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Blood, Vol. 93 No. 3 (February 1), 1999:
pp. 1118-1120
CORRESPONDENCE
Is the Oral Methionine Loading Test Insensitive to the
Remethylation Pathway of Homocysteine?
 |
LETTER |
To the Editor:
Methylenetetrahydrofolate reductase (MTHFR) is a key enzyme in the
remethylation pathway of homocysteine. The study by Girelli et
al1 showed that the C677T mutation of the gene encoding for
MTHFR is common in Italy, is not associated with coronary atherosclerotic disease, and influences the plasma levels of total homocysteine (tHcy) only in subjects who have low serum concentrations of folic acid. These findings are important confirmations of previous reports.2-6 In addition to the above issues, Girelli and
his coworkers addressed the problem of the influence of the MTHFR genotype on the increases of plasma tHcy levels after an oral methionine loading. The methionine loading test, which was originally developed to detect heterozygosity for the deficiency of
cystathionine- -synthase, a key enzyme in the trans-sulfuration
pathway of homocysteine,7 is useful not only to detect
subjects with defects of the trans-sulfuration pathway, but also to
identify subjects at risk for thrombosis.8,9 It has been
suggested that abnormal post-methionine loading (PML) increases in the
plasma levelsof tHcy reflect abnormalities of the trans-sulfuration
pathway of homocysteine metabolism and are insensitive to the
remethylation pathway, in contrast with fasting plasma levels of tHcy,
which are sensitive to the remethylation pathway.10,11 If
this hypothesis were true, the T/T mutation at nucleotide 677 of MTHFR,
which is associated with an enzyme activity of about 50% of normal,
should not influence the results of the methionine loading test.
Girelli et al commented that their results agree with this hypothesis,
because they showed that homozygosity for the C677Tmutation of MTHFR is
not associated with abnormally high PML tHcy increments.1
However, they found higher PML tHcy increments in individuals carrying
the mutant T/T genotype (25.5 µmol/L) than in those with normal (C/C)
or heterozygous (C/T) genotypes (23.6 µmol/L for both), although the
difference was not statistically significant (Table
1). In addition, they found that
individuals with low serum levels of folate, which plays an important
role in the remethylation pathway, tended to have higher PML tHcy
increments than those with high folate levels, independently of their
genotype (24.9 v 22.9 for C/C; 25.1 v 23.5 for C/T;
28.8 v 21.4 for T/T). In our opinion, these data do not allow a
definite conclusion regarding the influence of defects of the
remethylation pathway on the methionine loading test. At least four
additional reports with an experimental design similar to that of
Girelli et al have been published thus far.2,3,5,6 All of
them clearly showed that the fasting tHcy levels in plasma are
significantly higher in the T/T genotype of MTHFR than in the other
genotypes; however, the results of the methionine loading test were
less consistent. Table 1 shows that all the studies, perhaps with the
exception of that by Jacques et al,6 showed that the PML
increases of tHcy in plasma were higher in the T/T genotype of MTHFR
than in the normal C/C genotype, although the statistical significance
was reached in only two.2,5 Since the publication of our
study in 1997,2 we screened more individuals for MTHFR
genotype and the methionine loading test. Our most recent analysis of
the data obtained in 399 healthy controls and 250 patients with
previous episodes of arterial or venous thrombosis confirmed our
previous findings that the PML increases of tHcy levels in plasma are
significantly higher in individuals with the T/T genotype
(18.2 ± 7.5, arithmetic mean ± SD) than in those with the C/C
or the C/T genotypes of MTHFR (14.1 ± 5.6, P < .001). The differences remained highly statistically significant
(P < .001) also after adjustment for the serum levels of
folate and cobalamin, and the plasma levels of vitamin B6, which is the
essential cofactor in the trans-sulfuration pathway of
homocysteine.5 Among the 649 subjects studied, 142 had the
T/T genotype of MTHFR (80 controls and 62 cases); 14 of them (9.9%)
had high fasting tHcy levels, 15 (10.6%) had both high fasting levels
and high PML increases of tHcy, and 6 (4.2%) had high PML increases of tHcy. Therefore, the methionine loading test allowed the identification of 6 additional subjects with the MTHFR variant who had normal fasting
levels of tHcy.
In conclusion, the available experimental evidence indicates that the
fasting levels of tHcy in plasma are very sensitive to an abnormality
of the remethylation pathway of homocysteine, such as that associated
with the T/T genotype of MTHFR; however, they do not allow the
conclusion that the PML increases of tHcy levels in plasma only reflect
abnormalities of the trans-sulfuration pathway of homocysteine, because
they also tend to be abnormal in individuals with the T/T genotype of
MTHFR and in those with low serum levels of folic acid.
Marco Cattaneo
Rossana Lombardi
Anna Lecchi
Maddalena L. Zighetti
A. Bianchi Bonomi Hemophilia and Thrombosis Center
Department of Internal Medicine
IRCCS Ospedale Maggiore
University of Milano
Milano, Italy
| |
REFERENCES |
1.
