Clinica Chimica Acta 398 (2008) 134139

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Clinica Chimica Acta

j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / c l i n c h i m

Relationships between gene polymorphisms of folate-related proteins and vitamins

and metabolites in pregnant women and neonates
Fernanda R. Lopreato a, Sally P. Stabler c, Felipe R. Carvalho a, Rosario D.C. Hirata a, Mario H. Hirata a,
Dbora L. Robi a, Luiz F. Sampaio-Neto b, Robert H. Allen c, Elvira M. Guerra-Shinohara a,
a
b
c

Departamento de Anlises Clnicas e Toxicolgicas, Faculdade de Cincias Farmacuticas da Universidade de So Paulo, Brazil
Faculdade de Medicina, Pontifcia Universidade Catlica de So Paulo, Brazil
Department of Medicine, University of Colorado Health Sciences Center, Denver, CO, United States

a r t i c l e

i n f o

Article history:
Received 23 March 2008
Received in revised form 1 September 2008
Accepted 4 September 2008
Available online 11 September 2008
Keywords:
Cobalamin
Folate
Polymorphisms
Homocysteine
Pregnant women
Neonates

a b s t r a c t
Background: The methylenetetrahydrofolate reductase (MTHFR), glutamate carboxypeptidase II (GCPII)
and reduced folate carrier (RFC1) gene polymorphisms were associated with folate status. We investigated
the effects of these polymorphisms on serum folate (SF) and folate-related metabolites in mothers and their
neonates.
Methods: Cobalamin (Cbl), SF, total homocysteine (tHcy), methylmalonic acid (MMA), S-adenosylmethionine
(SAM), and S-adenosylhomocysteine (SAH) were measured in 275 healthy women and their neonates.
MTHFR C677T, GCPII C1561T and RFC1 A80G polymorphisms were determined by PCR-RFLP.
Results: Maternal tHcy was affected individually by MTHFR C677T and GCPII C1561T polymorphisms and by
combined genotypes MTHFR 677TT/GCPII 1561CC and MTHFR 677TT/RFC1 80AG. The MTHFR and RFC1
polymorphisms were not associated with variations in vitamins or SAM, SAH and MMA in neonates. Neonatal
tHcy was predicted directly by maternal tHcy and inversely by maternal SF, neonatal Cbl and neonatal RFC1
80G allele (AG+GG genotypes). Maternal MMA and SAM/SAH were predicted by creatinine and Cbl,
respectively. Neonatal MMA was predicted by maternal MMA and GCPII 1561T allele (CT+TT genotypes) and
by neonatal Cbl.
Conclusions: Maternal tHcy was affected by MTHFR C677T, RFC1 A80G and GCPII C1561T polymorphisms.
Maternal GCPII C1561T variant was associated with neonatal MMA. Neonatal RFC1 A80G polymorphism
inuenced tHcy in neonates.
2008 Elsevier B.V. All rights reserved.

1. Introduction
Folate and cobalamin (Cbl) are essential for nucleic acid synthesis
and are required for homocysteine metabolism and methylation
reactions [1]. Deciencies of these vitamins could impair maternal and
neonatal metabolisms [2]. Furthermore, maternal folate and Cbl
deciencies have been associated with abnormal pregnancy outcomes
and increased risk for birth defects [3].
Folates in nature are present as a mixture of pteroylglutamates that
differ in oxidation state, in one-carbon substitutions of pteridine
ring and by the number of glutamate residues attached to the paminobenzoylglutamate moiety [4]. Intestinal absorption of polyglutamyl folates involves cleavage of the glutamate side chain by folylpoly-glutamate carboxypeptidase (FGCP), an enzyme that is anchored to the
intestinal brush border membrane and it is encoded by the GCPII gene
Corresponding author. Department of Clinical Chemistry and Toxicology, Faculty of
Pharmaceutical Science, University of Sao Paulo, Av. Prof. Lineu Prestes, 580. CEP 05508900. Sao Paulo, SP, Brazil. Fax: +55 11 3813 2197.
E-mail address: emguerra@usp.br (E.M. Guerra-Shinohara).
0009-8981/$ see front matter 2008 Elsevier B.V. All rights reserved.
doi:10.1016/j.cca.2008.09.004

[5]. The monoglutamyl folate derivative is transported across the intestinal brush border by the reduced folate carrier (RFC1) that is a very
efcient transporter with high afnity to 5-methyltetrahydrofolate [6].
Two common single nucleotide polymorphisms (SNPs) in the GCPII
and RFC1 genes have been associated with the differences in folate
absorption and the transport through the cells affecting the bioavailability of the dietary folate [5,7].
The CGPII C1561T (H475Y) is a single nucleotide polymorphism
(SNP) located in the putative catalytic region of the FGCP enzyme and
GCPII 1561T allele has been associated with lower serum folate and
higher total homocysteine (tHcy) concentrations [5]. Nevertheless,
this nding was not conrmed in other studies [811]. In other reports
the GCPII 1561T allele was associated with high serum or red cell folate
concentrations [7,1214].
The RFC1 A80G SNP was associated with reduced RFC1-mediated
transport of folate through cell membranes that impairs the absorption of dietary folate [15]. It has been shown that infants with the RFC1
80GG genotype have 2.56-fold increased risk of neural tube defects
(NTDs) when compared to the AA genotype [16]. In addition, neonates
carrying the RFC1 80GG genotype whose mothers did not use

F.R. Lopreato et al. / Clinica Chimica Acta 398 (2008) 134139

periconceptional vitamin supplements containing folic acid had an

increased risk of neural tube defects (NTD) [16,17].
Folate homeostasis is also regulated by methylenetetrahydrofolate
reductase (MTHFR) that catalyses the conversion of 5,10 methylenetetrahydrofolate into 5-methyltetrahydrofolate, which provides the
carbon moiety for conversion of homocysteine into methionine. The
TT genotype of MTHFR C677T SNP has been associated with reduced
enzyme activity and increased tHcy concentrations [1821] and risk of
NTDs [2224].
The effects of GCPII C1561T and RFC1 A80G SNPs on folate and
folate-related metabolite concentrations in pregnant women and their
neonates remain to be elucidated. GCPII and RFC1 variants may affect
the folate bioavailability of the embryo development through the
placenta. This study investigated the relationship between MTHFR,
GCPII and RFC1 common polymorphisms and serum folate (SF), cobalamin (Cbl), total homocysteine (tHcy), methylmalonic acid (MMA),
S-adenosylmethionine (SAM), and S-adenosylhomocysteine (SAH)
concentrations in mothers and their neonates.
2. Materials and methods

