Chapter 12. Folic Acid

12 Folic Acid Lynn B Bailey CONTENTS Introduction 386 Chemistry 386 Structure and Nomenclature 386 Chemical Properties 387 Food Sources 387 Naturally Occurring Food Folate 387 Folic Acid in Fortified and Enriched Food Products 388 Effect of Folic Acid Fortification on Folate Intake and Status in the United States and Canada 388 Bioavailability 388 Methods 388 Food Folate 388 Biological Samples 389 Dietary Reference Intakes 389 Metabolism 390 Absorption and Transport 390 Intracellular Storage 391 Catabolism and Turnover 391 Excretion 392 Biochemical Functions 392 Overview of Biochemical Functions 392 Key Folate-Dependent Enzymatic Reactions Involved in One Carbon Metabolism 392 Serine–Glycine Interconversion 392 Formation of 5-Methyltetrahydrofolate 394 Homocysteine Remethylation 394 Formation of S-Adenosylmethionine 394 Conversion of S-Adenosylmethionine to S-Adenosylhomocysteine 395 Transsulfuration Pathway 395 Nucleotide Biosynthesis 396 Histidine Catabolism 396 Factors Affecting Folate Metabolism 396 Single Nucleotide Polymorphisms 396 Alcohol and Drugs 398 Deficiency 398 Biomarkers and Status Assessment 398 Serum and Red Blood Cell Folate Concentration 398 Homocysteine Concentration 399 ß 2006 by Taylor & Francis Group, LLC DNA Methylation 399 Hematological Indices 400 Abnormal Pregnancy Outcomes 400 Increased Risk of Birth Defects 400 Neural Tube Defects 400 Congenital Heart Defects 401 Increased Risk Chronic Disease 401 Vascular Disease 401 Cancer 402 Summary and Conclusions 403 References 403 INTRODUCTION The isolation, structure identification, and synthesis of folic acid, which took place in the 1940s, led to the widespread therapeutic use of this water-soluble vitamin for the treatment of megaloblastic anemia During the next 50 years, the basic aspects of folate metabolism and the biochemical functions were investigated and the key role of folate coenzymes in one carbon metabolism established Since the early 1990s, the links between folate intake and birth outcome or chronic disease risk were explored One of the most important public health discoveries of this century is that daily supplemental folic acid taken periconceptionally significantly reduces the risk of neural tube defects (NTDs) The conclusive evidence related to folic acid and NTD risk reduction led to the implementation of global public health policies including mandatory folic acid fortification in North America The identification of genetic polymorphisms that affect the structure–function of folate-related enzymes and proteins has served as the catalyst for the ongoing search for links between these polymorphisms and increased risk for birth defects or chronic disease This chapter highlights the current knowledge of folate nutrition including key aspects of the chemistry, food sources, intake recommendations, methods of analysis and status assessment, metabolism, biochemical functions, and genetic polymorphisms An overview of the association between folate status and health-related risks provides the foundation for new research efforts to address challenging new questions that have evolved from research efforts to date CHEMISTRY STRUCTURE AND NOMENCLATURE Folate consists of a family of compounds that differ in a variety of ways including the oxidation state of the molecule, the length of the glutamate side chain, and the specific one carbon units attached to the molecule The folate molecule, tetrahydrofolate (THF), is derived from 5,6,7,8-tetrahydropteroylglutamate, which consists of a 2-amino-4hydroxy-pteridine (pterin) moiety linked via a methylene group at the C-6 position to a p-aminobenzoylglutamic acid (pABG) (Figure 12.1) The pyrazine ring in THF is fully reduced at the 5,6,7, and positions and reduction at positions and only yields dihydrofolate The monoglutamate form of the vitamin contains one glutamic acid molecule, which can be converted to a glutamate chain by the addition of glutamate residues by g-peptide linkage In the majority of naturally occurring folates, the number of glutamate units in the side chain varies from to The fully oxidized monoglutamate form of the vitamin is referred to as folic acid and is the form used commercially in supplements and fortified foods In contrast to polyglutamyl folate, folic acid rarely occurs naturally in food Specific