Case Study:Folate Bioavailability

Jess GregoryFood Science & Human Nutrition Dept.

University of Florida

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.

5 or 10

Substituent (R) (methyl

)(formyl) (formimino)(methylene)(methenyl)

5

55 and 10

5 and 10

Position

Polyglutamyl Tetrahydrofolates

Folic (Pteroyl-L-Glutamic) Acid

OHCH2N C

OCH

COOHCH2 CH2 C

N

NN

N

H2N

NH O

NH

n

CCOOH

HCH2 CH2 COOH

HHH

H

5

10

CH3CHOCH=NHCH2CH

R

OHCH2 N C

OCH

COOHCH2 CH2 COOH

HN

NN

N

H2N

NH

(plasma?)

Pancreatic Juice

PG1

Dietary Folate

PGn

UrineFeces

Pool A Pool 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 MethodsRat 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.

Short-Term Kinetic Approaches

Pharmacokinetic Study of Levoleucovorin (15 mg doses)

DeVito et al., Clin. Pharmacy 1993

0

100

200

300

0 10 20 30

Time (h)

Plas

ma

5-M

ethy

l-TH

F (n

g/m

L ab

ove

basa

l)

oral

iv

AUC Study of Spinach Folate

Prinz-Langenohl et al., J. Nutr. 1999

0102030405060

0 5 10 15

600g Spinach

300g Spinach

0.4 mg FA

folate-free

Time, h

Plas

ma

Fola

te, n

mol

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

Isotopic Methods

Subject 12: 454 nmol/d

Time, d0 10 20 30 40 50 60

Enric

hmen

t, 2 H 2/(2 H 2 +

1 H)

0.0

0.1

0.2

0.3

0.4

0.5

Urinary Folate

Urinary ApABG

Subject 12: 454 nmol/d

Time, d0 10 20 30 40 50 60

Enric

hmen

t, 2 H 2/(2 H 2 +

1 H)

0.0

0.1

0.2

0.3

0.4

0.5

Urinary Folate

Urinary ApABG

Subject 12: 454 nmol/d

Time, d0 10 20 30 40 50 60

Enric

hmen

t, 2 H 2/(2 H 2 +

1 H)

0.0

0.1

0.2

0.3

0.4

0.5

Urinary Folate

Urinary ApABG

Subject 12: 454 nmol/d

Time, d0 10 20 30 40 50 60

Enric

hmen

t, 2 H 2/(2 H 2 +

1 H)

0.0

0.1

0.2

0.3

0.4

0.5

Urinary Folate

Urinary ApABG

Subject 12: 454 nmol/d

Time, d0 10 20 30 40 50 60

Enric

hmen

t, 2 H 2/(2 H 2 +

1 H)

0.0

0.1

0.2

0.3

0.4

0.5

Subject 12: 454 nmol/d

Time, d0 10 20 30 40 50 60

Enric

hmen

t, 2 H 2/(2 H 2 +

1 H)

0.0

0.1

0.2

0.3

0.4

0.5

O HC H2 N C

OCCO O HH

N

NN

N

H 2N

NH

H H

H

C D2 C D 2 CO O H

H

HH

2[Glu- H4]Folic Acid

O HCH 2 N C

OCH

CO O HHN

NN

N

H2N

NH

H H

HD

D

CH 2 C H 2 C O O H

[3',5'-2H2]Folic Acid

O HC H 2 N C

O

H

O O HHN

NN

N

H 2N

NH

H H

H

H2 H 2 O O H

H

HCC C C C

13[Glu- C5]Folic Acid

Time (h)0 2 4 6 8 10

Mol

ar R

atio

of P

lasm

a Fo

late

s

0

1

2

3

4

5

6

7

22H

13 C5

Rogers et al., J. Nutr. 1997

Short-Term Plasma Kinetics of Oral and IV Doses

(100 µg iv [2H2]FA)

(400 µg oral [13C5]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 1990Wei and Gregory, J. Agric. Food Chem. 1998

Bioavailability of [2H4]Mono and [2H2]Polyglutamyl Folate Tracers

Added to Selected Foods

Materials Tested: Orange juiceTomatoesLima beansCitric 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 OJTomatoes

Lima BeansCitra

teControl 2

Rat

io o

f urin

ary

d2/d

4-fo

late

s(%

of d

2-do

se)/(

% o

f 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 [13C5]FA (400 µg) in apple juice ± light breakfast; iv [2H2]FA (100 µg) bolus injection.

Collected urine 24 h. HPLC and GCMS analysis.

OH

CH2 N CO

CHCOOHH

NNN

NH2N

NH

H H

HD

DCH2 CH2 COOH

[3',5'-2H2]Folic Acid

OHCH2 N C

OH

OOHHNNN

NH2N

NH

H H

HH2 H2 OOH

H

H CC C C C

13[Glu- C5]Folic Acid

Bioavailability of Oral Folic Acid Effect of Food

Doses: oral [13C5]folic acid (400 µg) iv [2H2]folic acid (100 µg)

3

2

1.5

1

0.7

Without Food

WithFood

}~15%

Urin

ary

Fola

te E

xcre

tion

Rat

io(%

13C

5 dos

e / %

2 H2 d

ose)

Pfeiffer et al., AJCN, 1997

P = 0.085, n = 14.Means ± 95% CI.

Bioavailability of [13C5]Folic Acid Used in Cereal Food

FortificationSelected 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

Bioavailability of [13C5]Folic Acid in Fortified Cereal Grain Foods

0

0.5

1

1.5

2

2.5

3

Control White bread Wheat bread White rice Pasta

Excr

etio

n ra

tio(%

13C

dos

e / %

2 H d

ose)

Pooled SE = 0.33No sig. difference, P>0.05

Vehicle for oral [13C]folic acid

Simultaneous IV reference dose = [2H2]folic acid

Pfeiffer et al., AJCN, 1997No 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 [13C5]FA and [2H2]PteGlu5.

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/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.

Zinc Intake Does Not Significantly Affect Folate Bioavailability

Kauwell et al., AJCN 1995

Dietary Folate Equivalents: Application of Folate Bioavailabilityµg 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 NeedsAvailability 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.