135

2.2. Blood sampling and biochemical measurements

Blood was drawn using tubes containing K3EDTA for genetic analysis. An additional
sample of blood was collected into tubes without anticoagulant to measure Cbl, folate,
creatinine, AST, ALT and metabolites (tHcy, MMA, SAM and SAH). The blood samples were
cooled immediately at 4C and centrifuged within 1 h. The serum was frozen at 80 C and
shipped to the US on dry ice. The determination of metabolites was performed at the
University of Colorado Health Sciences Center, and the remaining analyses were performed
in the Hematology Laboratory, at the University of Sao Paulo, Brazil.
Serum folate concentration was determined by the ion capture method (IMx
System, Abbott Laboratories, Abbott Park, IL) and by the chemiluminescent method
(Immulite, DPC Medlab, Los Angeles, CA). Creatinine, AST and ALT were analyzed using
commercial kits (Dimension AR, Dade Behringer, Marburg, Germany). The measurements of tHcy and MMA were performed using stable isotope dilution capillary gas
chromatography/mass spectrometry [26,27]. SAM and SAH were measured using a
stable isotope dilution liquid chromatography/mass spectrometry method [28].
2.3. Genetic analysis
Genomic DNA was isolated from whole blood [29]. Genotyping for each
polymorphism was accomplished by PCR-RFLP as described previously for MTHFR
C677T [18], GCPII C1561T [5], and RFC1 A80G [30] using, respectively, the restriction
enzymes HinfI, AccI and HhaI. Restriction fragments were separated by agarose (2%) and
polyacrilamide (15%) gel electrophoresis. In addition, 20% of genetic analyses were
randomly repeated.

2.1. Subjects
2.4. Statistical analysis
Blood was collected from 405 pregnant women at 2 public hospitals in Sorocaba
City, Brazil, after admission for labor and up to 12 h before delivery, from April to May in
2001 and 2003. Neonate blood samples were collected directly from placenta veins
right after expulsion of the placenta.
All deliveries occurred within gestational age range of 3742 wk. We excluded 28
maternalfetus pairs because of one or more of the following reasons: metabolic
diseases, renal insufciency, multiple gestations, increased serum liver enzymes,
hemoglobinopathies (HbS and HbC trait, increased HbA2 and HbF concentrations), and
complications during the delivery, including the birth of 1 newborn with congenital
malformation. Women who took supplements during pregnancy (N = 94) and those
with no information about the use of supplements (N = 8) were also excluded from this
study.
Socioeconomic data (monthly per capita of family, schooling and occupation),
ethnic group, reproductive history, and vitamin supplement intake were obtained
through an interview. The group of pregnant women was separated in 3 sub-groups
(Caucasian, African and Asian-descents) according to ethnic morphological criteria, as
previously published [20,21,25]. The newborn's birth weight, height and Apgar score
were collected. The study protocol was approved by the Investigational Review Board of
both University of So Paulo (Brazil) and the University of Colorado, and written
informed consent was obtained from all participants prior to entering the study.

HardyWeinberg equilibrium test was determined for all genotypes using the Chi
square test. ShapiroWilk test was used to study if the continuous variables followed a
normal distribution. The distributions of serum Cbl, serum folate, tHcy, SAM, SAH and
MMA were all skewed. Antilog transformation was used to obtain geometric mean and
95% CI of vitamins and metabolite concentrations.
One-way analysis of covariance adjusted by age, number of deliveries, ethnic group
and monthly per capita income (ANCOVA) was used to compare the maternal
biochemical data from groups formed according to genotypes individually or in
combination of genotypes for MTHFR C677T, GCPII C1561T and RFC1 A80G polymorphisms. The effects of the variables: age, number of deliveries, ethnic group and monthly
per capita income on vitamin and metabolite concentrations were shown to be
signicant in a previous study [26]. However, especially for statistical analyses using the
maternal genotypes for RFC1 A80G and GCPII C1561T (individually or in combination of
genotypes), the ANCOVA was adjusted by the same covariates including the maternal
genotypes for MTHFR C677T polymorphism (CT+TT vs CC genotype). In multiple comparisons, TukeyKramer post-test was performed to determine signicant differences
among groups.
The ANOVA was used for comparing the effects of neonatal genotypes for MTHFR
C677T and the combination of genotypes of this polymorphism with GCPII C1561T or

Table 1
Geometric means (95% CI) of vitamin and metabolite concentrations according to genotypes for MTHFR C677T, GCPII C1561T and RFC1 A80G gene polymorphisms in mothers

Maternal genotypes
MTHFR C677T
CC
CT+TT
ANCOVA P value
GCPII C1561T
CC
CT+TT
ANCOVA P value
RFC1 A80G
AA
AG+GG
ANCOVA P value

Cbl (pmol/L)

Serum folate (nmol/L)

tHcy (mol/L)

SAM (nmol/L)

SAH (nmol/L)

SAM/SAH

MMA (nmol/L)

145 (137, 153)

132
140 (132, 149)
143
NS

13.7 (12.8, 14.6)

132
11.5 (10.8, 12.3)
141
0.002

6.3 (6.0, 6.7)

132
7.3 (6.8, 7.8)
142
0.001

82.9 (77.3, 88.7)

129
81.4 (77.3, 85.7)
139
NS

19.6 (18.3, 21.0)

129
18.9 (17.8, 20.0)
141
NS

4.2 (3.8, 4.7)

129
4.3 (4.0, 4.7)
139
NS

229 (210, 248)

116
237 (218, 259)
128
NS

143 (136, 149)

254
152 (138, 169)
16
NS

12.4 (11.8, 13.0)

252
14.3 (11.3, 18.3)
16
NS

6.9 (6.6, 7.2)

253
5.4 (4.7, 6.2)
16
0.027

81.8 (78.2, 85.4)

247
84.2 (71.8, 98.6)
16
NS

19.1 (18.2, 20.0)

249
19.6 (17.0, 22.6)
16
NS

4.3 (4.0, 4.6)

247
4.3 (3.4, 5.4)
16
NS

234 (220, 249)

225
229 (180, 293)
14
NS

145 (133, 158)

66
141 (135, 148)
209
NS0.839

13.7 (12.3, 15.2)

64
12.2 (11.5, 12.9)
209
NS

6.4 (5.9, 6.9)

65
7.0 (6.6, 7.3)
209
NS

80.0 (73.5, 87.2)

63
82.7 (78.8, 86.9)
205
NS

18.3 (16.6, 20.1)

65
19.5 (18.6, 20.5)
205
NS

4.4 (3.9, 5.0)

63
4.2 (3.9, 4.6)
205
NS

225 (201, 251)

56
236 (219, 253)
188
NS

All values are geometric mean; 95% CIs in parentheses.

