RENGE INTAKES
`
`FOR
`
`Thiamin, R1'bof1a‘v1'n,
`
`Niacin, Vitamin B5,
`
`Folate, V1'tam1'n B12,
`
`Pantoflienic Acid,
`
`Biotin, and Choline,a
`
`/
`
`A Report of the
`Standing Committee on the Scientific Evaluation
`of Dietary Reference Intakes and its
`Panel on Folate, Other B Vitamins, and Choiine and
`Subcommittee on Upper Reference Levels of Nutrients
`Food and Nutrition Board
`
`Institute of Medicine
`
`NATIONAL ACADEMY PRESS
`Washington, D.C.
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`i
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`'n..
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`NATIONAL ACADEMY PRESS - 2101 Constitution Avenue, N.W. 0 Washington, DC 20418
`
`NOTICE: The project that is the subject of this report was approved by the Governing Board
`of the National Research Council, whose members are drawn from the councils of the
`National Academy of Sciences, the National Academy of Engineering, and the Institute of
`Medicine. The members of the committee responsible for the report were chosen for their
`special competences and with regard for appropriate balance.
`
`This project was funded by the U.S. Department of Health and Human Services Oflice of
`Disease Prevention and Health Promotion, Contract No. 282960033, T0]; the National
`Institutes of Health Office of Nutrition Supplements, Contract No. N01-OD-4-2139, T024,
`the Centers for Disease Control and Prevention, National Center for Chronic Disease Preven-
`
`tion and Health Promotion, Division of Nutrition and Physical Activity; Health Canada; the
`Institute of Medicine; and the Dietary Reference Intakes Corporate Donors’ Fund. Contribu-
`tors to the Fund include Roche Vitamins Inc, Mead Johnson Nutrition Group, Daiichi Fine
`Chemicals, Inc, Kemin Foods. Inc. M8cM Mars. Weider Nutrition Group, and Natural Source
`Vitamin E Association. The opinions or conclusions expressed herein do not necessarily
`reflect those of the funders.
`
`Library of Congress Cataloging-in-Publication Data
`
`Dietary reference intakes for thiamin, riboflavin, niacin, vitamin B6, folate, vitamin
`B12, pantothenic acid, biotin, and choline / a report of the Standing Committee on the
`Scientific Evaluation of Dietary Reference Intakes and its Panel on Folate, Other B
`_Vitamins, and Choline and Subcommittee on Upper Reference Levels of Nutrients, Food
`and Ntttrition Board, Institute of Medicine.
`
`p. cm.
`Includes bibliographical references and index.
`ISBN 0-309-06554-2 (pbk.) — ISBN 0-309-06411-2 (case)
`1. Vitamin B in human nutrition. 2. Reference values (Medicine) 1. Institute of
`
`Medicine (U.S.). Standing Committee on the Scientific Evaluation of Dietary Reference
`Intakes. II. Institute of Medicine (U.S.). Panel on Folate, Other B Vitamins, and Choline.
`III. Institute of Medicine (U.S.). Subcommittee on Upper Reference Levels of Nutrients.
`
`Q_P772.V52 D53 2000
`612.3’99—dc2l
`
`UU-0285250
`
`Additional copies of this report are available from National Academy Press, 2101 Constitution
`Avenue, N.W., Lock Box 285, Washington, DC 20055. Call (800) 624-6242 or (202) 334-3313
`(in the Washington metropolitan area), or visit the NAP's on~line bookstore at http:/
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`
`For more information about the Institute of Medicine or the Food and Nutrition Board, visit
`
`the IOM home page at http://www.nas.edu/iom.
`
`Copyright 1998 by the National Academy of Sciences. All rights reserved.
`Printed in the United States of America
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`tures and religions since the beginning of recorded history. The image adopted as a logotype
`'~-' - --'-'v.v‘.<.z-1:: .-..
