Differences in Serum Folate Among Ethnic Groups

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Differences in Serum Folate Among Ethnic Groups

• By
• Stacey Renna

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Presentation Objectives

• Introduction
• Statement of the Problem
• Hypotheses
• Limitations/Delimitations
• Definition of Terms
• Literature Review
• Methodology and Procedures
• Results and Discussion

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Introduction

• Folate is a water-soluble vitamin
• Involved in accepting and donating one carbon
  units in amino acid metabolism and synthetic
  reactions
• Synthesis of certain amino acids
• Coenzyme Homocysteine to Methionine
• Methionine methyl donor for DNA synthesis
• Neural tube defects and cancer (low folate
  intake)
• Low folate results in increased homocysteine
• ? homocysteine has been linked to CVD and NTD
• Very few studies have explored differences in
  folate metabolism among ethnic groups

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Folate and HCY Metabolism
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Introduction

• This study will investigate serum folate
  concentrations of premenopausal women from three
  ethnic groups.
• Mexican American (n 12)
• African American (n 12)
• Caucasian (n 12)
• All subjects will be randomly assigned to receive
  a controlled folate intake of 400 ?g or 800 ?g
  Dietary Folate Equivalents per day

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Statement of the Problem

• To determine if there are differences in serum
  folate status response among Mexican American,
  African American, and Caucasian women.
• To compare serum folate concentrations of
  subjects from each ethnic/racial group after
  intakes of 400 ug DFE/day and 800 ug DFE/day.
• To determine whether the 1998 RDA for folate (400
  ug DFE/day) is sufficient for these racial/ethnic
  groups to maintain adequate folate status.

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Hypotheses

• Hypothesis 1
• There will be differences in serum folate
  concentrations in women (18-45 years) of
  different ethnic groups (Mexican American,
  African American, and Caucasian) after receiving
  the current RDA for folate (400ug DFE) or twice
  that level (800 ug DFE).
• Hypothesis 2
• The current RDA for folate of 400 ug DFE/d is
  not sufficient for all women of different ethnic
  groups to maintain adequate folate status.

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Limitations

• The results may not be representative of the
  entire population due to the small sample size
  and the limited geographic location.
• The results of the study may be affected by the
  personal lifestyle of the participating subjects.

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Delimitations

• Female
• Mexican American, African American, or Caucasian
• CC MTHFR Genotype
• 18-45 years
• Non-smoker
• No chronic substance or alcohol abuse

• Non anemic
• Healthy (no chronic disease)
• Non supplement user
• Non-pregnant
• Non-lactating
• Normal Blood Chemistry

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Definition of Terms

• Folate Folate exists in multiple forms with a
  pteroyglutamic structure in common. The main
  form of folate in the blood is 5-methyl-THF,
  which acts as a methyl donor in the synthesis of
  DNA, thymidylate and purines, as well as certain
  neurotransmitters, phospholipids, and hormones
• Folic Acid synthetic folate found in
  supplements.

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Definition of Terms

• Serum Folate folate found in the blood plasma.
  Normal range is gt6.8 nmol/L.
• Homocysteine a sulfur containing amino acid
  that is formed in the body during the synthesis
  of methionine. The homocysteine pathway is
  folate and vitamin B12 dependent. These
  coenzymes are required for the methylation of
  homocysteine to methionine.

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Definition of Terms

• Methyltetrahydrofolate Reductase (MTHFR)
• an FAD-dependent enzyme that catalyzes the
  conversion of 5,10-methylenetetrahydrofolate to
  5-methyl-THF
• Neural Tube Defects (NTD)
• A wide range of congenital malformations that
  result in a disturbance of neurulation. The
  neural tube marks the early formation of the CNS.
  Upper defects include anencephaly and cervical
  and thoracic spina bifida. Lower defects refer
  to lumbar and sacral spina bifida

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Definition of Terms

• Recommended Dietary Allowances (RDA) the levels
  of intake of essential nutrients, on the basis of
  scientific knowledge, that are judged by the Food
  and Nutrition Board to be adequate to meet the
  known nutrient needs of practically all healthy
  persons.
• Dietary Folate Equivalents (DFE) single value
  used to indicate folate intake from both dietary
  and supplemental sources. Because folic acid
  intake taken with food is 85 bioavailable but
  food folate is only 50 bioavailable, DFEs are
  used.
• 1 ug DFEs 1 ug of food folate 0.5 ug of folic
  acid taken on an empty stomach 0.6 ug of folic
  acid with meals.