ANOVA performed on the log-transformed variables.
The maternal genotypes for gene polymorphisms were tested with maternal biochemical variables and the models were adjusted by covariates: age, ethnic group, number of
deliveries and per capita income. Specially, for statistical analyses using the maternal genotypes for GCPII C1561T and RFC1 A80G gene polymorphisms, the models were adjusted by
the same covariates including the maternal genotypes for MTHFR C677T gene polymorphism (CT+TT vs CC).

136

F.R. Lopreato et al. / Clinica Chimica Acta 398 (2008) 134139

RFC1 A80G polymorphisms on vitamin and metabolite concentrations of neonates. The

ANCOVA was adjusted by genotypes for the MTHFR C677T polymorphism in models of
neonatal data that included genotypes for RFC1 A80G and GCPII C1561T individually or
in combination genotypes for the two polymorphisms.
The correlations between the maternal and neonatal concentrations of cobalamin,
serum folate, tHcy, MMA, SAM and SAH were determined by Pearson coefcient. To
assess the simultaneous relations between the various predictors of tHcy, MMA and
SAM/SAH ratio in mothers (as dependent variables), three models of stepwise multiple
linear regression analysis were used. The independent variables were: Cbl, serum folate,
creatinine, tHcy, SAM/SAH ratio, MMA and genotypes for MTHFR C677T, GCPII C1561T
and RFC1 A80G polymorphisms. The reference genotypes were: CC for MTHFR C677T,
CC for GCPII C1561T and AA for RFC1 A80G polymorphisms. The models were adjusted
by covariates: age, number of deliveries, ethnic group (Caucasian-descent vs Africandescent) and per capita income.
For neonatal dependent variables (tHcy, MMA and SAM/SAH), nine models of
stepwise multiple linear regression analysis were evaluated. In models 1, 4 and 7, the
independent variables were maternal Cbl, serum folate, creatinine, tHcy, genotypes CT+
TT vs CC for MTHFR C677T polymorphism; genotypes CT+TT vs CC for GCPII C1561T

polymorphism; and genotypes AG+GG vs AA for RFC1 A80G polymorphism. In models

2, 5 and 8, the independent variables were from neonates (Cbl, serum folate, genotypes
CT+TT vs CC for MTHFR C677T polymorphism; genotypes CT+TT vs CC for GCPII C1561T
polymorphism; and genotypes AG+GG vs AA for RFC1 A80G polymorphism). In models
3, 6 and 9, the independent variables were the maternal and neonatal variables used in
models 1 and 2. All statistical analyses were carried out using statistical analysis
software (Statistical Analysis System for Windows, ver. 8.02 SAS Institute Inc, Cary, NC),
with the level of signicance set at P b 0.05.

3. Results
3.1. Population characteristics
Of the 405 eligible subjects, 275 pregnant women met the entry
criteria of this study. Detailed descriptions of the pregnant women's
characteristics were published in a previous study [21]. Women with

Table 2
Geometric means (95% CI) of vitamin and metabolite concentrations according to combination of genotypes for MTHFR C677T and GCPII C1561T; and MTHFR C677T and RFC1 A80G;
and GCPII C1561T and RFC1 A80G gene polymorphisms in mothers
Cobalamin (pmol/L)

Serum folate (nmol/L)

tHcy (mol/L)

SAM (nmol/L)

SAH (nmol/L)

SAM/SAH ratio

MMA (nmol/L)

MTHFR C677T/GCPII C1561T

CC/CC
144 (136, 154)
122
CC/CT+TT
146 (122, 176)
8
CT/CC
145 (135, 156)
107
CT/CT+TT
166 (141, 196)
5
TT/CC
123 (110, 138)
25
TT/CT+TT
147 (89, 243)
3
ANCOVA P value
NS

13.5 (12.6, 14.5)a

122
16.9 (12.9, 22.2)a
8
12.0 (11.1, 13.0)a,b
105
14.2 (7.2, 27.9)a,b
5
9.6 (8.3, 11.3)b
25
9.4 (3.1, 28.5)a,b
3
0.001

6.3 (6.0, 6.7)a

122
5.7 (4.4, 7.3)a
8
7.1 (6.6, 7.5)a
106
5.3 (3.6, 7.8)a
5
9.6 (7.5, 12.3)b
25
4.9 (3.7, 6.4)a
3
b0.001

83.1 (77.2, 89.5)

119
79.3 (65.7, 95.7)
8
82.6 (77.9, 87.6)
104
79.2 (60.4, 103.8)
5
72.1 (65.8, 79.0)
24
109.2 (30.2, 394.5)
3
NS

19.5 (18.1, 21.0)

119
20.4 (15.9, 26.2)
8
19.2 (17.9, 20.5)
106
17.9 (13.2, 24.3)
5
17.1 (15.1, 19.3)
24
20.4 (10.5, 39.4)
3
NS

4.3 (3.8, 4.7)

119
3.9 (2.8, 5.4)
8
4.3 (4.0, 4.7)
104
4.4 (3.1, 6.3)
5
4.2 (3.6, 5.0)
24
5.4 (0.8, 35.4)
3
NS

231 (212, 252)

108
199 (138, 289)
6
233 (210, 257)
97
217 (123, 382)
5
257 (202, 327)
20
333 (126, 884)
3
NS

MTHFR C677T/RFC1 A80G

CC/AA
148 (132, 167)
38
CC/AG
142 (131, 153)
63
CC/GG
146 (129, 166)
31
CT/AA
147 (127, 170)
21
CT/AG
143 (128, 160)
54
CT/GG
144 (128, 162)
40
TT/AA
126 (86, 184)
7
TT/AG
120 (105, 136)
14
TT/GG
137 (112, 169)
7
ANCOVA P value
NS

14.4 (12.6, 16.5)a

38
13.4 (12.1, 14.9)a
63
13.3 (11.7, 15.2)a.b
31
13.3 (10.7, 16.7)a.b
19
12.0 (10.7, 13.6)a.b
54
11.5 (10.3, 12.8)a.b
40
10.8 (7.8, 15.0)a.b
7
10.3 (8.3, 12.7)a.b
14
7.5 (5.5, 10.3)b
7
0.006