`by the Institute of Medicine is based on a relief carving from ancient Greece, now held by the
`v
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`
`Folate
`
`SUMMARY
`
`Folate functions as a coenzyme in single-carbon transfers in the
`metabolism of nucleic and amino acids. The primary indicator
`used to estimate the Recommended Dietary Allowance (RDA) for
`folate is erythrocyte folate in conjunction with plasma homocys-
`teine and folate concentrations. The RDA for both men and women
`is 400 pg/ day of dietary folate equivalents (DFES). DFEs adjust for
`the nearly 50 percent lower bioavaiiabiiity of food folate compared
`with that of folic acid: 1 pg of dietary folate equivalent = 0.6 pg of
`folic acid from fortified food or as a supplement taken with meals
`= 1 pg of food folate = 0.5 pg of a supplement taken on an empty
`stomach, To reduce the
`of neural tube defects for women
`capable of becoming pregnant, the recommendation is to take
`400 pg of folic acid daily from fortified foods, supplements, or
`both in addition to consuming food folate from a varied diet. The
`evidence available on the role of folate in reducing the risk of
`vascular disease, cancer, and psychiatric and mental disorders is
`not sufficiently conclusive to use risk reduction of these condi-
`tions as a basis for setting the Estimated Average Requirement
`(EAR) and the RDA.
`
`In the U.S. adult population from 1988 to 1994, which was before
`cereal grains were fortified with folate, the reported median in-
`take of folate from food was approximately 250 pg/day, but this
`value imderesti mates current intake. The ninety-fi_ft_h percentile of
`intake from food and supplements was close to 900 ug/day overall
`
`196
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`_
`F—’.}LATE
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`1 0'7
`.1 .; :
`
`and nearly 1,700 pg/day for pregnant women. After the fortifica-
`tion of cereal grains with folate——which became mandatory for
`enriched grains in the United States as ofJanuary 1, 1998, and is
`nowauthorized in Canada—average intake of folate is expected to
`increase by about 80 to 100 pg/day for women and by more for
`men. The Tolerable Upper Intake Level (UL) for adults is set at
`1,000 pg/ day of folate from fortified food or as a supplement,
`exclusive or Iood tolate.
`
`BACKGROUND INFORMATION
`
`Folate is a generic term for this water-soluble B-complex vitamin,
`which functions in single-carbon transfer reactions and exists in
`many chemical forms (Wagner, 1996). Folic acid (pteroylmono-
`glutamic acid), which is the most oxidized and stable form of folate,
`occurs rarely in food but is the form used in vitamin supplements
`and in fortified food products. Folic acid consists of a p-aminoben-
`zoic acid molecule linked at one end to a pteridine ring and at the
`other end to one glutamic acid molecule. Most naturally occurring
`folates, called food folate in this report, are pteroylpolyglutamates,
`which contain one to six additional glutamate molecules joined in a
`peptide linkage to the 'y-carboxyl of glutamate.
`
`Function
`
`The folate coenzymes are involved in numerous reactions that
`involve (1) deoxyribonucleic acid (DNA) synthesis, which depends
`on a folate coenzyme for pyrimidine nucleotide biosynthesis (meth-
`ylation of deoxyuridylic acid to thymidylic acid) and thus is required
`for normal cell division; (2) purine synthesis (formation of glycina-
`mide ribonucleotide and 5~amino-4-imidazole carboxamide ribonu-
`cleotide); (3) generation of formate into the formate pool (and
`utilization of formate); and (4) amino acid interconversions, in-
`cluding the catabolism of histidine to glutamic acid, interconver—
`sion of serine and glycine, and conversion of homocysteine to me-
`thionine. Folate-mediated transfer of single—carbon units from
`serine provides a major source of substrate in single-carbon metab-
`olism. The conversion of homocysteine to methionine serves as a
`major source of methionine for the synthesis of S-adenosyl—met.hi0-
`nine, an important in vivo methylating agent (Wagner, 1996).
