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Vitamin Metabolism Laboratory, Jean Mayer U.S. Department of Agriculture Human Nutrition Research Center on Aging at Tufts University, Boston MA 02111
2To whom correspondence and reprint requests should be addressed.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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KEY WORDS: • folate • methylenetetrahydrofolic acid • formyltetrahydrofolic acid • homocysteine • polymorphism • humans
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, Mudd et al. 1972
, Rosenblatt 1989
, Wong et al. 1977
).
Kang et al. (1988)
first identified a common variant of
the MTHFR enzyme, characterized by reduced enzyme activity (
50%)
and greater sensitivity to heat inactivation compared with the normal
enzyme in vitro. When lymphocyte or fibroblast extracts containing this
variant enzyme were preincubated at 46°C for 5 min, the residual
enzyme activity was considerably lower than the normal enzyme; thus
this variant was named "Thermolabile MTHFR."
Upon isolation of the human cDNA for MTHFR (Goyette et al. 1994
), these in vitro properties of the thermolabile enzyme
were found to be due to a homozygous cytosine-to-thymidine transition
at nucleotide 677 (C677T), resulting in an alanine-to-valine
substitution (Frosst et al. 1995
). The prevalence of
this mutation was first described in French Canadians with an allele
frequency of 38% and homozygosity (i.e., T/T genotype) of 12%. More
recently, the frequency of the T allele has been determined in
Caucasians (36%), Asians (40%), and African Blacks (5%)
(Franco et al. 1998
).
To date, the activity of thermolabile MTHFR has been described only in
vitro. The intent of this study, therefore, was to determine whether
the presence of this polymorphism impairs the synthesis of
5-CH3-H4 folic acid in
vivo. We determined the rise in
5-CH3-H4 folic acid
concentration in plasma and urine after oral ingestion of
5-HCO-H4 folic acid in human subjects with either
the T/T (mutant) or C/C (wild-type) MTHFR genotype. Our approach was
based on previous studies (Nixon and Bertino 1972
,
Whitehead et al. 1972
), which have shown that such an
increase in plasma and urinary
5-CH3-H4 folic acid follows
the conversion of 5-HCO-H4 folic acid to
5-CH3-H4 folic acid, a
process that requires the action of MTHFR.
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SUBJECTS AND METHODS |
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TOPABSTRACTINTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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After an overnight fast, baseline blood and urine samples were
collected. A 5-mg oral dose of
(6R,S)-5-HCO-H4 folic acid
was then administered with water. Subsequent blood and urine samples
were collected at 30 min and 1 h postdose, then every hour for a
total of 7 h. Urine collection containers contained 1 gm sodium
ascorbate as a preservative and were kept at 4°C during the course of
the collection. Samples were immediately processed and stored at
-70°C until analysis. Fasting total plasma and RBC folates were
determined by a conventional microbial (Lactobacillus casei)
method using a 96-well plate (Tamura et al. 1990
). Fasting plasma total homocysteine was determined
by HPLC using the method of Araki and Sako (1987)
.
Specific forms of plasma and urine folates were analyzed by a combined
affinity/HPLC method (Bagley and Selhub 1997
) and are
described in more detail below. All chemicals were purchased from Sigma
Chemical (St. Louis, MO) unless otherwise indicated.
MTHFR genotype determination.
Analysis of the MTHFR C677T mutation was based on the method of
Frosst et al. (1995)
with minor modifications. Briefly,
DNA was isolated from peripheral leukocytes using an Invitrogen
Easy-DNA Kit (San Diego, CA). Polymerase chain reaction was
performed using genomic DNA and the primers 5'-TGAAGGAGAA GGTGTCTGCG
GGA-3' and 5'-AGGACGGTGC GGTGAGAGTG-3' to generate a 198-bp fragment.
The amplified product was digested with HinfI
restriction enzyme (Life Technologies, Grand Island, NY), which cleaves
only the mutant MTHFR allele into 175- and 23-bp fragments. Restriction
analysis was visualized by agarose gel electrophoresis with ethidium
bromide staining.
Analysis of plasma folates.