6.3 (5.6, 6.9)a

38
6.3 (5.7, 6.8)a
63
6.5 (5.7, 7.3)a
31
6.4 (5.6, 7.4)a
20
6.9 (6.2, 7.6)a
54
7.4 (6.7, 8.2)a,b
40
6.8 (4.6, 10.1)a,b
7
10.5 (7.0, 15.8)b
14
8.4 (5.4, 13.1)a,b
7
b0.001

77.4 (68.3, 87.9)

38
86.2 (79.1, 94.0)
61
83.2 (68.9, 100.4)
30
81.0 (72.8, 90.2)
18
84.7 (77.6, 92.2)
54
81.4 (73.0, 90.8)
40
92.4 (67.0, 127.6)
7
70.1 (60.5, 81.1)
13
70.8 (58.3, 86.1)
7
NS

19.5 (17.0, 22.2)

38
19.8 (17.7, 22.0)
61
19.5 (17.3, 21.9)
30
17.0 (14.6, 19.8)
20
19.6 (17.9, 21.3)
54
20.1 (17.6, 22.9)
40
16.0 (10.9, 23.5)
7
17.9 (15.3, 20.9)
13
18.0 (15.1, 21.4)
7
NS

4.0 (3.3, 4.7)

38
4.4 (3.8, 5.1)
61
4.3 (3.4, 5.4)
30
4.9 (4.1, 5.8)
18
4.3 (3.9, 4.8)
54
4.1 (3.4, 4.8)
40
5.8 (3.5, 9.5)
7
3.9 (3.1, 5.0)
13
3.9 (3.5, 4.4)
7
NS

223 (192, 260)

34
237 (208, 269)
56
219 (184, 260)
26
210 (178, 248)
17
249 (216, 287)
50
219 (184, 262)
38
295 (160, 543)
5
274 (197, 381)
12
228 (130, 399)
6
NS

GCPII C1561T/RFC1 A80G

CC/AA
143 (131, 158)
60
CC/AG
141 (132, 150)
120
CC/GG
145 (133, 157)
74
CT+TT/AA
169 (143, 200)
5
CT+TT/AG
142 (119, 170)
8
CT+TT/GG
155 (106, 226)
3
ANCOVA P value
NS

13.2 (11.8, 14.7)

58
12.4 (11.5, 13.4)
120
11.9 (10.9, 13.0)
74
19.1 (11.1, 32.6)
5
14.2 (10.4, 19.5)
8
9.1 (3.4, 24.4)
3
NS

6.5 (6.0, 7.1)

59
7.0 (6.4, 7.5)
120
7.1 (6.6, 7.7)
74
4.6 (3.6, 5.9)
5
5.7 (4.4, 7.4)
8
6.0 (3.8, 9.7)
3
NS

79.1 (72.5, 86.4)

57
83.1 (78.3, 88.1)
117
81.7 (74.3, 90.0)
73
96.3 (54.7, 169.3)
5
83.4 (71.7, 97.1)
8
68.9 (44.7, 106.1)
3
NS

18.4 (16.6, 20.4)

59
19.2 (17.9, 20.5)
117
19.6 (18.0, 21.3)
73
16.6 (13.2, 20.8)
5
20.8 (16.0, 27.1)
8
21.9 (16.1, 29.7)
3
NS

4.3 (3.8, 4.9)

57
4.3 (4.0, 4.8)
117
4.2 (3.7, 4.8)
73
5.8 (3.2, 10.7)
5
4.0 (2.9, 5.6)
8
3.2 (2.6, 3.8)
3
NS

232 (207, 261)

50
245 (223, 270)
109
217 (192, 245)
66
180 (112, 290)
5
263 (184, 375)
6
261 (60, 1138)
3
NS

All values are geometric means; 95% CIs in parentheses.

ANCOVA performed on the log-transformed variables. The models were adjusted by covariates: age, ethnic group, number of deliveries and per capita income. The statistical
analyses made using the haplotypes for GCPII C1561T and RFC1 A80G gene polymorphisms were adjusted by the same covariates including the maternal genotypes for MTHFR C677T
polymorphism (CT+TT vs CC).
Values in a row with different superscript letters are signicantly different, P b 0.05 (Tukey's test).

F.R. Lopreato et al. / Clinica Chimica Acta 398 (2008) 134139

renal disease were excluded from this study. The mean SD of serum
creatinine concentrations was 0.61 0.12 mg/dl.
The information about the weight, height and gender was not
available for 6 neonates. The mean SD of weight and height of the
neonates were 3209 458 g and 48.2 2.2 cm, respectively. The
frequencies of male and female neonates were, respectively, 46.8 and
53.2%. The bloods of fty four neonates were not collected because of
the loss of placentas.
The maternal and neonatal genotype distributions of the MTHFR
C677T, GCPII C1561T and RFC1 A80G were in HardyWeinberg
equilibrium (P N 0.05). The allele frequencies for MTHFR 677T, GCPII
1561T and RFC1 80G were, respectively, 31.1, 3.1 and 52.2% in mothers
and 28.3, 2.7 and 59.2% in neonates (data not shown).

Table 4
Association between maternal and neonatal biochemical and genetic variables and total
homocysteine (models 1, 2 e 3), methylmalonic acid (models 4, 5 and 6), and the ratio of
S-adenosylmethionine to S-adenosylhomocysteine (models 7, 8 and 9) in neonates by
stepwise multiple linear regression analysis
Models Dependent
variables (n)a
1