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`198
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`DIETARY REFERENCE INTAKES
`
`Physiology ofAbsorption, Zlfetabolism, and Excretion
`
`Absorption, Transport, and Storage
`
`Food folates (polyglutamate derivatives) are hydrolyzed to mono-
`glutarnate forms in th gut before absorption across the intestinal
`mucosa. This cleavage is accomplished by a 'y-glutamylhydrolase,
`more commonly called folate conjugase. The monoglutamate form
`of folate is actively transported across the proximal small intestine
`by a saturable pH-dependent process. When phannacological doses
`of the monoglutamate form of folate are consumed, it is also ab-
`sorbed by a nonsaturable mechanism involving passive diffusion.
`Monoglutamates, mainly 5-methyl-tetrahydrofolate, are present in
`the portfl circulation. Much of this folate can be taker: up by the
`liver, where it is metabolized to polyglutamate derivatives and re-
`tained or released into the blood or bile. Approximately two-thirds
`of the folate in plasma is protein bound. A variable proportion of
`plasma folate is bound to low~affinity protein binders, primarily al-
`bumin, which accounts for about 50 percent of bound folate. Low
`levels of high-affinity folate binders are also present in plasma.
`Cellular transport of folate is mediated by a number of different
`folate transport systems, which can be characterized
`either mem-
`brane carriers or folate-binding protein-mediated systems. These
`transport systems are not saturated by folate under physiological
`conditions, and folate influx into tissues would be expected after
`any elevation in plasma folate after supplementation.
`Folate concentr-ations in liver of 4.5 to 10 pg/g were reported
`after liver biopsies (Whitehead, 1973). Because the adult male liver
`weighs approximately 1,400 g, the total quantity of folate in the
`liver would be approximately 6 to l4 mg. If the liver is assumed to
`contain 50 percent of the body stores of folate, the estimated total
`body folate store would be 12 to 28 mg. Using the same assumption,
`Hoppner and Lampi (1980) determined average liver folate con-
`centrations to be approximately 8 1137’g (range 3.6 to" 14.8 pg/g)
`after autopsy; the liver folate content would be approximately 11
`mg and total body folate 22 mg.
`
`Metabolism and Excretion
`
`Before being stored in tissue or used as a coenzyme, folate mono-
`glutamate is converted to the polyglutamate form by the enzyme -
`folylpolyglutamate synthetase. When released frorn tissues into cir-
`culation, folate polyglutamates are reconverted to the mono-
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`glutamate form by yhglutamylhydrolase. Folates must be reduced
`enzymatically and resynthesized to the polyglutamate form to func-
`tion in single-ca.rbon tra_n_sfer reactions,
`The metabolic interrelationship between folate and vitamin B12
`may explain why a single deficiency of either vitamin leads to the
`same hematological changes. Both folate and vitamin B12 are re-
`quired for the formation of 5,10-methylenetetrahydrofolate and in-
`volved in thymidylate synthesis by way of a vitamin B12-containing
`enzyme. The formation of 5,10-methylene tetrahydrofolate depends
`on the regeneration of the parent compound (tetrahydrofolate) in
`the homocysteine-to-methionine conversion. This reaction involves
`the removal of a methyl group from methyl folate and the delivery
`of this group to homocysteine for the synthesis of methionine.
`Folate is involved as a substrate (5-methyl-tetrahydrofolate) and vi-
`tamin B12 as a coenzyme. The 5,10-methylenetetrahydrofolate deliv-
`ers its methyl group to deoxyuridylate to convert it to thymidylate
`for incorporation into DNA. In either a folate or vitamin B12 defi-
`ciency, the megaloblastic changes occurring in the bone marrow
`and other replicating cells result from lack of adequate 5,10-methylene
`tetrahydrofolate.
`The major route of whole-body folate turnover appears to be via
`catabolism to cleavage products. The initial step in folate catabolism
`involves the cleavage of intracellular folylpolyglutmate at the C9-N10
`bond, and the resulting paminobenzoylpolyglutamates are hydro-
`lyzed to the monoglutamate, which is N-acetylated before excretion.
