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*
Northern Ireland Centre for Diet and Health (NICHE), School of Biomedical Sciences, University of Ulster, Coleraine, Northern Ireland and the Departments of
Clinical Medicine and
**
Biochemistry, Trinity College, Dublin, Ireland.
1To whom correspondence should be addressed.
 |
ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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0.001), and those in the
bottom quartile almost doubled their methionine intake, from 1155
± 401 to 2112 ± 379 mg/d (P 0.001).
Despite these changes in methionine intake, no corresponding changes in
plasma tHcy were observed. These results suggest that in the absence of
an obvious deficiency of relevant B-vitamins, fasting plasma tHcy
is unaffected by intermediate-term fluctuations (up to 100% of
usual intake) in dietary methionine.
KEY WORDS: • dietary intervention • methionine • homocysteine • humans
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, Hankey and Eikelboom 1999
). Two principal
pathways of homocysteine metabolism exist (Finkelstein 1990
): remethylation to methionine, requiring both
5-methyl-tetrahydrofolate and methylcobalamin, or catabolism to
cysteine requiring pyridoxal phosphate (PLP; vitamin B-6). Because of
the key roles of folate and vitamins B-12 and B-6 in the metabolism of
homocysteine, it is not surprising that a deficiency of one or more of
them may account for up to two thirds of all cases of moderate
hyperhomocysteinemia (Selhub et al. 1993
).
The possible influence of nutrients other than the B vitamins
on plasma tHcy is less well understood. Methionine, an essential amino
acid found in foods rich in animal protein, is one of the principal
dietary sources of homocysteine. The association between tHcy and
dietary methionine intake has been investigated in previous studies
(Shimakawa et al. 1997
, Verhoef et al. 1996
). Shimakawa and colleagues (1997) examined the
relationship between tHcy and dietary intakes of nutrients of relevance
to homocysteine metabolism in the Atherosclerosis Risk in Communities
(ARIC) study. Methionine intake (assessed using a food-frequency
questionnaire; FFQ) showed no association with baseline plasma tHcy in
either case (subjects with atherosclerosis, n = 318, or
control subjects, n = 322). Similarly, Verhoef and
colleagues (1996) reported no correlations between methionine intake
and tHcy in 130 patients, with a first myocardial infarction, who were
participants in a case-control study examining the association
between tHcy, B-vitamins and the risk of myocardial infarction.
There was, however, a significant correlation between tHcy and dietary
methionine in control subjects (n = 118) in this study,
but contrary to what might have been expected, the correlation was
negative (r = -0.22, P = 0.01). One
possible explanation for such a finding, although not suggested by the
authors, is that dietary methionine may have been a marker for another
dietary variable known to be protective (e.g., vitamin B-12). This
theory is supported by the findings of Mann and co-workers (1999),
who compared free-living habitual meat-eaters and habitual
vegetarians with respect to B-vitamins and tHcy. Results indicated
that tHcy concentrations increased in a step-wise manner from high
meat-eaters up to vegans, with a corresponding progressive lowering
of serum vitamin B-12 concentrations as the consumption of animal
products decreased.
Several studies (Guttormsen et al. 1994
, Ubbink et al. 1992
) have investigated the acute response of tHcy to a
high methionine meal and have reported the maximum effect occurring
8 h after ingestion of the meal. More recently, Chambers et al. (1999)
demonstrated some evidence of disturbances in endothelial
function in response to a high methionine meal. To the best of our
knowledge, the effect of an intermediate or chronic increase in
methionine intake has, however, been investigated in only one study to
date (Andersson et al. 1990
). A methionine load test was
performed in healthy subjects before and after a 14-d intervention
study with supplementary methionine (
25 mg ·
kg-1 · d-1). The
authors concluded that the variations in daily methionine intake did
not seem to affect the result of methionine loading or fasting
concentrations of methionine or homocysteine (Andersson et al. 1990
).