Girelli D, Friso S, Trabetti E, Olivieri O, Russo C, Pessotto R, Faccini G, Pignatti PF, Mazzucco A, Corrocher R:
Methylenetetrahydrofolate reductase C677T mutation, plasma homocysteine, and folate in subjects from Northern Italy with or without angiographically documented severe coronary atherosclerotic disease: Evidence for an important genetic-environmental interaction.
Blood
91:4158, 1998[Abstract/Free Full Text]
2.
Cattaneo M, Tsai MY, Bucciarelli P, Taioli E, Zighetti ML, Bignell M, Mannucci PM:
A common mutation in the methylene-tetrahydrofolate reductase gene (C677T) increases the risk for deep-vein thrombosis in patients with mutant factor V (factor V:Q506).
Arterioscler Thromb Vasc Biol
17:1662, 1997[Abstract/Free Full Text]
3.
Legnani C, Palareti G, Grauso F, Sassi S, Grossi G, Piazzi S, Bernardi F, Marchetti G, Ferraresi P, Coccheri S:
Hyperhomocyst(e)inemia and a common methylenetetrahydrofolate reductase mutation (Ala223Val MTHFR) in patients with inherited thrombophilic coagulation defects.
Arterioscler Thromb Vasc Biol
17:2924, 1997[Abstract/Free Full Text]
4.
Brattström L:
Common mutation in the methylenetetrahydrofolate reductase gene offers no support for mild hyperhomocysteinemia being a causal risk factor for cardiovascular disease.
Circulation
96:3805, 1997
5.
Verhoef P, Kok FJ, Khuijtmans LAJ, Blom HJ, Refsum H, Ueland PM, Kruyssen DACM:
The 677C  T mutation in the methylenetetrahydrofolate reductase gene: Associations with plasma total homocysteine levels and risk of coronary atherosclerotic disease.
Atherosclerosis
132:105, 1997[Medline]
[Order article via Infotrieve]
6.
Jacques PF, Bostom AG, Williams RR, Ellison RC, Eckfeldt JH, Rosenberg IH, Selhub J, Rozen R:
Relation between folate status, a common mutation in methylenetetrahydrofolate reductase, and plasma homocysteine concentrations.
Circulation
93:7, 1996[Abstract/Free Full Text]
7.
Refsum H, Ueland PM, Nygárd O, Vollset SE:
Homocysteine and cardiovascular disease.
Annu Rev Med
49:31, 1998[Medline]
[Order article via Infotrieve]
8.
Cattaneo M, Martinelli I, Mannucci PM:
Hyperhomocysteinemia as a risk factor for deep-vein thrombosis.
N Engl J Med
335:974, 1996[Free Full Text] (letter)
9.
D'Angelo A, Fermo I, Mazzola G, Paroni R, Di Minno G, Viganó D'Angelo S:
Isolated methionine intolerance is a risk factor for early-onset venous thromboembolic disease.
Neth J Med
52:S38, 1998 (abstr, suppl)
10.
Selhub J, Miller JW:
The pathogenesis of homocysteinemia: Interruption of the coordinate regulation by S-adenosylmethionine of the remethylation and transsulfuration of homocysteine.
Am J Clin Nutr
55:131, 1992[Abstract/Free Full Text]
11.
Miller JW, Nadeau MR, Smith D, Selhub J:
Vitamin B-6 deficiency vs folate deficiency: Comparison of responses to methionine loading in rats.
Am J Clin Nutr
59:1033, 1994[Abstract/Free Full Text]
Response
We appreciated the comments of Dr Cattaneo et al, which raise the
interesting problem of the factors influencing the PML
hyperhomocysteinemia, although it was not the main object of our
study.1 We also cited the only two studies published at
that moment on the relationship between the C677T mutation and PML tHcy
levels,2,3 pointing out their conflicting results. The
thorough update by Dr Cattaneo et al adds uncertainty to this matter.
The discrepancies may be partly caused by differences in the duration
of PML test (4 hours2,4,5 or 6 hours,1-3
reflecting a different stage in plasma tHcy clearance) and/or,
more remarkably, in the statistical analysis used. For example,
we1 and others2,5 compared the three MTHFR
genotypes by one-way ANOVA followed by Tukey's post-hoc comparison of
the means, which is a more conservative approach. On the other hand, Cattaneo et al4 used the t-test between the T/T
group and the C/C plus C/T combined group. We also revalued an
increased number of subjects (n = 517), confirming the trend toward
higher PML increases in the T/T group than in other genotypes, again
without reaching the statistical significance (23.3, 23.6, and 25.9 µmol/L in C/C, C/T, and T/T, respectively; ANOVA: F = 1.9,
P = .15; Tukey's test: T/T v C/T, P = .19;
T/T v C/C, P = .15). In our report, we concluded that
our results were consistent with the generally accepted view that an
abnormal PML increase in tHcy levels would primarily reflect
abnormalities in the transsulfuration pathway. However, this does not
exclude in principle an association between an impaired remethylation
and the PML tHcy levels, whereas it is clearly not as strong as that
observed with fasting Hcy. This could be due to at least two reasons.
