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Articles |
Departments of
1
Pathology and Laboratory Medicine and
2
Internal Medicine, University Hospital Groningen, NL-9700 RB Groningen, The Netherlands.
aAddress correspondence to this author at: Department of Pathology and Laboratory Medicine, University Hospital Groningen, CMC-V, Room Y1.165, PO Box 30.001, NL-9700 RB Groningen, The Netherlands. Fax 31-50-3612290; e-mail m.r.fokkema{at}path.azg.nl.
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Abstract |
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 Top Abstract IntroductionSubjects and MethodsResultsDiscussionReferences |
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Methods: Healthy adults (n = 101) received folic acid (5 mg/day) and vitamin B12 (1 mg/day) for 2 weeks and the same dosages of folic acid and vitamin B12 plus vitamin B6 (1 mg · kg-1 · day-1) during the following 2 weeks. Hcy concentrations, both fasting and 6-h post-methionine load, were determined at baseline and after 4 weeks.
Results: Baseline (4 weeks) fasting and 6-h postload Hcy reference values were 4.7–14.6 (4.1–9.3) and 18.8–49.7 (12.9–35.1) µmol/L, respectively. Mean fasting and 6-h postload Hcy decreased after 4 weeks of vitamin supplementation by 3.5 µmol/L (33.5%) and 8.5 µmol/L (26.3%), respectively. The percentages of subjects exhibiting significant decreases in fasting Hcy following vitamin supplementation were 88% (all subjects), 92% (non-vitamin users), and 72% (vitamin users). The prevalences of hyperhomocysteinemia with use of ECAP cutoff values were 29% for all groups, 29% for men, 27% for premenopausal women, and 53% for postmenopausal women. With vitamin-optimized cutoff values, prevalences were 58%, 58%, 76%, and 89%, respectively. Use of vitamin-optimized cutoff values increased the diagnostic value of fasting Hcy and decreased that of a 6-h postload Hcy compared with use of ECAP cutoff values.
Conclusions: Use of vitamin-optimized cutoff values gives rise to high hyperhomocysteinemia pretest probabilities in the general population and, therefore, precludes any meaningful role for Hcy testing. Future demonstration of a beneficial effect of decreasing Hcy on CVD risk would justify use of vitamin-optimized cutoff values for assessment of CVD risk.
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Introduction |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Plasma Hcy concentrations depend on genetic, environmental, and other factors, such as kidney function (1). Some vitamins are cofactors or cosubstrates in the metabolic clearance of Hcy (1), and it has repeatedly been demonstrated for populations in many countries that both fasting and 6-h postload plasma Hcy concentrations decrease with administration of vitamin B12, vitamin B6, and particularly folic acid in both healthy subjects and patients with CVD (9)(10)(11). Reaching optimal status for these vitamins in terms of their influence on plasma Hcy may, therefore, constitutes the easiest and least expensive way to decrease plasma Hcy concentrations and thereby possibly reduce CVD risk. From the clinical chemical point of view, such an approach would require the use of Hcy reference values at optimized vitamin status, as suggested previously by Ubbink et al. (12).
We determined fasting and 6-h postload Hcy reference values at optimized folate, vitamin B12, and vitamin B6 status (hereafter referred to as "vitamin-optimized Hcy reference values") and investigated their influence on the prevalence of hyperhomocysteinemia in 101 healthy Dutch adults. The outcome was compared with the prevalence calculated from the use of ECAP cutoff values. In contrast to our previous study (13), we also determined 6-h postload Hcy reference values, coadministered vitamin B12, and supplemented the three vitamins for a longer time period.
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Subjects and Methods |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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18 years) were recruited from
hospital employees and from students of the Pharmacy and Medicine
Departments of the Groningen University Hospital. Subjects >50 years
were recruited from sport and community center visitors. Exclusion
criteria were pregnancy, psoriasis, epilepsy, kidney insufficiency,
increased serum liver enzymes, CVD, and use of drugs that interfere
with folic acid, vitamin B12, or vitamin
B6 metabolism. The study protocol was approved by
the medical ethics committee of the Groningen University Hospital and
was in agreement with local ethics standards and the Helsinki
Declaration of 1975, as revised in 1996.
