Case Study: Folate Bioavailability

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

2. Folate BioavailabilityOutline: • 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. O H C O O H O H H C N C C H C H C O O H N C H N 2 2 2 N H Folic (Pteroyl-L-Glutamic) Acid H N N N 2 5 or 10 Polyglutamyl Tetrahydrofolates O H C O O H O H C O O H C N C C H C H C N C C H C H C O O H R N O H 2 2 2 1 0 2 H H N C H 2 n N 5 H Position Substituent (R) H H N N N H 2 C H (methyl) 5 H 3 (formyl) C H O (formimino) C H = N H 5 (methylene) C H 5 and 10 2 C H (methenyl) 5 and 10

4. D i e t a r y F o l a t e Pa n c r e a t i c (plasma?) J u i c e P G n Bile Po o l A Po o l B ( t i s s u e s ) P G 1 U r i n e Fe c e s ( f o l a t e s & c a t a b o l i t e s )

5. Folate Absorption • 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.

6. Factors Affecting Bioavailability -Form of Folate: • 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.

7. Bioavailability of Folate in Food • 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).

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. 1999.

11. Short-Term Kinetic Approaches

12. 300 200 oral Plasma 5-Methyl-THF (ng/mL above basal) iv 100 0 0 10 20 30 Time (h) Pharmacokinetic Study of Levoleucovorin (15 mg doses) DeVito et al., Clin. Pharmacy 1993

13. AUC Study of Spinach Folate 60 50 600g Spinach 40 300g Spinach 30 Plasma Folate, nmol/L 0.4 mg FA 20 folate-free 10 0 0 5 10 15 Time, h Prinz-Langenohl et al., J. Nutr. 1999

14. Isotopic Methods • 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.

15. Isotopic Methods • 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. [14C]Folates may be applicable in some human studies (accelerator MS – Clifford et al. 1999).

16. H H O H C O O H O H H C N C C D C D C O O H N C H N 2 2 2 N H H H H N N N H 2 2 [ G l u - H ] F o l i c Ac i d 4 D H O H C O O H O H H C N C C H C H C O O H N C H N 2 2 2 N H H D H N N N H 2 2 i [ 3 ' , 5 ' - H ] F o l i c Ac d 2 H H O H C O O H O H H C N C C H C H C O O H N C H N 2 2 2 N H H H H N N N H 2 1 3 [ G l u - C ] F o l i c Ac i d 5

17. 2 H 13 C 5 Short-Term Plasma Kinetics of Oral and IV Doses 7 6 5 4 Molar Ratio of Plasma Folates (100 µg iv [2H2]FA) 3 2 2 (400 µg oral [13C5]FA) 1 0 0 2 4 6 8 10 Time (h) Rogers et al., J. Nutr. 1997 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 [2H4]Mono and [2H2]Polyglutamyl Folate Tracers Added to Selected Foods Materials Tested: Orange juice Tomatoes Lima beans Citric acid (oj equivalent) Wei et al., J. Nutr. 1996 (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. Relative Bioavailability of Mono- & Polyglutamyl Folates: Food Effects Urinary d2/d4 Folate Excretion Ratio 1.4 1.2 a a 1.0 a a a 0.8 (% of d2-dose)/(% of d4-dose) Ratio of urinary d2/d4-folates b 0.6 0.4 0.2 0.0 OJ Citrate Control 2 means ± SD Tomatoes Control 1 Lima Beans Wei et al., J. Nutr. 1996

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. 1998 • 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. D H O H C O O H O H H C N C C H C H C O O H N C H N 2 2 2 N H D H H N N N H 2 2 [ 3 ' , 5 ' - H ] F o l i c Ac i d 2 H H O H C O O H O H H N C H N C N C C H C H C O O H 2 2 2 N H H H H N N N H 2 3 1 [ G l u - C ] F o l i c Ac i d 5 Bioavailability of Supplemental Folic Acid Consumed with Food • Subjects given [13C5]FA (400 µg) in apple juice ± light breakfast; iv [2H2]FA (100 µg) bolus injection. • Collected urine 24 h. HPLC and GCMS analysis.

26. Bioavailability of Oral Folic Acid Effect of Food 3 2 } ~15% 1.5 Urinary Folate Excretion Ratio (% 13C5 dose / % 2H2 dose) 1 0.7 Without Food With Food P = 0.085, n = 14. Means ± 95% CI. Doses: oral [13C5]folic acid (400 µg) iv [2H2]folic acid (100 µg) Pfeiffer et al., AJCN, 1997

27. Bioavailability of [13C5]Folic Acid Used in Cereal Food Fortification • Selected foods experimentally fortified with 13C-labeled folic acid at FDA fortification level. • Single serving given to subjects to provide 50-100 µg dose. • Simultaneous IV reference dose of [2H2]folic acid. • Analysis of 48-h urinary folate excretion. Pfeiffer et al., AJCN 1997

28. Bioavailability of [13C5]Folic Acid in Fortified Cereal Grain Foods Simultaneous IV reference dose = [2H2]folic acid 3 Excretion ratio (%13C dose / % 2H dose) Pooled SE = 0.33 No sig. difference, P>0.05 2.5 2 1.5 1 0.5 0 Control White bread Wheat bread White rice Pasta Vehicle for oral [13C]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 [13C5]FA and [2H2]PteGlu5. • Measured excretion of both forms of labeled folate in collected urine. Boddie et al., presented at EB99

30. Zinc Intake Does Not Significantly Affect Folate Bioavailability Urinary d2-Folate Excretion 3.5 mg Zn/d 14.5 mg Zn/d % of total folate intake 26.7 ± 7.3 23.8 ± 9.8 % of total urinary folate 47.0 ± 5.1 46.6 ± 6.5 % of oral d2-FA dose 36.3 ± 10.0 32.4 ± 13.3 Mean ± SD, n=6; no sig. differences. 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. Summary: Folate Bioavailability • 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.

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.