2
3

3.2. Effects of gene polymorphisms on vitamin and metabolite concentrations

Pregnant women carrying the MTHFR 677T allele (CT+TT genotypes) had lower serum folate levels and higher tHcy concentrations
than CC genotype carriers (Table 1). However, these alterations were
not observed in neonates carrying the MTHFR 677T allele (data not
shown).
Higher tHcy was found in pregnant women carrying the GCPII
1561CC genotype when compared with those carrying the GCPII 1561T
allele (CT+TT genotypes) (Table 1). No difference was found in neonatal
Cbl, serum folate, tHcy, SAM, SAH and MMA concentrations according
to genotypes for GCPII C1561T polymorphism (data not shown).
Alterations in vitamin and metabolite concentrations were not
associated with the RFC1 A80G SNP in pregnant women (Table 1) and
neonates (data not shown).
The effects of combined genotypes for MTHFR C677T and GCPII
C1561T, MTHFR C677T and RFC1 A80G, and RFC1 A80G and GCPII
C1561T on vitamin and metabolite concentrations in mothers are
shown in Table 3. The ANCOVA adjusted by covariates (age, number of
deliveries, ethnic group and monthly per capita income) showed that
pregnant women with the combination of genotypes MTHFR 677TT/
GCPII 1561CC had lower serum folate concentration than those with
677CC/1561CC and 677CC/1561CT+TT. In addition, higher tHcy
concentration was found in pregnant women with 677TT/1561CC
genotypes when compared with the other combinations of genotypes
(Table 2). No difference was observed between neonatal vitamin and
metabolite concentrations according to neonatal combination genotypes for MTHFR C677T and GCPII C1561T variants (data not shown).
The mothers with the combination of genotypes MTHFR 677TT/
RFC1 80GG had lower serum folate concentration than those with

Table 3
Association between biochemical variables and genotypes for MTHFR C677T, GCPII
C1561T and RFC1 A80G gene polymorphisms as independent variables in three models
of multiple linear regression analysis with stepwise
Models

Dependent
variables (n)a

Independent variablesa

Beta (EP)

Partial
R2

P
value

Maternal tHcy
(n = 258)

1.36 (0.15)
0.35 (0.05)
0.04 (0.02)

0.1518
0.0139

b0.001
b0.001
0.034

Maternal MMA
(n = 231)
Maternal SAM/
SAH (n = 252)

Intercept
Maternal serum folate
Maternal MTHFR 677T
allele (CT+TT genotypes)
Intercept
Maternal creatinine
Intercept
Maternal Cbl

3.10 (0.20)
0.39 (0.16)
0.31 (0.26)
0.22 (0.09)

0.0241

0.0205

b0.001
0.015
NS
0.021

The three models were adjusted by maternal age, ethnic group, number of deliveries
and per capita income.
B = regression parameter estimate; SE = standard error (SE); Cbl: cobalamin; tHcy: total
homocysteine; SAM: S-adenosylmethionine; SAH: S-adenosylhomocysteine.
Independent maternal variables: Cbl, serum folate, creatinine, genotypes CT+TT vs CC
for MTHFR C677T polymorphism; genotypes CT+TT vs CC for GCP2C1561T
polymorphism; and genotypes AG+GG vs AA for RFC1 A80G polymorphism.
a
Log transformed variable.

137

8
9

Neonatal tHcy
(n = 217)

Independent variablesa

Intercept
Maternal tHcy
Maternal Cbl
Maternal serum folate
Neonatal tHcy Intercept
(n = 115)
Neonatal Cbl
Neonatal tHcy Intercept
(n = 112)
Maternal tHcy
Maternal serum folate
Neonatal Cbl
Neonatal RFC1 80G allele
(AG+GG genotypes)
Neonatal MMA Intercept
(n = 193)
Maternal MMA
Maternal GCPII 1561T
allele (CT+TT genotypes)
Maternal Cbl
Neonatal MMA Intercept
(n = 114)
Neonatal Cbl
Neonatal serum folate
Neonatal MMA Intercept
(n = 112)
Maternal MMA
Neonatal Cbl
Maternal GCPII 1561T
allele (CT+TT genotypes)
Neonatal SAM/ Intercept
SAH (n = 113)
Maternal SAM/SAH
Maternal serum folate
Neonatal SAM/ Intercept
SAH (n = 115)
Neonatal serum folate
Neonatal SAM/ Intercept
SAH (n = 113)
Maternal SAM/SAH
Maternal tHcy
Neonatal serum folate

Beta (EP)

Partial R2 P value

0.63 (0.13)
0.56 (0.05)
0.11 (0.05)
0.09 (0.04)
2.90 (0.34)
0.21 (0.06)
1.99 (0.34)
0.57 (0.06)
0.12 (0.05)
0.17 (0.05)
0.13 (0.06)

0.4632
0.0156
0.0101

0.0918

0.4290
0.0836
0.0372
0.0192

b 0.001
b 0.001
0.035
0.041
b 0.001
b 0.001
b 0.001
b 0.001
0.028
b 0.001
0.028

1.61 (0.16)
0.53 (0.04) 0.4896
0.11 (0.04) 0.0280

b 0.001
b 0.001
0.001

0.14 (0.05)
6.17 (0.47)
0.23 (0.07)
0.26 (0.08)
3.44 (0.43)
0.54 (0.05)
0.14 (0.05)
0.23 (0.09)

0.0172

0.0842
0.0749

0.5051
0.0295
0.0287

0.009
b 0.001
0.001
0.002
b 0.001
b 0.001
0.007
0.014

1.28 (0.17)
0.42 (0.06)
0.23 (0.08)
0.41 (0.32)
0.28 (0.09)
0.71 (0.30)
0.38 (0.06)
0.24 (0.08)
0.17 (0.08)

0.2717
0.0506

0.0802

0.2717
0.0506
0.0296

b 0.001
b 0.001
0.004
0.201
0.002
0.021
b 0.001
0.003
0.026

B = regression parameter estimate; SE = standard error (SE); Cbl: cobalamin; tHcy: total
homocysteine; SAM: S-adenosylmethionine; SAH: S-adenosylhomocysteine.
Model 1: independent maternal variables used: Cbl, serum folate, creatinine, tHcy,
genotypes CT+TT vs CC for MTHFR C677T gene polymorphism; genotypes CT+TT vs CC
for GCP2C1561T gene polymorphism; and genotypes AG+GG vs AA for RFC1 A80G gene
polymorphism.
Model 2: Independent neonatal variables used: Cbl, serum folate, genotypes CT+TT vs
CC for MTHFR C677T gene polymorphism; genotypes CT+TT vs CC for GCP2C1561T gene
polymorphism; and genotypes AG+GG vs AA for RFC1 A80G gene polymorphism.
Model 3: independent variables are maternal and neonatal variables in models 1 and 2.
Model 4: independent maternal variables used in model 1.
Model 5: independent neonatal variables used in model 2.
Model 6: independent variables are maternal and neonatal variables in models 1 and 2.
Model 7: independent maternal variables used in model 1.
Model 8: independent neonatal variables used in model 2.
Model 9: independent variables are maternal and neonatal variables in models 1 and 2.
a
Log transformed variable.