study design
Indices of kidney and liver pathology and hematological
abnormalities were investigated at baseline by measurement of serum
creatinine, aspartate aminotransferase, alanine aminotransferase,
-glutamyltranspeptidase, and alkaline phosphatase, and standard
hematological indices (erythrocytes, leukocytes, platelets, hemoglobin,
hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, and
mean corpuscular hemoglobin concentration). Anthropometric data,
menopausal status, and supplemental vitamin intake were assessed by
questionnaire. The total study duration was 4 weeks. The volunteers
took folic acid and vitamin B12 during the first
2 weeks, and folic acid, vitamin B12, and vitamin
B6 during the following 2 weeks. The dosages were
5 mg/day for folic acid [12.5 times the Recommended Dietary Allowance
(RDA)], 1 mg/day for vitamin B12 (417 times the
RDA) and 1
mg · kg-1 · day-1
for vitamin B6 (46–60 times the RDA; maximum,
100 mg/day). The participants were asked to take the supplements at
breakfast and to consume protein-poor meals on the day before sampling
(i.e., at baseline and after 4 weeks). No supplements were taken on the
sampling days. Blood was collected in the fasting state (>10 h
fasting) at baseline and after 4 weeks for the analyses of plasma Hcy
(fasting Hcy), serum folate, serum vitamin B12,
and whole-blood vitamin B6. MLTs were performed
at baseline and after 4 weeks by administration of 0.1 g of
methionine per kg of body weight, suspended in orange juice. Blood
samples were taken 6 h after methionine ingestion for the analysis
of plasma Hcy (6-h postload Hcy). The participants were instructed to
consume only protein-poor food during the test.
analytical methods
Blood and EDTA-blood were collected. EDTA-blood was immediately
placed in melting ice. Both samples were processed within 1 h to
serum and EDTA-plasma. Serum folate and vitamin
B12 were determined by an immunofluorometric
method (Autodelfia; Wallac Oy). Whole-blood vitamin
B6 was determined by HPLC (14). Plasma
Hcy was determined by an immunochemical method (IMx; Abbott
Laboratories). This method measures plasma total Hcy, which is the sum
of both reduced and oxidized forms in the circulation. Standard
hematological indices were determined with a Coulter STKS (Coulter
Corporation). Serum creatinine, aspartate aminotransferase, alanine
aminotransferase, -glutamyltranspeptidase, and alkaline phosphatase
were determined with a MEGA (Merck).
data evaluation and statistics
Data were evaluated on the basis of intention to treat and were
processed with Microsoft Excel. Reference intervals for fasting and 6-h
postload Hcy concentrations were calculated according to methods
recommended by the IFCC (15) by taking the central 95%
interval of the data. Vitamin-optimized cutoff values for the
establishment of hyperhomocysteinemia were calculated for the total
group, for men, and for pre- and postmenopausal women separately.
Cutoff values were placed at the 97.5th percentiles. ECAP cutoff values
for fasting and 6-h postload Hcy were 12.1 and 38.0 µmol/L (total
group), 11.9 and 39.8 µmol/L (men <45 years), 12.6 and 38.0 µmol/L
(men 45 years), 10.4 and 34.1 µmol/L (women <45 years), and 11.4
and 36.9 µmol/L (women 45 years), respectively (1).
Statistical analyses were performed with Microsoft SPSS 9.0. Associations were analyzed by Spearman rank tests, longitudinal changes in vitamin and Hcy concentrations by the paired Student t-test (gaussian distributions) or by Wilcoxon signed-ranks tests (nonparametric distributions). P <0.05 was considered significant.
Individual fasting Hcy decreases were considered significant when individual percentile changes exceeded 2.77 x CVanal,biol (i.e., significantly higher change than the combined intraassay analytical and mean intraindividual biological variations). On the basis of a mean CVbiol of 7.03% (16) and a CVanal of 1.85% (our observations), the CVanal,biol was estimated to be 7.27%. The least significant change in plasma Hcy was therefore calculated to be 20.1%. Because there are no reliable data on the biological variation of a 6-h postload Hcy, the least significant 6-h postload Hcy change could not be determined.
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Results |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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plasma Hcy reference values before and after vitamin
supplementation
The circulating vitamin and Hcy concentrations and Hcy reference
values before and after vitamin supplementation are shown in Table 1
. Before supplementation, men and premenopausal women had
different fasting Hcy (P <0.05). Men and pre- and
postmenopausal women differed in fasting Hcy after supplementation
(P <0.001) and in 6-h postload Hcy both before
(P <0.01) and after (P <0.0001)
supplementation. Fasting Hcy was independent of age in men and women
before supplementation, but was age dependent after supplementation in
men (r = 0.484; P = 0.001) and all
women combined (r = 0.345; P = 0.016).
The 6-h postload Hcy was age dependent in all women combined, both
before (r = 0.399; P = 0.003) and after
(r = 0.433; P = 0.002) supplementation.