`Folate freely enters the glomerulus and is reabsorbed in the prox-
`imal renal tubule. The net effect is that most of the secreted folate
`is reabsorbed. The bulk of the excretion products in humans are
`folate cleavage products. Intact urinary folate represents only a very
`small percentage of dietary folate. Biliary excretion of folate has
`been estirnated to be as high as 100 pg,/ day (Herbert and Das, 1993;
`Whitehead, 1986); however, much of this is reabsorbed by the small
`intestine (Weir et al., 1985). Fecal folate losses occur, but it is diffi-
`cult to distinguish actual losses from losses of folate synthesized by
`the intestinal microflora (K_t'u.m.diec.k et. al-, .1978) _
`
`- -
`
`fl
`1..
`
`- -
`
`Clinical Effects ofInadequate Intake
`.....-
`.,
`--
`..
`-
`olate intake -.rst leads to a decrease in serum folate
`
`concentration, then to a decrease in erythrocyte folate concentra-
`tion, a rise in homocysteine concentration, and megaloblastic
`changes in the bone marrow and other tissues with rapidly dividing
`\/\l&4\\IO
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`=
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`200
`
`DIETARY REFERENCE INTAKES
`
`‘Nithi weeks of the development of early rnorphologicfl abnor-
`malities in the marrow, subtle changes appear in the peripheral
`blood (Eichner et al., 1971) when hypersegmentation of the neutro-
`phils becomes apparent. The peripheral blood picture is variable
`before the devel pment
`f a clearly increased mean cell v lnme or
`anemia (Lindenbaum et al., 1988). In some deficient individuals,
`macrocytes and macroovalocytes are seen on blood smears, but in
`others the erythrocytes may show only minimal anisocytosis or no
`abnormalities. When fol te supply to the bone marrow becomes
`rate limiting for erythropoiesis, macrocytic cells are produced. How-
`ever, because of the 120-day lifespan of normal erythrocytes, macro-
`cytosis is not evident in the early stages of folate~deficient megalo~
`blastosis.
`As folate depletion progresses further, the mean cell volume in-
`creases above normal. Neutrophil hypersegmentation (defined as
`more than 5 percent five-lobed or any six-lobed cells per 100 granu-
`loqrtes) is typically present in me peripheral blood at this stage of
`macrocytosis and the neutrophil lobe average is elevated.
`Macrocytic anemia then develops, as first evidenced by a depres-
`sion of the erythrocyte count. Eventually, all three measures of ane-
`(hematocrit, hemoglobin concentration, and erythr cyte con-
`centration) are depressed. At
`this point, macroovalocytes and
`macrocytes are usually detectable in the peripheral blood, and hy-
`persegmentation is more impressive (Lindenbaum et al., 1988).
`Because the onset of anemia is usually gradual, compensating car-
`diopulmonary and biochemical mechanisms provide adaptive ad-
`justments to the diminished oxygen-carrying capacity of the blood
`until anemia is moderate to severe. Symptoms of weakness, fatigue,
`difficulty concentrating,
`irritability, headache, palpitations, and
`shortness of breath therefore typically appear at an advanced stage
`of anemia. They may be seen at milder degrees of anemia in some
`patients, especially the elderly (Lindenbaum et al., 1988). Atrophic
`glossitis may also or'.r_'.I_1r (Savage et. al.., 1994.).
`
`SELECTION OF INDICATORS FOR ESTIMATING
`THE REQUIREMENT FOR FOLATE
`
`The primary indicator selected to determine folate adequacy is
`erythrocyte folate, which reflects tissue folate stores, as described in
`detail below. For some life stage or gender groups, this is used in
`conjunction with plasma homocysteine (which reflects the extent of
`the conversion of homocysteine to methionine) and plasma or se-
`rum folate. Other indicators are discussed briefly below; risk reduc-
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`F0l..AJ!.‘._.
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`E201
`
`tion of chronic disease or developmen.tal abnormalities is covered
`in detail in a later section.