CC/AA and CC/AG genotypes (Table 2). Higher values of tHcy were
found in pregnant women carrying the combination of genotypes TT/
AG when compared with CC/AA, CT/AA, CC/AG and CT/AG carriers
(Table 2). However, the combination of genotypes for MTHFR C677T
and RFC1 A80G polymorphisms was not associated with any alterations in vitamin and metabolite concentrations in neonates (data not
shown).
No alterations in vitamin and metabolite concentrations were
found in mothers according to combinations of genotypes for GCPII
C1561T/RFC1 A80G polymorphisms (Table 2). Neonates carrying GCPII
1561CT/RFC1 80AA had higher serum folate concentrations than other
combinations of genotypes (data not shown). However, no alterations
in Cbl and metabolite concentrations were found in neonates according to combinations of genotypes for GCPII C1561T and RFC1 A80G
polymorphisms (data not shown).

138

F.R. Lopreato et al. / Clinica Chimica Acta 398 (2008) 134139

3.3. Biochemical and genetic determinants for tHcy, MMA and SAM/SAH
ratio
Strong correlations were observed between the following maternal
and neonatal variables cobalamin (r = 0.553, P b 0.001), tHcy (r = 0.680,
P b 0.001) and MMA (r = 0.700, P b 0.001). Weaker correlations were
observed between maternal and neonatal serum folate (r = 0.180,
P = 0.009), SAM (r = 0.254, P = 0.002), SAH (r = 0.365, P b 0.001) and
SAM/SAH (r = 0.439, P b 0.001).
Results from the multiple linear regression analysis with stepwise
criteria of maternal genetic and metabolic variables are shown in
Table 3. Maternal serum folate and MTHFR 677T allele (CT+TT
genotypes) were, respectively, inverse and direct predictors for tHcy
concentrations. The maternal MMA concentrations were predicted
only by maternal serum creatinine (partial R2 = 0.0241, P = 0.015).
Maternal SAM/SAH was predicted by maternal cobalamin concentration (P = 0.021).
Neonatal variables were also analyzed by multiple linear regression analysis with stepwise criteria, as shown in Table 4. Maternal
tHcy, maternal cobalamin and maternal serum folate concentrations
predicted the neonatal tHcy concentrations (Model 1, Table 4). In
model 2, which tested only neonatal independent variables, the
neonatal cobalamin was inversely associated with neonatal tHcy
concentrations. Model 3 explores the maternal and neonatal independent variables from models 1 and 2. Model 3 showed that
maternal tHcy was directly associated with neonatal tHcy concentrations. However, maternal serum folate, neonatal Cbl and neonatal
RFC1 80G allele (AG+GG genotypes) were inversely associated with
neonatal tHcy concentrations (Table 4).
The predictors of neonatal MMA concentrations were maternal
MMA, maternal GCPII 1561T allele (CT+TT genotypes) and maternal
cobalamin concentrations in model 4, neonatal cobalamin and
neonatal serum folate in model 5. In model 6, the maternal MMA
was associated directly and neonatal cobalamin concentrations and
maternal genotypes CT+TT for GCPII polymorphism) were associated
inversely with neonatal MMA (Table 4).
The determinants for neonatal SAM/SAH were maternal SAM/SAH
and maternal serum folate concentration in model 7, neonatal serum
folate concentration in model 8. In model 9, the maternal SAM/SAH,
maternal tHcy and neonatal serum folate concentrations were
selected as determinants for neonatal SAM/SAH (Table 4).
4. Discussion
This is the rst study to examine a possible interrelationship of
folate, cobalamin and the vitamin dependent metabolite concentrations with GCPII and RFC1 common variants in healthy mothers and in
their neonates. Serum MMA was used as a marker of impaired
cobalamin status and the serum tHcy and SAM/SAH ratio were also
used as markers of folate and cobalamin deciencies.
The frequencies of the MTHFR 677T and GCPII 1561T alleles in
mothers and neonates were similar to those reported in different
populations [7,9,10,11,19,22,24,30].
In the present study, the MTHFR 677T allele was associated with
low serum folate and high tHcy concentration in the mothers,
however, these effects were not found in neonates carrying MTHFR
677T allele. In neonates, tHcy concentration was predicted directly by
maternal homocysteine and inversely by maternal serum folate
concentrations and neonatal Cbl concentrations and RFC1 80G allele
(AG+GG genotypes), but not by MTHFR G677T variant. It is noteworthy
that the active transport of folate and cobalamin through the placenta
is consistent with higher concentrations of these vitamins in the fetus
than in the mother. Therefore, the fetus may be able to metabolize
homocysteine more efciently than the mother, despite the overwhelmingly greater blood volume of the mother and therefore
independent of the presence of the MTHFR 677T allele [31].