Clearly, the relationship between methionine intake and tHcy requires further clarification. The aim of this study, therefore, was to examine the response of tHcy to altered dietary methionine in a group of healthy men.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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A total of 52 healthy nonvegetarian male volunteers aged 19 to 29 y were randomly selected from the student population at the University of Ulster to complete a FFQ, which focused on foods rich in methionine and was designed specifically to assess habitual dietary methionine intake. Following analysis of the dietary data, subjects in the top and bottom quartiles for methionine intake were selected to participate in an intervention study. Prior to the study, which received ethical approval from the Research Ethical Committee at the University of Ulster, subjects were asked to complete a brief medical questionnaire and give their written informed consent. At the time of recruitment and throughout the study period, none of the subjects was known to be taking any form of medication, B-vitamin supplement or foods fortified with B-vitamins.
Study design.
The study consisted of two simultaneous cross-over intervention
trials, running in parallel, preceded by a 7-d baseline period during
which subjects continued to eat their usual diets (Fig. 1
). During the baseline period and throughout the study, selected
subjects (n = 26) were provided with 7-d food records
and asked to record on a daily basis all food and beverages consumed.
Following baseline (week 1), group A (subjects in the top quartile for
habitual methionine intake; n = 14) were randomly
divided into two subgroups to receive either a low-methionine
intervention diet for 1 wk followed by control diet for another week
(A1; n = 7) or vice-versa (A2; n = 7). Simultaneously, group B (subjects in the bottom quartile for
habitual methionine intake; n = 12) were randomly
divided into 2 subgroups to receive either a high-methionine
intervention diet for 1 wk followed by a control diet (B1; n
= 6) for 1 wk or vice-versa (B2; n = 6). High
methionine foodstuffs included beef, chicken, fish, eggs, cheese and
ham, and low methionine foods consisted of commercially prepared
vegetarian meals, including pasta dishes and curries containing soy
protein, tofu and beans.
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Blood sampling and analysis.
Venous blood samples (20 mL) were collected from fasting subjects. Whole blood was collected in evacuated tubes containing EDTA for tHcy, whole blood folate and full blood count analysis and in nonheparinized integrated serum separator tubes for SF analysis. Samples for homocysteine analysis were placed immediately on ice and centrifuged within 1 h. After centrifugation at 2000 x g for 15 min, plasma and serum samples were stored at -20°C until analyzed. Lysates of RCF were prepared by dilution of whole blood collected into EDTA (1:10) with freshly prepared ascorbic acid (10 g/L). Aliquots were mixed thoroughly on a roller mixer, incubated at 37°C for 30 min and stored at -20°C. Full blood count analysis was carried out by automated Coulter counter at Causeway Laboratories (Coleraine, Northern Ireland) on the day of sample collection.
Plasma samples were analyzed for tHcy by isocratic high performance
liquid chromatography (HPLC; Ubbink et al. 1991
) using
the fluorescent conjugate (SBD-F; Araki and Sako 1987
).
The microbiological assay was used to determine SF and RCF using the
microtiter assay procedure of Molloy & Scott (1997)
and serum vitamin
B-12 concentration by the method described by Kelleher and O’Broin (1991)
with no modifications. PLP was determined by HPLC using
fluorescent detection (Bates et al. 1999
).
Dietary assessment.
Two dietary methods were used in the present investigation for distinct
purposes. The FFQ, which focused on foods rich in methionine, was used
only on the initial screening sample to divide subjects into quartiles
of habitual methionine intake. The 7-d food record method was used to
obtain detailed data for specific time periods during the 3-wk
cross-over study, and portion sizes were subsequently quantified
using published data (Crawley 1993
). For both
methods, nutrient intakes were calculated using the nutrient analysis
program Comp-Eat (Nutritional Services, London, UK), based
on McCance and Widdowson’s The Composition of Foods (Holland et al. 1992
), which had been updated to include the most recent
data available on the methionine content of foods.