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Literature Review
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Folate and Heart Disease

• There is an inverse relationship between folate
  and homocysteine
• Heart disease has been linked to ? homocysteine
  and ? serum and RBC folate.
• Increased folic acid intake reduces homocysteine
  concentrations

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Folate and Cancer

• Sub optimal folate levels have been linked to
  certain forms of cancer colon, cervical, breast
• Giovannucci et al. 1998 (colon cancer)
• found that women who took multivitamins
  containing folic acid for fourteen years or less
  had a higher risk of developing colon cancer
  compared to women who took the supplements for
  more than fifteen years. It was also found that
  folate obtained from dietary sources was related
  to a modest reduction in colon cancer.

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Folate and Cancer

• Folate and Cervical Cancer
• Many studies mixed results
• One study found improvement in cervical dysplasia
  with folate supplementation (Butterworth et al.
  1992)
• More recently (Weinsteine et al. 2001)
• concluded that low serum and RBC folate were
  moderately, but not significantly, associated
  with invasive cervical cancer risk.

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Folate and Cancer

• Folate and Breast Cancer
• Zhang et al. 1999
• found that folate intake was not associated with
  overall risk of breast cancer.
• found that women who consumed at least 15 gm/day
  of alcohol and had low folate intake were at a
  higher risk of obtaining breast cancer.

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Folate and Neural Tube Defects

• Czeizel Dudas 1992, Laurence et al. 1981,
  Smithells et al. 1983, Kirke et al. 1992, Werler
  et al. 1993
• periconceptional administration of multivitamins
  (containing folic acid) or folic acid alone
  prevents the occurrence and recurrence of neural
  tube defects

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Folate and Neural Tube Defects

• Kirke et al. 1993, Molloy et al. 1998
• Low plasma folate levels are independent risk
  factors for neural tube defects
• Vanderput et al. 1997, Mills et al. 1995,
  Steeger-Theunissen et al. 1994
• homocysteine levels are significantly higher in
  women giving birth to babies with NTD compared to
  controls

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Folate and Neural Tube Defects

• Fleming 2001
• The mechanism by which neural tube defects can be
  prevented by folate supplementation is still
  unclear
• Two proposed mechanisms
• Scott et al. 1994, Mills et al. 1996
• Correct a deficiency in maternal folate levels
• Overcome an intrinsic abnormality of folate
  metabolism

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Changes in the RDA for Folate

• 1989
• RDA adult women 180 ?g DFE/d
• Pregnant women 400 ?g DFE/d
• 1992
• U.S. Public Health Service recommended that women
  of childbearing age consume 400 ug of folic acid
  supplement to reduce the risk of NTDs

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Changes in the RDA for Folate

• 1996
• FDA conducted an investigation of folate and NTDs
• 1998
• The FDA mandated the addition of folic acid to
  enriched cereal-grain products in an effort to
  help women of childbearing age reduce the risk of
  having a neural tube defect-affected offspring
  (FDA 1996).
• Current RDA for women 400 ?g DFE/d
• Pregnant women 600 ?g DFE/d

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Ethnic Differences in Folate Metabolism

• Limited research
• Studies relating folate to ethnicity were not
  conducted under controlled conditions
• Fanelli-Kuczmarski 1990
• 11.9 of Mexican American, 10.1 of Cuban women,
  and 8.1 of Puerto Rican women had low serum
  folate
• education and poverty level did not significantly
  affect folate status

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Ethnic Differences in Folate Metabolism

• NHANES III
• Sub optimal folate levels are more common among
  African Americans and Mexican Americans compared
  to Caucasians
• Ford and Bowman in 1999
• White men had ? serum and red blood cell folate
  concentrations than did A A men and MA men
• White women had ? serum and red blood cell folate
  concentrations than did A A women and MA women
• no linear trends between folate concentrations
  and education

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Folate and the MTHFR Genotype

• Frost et al. 1995
• common polymorphism (C677T) in the MTHFR gene
• Brattstrom et al. 1998
• homozygous (T/T) for the MTHFR genotype have
  reduced MTHFR activity
• ? plasma homocysteine and ? folate
• Jaques et al. 1996, Rosenberg and Rosenberg 1998
• T/T genotype may require more folate

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Serum Folate as an Assessment of Folate Status

• Herbert 1990
• Serum folate concentration represents folate
  status influenced by recent dietary intake
• Serum folate levels drop as the need for folate
  increases
• Inadequate folate will cause serum folate
  concentrations to decline

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Methodology and Procedures
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Subjects

• Same criteria described in delimitations
• Staff and student from Cal Poly and Mt. SAC
• Posting of flyers on campus at off-campus housing
• Phone screenings
• Blood screening for CC genotype at Cal Poly
  Pomona Student Health Center
• Informed consent
• Incentives money (1400), saved time and money,
  foodservice, appealing menu.