It has been suggested that homocysteine activates the heterogeneous nuclear ribonucleoprotein E1 that triggers biosynthesis and
translational up-regulation of folate receptors [32]. Therefore, the upregulation of folate receptors by homocysteine may be what maintains
the adequate folate levels in the fetus. However, it remains to be
demonstrated whether human placental folate receptors are also upregulated during folate deciency in vivo [33].
The lack of association between the RFC1 A80G polymorphism and
changes in serum folate and tHcy concentrations in mothers and
neonates, when the polymorphism was evaluated alone, may not be
limited to the Brazilian population, since frequencies of the polymorphism studied in our population are similar to those for other
populations [11,15,17].
The MTHFR 677TT and RFC1 80GG and AG combined genotypes
were associated with reduced maternal folate or increased tHcy,
respectively, in this study. The interaction effect of MTHFR 677TT/RFC1
80GG genotypes on tHcy concentrations was previously described in
unselected subjects from France [15] and in healthy volunteers from
the United Kingdom [11]. Even though the RFC1 polymorphism was
not selected as a predictor of maternal tHcy by multiple linear
regression analysis, it may contribute to the deleterious effect of the
MTHFR 677TT genotype on folate metabolism. However, the RFC1
80G allele (AG+GG genotypes) was a predictor for reduced neonatal
tHcy concentrations. This nding indicates that the neonatal RFC1 AA
genotype is associated with increased neonatal tHcy concentrations,
which is an unexpected result when compared with the role of the
RFC1 A80G variant in mothers. The inuence of the RFC1 A80G variant
in maternal-to-fetal folate transfer and neonatal folate homeostasis
should be conrmed in studies with larger samples.
The association between the GCPII 156T allele (CT+TT genotypes)
and low tHcy concentrations in mothers suggests that this allele may
confer lesser risk for neonates than the common allele. Similar results
were found in a study conducted with neural tube defect patients and
their parents from the Netherlands [13] and in a larger study
conducted with 2734 DNA samples from the 19971999 Hordaland
Homocysteine Study, in Norway [34].
It has been suggested that the GCPII 1561T allele enhances the
FGCP deconjugation activity that increases folate bioavailability and
reduces plasma homocysteine [13]. However in our study, no
relationship was found between GCPII C1561T variant and serum
folate in mothers, as it has been previously described in other crosssectional studies [811]. The lack of association between the C1561T
SNP and serum folate may have been inuenced by the lack of folate
supplementation by Brazilian women during their pregnancy and
absence of mandatory food fortication with folic acid in Brazil until
2004.
Interesting interaction effects of the MTHFR 677TT/GCPII 1561CC
combined genotypes on tHcy and serum folate concentrations were also
found in mothers. This genotype combination may result in lower folate
bioavailability and reduced production of 5-methyltetrahydrofolate,
form used in methionine synthesis, by both impaired MTHFR and FGCP
enzymatic activities found in mothers carrying the MTHFR 677TT/GCPII
1561CC genotypes.
Neonates carrying the combination of genotypes GCPII 1561CT/
RFC1 80AA had higher serum folate than other combinations of
genotypes for two polymorphisms. This nding suggests that the folate
absorption could be facilitated in neonate carriers of this combination
of genotypes. Considering the low frequency of the GCPII 1561T allele,
this result should be conrmed in studies with larger samples.
Elevated MMA concentration is a sensitive marker for functional
cobalamin deciency [35]. In our study, cobalamin concentrations or
genetic variants were not independent predictors of MMA concentrations in mothers, in contrast to creatinine. Nevertheless, in neonates,
the MMA concentrations were predicted directly by maternal MMA
concentration and inversely by maternal GCPII T allele (CT+TT genotypes) and neonatal cobalamin concentration.

F.R. Lopreato et al. / Clinica Chimica Acta 398 (2008) 134139

In a previous study, we found that patients with severe cobalamin

decient megaloblastic anemia had low values of SAM/SAH ratio, which
was corrected with Cbl therapy [36]. Lower SAM/SAH ratio was
previously found in pregnant women in the lowest cobalamin quartile
(102 pmol/L) and their neonates [2]. In the present study, cobalamin
was the only predictor of maternal SAM/SAH ratio conrming its
relevance as biomarker of cobalamin deciency as previously suggested
[2]. Nevertheless, the SAM/SAH ratio in neonates was predicted directly
by maternal SAM/SAH ratio and neonatal serum folate concentrations
and inversely by maternal tHcy concentrations. This result shows that
the MTHFR C677T, GCPIIC1561T and RFC1 A80G polymorphisms were
not associated with alterations in SAM/SAH ratios in neonates.
One of strengths of our study is our access to maternal and neonatal concentrations of cobalamin, serum folate, tHcy, MMA, SAM and
SAH and maternal and neonatal genotypes for MTHFR C677T, GCPII
C1561T and RFC1 A80G polymorphisms. Furthermore, the statistical
analysis of maternal data were adjusted for potential confounding by
age, ethnic group, number of deliveries, per capita income and MTHFR
C677T genotype. Conversely, a potential limitation is that the dietary
intake of mothers was not evaluated in this study.
We conclude that maternal tHcy was affected by MTHFR C677T,
RFC1 A80G and GCPII C1561T polymorphisms. Maternal GCPII
C1561T variant was associated with neonatal MMA. Neonatal RFC1
A80G polymorphism inuenced tHcy in neonates.
Additional studies in larger populations are required to conrm the
effects of the described genotype interactions on maternal and
neonatal folate-related metabolites and the consequences of these
alterations in mother and child health.
Acknowledgments
This study was nancially supported by Fundao de Amparo
Pesquisa do Estado de So Paulo FAPESP, Brazil (Proc. 01/09836-7
and 04/00740-5) and NIA-AG09834 (SPS). Fernanda R Lopreato and
Felipe R Carvalho had fellowships of CNPq and FAPESP, respectively.
We thank the Hospital Regional of Conjunto Hospitalar de Sorocaba
and Hospital Santa Lucinda and the pregnant women who participated in the study. We thank Dr Manuela Fdinger and Dr Corinna C
Eberle from Institute of Medical and Chemical Laboratory Diagnostics,
University of Vienna, Austria for providing the DNA samples with
GCPII 1561 CT and TT genotypes.
References
[1] Chanarin I. The megaloblastic Anemias. 3rd edn. London: Blackwell Scientic
Publications; 1990.
[2] Guerra-Shinohara EM, Morita OE, Peres S, et al. Low ratio of S-adenosylmethionine/
S-adenosylhomocysteine is associated with vitamin deciency in Brazilian
pregnant women and newborns. Am J Clin Nutr 2004;80:131221.
[3] Allen LH. Multiple micronutrients in pregnancy and lactation: an overview. Am J
Clin Nutr 2005;81:1206S12S.
[4] Gregory III JF. Bioavailability of folate. Eur J Clin Nutr 1997;51(suppl 1):S549.
[5] Devlin A, Ling E, Peerson J, et al. Glutamate carboxypeptidase II: a polymorphism
associated with lower levels of serum folate and hyperhomocysteinemia. Hum Mol
Genet 2000;9:283744.
[6] Antony AC. The biological chemistry of folate receptors. Blood 1992;79:280720.
[7] Lievers K, Kluijtmans L, Boers G, et al. Inuence of a glutamate carboxypeptidase II
(GCP II12 levels and its relationship to cardiovascular disease risk. Atherosclerosis
2002;164:26973.
[8] Chen J, Kyte C, Valcin M, et al. Polymorphisms in the one-carbon metabolic
pathway, plasma folate levels and colorectal cancer in a prospective study. Int J
Cancer 2004;110:61720.
[9] Morin I, Devlin A, Leclerc D, et al. Evaluation of genetic variants in the reduced
folate carrier and in glutamate carboxypeptidase II for spina bida risk. Mol Genet
Metab 2003;79:197200.
[10] Relton CL, Wilding CS, Pearce MS, et al. Genegene interaction in folate-related
genes and risk of neural tube defects in a UK population. J Med Genet 2004;41:
25660.
[11] Devlin A, Clark R, Birks J, Evans J, Halsted C. Interactions among polymorphism in
folate-metabolizing genes and serum total homocysteine concentrations in a
healthy elderly population. Am J Clin Nutr 2006;83:70813.