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During the supplementation period, circulating vitamin concentrations increased, whereas fasting and 6-h postload Hcy concentrations decreased in the total study group and in each of the three subgroups. The mean (± SD) decreases in fasting Hcy and 6-h postload Hcy during the study were 3.5 ± 2.1 µmol/L (33.5% ± 13.5%) and 8.5 ± 5.9 µmol/L (26.3% ± 14.7%), respectively, for the total study group. The percentage of subjects with significantly decreased fasting Hcy after vitamin supplementation was 88%. Forty-five percent (5 of 11) of the subjects who did not exhibit a Hcy decrease used vitamin supplements at baseline. The percentage of vitamin supplement users in the group that did exhibit a Hcy decrease was 16% (13 of 82). Hcy decreased significantly in 92% (69 of 75) of the participants who did not use vitamin supplements at baseline and in 72% (13 of 18) of the subjects who did use vitamin supplements at baseline. It should be noted that supplemental vitamin intake was highly variable but generally low. Reference values for baseline fasting and 6-h postload Hcy were 4.7–14.6 and 18.8–49.7 µmol/L, respectively, for the total study group. Vitamin-optimized fasting and 6-h postload Hcy reference values were 4.1–9.3 and 12.9–35.1 µmol/L, respectively.
hyperhomocysteinemia prevalence at different Hcy cutoff values
The consequences of the use of different Hcy cutoff values for the
diagnosis of hyperhomocysteinemia are depicted in Table 2
and Fig. 1
. When we applied the whole group and subgroup-specific
vitamin-optimized cutoff values (Table 1
) for evaluation of fasting and
6-h postload Hcy at baseline, 58% of the total group, 58% of the men,
76% of the premenopausal women, and 89% of the postmenopausal women
had increased values. With ECAP cutoff values, these percentages were
29% for the total group, 29% for the men, 27% for the premenopausal
women, and 53% for the postmenopausal women.
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diagnostic value of fasting and 6-h postload Hcy at ECAP and vitamin-optimized cutoff values
The use of ECAP or vitamin-optimized cutoffs had different impacts
for a fasting Hcy and 6-h postload Hcy on the diagnosis of
hyperhomocysteinemia (Table 2
). The use of vitamin-optimized reference
values produced a sharp decrease in the percentage of subjects who
could be diagnosed as hyperhomocysteinemic only on the basis of a 6-h
postload Hcy. The percentages decreased from 14% to 2% for the total
group, from 8% to 2% for men, from 3% to 0% for premenopausal
women, and from 42% to 5% for postmenopausal women. On the other
hand, the use of vitamin-optimized reference values increased the
percentages of subjects who could be diagnosed only on the basis of a
fasting Hcy. The percentages increased from 10% to 29% for the total
group, from 17% to 29% for men, from 15% to 55% for premenopausal
women, and from 0% to 63% for postmenopausal women.
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Discussion |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Vitamin-optimized Hcy reference values are independent from the background intake of vitamins via the diet or supplements, which is probably at the base of the encountered international similarities. Until the beneficial effect of decreasing Hcy on CVD risk is confirmed, one may use these upper limits of the reference intervals as cutoff values for both the diagnosis and treatment of hyperhomocysteinemia because treatment consists of supplementation with folic acid, vitamin B12, and vitamin B6. The use of the upper limit of the vitamin-optimized fasting Hcy reference interval (i.e., 9.3 µmol/L) is in line with the <10 µmol/L recommendation of the American Heart Association for subjects with increased CVD risk and makes the presence of (sub)clinical deficiencies of the three B vitamins unlikely.
We used relatively high, nontoxic vitamin dosages to obtain new, vitamin-optimized, plasma Hcy steady-state values by rapid means. The metaanalysis of the Homocysteine Lowering Trialists’ Collaboration (9) and the study of Lobo et al. (10) indicated that a daily folic acid dose of 400–500 µg in combination with vitamin B12 and vitamin B6 is equally effective compared with folic acid dosages up to 5 mg/day. These studies indicate that lower vitamin dosages would have led to similar results and that there is no support for any concern that pharmacological dosages produce nonphysiologically low Hcy values.
Compared with ECAP cutoff values, the use of vitamin-optimized cutoff
values increased the diagnostic value of a fasting Hcy and decreased
the diagnostic value of a 6-h postload Hcy for the establishment of
hyperhomocysteinemia (Table 2
). The remaining very low prevalence of an
isolated abnormal 6-h postload Hcy does not seem to justify the use of
a MLT when vitamin-optimized cutoff values are used. It should also be
noted that a MLT is not necessarily harmless
(20)(21).