Statistical analysis.
Results are presented as mean ± SD. Differences between groups, carry-over and period effects were determined using t tests for unpaired data (P < 0.05). Pearson product moment correlation statistics were used to calculate correlations. The Data-Desk statistics program for Windows (version 6; Data Description, Ithaca, NY) was used for all statistical analyses.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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As determined by the FFQ, the dietary methionine intake for the 52
subjects screened was 2331 ± 911 mg/d, with values ranging from
488 to 4978 mg/d. Subjects in the top quartile for methionine intake
had a significantly higher mean intake (P 0.001;
Table 1
) than the total screened sample (n = 52) and those in
the bottom quartile for methionine intake.
|
; there was no significant difference in age or body mass index between
the groups. Assessment of dietary intakes based on detailed 7-d food
records completed over the baseline period revealed a significantly
higher intake (P 0.01) of methionine in group A than
in group B, consistent with the initial assessment of dietary intake
based on the FFQ. For the total intervention sample (n
= 24), the mean dietary methionine intake was 1552 mg/d, which
compares well with that reported by Lucock and colleagues (1996) for
men of a similar age. Methionine intakes assessed by the two
alternative dietary methods were significantly correlated (r
= 0.414, P 0.05).
|
0.05)
than those in group B, but when protein was expressed as a percentage
of energy, no difference between groups was noted (Table 2)
. All
subjects had normal B-vitamin status as determined from dietary and
laboratory data, and no significant difference in vitamin intake or
status between groups was noted (Table 2)
, with the exception of serum
vitamin B-12 concentration, which was significantly higher in group A.
SF correlated negatively with plasma homocysteine (r = -0.447, P = 0.0284). No correlation was noted between
plasma homocysteine and RCF, serum vitamin B-12 or any of the dietary
variables including dietary methionine. The mean plasma homocysteine
concentration reported for the groups combined (n = 24)
was 8.27 µmol/L, with values ranging from 4.06 to 25.16
µmol/L; the two groups did not differ at baseline and were
well within the normal range. Effects of an altered dietary methionine intake.
In group A (subjects with a high baseline dietary methionine intake),
intervention with a low methionine diet resulted in a significant
(P 0.001) decrease in dietary methionine intake
compared with the control diet (Table 3
). Despite this, no corresponding response was observed in tHcy
concentration; values after consumption of control and intervention
diets were not different (Table 3)
. As a result of decreasing
methionine intake, there were decreases (P 0.001) in
dietary energy, protein, percentage of energy from protein, percentage
of energy from fat and intakes of vitamins B-12 and B-6. No change in
dietary folate intake, SF or plasma PLP concentration was observed
during intervention. Not surprisingly, the intervention diet also
significantly increased (P 0.001) the percentage of
energy obtained from carbohydrates compared with the control diet,
because carbohydrate foods were specifically incorporated as
replacement foods for those high in protein.
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0.001) in dietary
methionine was achieved by subjects in group B (subjects with a low
baseline dietary methionine) in response to intervention with a high
methionine diet (Table 3)
. Once again, no significant change in tHcy
was observed. However, the increased dietary methionine intake did
significantly (P 0.05) increase dietary protein,
percentage of energy from protein, energy (P 0.05),
percentage of energy from fat, intakes of vitamin B-12 and B-6 and
plasma PLP concentration (Table 3)
. Intervention with a high methionine
diet also resulted in a small but significant (P 0.05) increase in folate intake, although no change was observed in SF
concentration. |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Furthermore, our results are
consistent with the more recent findings reported by Mann and
colleagues (1999) who found no association between tHcy and dietary
methionine in a cross-sectional comparison of free living
meat-eaters and habitual vegetarians.
Although our study was not designed to identify determinants of tHcy,
we observed a significant relationship between SF and tHcy at baseline,
supporting folate as the major nutritional determinant of homocysteine.