For the extraction of plasma folate, an aliquot of plasma (0.2 mL) was
added to 0.8 mL of a high pH (9.4) buffer containing 0.1 mol/L
potassium tetraborate, 2 g/L triton X-100, 1 g/L sodium ascorbate and
10.9 nmol/L of an internal standard, 5-ethyltetrahydrofolic acid
(5-CH2CH3-H4 folic acid),
synthesized in our laboratory (see below). This solution was heated at
100°C for 25 min, then centrifuged at 16,000 x g. We used a Gilson Aspec XL solid phase extractor
(Middletown, WI) to perform the following step sequence: 0.8-mL aliquot
of supernatant was applied to a 1-mL capacity affinity column
containing 0.2 mL Sepharose matrix bound to purified milk folate
binding protein (Selhub et al. 1988
); the affinity
column was washed with a 1 mol/L potassium phosphate solution (pH 7)
and water; folates were eluted with 0.7 mL of 20 mmol/L trifluoroacetic
acid and 1 g/L ascorbic acid into a tube containing 1 mol/L piperazine
(0.01 mL), which allowed neutralization of the eluant to pH 7; 0.5 mL
of eluate was injected onto an ODS Hypersil 250 x 4.6 mm
(Keystone, Bellefonte, PA) analytical column. We used an isocratic
system (30 mmol/L potassium phosphate, 115 mL/L acetonitrile, pH 2.4)
to separate methyl- from ethyltetrahydrofolic acid. Fluorescence
(Applied Biosystems 980 detector, Foster City, CA) was used for
detection because of its high sensitivity for
5-CH3-H4 folic acid (Gregory et al. 1984
). Millenium software (Millipore Corporation, Milford, MA)
was used for peak integration.
Synthesis of 5-ethyltetrahydrofolic acid standard.
The measurement of plasma folates was automated such that the affinity
column eluate was injected immediately onto the analytical column by
the Gilson instrument. Therefore, it was necessary to use an internal
standard that would allow quantification of folate recovery from the
affinity column during the HPLC phase of folate determination. For this
purpose, we synthesized 5-CH2CH3-H4
folic acid, which was detectable by fluorescence and did not coelute
with 5-CH3-H4 folic acid, the predominant
folate form in plasma. One milligram of tetrahydrofolic acid (1 mL of a
2 mmol/L solution) was added to a tube containing acetaldehyde (0.05
mL) and 2-mercaptoethanol (0.02 mL). This solution was cooled on ice;
solid potassium borohydride (25 mg) was added, and the tube was capped
with a rubber stopper in which a syringe needle was inserted. The tube
was incubated at 37°C for 1 h. The solution was cooled, then
neutralized with glacial acetic acid. The newly synthesized
5-CH2CH3-H4 folic acid was purified
by the ion-pair HPLC method described by Selhub (1989)
.
Analysis of urine folates.
Folates were analyzed in urine samples collected at baseline and in the
urine pooled for each subject from the cumulative 7-h collection after
5-HCO-H4 folic acid ingestion. The same affinity/HPLC
principle was used as for plasma folate determination; however, urine
folate analysis was performed using a nonautomated HPLC system to
accommodate a larger sample volume. This larger volume enabled the
detection of post-dose accumulation of 5-HCO-H4 folic
acid by UV, which has weak fluorescence. Compared with fluorescence,
however, the limits of detection for 5-CH3-H4
folic acid by UV light are 1000-fold lower (Bagley and Selhub 1997
, Gregory et al. 1984
), resulting in the
inability to detect folates in the urine samples collected at baseline.
An aliquot of urine (5 mL) was heat-extracted with 5 mL of the same
high pH buffer used for plasma folate analysis. After centrifugation,
the supernatant fraction was mixed with [3H]-folic acid
tracer for quantification of folate recovery (Varela-Moreiras et al. 1992
), then applied onto a 1-mL (bed volume)
sepharose-folate binding protein column. After being washed with 1
mol/L potassium phosphate (pH 7) and water, the column was eluted with
3 mL of an acid solution (20 mmol/L trifluoroacetic acid, 1 g/L
ascorbic acid). The acid eluate was neutralized and a portion (2 mL)
was applied onto a Bio-sil ODS-5S 150 x 4 mm (Bio-Rad,
Richmond, CA) analytical column connected to a Hewlett Packard 1090
HPLC system (Palo Alto, CA). Folate forms were separated using a
tetrabutyl ammonium phosphate (4.5 mmol/L)/sodium chloride (22.7
mmol/L) solution buffered to pH 6.8 with an acetonitrile gradient. In
this HPLC system, individual folate forms are identified on the basis
of retention time and their specific spectra. Folate quantification is
based on peak areas and peak activity coefficient determined for each
folate form (Selhub 1989
). Sample peaks were integrated
by Hewlett Packard Chemstation software v.A.02.02 (Avondale, PA).
Statistical analysis.
