The Journal of Nutrition
Commentary
Factors Associated with Different Forms of
Folate in Human Serum: The Folate Folio
Continues to Grow
Joshua W Miller
Department of Nutritional Sciences, School of Environmental and Biological Sciences, Rutgers, The State University of New Jersey, New
Brunswick, NJ, USA
The long and storied history of the B vitamin, folate, began
in the 1930s when Wills (1) and Stokstad and Manning (2)
demonstrated that a factor in yeast could prevent megaloblastic
anemia of pregnancy and support the growth of chickens,
respectively. Later, Mitchell et al. (3) isolated the same substance
from spinach that could support the growth of bacteria. Initially
termed “Wills factor” and “factor U,” this substance came to be
known as “folic acid,” stemming from folium, the Latin word
for leaf (3). Since then the folio of folate research has come to
include seminal works describing the roles of the different forms
of reduced folates in the canonical one-carbon metabolism
pathways that underlie DNA and RNA synthesis and myriad
methylation reactions, the groundbreaking development of
chemotherapeutic antifolate drugs, and the highly successful
public health intervention of folic acid fortification (4).
Although fully implemented in the United States and Canada
>20 y ago, and now in >80 countries around the world,
folic acid fortification has been controversial. For its intended
purpose—reducing the incidence of neural tube defects (NTDs)
such as spina bifida and anencephaly—folic acid fortification
has undoubtedly been very effective. Estimates of reductions
in the rates of NTDs range from ∼20% to >50% depending
on how they are diagnosed and counted, the type of defect,
and the rates before the initiation of fortification within
a specific location (5). However, it is recognized that folic
acid is a synthetic form of folate not found in unfortified
food sources. Although folic acid is readily absorbed and
converted into active forms of folate in one-carbon metabolism
reactions, the potential for exposure to excess amounts of a
synthetic substance to have unintended metabolic and clinical
consequences seems to be a reasonable issue to address. This is
particularly important because fortification necessarily exposes
entire populations to folic acid through the food supply in order
to prevent what amounts to a few thousand NTDs per year.
So far, most of the scientific evidence suggesting adverse
effects of exposure to excess folic acid is observational,
circumstantial, or theoretical. Examples include possible in-
creased risk of disrupted metabolism, anemia, and cognitive
The author reported no funding received for this study.
Author disclosures: The author reports no conflicts of interest.
Address correspondence to JWM (e-mail: jmiller@sebs.rutgers.edu).
Abbreviations used: LC/MS-MS, LC–tandem MS; MeFox, 4α-hydroxy-5-
methyltetrahydrofolate; NTD, neural tube defect; UMFA, unmetabolized folic
acid.
Manuscript received November 27, 2019. Initial review completed December 24, 2019. Revision accepted February 7, 2020.
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See corresponding article on page 851.
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impairment in older adults with low vitamin B-12 status
(6–8), increased susceptibility to insulin resistance in developing
children exposed in utero (9), promotion of tumor growth
(10, 11), impaired immune function (12), and interference with
the efficacy of antifolate drugs (13). However, there is a lack
of a clear mechanism or mechanisms by which excess folic
acid might induce adverse effects, and there is no “smoking
gun” randomized control trial proving that adverse effects
occur. Moreover, there is no clear understanding of whether
adverse effects would be confined to only excess folic acid
exposure, or result from excess exposure to all forms of folate.
Recognizing these holes in the literature, the US NIH hosted a
workshop in the summer of 2019 titled “Metabolic Interactions
between Folic Acid Excess and Vitamin B12 Deficiency” with
the objective of developing a basic, translational, and clinical
research agenda to address these issues (14). A white paper from
this meeting is expected to be published later in 2020.
It is in this context that we consider the report by
Fazili et al. (15) in this issue of the Journal. In order
to ultimately address some of the fundamental questions
raised in the NIH workshop regarding folic acid, folates,
and their metabolic, physiologic, and clinical effects, basic
information must be obtained, some of which is provided in
the Fazili et al. (15) publication. Using state-of-the-art LC–
tandem MS (LC/MS-MS), they measured 5 biologically active
forms of folate [tetrahydrofolate, 5-methyltetrahydrofolate,
5-formyltetrahydrofolate, 5,10-methenyltetrahydrofolate, and
unmetabolized folic acid (UMFA)] and 1 inactive oxidative
product [4α-hydroxy-5-methyltetrahydrofolate (MeFox)] in
fasted serum samples from the postfortification 2011–2016 US
NHANES. They also assessed associations of the various forms
of folates and total folate with demographic, physiologic, and
lifestyle factors. The total number of subjects in the study
sample was 10,070, and included individuals from 1 y to >70 y
of age, and representative proportions of Hispanics and non-
Hispanic Asians, blacks, and whites.
As might be expected, a variety of differential associations
with several factors were observed for the different forms of
folate, and readers are referred to the article for specific details.
Of particular note was serum UMFA, which was detected in
practically all of the samples (95% reference interval: 0.27,
3.24 nmol/L). It is known that UMFA is metabolized fairly
quickly, and thus detection in fasted samples (defined as no food
for ≥8 h before blood draw) from almost all subjects suggests
Copyright  C The Author(s) 2020.
First published online March 2, 2020; doi: https://doi.org/10.1093/jn/nxaa046.
a significant amount of exposure to folic acid. Moreover,
serum concentrations of UMFA were highest in older adults
(age 60 y and older) and were higher in adults with chronic
kidney disease than in healthy individuals. These findings
indicate that renal clearance is an important process for
reducing the overall exposure to UMFA.
Another interesting finding is the ubiquity of the inactive
metabolite, MeFox. Like UMFA, MeFox was detected in
practically all of the samples (95% reference interval: 0.38,
4.39 nmol/L). Also like UMFA, MeFox concentration was
highest in older adults, as well as in individuals with chronic
kidney disease. These observations are noteworthy because a
previous study (16) suggested that MeFox is a by-product of
oxidation of 5-methyltetrahydrofolate post–blood collection.
The Fazili et al. (15) study suggests that at least some of the
MeFox is produced by oxidative processes in vivo before blood
draw. Elucidating these oxidative processes and determining
their clinical significance, if any, will be of great interest.
Considered on its overall merits, the Fazili et al. (15) study
is perhaps not overly groundbreaking. It is limited in that it is a
cross-sectional observational study and as such does not address
causal relations. It also does not take into consideration com-
mon genetic polymorphisms that might be expected to affect the
metabolism of folates, such as the methylenetetrahydrofolate
reductase C677T polymorphism. Nonetheless, the study lays a
strong foundation for future research. First, it establishes the
reliability and utility of gold-standard LC/MS-MS methodology
for simultaneous measurement of 6 forms of folate; and second,
it provides insight into potential confounding variables that
should be accounted for in future cross-sectional, longitudinal,
and folic acid supplementation studies designed to assess the
associations of the various forms of folate with physiological
and clinical outcomes. It thus represents an important addition
to the folate folio.
Acknowledgments
The sole author was responsible for all aspects of this
manuscript.
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Commentary 651