First, marginal deficiencies of folate and vitamin B6 are often
correlated,6 so that if you have an impaired remethylation
because of inadequate folate status, probably you will also see an
impaired transsulfuration. Second, according to Selhub and Miller's
hypothesis,7 remethylation and transsulfuration are
coordinately regulated so that a defect in one pathway will lead to the
impairment of the other. A key role is played by S-adenosylmethionine
(SAM), the most important intermediate in the synthesis of homocysteine
from methionine, which is both an allosteric inhibitor of MTHFR and an
activator of cystathionine--synthase (CBS). In subjects with fasting
hyperhomocysteinemia because of a primary impairment of the
remethylation pathway (ie, defective MTHFR, folate deficiency, or both)
the decreased de novo synthesis of methionine implies a decreased
cellular SAM concentration, ultimately leading to a suboptimal
activation of CBS. So, it is conceivable that these subjects also tend
to have slightly increased PML tHcy value because of a basal defect in the transsulfuration, which is partly counterbalanced by the
PML-induced increase of SAM. However, from a practical standpoint this
phenomenon may not be so relevant, as the determination of fasting tHcy
is clearly sufficient to detect subjects with abnormalities in the remethylation. Thinking in terms of vascular risk, the PML test is
important mainly for detecting subjects with isolated PML
hyperhomocysteinemia, ie, a subject who could be at risk but would not
be diagnosed with fasting tHcy determination alone. It was previously
reported that this subgroup may represent up to 40%8 of
the whole hyperhomocysteinemic population, and our recent
analysis9 is in keeping with this view. In our opinion, it
is especially in this subgroup that we need to learn much more about
the determinants of PML tHcy by means of further comprehensive studies
including all potential genetic and environmental factors (CBS
polymorphisms, vitamin B6 status, and others).
Domenico Girelli
Oliviero Olivieri
Carla Russo
Roberto Corrocher
Department of Clinical and Experimental Medicine
University of
Verona
Verona, Italy
| |
REFERENCES |
1.
Girelli D, Friso S, Trabetti E, Olivieri O, Russo C, Pessotto R, Faccini G, Pignatti PF, Mazzucco A, Corrocher R:
Methylenetetrahydrofolate reductase C677T mutation, plasma homocysteine and folate, in subjects from Northern Italy with or without angiographically documented severe coronary atherosclerotic disease. Evidence for an important genetic-environmental interaction.
Blood
91:4158, 1998
2.
Jacques PF, Bostom AG, Williams RR, Ellison RC, Eckfeldt JH, Rosenberg IH, Selhub J, Rozen R:
Relation between folate status, a common mutation in methylenetetrahydrofolate reductase, and plasma homocysteine concentrations.
Circulation
93:7, 1996
3.
Verhoef P, Kok FJ, Kluijtmans LAJ, Blom HJ, Refsum H, Ueland PM, Kruyssen DACM:
The 677 C T mutation in the methylenetetrahydrofolate reductase gene: Associations with plasma total homocysteine levels and risk of coronary atherosclerotic disease.
Atherosclerosis
132:105, 1997
4.
Cattaneo M, Tsai MY, Bucciarelli P, Taioli E, Zighetti M, Bignell M, Mannucci PM:
A common mutation in the methylenetetrahydrofolate reductase gene (C677T) increases the risk for deep-vein thrombosis in patients with mutant factor V (Factor V:Q506).
Arterioscler Thromb Vasc Biol
17:1662, 1997
5.
Legnani C, Palareti G, Grauso F, Sassi S, Grossi G, Piazzi S, Bernardi F, Marchetti G, Ferraresi P, Coccheri S:
Hyperhomocyst(e)inemia and a common methylenetetrahydrofolate reductase mutation (Ala223Val MTHFR) in patients with inherited thrombophilic coagulation defects.
Arterioscler Thromb Vasc Biol
17:12924, 1997
6.
Stampfer MJ, Willer WC:
Homocysteine and marginal vitamin deficiency. The importance of adequate vitamin intake.
JAMA
270:2726, 1993[Abstract/Free Full Text]
7.
Selhub J, Miller JW:
The pathogenesis of homocysteinemia: Interruption of the coordinate regulation by S-adenosylmethionine of the remethylation and transsulfuration of homocysteine.
Am J Clin Nutr
55:131, 1992
8.
Bostom AG, Jacques PF, Nadeau MR, Williams RR, Ellison RC, Selhub J:
Post-methionine loading hyperhomocysteinemia in persons with normal fasting total plasma homocysteine: Initial results from the NHLBI Family Heart Study.
Atherosclerosis
116:147, 1995[Medline]
[Order article via Infotrieve]
9.
Girelli D, Friso S, Olivieri O, Russo C, Trabetti E, Pessotto R, Faccini G, Zenari ML, Grazioli S, Minguzzi D, Pignatti PF, Mazzucco A, Corrocher R:
Post-methionine loading hyperhomocysteinemia in patients with angiographically documented coronary artery disease.
Br J Haematol
102:251, 1998
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