The area under the curve (AUC) created by the rise and fall in plasma 5-CH3-H4 folic acid over 7 h after the 5-HCO-H4 folic acid dose was calculated for each subject by the trapezoidal rule. The paired Student’s t test was then used to compare the mean differences in folate status indices, the AUC for each genotype and total urinary 5-CH3-H4 folic acid excreted during the 7-h testing period. ANOVA was used to test for interactions of age or sex with either genotype or the plasma 5-CH3-H4 folic acid AUC after the 5-HCO-H4 folic acid dose. All statistical analyses were performed with Systat 5.2.1 Software for Macintosh (Evanston, IL). Differences were significant at P < 0.05. Values are expressed as means ± SD.
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RESULTS |
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TOPABSTRACTINTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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).
|
); 1 h after the 5-mg oral dose of 5-HCO-H4 folic
acid, the 5-CH3-H4 folic
acid peak height rose fivefold over baseline. The
5-CH3-H4 folic acid peak
eluted at 5.6 min and was equivalent to 20 nmol/L at baseline and
100 nmol/L at 1 h.
|
). The mean increase in the AUC for plasma
5-CH3-H4 folic acid was
424.5 ± 140.3 h·nmol/L in the T/T genotype subjects and 424.1
± 202.4 h·nmol/L in the C/C genotype group.
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). However, both folate forms were detectable in the total volume of
urine collected during the 7 h after the oral
5-HCO-H4 folic acid dose. In this subject, the
cumulative 5-HCO-H4 folic acid peak area at a
retention time of 18.8 min was equivalent to 1.4 µmol, and
the 5-CH3-H4 folic acid
peak area at a retention time of 19.5 min was equivalent to 2.3
µmol.
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). These quantities represent 45% of the biologically active
(6S)-5-HCO-H4 folic acid administered.
Total urinary excretion of 5-HCO-H4 folic acid
accounted for an additional 10% of the total dose. Mean urinary
excretion of 5-HCO-H4 folic acid in the T/T
genotype subjects, 1.0 ± 0.7 µmol, was not different
from that in the C/C genotype subjects, 1.0 ± 0.6
µmol.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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showed that ingestion of
5-HCO-H4 folic acid (2 mg) is associated with a
prompt rise of 5-CH3-H4
folic acid in the portal and peripheral blood. The fact that this rise
in 5-CH3-H4 folic acid was
seen in the portal blood, i.e., before the ingested folate reached the
liver, is evidence that the intestinal tissue has the capacity of
converting 5-HCO-H4 folic acid to
5-CH3-H4 folic acid. This
capacity of the intestine to metabolize folates to the 5-methyl-
derivative has also been demonstrated in ex vivo studies using rat
intestinal tissue (Selhub et al. 1973
, Strum 1977
).
A second study by Nixon and Bertino (1972)
used
5-[14C]-HCO-[3H]-H4
folic acid (0.18–2.65 mg) to show that ingestion of this folate is
associated with the prompt appearance of radioactivity in peripheral
plasma that is proportional to the amount ingested and consists
primarily (90%) of
5-CH3-[3H]-H4
folic acid. These data indicate that in the course of absorption, there
is a loss of the formyl group and subsequent acquisition of a new
one-carbon unit, which is then reduced to the 5-methyl- derivative.
That study also showed that after ingestion of the radiolabeled
5-[14C]-HCO-[3H]-THF,
the pattern of urinary folate excretion is characterized by an initial
rise in
5-[14C]-HCO-[3H]-THF
followed by a rise in
5-CH3-[3H]-H4
folic acid that is proportional to the levels in the plasma
(Nixon and Bertino 1972
). These data indicate that the
source of these urinary folates is the ingested dose.
Two additional studies by McGuire et al. (1987)
and
Straw et al. (1984)
investigated the
pharmacokinetics of orally ingested
(6R,S)-5-HCO-H4 folic acid
at high doses. Assuming that only the natural (6S)-isomer
possesses biologic activity (Keresztesy and Silverman 1951
), they showed that essentially 100% of the stereoactive
(6S)-isomer was absorbed, with >90% of the stereoactive
dose represented in the plasma as
5-CH3-H4 folic acid after
oral ingestion of a 20- to 25-mg dose.