`
`Erythrocyte Folate
`
`Because folate is taken up only by the developing erythrocyte in
`the bone marrow and not by the circulating mature erythrocyte
`during its 120-day lifespan, erythrocyte folate concentration is an
`indicator of long-term status. Erythrocyte folate concentration was
`shown to be related to tissue stores by its correlation, although weak,
`with liver folate concentration determined by biopsy in the same
`individual in a study of 45 subjects (Wu et al., 1975).
`Erythrocyte folate concentration does not reflect recent or tran-
`sient changes in dietary folate intake. A value of 305 nmol/L (140
`ng/mL) of folate was chosen as the cutoff point for adequate folate
`status on the basis of the following experiments: On a diet contain-
`ing only 5 pg/day of folate, the appearance of hypersegmented neu~
`trophils in the peripheral blood of one subject coincided with the
`approximate time when the erythrocyte folate concentration de-
`creased to less than 305 nmol/L (140 ng/mL) (Herbert, 1962a).
`On a diet containing less than 20 pg/day of folate, the appearance
`of hypersegmented neutrophils in two subjects preceded the reduc-
`tion in erythrocyte folate to concentrations below 305 nmol/L (140
`ng/mL) by about 2 weeks (Eichner et aL, 1971). In a group of 40
`patients with megaloblastic anemia caused by folate deficiency, 100
`percent had erythrocyte folate values less than 305 nmol/L (140
`ng/mL); values were the lowest in the most anemic subjects and the
`highest mean lobe counts occurred in the subjects with the lowest
`erythrocyte folate concentrations (Hofibrand et al., 1966). All 238
`pregnant women with erythrocyte folate concentrations below 327
`nmol/L (150 ng/mL) were found to have megaloblastic marrow
`(Varadi et al., 1966). Eight subjects with erythrocyte folate of less
`than 305 nmol/L (140 ng/mL) had eight- to ninefold greater in-
`corporation of uracil into DNA than did 14‘ control subjects and
`had a threefold increase in frequency of cellular micronuclei (a
`, measure of DNA and chromosome damage); folate supplementa-
`tion reduced the abnormalities (Blount et a1., 1997).
`
`In this report, plasma homocysteine concentration refers to total
`homocysteine concentration. Plasma homocysteine concentration
`increases when inadequate quantities of folate are available to do-
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`202
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`DIETARY REFERENCE INTAKES
`
`nate the methyl. group that is required to convert homocystein to
`methionine. Controlled metabolic and epidemiological studies pro-
`vide evidence that plasma homocysteine rises with reductions in
`blood folate indices. Different cutoff values have been used by vari-
`ous investigators to define elevated homocysteine concentrations.
`The cutoff value for plasma homocysteine cited most often is great-
`er than 16 pmol/L, but 14 pmol/L (Selhub et al., 1993) and 12
`pmol/L (Rasmussen et al., 1996) have also been used. Ubbink and
`coworkers (1995a) used a prediction model to define a reference
`range as 4.9 to 11.7 umol/L. Other investigators have proposed age-
`and gender-specific reference intervals (Rasmussen et al., 1996).
`Many investigators have reported that plasma homocysteine is sig-
`nificantly elevated in_ i..n.r_livid.u.a.ls who have been diagnosed as fol te
`deficient on the basis of established serum folate, plasma folate, or
`erythrocyte folate norms (Allen et al., 1993; Chadefaux et al., 1994;
`Curtis et al., 1994; Kang et al., 1987; Savage et al., 1994; Stabler et
`al., 1988; Ubbink et al., 1993),
`The evidence supporting the use of homocysteine as an ancillary
`indicator of folate status is summarized as follows:
`
`0 In 10 young men, folate depletion led. t- a -ise in plasma homo-
`cysteine and a decrease in plasma folate (]acob et al., 1994).
`0 In young women, a folate intake equivalent to 320 pg/day of
`dietary folate equivalents was associated with elevated plasma homo-
`cysteine (greater than 14 pmol/L); at this level of intake plasma
`homocysteine concentrations were inversely associated with eryth-
`rocyte and serum folate concentrations (O’Keefe et al., 1995).