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Diet and Supplements

• Controlled low folate diet for first 7 weeks
• 5 day cycle menu, 140 ?g DFE/d
• Some items boiled 3 times
• Food portioned out to nearest gram
• Low folate diet folic acid supplement (final 7
  weeks)
• Total folate 400 ?g or 800 ?g

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Diet and Supplements

• RDA for all nutrients in diet excluding folate
• Vitamins lost through boiling will be provided
• Multi-mineral
• Folate free multivitamin
• Vitamin K
• Choline
• Additional calories can be obtained through
  jello, cool whip, and sodas

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Metabolic Kitchen

• Meals will be prepared by Cal Poly students
• Food Safety and Sanitation Standards
• Subjects eat breakfast and dinner in kitchen
• Lunches are To Go
• 14 additional To Go meals will be allowed
• No more than 3 consecutive days

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Blood Draws and Processing

• Fasting Blood sample will be taken each week
• Certified phlebotomist
• 13 ml Tiger Top SST tube (serum folate analysis)
• 5 ml EDTA tube (hematocrit analysis)
• Within one hour of collection, the blood will be
  transported to the Cal Poly Pomona Foods and
  Nutrition Laboratory where it will be processed
  immediately. Serum will be stored at -20?C until
  testing.

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Microbiological Assay

• Lactobacillus casei
• Organism grows on folate
• Add serum folate and determine growth
• The more turbidity the more folate

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Statistical Methods

• Determination of Sample Size
• Determined from data from a similar study
  (OKeefe et al. 1992)
• Effect Size Power Subject
• OKeefe Study 0.57 0.80 6
• My calculation 0 .80 0.80 12
• Testing of Serum Folate
• Multivariate analysis (MANOVA)
• Comparisons will be made between initial, final,
  and overall means.

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Statistical Methods
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Expected Results

• There will be significant differences in serum
  folate concentrations among the three ethnic
  groups.
• Mexican American and African American women will
  have lower serum folate levels compared to
  Caucasian women.
• The current RDA for folate is not adequate to
  meet the needs of all women of different ethnic
  groups.

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Actual Results

• There was no significant difference in serum
  folate concentrations in women (18-45 years) of
  different ethnic groups (Mexican American,
  African American, and Caucasian) after receiving
  the current RDA for folate (400ug DFE) or twice
  that level (800 ug DFE).
• The current RDA for folate of 400 ug DFE/d is
  sufficient to meet the requirements of all women
  of different ethnic groups.
• Reject both hypotheses

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Discussion of Results
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Discussion of Results

• The obtained results may actually have been
  false, and a Type II error may have been
  committed.
• The hypotheses may have been rejected when they
  were actually true.
• Due to threats to the internal and external
  validity

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Discussion of Results Threats to Internal
Validity

• History
• Unplanned events could have significantly
  effected the results.
• The 14 meals To Go meals in addition to
  lunches, provided 14 possibilities of error in
  preparation for each subject.
• 14 unmonitored possibilities for each subject to
  not completely finish their meals
• Due to the personal and social lives of the
  subjects, the participants may have acted upon
  temptations to cheat and consume food and
  beverages outside of the study

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Discussion of Results Threats to Internal
Validity

• History Continued
• The company that manufactured the pita bread went
  bankrupt.
• . It took several weeks to find another item of
  similar folate content. During that time, extra
  low folate calories were provided with additional
  popcorn, rice cakes, and granola bars.

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Discussion of Results Threats to Internal
Validity

• Instrumentation and Testing Procedures
• Test tubes kept breaking inside the centrifuge
  and blood was lost.
• A new centrifuge had to be ordered, but the past
  model was discontinued. For the second half of
  the study, a completely different model of
  centrifuge was used for blood processing
  procedures.

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Discussion of Results Threats to Internal
Validity

• Instrumentation and Testing Procedures
• The phlebotomist was unable to extract enough
  blood from some of the subjects.
• The pipettes were not properly calibrated when
  the folic acid supplementation was produced.
• Thus, the subjects were not receiving the correct
  amount of folic acid in addition to their dietary
  folate to accurately make either 400 ug DFE or
  800 ug DFE.

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Discussion of Results Threats to Internal
Validity

• Instrumentation and Testing Procedures
• Laboratory technicians involved in the blood
  processing procedures changed weekly.
• There may have been discrepancy in the blood
  processing from week to week.
• Experimental Mortality
• Experimental mortality did not affect the
  internal validity of the experiment. If a
  subject decided to drop out, a replacement was
  found.

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Discussion of Results Threats to External
Validity

• Laboratory Based Research Project
• It is not representative of the entire population
  
• The sample size was much too small to apply it to
  a larger population.
• Sample was taken from one geographical location.

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Any Questions????