The present study is consistent with these previous studies in that the
oral administration of 5-HCO-H4 folic acid was
associated with a prompt rise of
5-CH3-H4 folic acid
concentrations in both plasma and urine. In plasma, this rise peaked
between 1 and 3 h at which time the concentrations were
approximately fivefold greater than baseline. In the urine,
5-CH3-H4 folic acid and
5-HCO-H4 folic acid were initially undetectable
but were markedly elevated after the oral dose. For the
5-CH3-H4 folic acid, the
amount excreted was 45% of the stereoactive ingested dose. The
amount of 5-HCO-H4 folic acid excreted
represented an additional 10% of the unmetabolized total dose.
Our data showed no difference between the C/C and T/T MTHFR genotypes with respect to the rise of 5-CH3-H4 folic acid concentrations in plasma and urine after the oral dose of 5-HCO-H4 folic acid. This lack of difference between genotypes suggests that the activity of the mutant MTHFR variant was sufficient under the described conditions to prevent us from observing any decrease in the appearance of 5-CH3-H4 folic acid. Several explanations can be used to interpret these results.
One possibility is that the T/T MTHFR variant was protected from
inactivation in the presence of adequate folate substrate
concentrations. Such an interpretation has been derived from recent
epidemiologic studies (Christensen et al. 1997
,
Jacques et al. 1996
, Schwartz et al. 1997
, Verhoef et al. 1997
), which show that
plasma total homocysteine in persons with the T/T MTHFR genotype is
elevated only in those with a low folate status. When folate status is
adequate, total homocysteine levels are not different from those in
persons with the wild-type genotype. Because homocysteine is a
substrate for methylation by
5-CH3-H4 folic acid, the
level of plasma homocysteine in individuals with the T/T MTHFR genotype
serves to indicate whether the T/T MTHFR variant is capable of meeting
the cellular demands for
5-CH3-H4 folic acid. Thus,
when plasma homocysteine in homozygotes for the MTHFR C677T transition
is higher than in control subjects with the wild-type genotype, it
is most likely due to an impairment of activity of the thermolabile
enzyme. In this study, all subjects had adequate folate status as
assessed by plasma and RBC folate; notably, all blood homocysteine
concentrations were normal. Whether the rate of conversion of ingested
5-HCO-H4 folic acid to
5-CH3-H4 folic acid will
differ in homozygotes for the C677T transition with inadequate folate
status remains to be determined.
An alternate explanation is that the 5-mg dose was inadequate (i.e., too high or too low) to discriminate between the two MTHFR phenotypes. However, a recent study in our laboratory (Lathrop et al., unpublished data) showed that compared with the wild-type enzyme, the in vitro activity of the T/T MTHFR variant from extracts of lymphocytes remains depressed at a range of added substrate.
It is also possible that the in vivo expression of the T/T MTHFR
genotype differs among various tissues. We recently determined, in
these same subjects plus seven others, the distribution of folate forms
in RBC (Bagley and Selhub 1998
). In individuals with
wild-type MTHFR, RBC contained exclusively
5-CH3-H4 folic acid
polyglutamates. However in most individuals with the T/T MTHFR variant,
RBC contained, in addition to
5-CH3-H4 folic acid
polyglutamates, formylated tetrahydrofolic acid polyglutamates in a
proportion that ranged from 0 to 58% of total folates. In all
subjects, total RBC folate was determined to be within a normal range
and did not differ between genotypes. These data suggest that
irrespective of folate status, a process occurs during RBC maturation
in which the conversion of folates to
5-CH3-H4 folic acid by the
T/T MTHFR variant becomes impaired. Our present study, which contrasts
with this observation, implies that in those tissues responsible for
the conversion of the ingested 5-HCO-H4 folic
acid to 5-CH3-H4 folic acid
(e.g., intestine and liver), the T/T MTHFR variant does not exhibit
impaired activity. This possibility of a tissue difference in the in
vivo expression of the T/T MTHFR genotype remains to be explored.
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FOOTNOTES |
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3 Abbreviations used: AUC, area under the concentration curve by time; C/C, homozygous for the wild-type allele of the methylenetetrahydrofolate reductase gene; 5-CH2CH3-H4 folic acid, 5-ethyltetrahydrofolic acid; 5-CH3-H4 folic acid, 5-methyltetrahydrofolic acid; C677T, cytosine to thymidine transition at nucleotide 677; 5-HCO-H4 folic acid, (6R,S)-5-formyltetrahydrofolic acid; MTHFR, methylenetetrahydrofolate reductase; T/T, homozygous genotype for the C677T transition in the MTHFR gene.
Manuscript received February 1, 2000. Initial review completed March 6, 2000. Revision accepted May 22, 2000.
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TOPABSTRACTINTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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