Case%20Study:

1

• Case Study
• Folate Bioavailability

Jess Gregory Food Science Human Nutrition
Dept. University of Florida
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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.

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O
H
O
C
O
O
H
H
H
C
H
N
C
C
C
H
C
H
C
O
O
H
N
N
2
2
2
N
H
Folic (Pteroyl-L-Glutamic) Acid
N
N
H
N
2
Polyglutamyl Tetrahydrofolates
O
C
O
O
H
H
O
H
C
O
O
H
N
C
C
C
H
C
H
C
N
N
C
C
H
C
H
C
O
O
H
R
O
H
2
2
2
2
1
0
H
H
C
H
N
2
n
N
5
H
Position
Substituent (R)
H
N
N
H
N
2
H
(methyl)
5
H
C
H
3
(formyl)

• 5 or 10

C
H
O
(formimino)
C
H

N
H
5
(methylene)
5 and 10
C
H
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

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

• (plasma?)

1
U
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Fe
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f
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c
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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 (gt 5-10 µM),
  absorption by passive diffusion predominates.

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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.

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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).

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• 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.

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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 (lt 300 µg).

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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.

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Short-Term Kinetic Approaches
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Pharmacokinetic Study of Levoleucovorin (15 mg
doses)
DeVito et al., Clin. Pharmacy 1993
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AUC Study of Spinach Folate
60
50
600g Spinach
40
300g Spinach
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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
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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.

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Isotopic Methods

• Intrinsic/extrinsic labeling questions. Added
  tracers probably dont 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. 14CFolates may be
  applicable in some human studies (accelerator MS
  Clifford et al. 1999).

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(No Transcript)
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Short-Term Plasma Kinetics of Oral and IV Doses
7
6
5
4
Molar Ratio of Plasma Folates
3
(100 µg iv 2H2FA)
2
2
(400 µg oral 13C5FA)
1
0
0
2
4
6
8
10
Time (h)
Rogers et al., J. Nutr. 1997
Mean SEM, n4.
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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.

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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
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Bioavailability of 2H4Mono and
2H2Polyglutamyl Folate Tracers Added to
Selected Foods
Materials Tested Orange juice Tomatoes Li
ma beans Citric acid (oj equivalent)
Wei et al., J. Nutr. 1996
(Prior saturation of subjects)
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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.

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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
Tomatoes
means SD
Control 1
Lima Beans
Wei et al., J. Nutr. 1996
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Conclusions Conjugase Inhibition and In Vitro
Screening to Predict Food Folate Bioavailability

• Hypothesis rejected. Its 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.

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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).

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Bioavailability of Supplemental Folic Acid
Consumed with Food

• Subjects given 13C5FA (400 µg) in apple juice
  light breakfast iv 2H2FA (100 µg) bolus
  injection.
• Collected urine 24 h. HPLC and GCMS analysis.

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Bioavailability of Oral Folic Acid Effect of Food
3
2

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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 13C5folic acid (400 µg)
iv 2H2folic acid (100 µg)
Pfeiffer et al., AJCN, 1997
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Bioavailability of 13C5Folic 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 2H2folic
  acid.
• Analysis of 48-h urinary folate excretion.

Pfeiffer et al., AJCN 1997
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Bioavailability of 13C5Folic Acid in Fortified
Cereal Grain Foods
Simultaneous IV reference dose 2H2folic acid
3
Excretion ratio (13C dose / 2H dose)
Pooled SE 0.33 No sig. difference, Pgt0.05
2.5
2
1.5
1
0.5
0
Control
White bread
Wheat bread
White rice
Pasta
Vehicle for oral 13Cfolic acid
Pfeiffer et al., AJCN, 1997
No saturation of subjects.
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Folate Bioavailability and NTD Risk

• Previously suggested that women at higher risk of
  NTD may malabsorb food folate.
• We tested whether cases and controls (n11 each)
  handled mono and polyglutamyl folates
  differently.
• Folate saturated women given single combined dose
  of 13C5FA and 2H2PteGlu5.
• Measured excretion of both forms of labeled
  folate in collected urine.

Boddie et al., presented at EB99
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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, n6 no sig. differences.
Kauwell et al., AJCN 1995
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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
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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.

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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.