`0 In a cross-sectional analysis involving elderly individuals, plasma
`homocysteine exhibited a strong inverse associat.l'.on with plasma
`folate after age, gender, and intakes of other vitamins were con-
`trolled for (Selhub et al., 1993); homocysteine values appeared to
`plateau at folate intakes greater than approximately 350 to 400 pg/
`day. A meta-analysis by Boushey and colleagues (1995) supports the
`existence of a plateau when adequate folate is consumed.
`
`Thus, in studies of different types, a similar inverse relationship
`between folate intake and plasma homocysteine values is seen for
`pre- and postmenopausal women, adult men, and the elderly.
`Ward and colleagues (1997) supplemented each of 30 male sub-
`jects with 100, 200, or 400 pg of folate. The men were consuming a
`regular diet that averaged 281 ug/day of folate. Plasma homocys-
`teine, serum folate, and erythrocyte folate were assessed before, dur-
`ing, and 10 weeks after intervention. Results, expressed as tertiles of
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`baseline plasma homocysteine, showed significant homocysteine
`lowering in the top (mean 11 pmol/L) and middle (mean 9 pmol/
`L) hornomstein tertile° ‘wt not in the bottom tertile (mean '.7
`\.]|Jl.
`0 IJ\I-la
`. pmol/L). All baseline homocysteine values were within the normal
`range; the highest was 123 pmol/L. Of the three folate doses, 200
`pg appeared to be as effective as 400 pg whereas 100 pg was less
`eflective at lowering hornocysteine. These data suggest that there is
`a concentration of plasma homocysteine below which folate has no
`further lowering effect.
`Maternal hyperhomocysteinemia has been implicated as a risk fac-
`tor for complications during pregnancy (Burke et al., 1992; Goddijn-
`Wessel et al., 1996; Rajkovic et al., 1997; Steegers-Theunissen et al.,
`1992, 1994; Wouters et al., 1993), but the relationship between
`folate intake and the complications has not been established.
`Although plasma homocysteine is a .sens_i_ti_ve indicator of folate
`status, it is not a highly specific one: it can be influenced by vitamin
`B12 status (Stabler et al., 1996), vitamin B6 status (Ubbink et al.,
`1995a), age (Selhub et al., 1993), gender (Selhub et al., 1993), race
`(Ubbink et al., 199.513), some genetic abnormalities (e.g., methyl-
`tetrahydrofolate reductase deficiency) (lacques et al., 1996; Malinow
`et al., 1997), and renal insufficiency (Hultberg et al., 1993). Thus,
`plasma homocysteine alone is not an acceptable indicator on which
`to base the folate requirement.
`-
`Knowledge of the relationships of folate, homocysteine, and risk
`of vascular disease was judged too weak to use as the basis for deriv-
`ing the Estimated Average Requirement (EAR) for folate. This top-
`ic is described in more detail in “Reducing Risk of Developmental
`Disorders and Chronic Degenerative Disease.”
`
`Serum Folate
`
`A serum folate concentration of less than 7 nmol/L (3 ng/mL)
`indicates negative folate balance at the time the blood sample was
`drawn (Herbert, 1987). In all the experimental studies of human
`volunteers subjected to folate deprivation, a decrease in the serum
`folate concentration, usually occurring within 1 to 3 weeks, was the
`first event (Eichner and Hillman, 1971; Eichner et al., 1971; Halsted
`et al., 1973; Herbert 1962a; Sauberlich et al., 1987). This initial
`period of folate deprivation is followed by weeks or months when
`the serum folate concentration is low but there is no other evidence
`of deficiency. The circulating folate concentration may also be de-
`pressed in situations in which there is no detectable alteration in
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`204
`
`DIETARY REFERENCE INTAKES
`
`acute alcouoi ingestion (Eicii iei
`
`»
`
`‘
`
`to-tfl body folate, sucu
`Hillman, 1973).