No other significant baseline correlation was observed between
homocysteine concentration and recognized determinants of tHcy. In
agreement with the observations of Shimakawa et al. (1997)
and Verhoef et al. (1996)
this study showed no correlation between tHcy and dietary
methionine intake. An inverse association has been observed between
tHcy and protein intake by Stolzenberg-Solomon et al. (1999)
. The
authors speculated that this might be reflecting a more efficient
catabolism of homocysteine in response to high protein plus high
methionine intake as has been demonstrated in animal studies. They
alternatively suggest that dietary protein may have been acting as a
proxy for dietary vitamin B-12.
A limitation of the current study is that variables other than the main
dietary variable under investigation (i.e., methionine) were changed as
part of the intervention. The only relevant changes, however, are those
that could affect the homocysteine response. No change was observed in
SF concentration in either of the intervention groups, although a
significant increase in folate intake was observed in group B subjects
who consumed the high methionine intervention diet. Changes were also
observed in the intake of vitamins B-12 and B-6 as a result of the
intervention protocol. In subjects who received the low methionine
diet, intake of vitamins B-12 and B-6 decreased significantly. This is
not surprising because the foods restricted during the dietary
methionine intervention (i.e., high animal protein foods) are excellent
sources of vitamin B-12, and to a lesser extent, vitamin B-6.
Conversely, in subjects who received the high methionine diet, vitamin
B-12 and B-6 intakes increased, although no differences were noted in
this group when vitamin B-6 intakes were expressed relative to protein
intakes. The significant increase in PLP in response to the high
methionine intervention, yet lack of response to the low methionine
intervention, may reflect a lowered vitamin B-6 requirement during
periods of decreased methionine (protein) intake (Food and Nutrition Board 1998
).
In the present study, we screened a group of 52 healthy subjects for
habitual methionine intake using a specifically designed FFQ based on
the major food sources of methionine. This would not be the preferred
method for assessment of habitual dietary intake, but it is recognized
as a useful method for classifying subjects into appropriate dietary
categories (Levine and Morgan 1991
). This was found to
be the case. Subjects identified from the original cohort of 52 as
being in the top quartile for habitual dietary methionine intake by the
FFQ were also found to have significantly higher methionine intakes
compared with the bottom quartile during the baseline period when
assessed using the more detailed method of diet records.
Several claims have been made of a possible influence of a high dietary
methionine intake as a contributor to the progression of
arteriosclerosis by increasing homocysteine (McCully 1983
, Shapira et al. 1997
, Toborek and Hennig 1996
). In addition, the potential influence of
methionine intake on one-carbon metabolism has been highlighted
by Chen et al. (1996)
. Previously, the acute elevation in tHcy in
response to a high methionine meal was investigated (Guttormsen et al. 1994
), but to the best of our knowledge, this is the
first intervention study to address the response of homocysteine to
intermediate-term changes in dietary methionine. From the results
obtained here, both during the baseline period and in response to
intervention, the present study provides strong evidence suggesting
that in the absence of suboptimal status of one of the B-vitamins
involved in homocysteine metabolism fasting tHcy concentration is
unaffected by fluctuations (up to 100% of usual diet) in dietary
methionine. The possibility cannot be ruled out, however, that dietary
methionine may become a more important determinant of fasting plasma
homocysteine in those with suboptimal status of one or more of the
B-vitamins.
|
FOOTNOTES |
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3 Abbbreviations used: CD, control diet; FFQ, food-frequency questionnaire; HM, high methionine; LM, low methionine; PLP, pyridoxal phosphate; RCF, red-cell folate; SF, serum folate; SBD-F, ammonium-7-flurobenzone-2-oxa-1,3-diazole-4-sulfonate; tHcy, total homocysteine.
Manuscript received May 9, 2000. Initial review completed May 29, 2000. Revision accepted July 20, 2000.
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