139

[12] Vargas-Martinez C, Ordovas JM, Wilson PW, Selhub J. The glutamate carboxypeptidase gene II polymorphism does not affect folate status in the Framingham
Offspring Cohort. J Nutr 2002;132:11769.
[13] Afman LA, Trijbels FJM, Blom HJ. The H475Y polymorphism in the glutamate
carboxypeptidase II gene increases plasma folate without affecting the risk for
neural tube defects in humans. J Nutr 2003;133:757.
[14] Melse-Boonstra A, Lievers K, Blom H, Verhoef P. Bioavailability of polyglutamyl
folic acid and relative to that of monoglutamyl folic acid in subjects with different
genotypes of the glutamate carboxypeptidase II gene. Am J Clin Nutr 2004;80:
7004.
[15] Chango A, Emery-Fillon N, Courcy G, et al. A polymorphism (80 A G) in the reduced
folate carrier gene and its associations with folate status and homocysteinemia.
Mol Genet Metab 2000;70:3105.
[16] Pei L, Zhu H, Ren A, Li Z, et al. Reduced folate carrier gene is a risk factor for neural
tube defects in a Chinese population. Birth Defects Res 2005;73:4303.
[17] Shaw GM, Lammer EJ, Zhu H, Baker MW, Neri E, Finnell RH. Maternal periconceptional
vitamin use, genetic variation of infant reduced folate carrier (A80G), and risk of spina
bida. Am J Med Genet 2002;108:16.
[18] Frosst P, Blom HJ, Milos R, et al. A candidate genetic risk factor for vascular disease:
a common mutation in methylenetetrahydrofolate reductase (MTHFR). Nat Genet
1995;10:11113.
[19] Russo GT, Friso S, Jacques PF, et al. Age and gender affect the relation between
methylenetetrahydrofolate reductase C677T genotype and fasting plasma homocysteine concentrations in the Framingham Offspring Study Cohort. J Nutr 2003;133:
341621.
[20] Pereira AC, Schettert IS, Morandini Filho AAF, Guerra-Shinohara EM, Krieger JE.
Methylenetetrahydrofolate reductase (MTHFR) C677T gene variant modulates the
homocysteine folate correlation in a mild folate-decient population. Clin Chim
Acta 2004;340:99105.
[21] Barbosa PR, Stabler SP, Machado ALK, et al. Association between decreased vitamin
levels and MTHFR, MTR and MTRR gene polymorphisms as determinant for
elevated total homocysteine concentrations in pregnant women. Eur J Clin Nutr
2008;62:101021.
[22] Christensen B, Arbour L, Tran P, Leclerc D, Sabbaghian N, Platt R, et al. Genetic
polymorphisms in methylenetetrahydrofolate reductase and methionine
synthase, folate levels in red blood cells, and risk of neural tube defects. Am J
Med Genet 1999;84:1517.
[23] Wilson A, Platt R, Wu Q, Leclerc D, Christensen B, Yang H, et al. A common variant
in methionine synthase reductase combined with low cobalamin (vitamin B12)
increases risk for spina bida. Molec Genet Metab 1999;67:31723.
[24] Cunha ALA, Hirata M, Kim CA, Guerra-Shinohara EM, Nonoyama K, Hirata RDC.
Metabolic effects of C677T and A1298C mutations at the MTHFR gene in Brazilian
children with Neural Tube Defects. Clin Chim Acta 2002;318:13943.
[25] Barbosa PR, Stabler SP, Trentin R, et al. Evaluation of nutritional and genetic
determinants of total homocysteine, methylmalonic acid and S-adenosylmethionine/
S-adenosylhomocysteine values in Brazilian childbearing-age women. Clin Chim Acta
2008;388:13947.
[26] Stabler SP, Marcell PD, Podell ER, et al. Assay of methylmalonic acid in the serum of
patients with cobalamin deciency using capillary gas chromatographymass
spectrometry. J Clin Invest 1986;77:16102.
[27] Stabler SP, Marcell PD, Podell ER, Allen RH, Savage DG, Lindenbaum J. Elevation of total
homocysteine in the serum of patients with cobalamin or folate deciency detected
by capillary gas chromatographymass spectrometry. J Clin Invest 1988;81:46674.
[28] Stabler SP, Allen RH. Quantication of serum and urinary S-adenosylmethionine
and S-adenosylhomocysteine by stable-isotope-dilution liquid chromatography
mass spectrometry. Clin Chem 2004;50:36572.
[29] Salazar LA, Hirata MH, Cavalli AS, Machado MO, Hirata RDC. Optimized procedure
for DNA isolation from fresh and cryopreserved clotted human blood useful in
clinical molecular testing. Clin Chem 1998;44:174850.
[30] Fdinger M, Dierkes J, Skoupy S, et al. Effect of glutamate carboxypeptidase II and
reduced folate carrier polymorphisms on folate and total homocysteine concentrations in dialysis patients. J Am Soc Nephrol 2003;14:13149.
[31] Molloy AM, Mills JL, McPartlin J, Kirke PN, Scott JM, Daly S. Maternal and fetal
plasma homocysteine concentrations at birth: the inuence of folate, vitamin B12,
and the 5,10-methylenetetrahydrofolate reductase 677(T variant. Am J Obstet
Gynecol 2002;186:499503.
[32] Antony AC, Tang YS, Khan RA, et al. Translational upregulation of folate receptors is
mediated via RNA-heterogeneous nuclear ribonucleoprotein E1 interactions. J Clin
Invest 2004;113:285301.
[33] Antony AC. In utero physiology: role of folic acid in nutrient delivery and fetal
development. Am J Clin Nutr 2007;85:598S603S.
[34] Halsted CH, Wong DH, Peerson JM, et al. Relations of glutamate carboxypeptidase
II (GCPII) polymorphisms to folate and homocysteine concentrations and to scores
of cognition, anxiety, and depression in a homogeneous Norwegian population:
the Hordaland Homocysteine Study. Am J Clin Nutr 2007;86:51421.
[35] Allen RH, Stabler SP, Savage DG, Lindenbaum J. Metabolic abnormalities in
cobalamin vitamin B12) and folate deciencies. FASEB J 1993;7:134453.
[36] Guerra-Shinohara EM, Morita OE, Pagliusi RA, et al. Elevated serum S-adenosylhomocysteine in cobalamin decient megaloblastic anemia. Metabolism 2007;56:
33947.