one ß 2006 by Taylor & Francis Group, LLC p-Aminobenzoic acid Pteridine H N H2N N O 23 HN Glutamic acid NH CH2 COOH O 10 C N H N H O CH CH2 CH2 C γ COOH HN Glutamic acid CH CH2 CH2 Folate coenzymes (Polyglutamate THF) One carbon units (N-5, N-10, or N-5 and N-10 positions) Methyl C OH O γ −Carboxy amide linked polypeptide chain Glutamic acid residue n = 2–11 CH3 Methylene – CH2 Methyenyl – CH Formyl – CH Formimino – CH NH FIGURE 12.1 Folic acid structure Folic acid consists of a pteridine ring linked to p-aminobenzoic acid joined at the other end to a molecule of glutamic acid Food folates exist in various forms, containing different numbers of additional glutamate residues joined to the first glutamate The folate or folic acid structure can vary by reduction of the pteridine moiety to form dihydrofolic acid and tetrahydrofolic acid (THF), elongation of the glutamate chain, and substitution of one carbon units to the polyglutamated form of the THF molecule carbon units that can be added at either or both of the N-5 or N-10 positions of the polyglutamyl form of the THF molecule include methyl (CH3), methylene (ÀÀCH2ÀÀ), methenyl (ÀÀCH¼ ¼), formyl (ÀÀCH¼ ¼), or formimino (ÀÀCH¼ ¼NH) groups CHEMICAL PROPERTIES The molecular weight of folic acid is 441.4, and although it is described as ‘‘water soluble,’’ the acid form is only slightly soluble in water in contrast to the salt form, which is quite soluble The THF molecule is labile in solution due to sensitivity to oxygen, light, and extremes in pH In oxygenated solutions, THF breaks down to form pterin-6-carboxaldehyde, H2 pterin, pterin, and xanthopterin The molecule is rapidly cleaved at the C-9–N-10 bond forming pABG.1 In contrast to THF and N-10-substituted THF, which are unstable in the presence of oxygen, folic acid and THF substituted at N-5 (or N-5, N-10) are relatively stable when exposed to oxygen Instability to light is a consistent feature of all forms of folate FOOD SOURCES NATURALLY OCCURRING FOOD FOLATE Folate that occurs naturally in the diet, referred to in this chapter as food folate, is concentrated in select foods including orange juice, strawberries, dark green leafy vegetables, peanuts, and dried beans such as black beans and kidney beans.2 Meat in general is not a good source of folate, with the exception of liver ß 2006 by Taylor & Francis Group, LLC FOLIC ACID IN FORTIFIED AND ENRICHED FOOD PRODUCTS In addition to food folate, ready-to-eat breakfast cereals contribute significantly to folate intake in the United States since the majority of breakfast cereals in the United States marketplace contain ~100 mg per serving of folic acid and a smaller number contain 400 mg per serving.3 Folic acid is an added ingredient in a large number of other food products including meal replacement and infant formulas, and an increasing number of ready-to-eat breakfast cereals, nutritional bars, and snack foods In the United States, all ‘‘enriched’’ cereal grain products (e.g., bread, pasta, flour, breakfast cereal, and rice) and mixed food items containing these grains are required by the Food and Drug Administration to be fortified with folic acid for the purpose of reducing the risk of NTDs.4 Although the effective date was January 1, 1998, the majority of food manufacturers had implemented folic acid fortification by mid-1997 Mandatory fortification has also been implemented in select countries in addition to the United States including Canada,5 Chile,6 and some Latin American countries.7 EFFECT OF FOLIC ACID FORTIFICATION UNITED STATES AND CANADA ON FOLATE INTAKE AND STATUS IN THE Folic acid fortification has had a significant impact on folate status in the United States and Canada.8,9 In the United States, recently reported nationally representative data from the National Health and Nutrition Examination Survey (NHANES) (1999–2000) were compared with that from the prefortification period (NHANES III 1988–1994).8 The median serum folate concentration increased more than twofold (from 12.5 to 32.2 nmol=L) and the median red blood cell (RBC) folate concentration increased from 392 to 625 nmol=L from NHANES III (1988–1994) to NHANES (1999–2000) In Canada, the mean RBC folate concentration rose from 527 nmol=L during the prefortification period to 741 nmol=L postfortification in women of reproductive age.9 