`In population surveys it is generally assumed that measuring
`serum folate alone does not differentiate between what may be a
`transitory reduction in folate intake or chronic folate deficiency
`accompanied by depleted folate stores and functional changes.
`Serum or plasma folate is, however, considered a sensitive indicator
`of dietary folate intake, as illustrated by the report ofJacques and
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`of folate intake assessed in a study of 140 people. In a controlled
`metabolic study, repeated measures over time in the same individual
`do reflect changes in status. Serum folate concentration may be a
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`VVU1 L.llW.l1.llL \.lld.s11UOtlL |.CDL 11. Ll.-SCLI. 4.1.13.1 l.l.ll.C.l PLCLCLL LUIICLLI)’ 111 LlJl.1"
`junction with other folate status indices (Lindenbaum et al., 1988).
`
`Urinary Folate
`
`Data from a metabolic study in which graded doses of folate were
`fed showed that urinary folate is not a sensitive indicator of folate
`status (Sauberlich et al., 1987). In that study, approximately 1 to 2
`percent of di tary folate was excreted intact in the urine; excretion
`continued even in the face of advanced folate depletion. Other
`reports indicate that daily folate excretion on a normal diet ranges
`from 5 to 40 ],lg/day (Cooperman et al., 1970; Retief, 1969; Tamura
`KI Ink} A\\’ LOIKL,
`and Qfnlrcfatq
`.
`The major route of whole-body folate turnover is by catabolisrn
`and cleavage of the C9—N10 bond producing pteridines and p~amino—
`benzoylglutamate (pABG)
`(Knimdieck et al. 1978; Saleh et al.,
`1982). Before excretion from the body, most pABG is N—acetylated
`to acetamidobenzoylglutamate (apABG). It is not known whether
`folate coenzymes are catabolized and excreted or whether they are
`recycled after metabolic utilization. In a study designed to estimate
`the folate requirements of pregnant and nonpregnant women, Mc-
`Partlin and coworkers 1993
`uantified the urin
`excretion of
`.
`fry .
`.
`C1
`pABG and apABG as a measure of daily folate utilization. This ap-
`proach does not take into account endogenous fecal folate loss,
`which may be substantial (Ksumdieck et al., 1978); thus, quantita-
`tion of urinary catabolites alone may result in an underestimation
`of the requirement.
`
`Indicators ofHematological Status
`
`The appearance of hypersegmented neutrophils, macrocytosis,
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`and other abnormal hematological findings occurs late in the de-
`velopment of deficiency (see “Clinical Effects of Inadequate In-
`take”). Thus, hematological findings were not used to derive the
`EAR.
`
`Risk ofNeural Tube Defects and of Chronic Degenerative Diseases
`
`The role of folate in the prevention of neural tube defects (NTDS)
`was very carefully considered, but not in the context of setting an
`EAR. Although the evidence is strong that the risk of having a fetus
`with an NTD decreases with increasing intake of folate during the
`periconceptional period (about 1 month before to 1 month after
`conception), this type of risk reduction was judged inappropriate
`for use as an indicator for setting the EAR for folate for women of
`childbearing age. There are several reasons for this. The popula-
`tion at risk is all women capable of becoming pregnant, but only
`those women who become pregnant would benefit from an inter-
`vention aimed at reducing NTD risk. The risk of NTD in the U.S.
`population is about 1 per 1,000 pregnancies, but the critical period
`for prevention—the periconceptional period——-is very short. The
`definition of EAR, which indicates that half of the individuals in the
`population have intakes sufficient to meet a particular criterion,
`does not accommodate NTD prevention as an appropriate criterion.
`Because of the importance of this topic,- it is covered separately in
`the later section “Reducing Risk of Developmental Disorders and
`Chronic Degenerative Disease.”
`The possible use of criteria involving reduction of risk of vascular
`disease, certain types of cancer, and psychiatric and mental dis-
`orders was also carefully considered. The evidence was not judged-
`suflicient to use prevention of any chronic disease or condition as a
`criterion for setting the EAR; this evidence is also presented in the
`section “Reducing Risk of Developmental Disorders and Chronic
`Degenerative Disease.”
