Chem. 1998

20 Bioavailability of [ 2 H 4 ]Mono and [ 2 H 2 ]Polyglutamyl Folate Tracers Added to Selected Foods Materials Tested:Orange juice Tomatoes Lima beans Citric acid (oj equivalent) Wei et al., J. Nutr (Prior saturation of subjects)

21 Saturation Regimen (mod. from Tamura & Stokstad 1973)  10 mg/d folic acid first week.  2 mg/d folic acid for remainder of study (except on days of labeled folate administration).  This procedure gives relatively constant, yet highly elevated, folate status of subjects. Enhances and normalizes urinary excretion of tracers.

22 Urinary d2/d4 Folate Excretion Ratio Control 1 OJ Tomatoes Lima Beans Citrate Control 2 Ratio of urinary d2/d4-folates (% of d2-dose)/(% of d4-dose) a b a a a a Wei et al., J. Nutr Relative Bioavailability of Mono- & Polyglutamyl Folates: Food Effects means ± SD

23 Conclusions: Conjugase Inhibition and In Vitro Screening to Predict Food Folate Bioavailability  Hypothesis rejected. It’s not that simple.  This in vivo protocol works well.  The human digestive system has sufficient excess conjugase activity to overcome some much of the inhibition encountered.

24 A Promising In Vitro Method: Seyoum & Selhub, J. Nutr  Combined test of folate stability (ie simulating conditions of GI tract) and extent of PPH digestibility in vitro.  Moderate correlation of “bioavailability index” and in vivo results of Tamura and Stokstad (1973).

25 Bioavailability of Supplemental Folic Acid Consumed with Food  Subjects given [ 13 C 5 ]FA (400 µg) in apple juice ± light breakfast; iv [ 2 H 2 ]FA (100 µg) bolus injection.  Collected urine 24 h. HPLC and GCMS analysis. OH CH 2 N C O C H COOH H N NN N H 2 N N H H H HD D CH 2 CH 2 COOH [3',5'- 2 H 2 ]Folic Acid OH CH 2 NC O H OOH H N NN N H 2 N N H H H H H 2 H 2 OOH H H C CCCC 1 3 [Glu-C 5 ]Folic id

26 Bioavailability of Oral Folic Acid Effect of Food Doses: oral [ 13 C 5 ]folic acid (400 µg) iv [ 2 H 2 ]folic acid (100 µg) Without Food With Food } ~15% Urinary Folate Excretion Ratio (% 13 C 5 dose / % 2 H 2 dose) Pfeiffer et al., AJCN, 1997 P = 0.085, n = 14. Means ± 95% CI.

27 Bioavailability of [ 13 C 5 ]Folic Acid Used in Cereal Food Fortification  Selected foods experimentally fortified with 13 C-labeled folic acid at FDA fortification level.  Single serving given to subjects to provide µg dose.  Simultaneous IV reference dose of [ 2 H 2 ]folic acid.  Analysis of 48-h urinary folate excretion. Pfeiffer et al., AJCN 1997

28 Bioavailability of [ 13 C 5 ]Folic Acid in Fortified Cereal Grain Foods ControlWhite breadWheat breadWhite ricePasta Excretion ratio (% 13 C dose / % 2 H dose) Pooled SE = 0.33 No sig. difference, P>0.05 Vehicle for oral [ 13 C]folic acid Simultaneous IV reference dose = [ 2 H 2 ]folic acid Pfeiffer et al., AJCN, 1997 No saturation of subjects.

29 Folate Bioavailability and NTD Risk  Previously suggested that women at higher risk of NTD may malabsorb food folate.  We tested whether cases and controls (n=11 each) handled mono and polyglutamyl folates differently.  Folate saturated women given single combined dose of [ 13 C 5 ]FA and [ 2 H 2 ]PteGlu 5.  Measured excretion of both forms of labeled folate in collected urine. Boddie et al., presented at EB99

30 Urinary d2-Folate Excretion 3.5 mg Zn/d14.5 mg Zn/d % of total folate intake 26.7 ± ± 9.8 % of total urinary folate 47.0 ± ± 6.5 % of oral d2-FA dose 36.3 ± ± 13.3 Mean ± SD, n=6; no sig. differences. Zinc Intake Does Not Significantly Affect Folate Bioavailability Kauwell et al., AJCN 1995

31 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.  85% bioavailability of added folic acid.  Thus, synthetic is 85/50 times more available. Institute of Medicine, 1998

32  Bioavailability of dietary folate is incomplete.  Short-term trials in humans are feasible using non-labeled folate in some cases.  Stable isotope methods yield important information regarding folate bioavailability, but may not predict bioavailability of naturally occurring folates. Summary: Folate Bioavailability

33 Research Needs  Availability of naturally occurring folate in typical human diets and important food sources.  Better understanding of mechanisms involved.  Impact of various diets on folate status of populations.  Validated alternative techniques for assessment of bioavailability in whole diets and specific foods.  Impact and alternatives in fortification techniques.


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