BIOAVAILABILITY The bioavailability of folate may be defined as the portion of the nutrient that is physiologically available, which is influenced by numerous factors including but not limited to the following: (a) chemical form of folate; (b) food matrix; (c) the chemical environment in the intestinal tract; and (d) factors affecting the metabolic fate postabsorption.10,11 Reported estimates of folate bioavailability are quite variable due in part to differences between experimental approaches and analytical methodologies used.11–14 The blood folate response to folic acid is greater than that observed in response to food folate.10,11 The average bioavailability of food folate has been estimated to be no more than 50% relative to folic acid consumed alone in a fasting condition.15 When folic acid is consumed with a light meal, the bioavailability is ~85% that of supplemental folic acid taken alone when fasting.16 METHODS FOOD FOLATE Food folate has historically been measured by a wide range of methods including microbiological assay, radiobinding or radiometric assay, and fluorometric, electrochemical, or spectrophotometric methods, with some methods in combination with high-performance liquid chromatography (HPLC).17 A review of the interlaboratory variation using many of these different methods has been published.18 ß 2006 by Taylor & Francis Group, LLC The microbiological assay has been considered to be one of the best and most versatile methods for the determination of food folate for the past half century Lactobacillus casei subspecies rhamnosus (ATCC 7469) has been the most widely used microorganism since it responds almost equally to the widest variety of folate derivatives.19 Folate values using the microbiological assay are currently obtained after heat extraction to release folate from folate-binding protein or the food matrix in the presence of a reducing agent such as ascorbic acid, followed by trienzymatic extraction and deconjugation.20,21 This trienzyme procedure involves a combination of protease and a-amylase treatments to release folate bound to matrices of proteins and polysaccharides, respectively, in addition to pteroyl-g-glutamyl carboxypeptidase (folate conjugase, EC 3.4.19.9), which hydrolyzes folate polyglutamates to folate with shorter glutamyl residues that can be used by the microorganisms.19 Key studies have confirmed that the trienzyme assay results in significantly higher folate values compared with conjugase treatment alone and that the higher values are not an artifact of the assay procedure.22,23 Based on the results of a recent collaborative study, the microbiological assay with trienzyme extraction has been recommended for adoption as the official AOAC method.24 Food folate can also be measured by HPLC methods25,26 and procedures are also available to either allow the identification of specific one carbon derivatives of folate27 or characterize the length of the polypeptide chain.28,29 To identify the one carbon entities, folates are treated with folate conjugase to convert the polyglutamyl folates to monoglutamates and then separated by reverse phase HPLC.27 To identify polyglutamate distributions, folates can be cleaved at the C-9–N-10 bond to yield ( pABPG) derivatives, which can be separated and identified by HPLC analysis.28,29 The analysis of one carbon derivatives and polyglutamate chain length is complicated by the large number of folate derivatives present in food To address this challenge, Selhub and coworkers30 developed an affinity method using immobilized milk folate–binding protein to purify the extracted folates before reverse phase HPLC analysis This method allows quantitation of individual folates in foods and can also be applied to tissues BIOLOGICAL SAMPLES Quantitation of folate in biological specimens includes microbiological growth procedures, protein–ligand-binding methods, chromatographic and mass spectrometric methods.17,31 Several liquid chromatography–tandem mass spectrometry methods have been developed for the analysis of clinical specimens.32,33 The classical L casei method for measurement of both serum and RBC folate concentrations has been used in research laboratories for more than 50 years When the microbiological assay is used, the standard cutoff to define inadequate folate status for serum folate is