`
`MET}-IODOLOGICAL. ISSUES
`
`Measurement ofBlood Folate Values
`
`Substantial variation within and across methods was evident from
`the results of an international comparative study of the analysis of
`serum and whole—blood folate (Gunter et al., 1996). Results for
`whole—blood pools were more variable than for serum pools. The
`authors concluded that folate concentrations measured in one lab-
`
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`
`DIETARY REFERENCE INTAKES
`
`oratory cannot be conipared reliably with those measured in anoth-
`er laboratory without considering interlaboratory differences and
`that comparing data for different study populations measured by
`different methods is difficult.
`The Bio-kd Quantaphase Radioassay was used for the first 4 years
`of the Third National Health and Nutrition Examination Survey
`(NHANES III)
`(1988-1991). In 1991 it was determined that the
`Bio-Rad radioassay gave results that were 30 percent too high when
`external, purified pteroylglutalnic acid (PGA) standard solutions
`were measured. The Bio-Rad assay was then recalibrated by using
`calibrator solutions of PGA concentrations of 2.3, 5.7, 11, 22.6, and
`45 nmol/L (1.0, 2.5, 5.0, 10.0, and 20.0 ng/mL). The net effect of
`this recalibration was the expected 30 percent reduction in the mea-
`sured folate concentrations of a sample. An analysis by another ex-
`pert panel (LSRO/FASEB, 1994) provides further information. The
`NHANES III laboratory conducted a 19-day comparison study of
`NI-L’—‘.NF.S III serum md erythrocyte specimens using the original
`and recalibrated Bio-Rad kits and confirmed the 30 percent reduc-
`tion. Through the use of a regression equation developed from the
`comparison study, the correction was applied to the NHANES III
`data generated with the original assay (LSRO/FASEB, 1994).
`The NHANES III data (Appendix K) have been corrected for this
`method problem associated with inappropriate calibration. Data
`from NHANES III are believed to “provide as accurate and precise
`an estimation of serum and REC [red blood cell] folate levels in the
`United States population as is possible until a definitive method has
`been developed and [this should be considered] as a stand—alone
`data set, without applying cutoffs established using other laboratory
`methods” (EIW. Gunter, Division of Environmental I-Iealm Labora-
`tory Sciences, National Center for Environmental Health, Centers
`for Disease Control and Prevention, personal communication,
`1997).
`Earlier, after NHANES II, similar issues were adwess d by a Life
`Sciences Research Office expert panel (LSRO/FASEB, 1984) . Such
`an effort is even more warranted related to NHANES III because
`this survey (unlike NHANES II) had been designed to provide an
`assessment srf folate st....1s of the entire U.S. population.
`
`Measurement and Reporting ofFood Folate
`
`It is recogni
`zed that food folate composition data contained in
`currently u ed databases provide inaccurate estimations of folate
`intake of the U.S. population. Because of the limitations of tradi-
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`roulrs
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`_
`
`tional analytical methods used in generating the food composition
`data for folate, the database values underestimate actual folate con-
`fnnf Drnlxlpmc urifln H19 he-2rl1'finnal rnpflanrlc inr-lnrlp inrnrnnlnfp rp.
`%vA-J\oI L L \IKIL\1LJ.lI-I "ALLA \nLA‘4 \nL.£\.LnI-‘IAQLLZL flL&€ELLKIf\' LLLKILJT LL§E\ILflLtIA€§€
`Q %
`lease of folate from the food matrix and possibly incomplete hy-
`drolysis of polyglutamyl folates before quantitation. For example,
`buffer solutions widely used for sample homogenization in food
`analysis have been shown to yield incomplete recovery relative to a
`more effective extraction buffer (Gregory et al., 1990). As much as
`a twofold greater folate concentration is obtained when an im-
`proved extraction procedure rather than an older procedure is used
`in the analysis of foods such
`green peas and liver (Tamura et al.,
`1997).
`The use of a trienzyme approach (a