Twenty-nine percent of our healthy volunteers had hyperhomocysteinemia according to the ECAP cutoff values, which is in excellent agreement with the 30% of controls with hyperhomocysteinemia in the ECAP study (3). The hyperhomocysteinemia prevalence increased to 58% with use of vitamin-optimized cutoff values. When we considered men and pre- and postmenopausal women separately, the prevalences were 29%, 27%, and 53%, based on the ECAP cutoffs, and 58%, 76%, and 89%, based on vitamin-optimized cutoff values, respectively. These pretest probabilities may underscore the situation when the goal is to reach the lowest Hcy concentrations because at least 88% of the subjects had significantly decreased fasting Hcy after folic acid, vitamin B12, and vitamin B6 supplementation. The probability was even higher (92%) for subjects who did not take vitamin supplements. These high pretest probabilities preclude any meaningful role for the ordering of a Hcy test in the general population because this would require a pretest probability in the 25–65% range, according to the rules of "evidence based laboratory medicine" (22).
Decreasing Hcy by the use of vitamin supplements is advised for
hyperhomocysteinemic CVD patients and patients at high CVD risk because
of its expected beneficial effect (1)(4). In
view of the lack of evidence that decreasing Hcy decreases CVD risk,
there is at present no indication for ordering a Hcy test for CVD
prevention in the general population. Any future confirmation of a
beneficial effect of decreasing Hcy on CVD risk could, from the public
health point of view, be translated into adjustment of RDAs,
improvement of B-vitamin status through food fortification, and
concomitant implementation of vitamin-optimized reference values.
Fortification of grain products in the United States with 140 µg of
folic acid/100 g has changed the mean fasting plasma Hcy from 10.1 to
9.4 µmol/L in non-B-vitamin users and from 7.9 to 8.5 µmol/L in
B-vitamin users (23). It concomitantly changed the
percentage of subjects with plasma Hcy above the 9.3 µmol/L
vitamin-optimized cutoff value from 
63% to 52% (non-B-vitamin
users) and from 33% to 37% (B-vitamin users). These data show, from a
clinical chemical point of view, that the present fortification dosage,
or the choice of fortified food products (24), in the United
States is inadequate to prevent a large increase of the number of Hcy
tests from the moment that a causal relationship between
hyperhomocysteinemia and CVD is established. An explosion of Hcy test
requests can be avoided only when the daily supplemental B-vitamin
intake of the general population reduces the probability to <25% that
a random healthy subject has a fasting Hcy >9.3 µmol/L. It seems
that Hcy testing at a pretest probability of <25% in the general
population will be restricted to risk assessment of CVD patients and
relatives with high CVD risk, to diagnosis of inborn errors in
methionine and vitamin B12 metabolism, and to
assessment of vitamin status in subjects at risk or suspected of low
vitamin status (e.g., malabsorption syndrome, poor diet, and certain
drugs) (4).
In conclusion, there is remarkable uniformity in vitamin-optimized fasting Hcy reference values among different authors. Use of vitamin-optimized cutoff values increases the diagnostic value of a fasting Hcy and decreases the diagnostic value of a 6-h postload Hcy when compared with the use of ECAP cutoff values. With vitamin-optimized reference values, there does not seem to be a remaining role for the MLT. Current use of vitamin-optimized cutoff values offers no indications for ordering of a Hcy test: the pretest probability for the diagnosis hyperhomocysteinemia amounts to 58–89%, depending on gender and menopausal status, and the pretest probability of a Hcy decrease after supplementation with folic acid, vitamin B12, and vitamin B6 is 88% in apparently healthy subjects. Demonstration of a beneficial effect of decreasing Hcy on CVD risk would justify augmentation of the population’s B-vitamin status and the concomitant implementation of vitamin-optimized Hcy cutoff values. Proper augmentation of vitamin B status would drastically lower the prevalence of hyperhomocysteinemia, with remaining indications for a Hcy test for CVD patients and relatives with high CVD risk, and the diagnosis of certain inborn errors and subjects suspected or at risk of low B-vitamin status.
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Acknowledgments |
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Footnotes |
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References |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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The following articles in journals at HighWire Press have cited this article:
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  H. Refsum, A. D. Smith, P. M. Ueland, E. Nexo, R. Clarke, J. McPartlin, C. Johnston, F. Engbaek, J. Schneede, C. McPartlin, et al. Facts and Recommendations about Total Homocysteine Determinations: An Expert Opinion Clin. Chem., January 1, 2004; 50(1): 3 - 32. [Abstract] [Full Text] [PDF]
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