Presentation on theme: "Case Study: Folate Bioavailability Jess Gregory Food Science & Human Nutrition Dept. University of Florida."— Presentation transcript:
Case Study: Folate Bioavailability Jess Gregory Food Science & Human Nutrition Dept. University of Florida
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.
5 or 10 Substituent (R) (methyl) (formyl) (formimino) (methylene) (methenyl) 5 5 5 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
(plasma?) Pancreatic Juice PG 1 Dietary Folate PG n Urine Feces Pool A ol B (tissues) Bile (folates & catabolites)
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
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:
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
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.
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).
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.
Pharmacokinetic Study of Levoleucovorin (15 mg doses) DeVito et al., Clin. Pharmacy 1993 0 100 200 300 0102030 Time (h) Plasma 5-Methyl-THF (ng/mL above basal) oral iv
AUC Study of Spinach Folate Prinz-Langenohl et al., J. Nutr. 1999 0 10 20 30 40 50 60 051015 600g Spinach 300g Spinach 0.4 mg FA folate-free Time, h Plasma Folate, nmol/L
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
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
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
Time (h) 0246810 Molar Ratio of Plasma Folates 0 1 2 3 4 5 6 7 2 2 H 13 C 5 Rogers et al., J. Nutr. 1997 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.
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.
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
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. 1996 (Prior saturation of subjects)
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.
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) 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 a b a a a a Wei et al., J. Nutr. 1996 Relative Bioavailability of Mono- & Polyglutamyl Folates: Food Effects means ± SD
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.
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).
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
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) 3 2 1.5 1 0.7 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.
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 50- 100 µg dose. Simultaneous IV reference dose of [ 2 H 2 ]folic acid. Analysis of 48-h urinary folate excretion. Pfeiffer et al., AJCN 1997
Bioavailability of [ 13 C 5 ]Folic Acid in Fortified Cereal Grain Foods 0 0.5 1 1.5 2 2.5 3 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.
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
Urinary d2-Folate Excretion 3.5 mg Zn/d14.5 mg Zn/d % of total folate intake 26.7 ± 7.323.8 ± 9.8 % of total urinary folate 47.0 ± 5.146.6 ± 6.5 % of oral d2-FA dose 36.3 ± 10.032.4 ± 13.3 Mean ± SD, n=6; no sig. differences. Zinc Intake Does Not Significantly Affect Folate Bioavailability Kauwell et al., AJCN 1995
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
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
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.