Plasma homocysteine in adolescents depends on the interaction between methylenetetrahydrofolate reductase genotype, lipids and folate: a seroepidemiological study

In addition to being the principal cause of premature death in most European countries, cardiovascular diseases (CVDs) are a major cause of disability, and, by extension, increased health care cost [1]. Such diseases are generally due to a combination of several risk factors [2]. In 1996, the 27^th Bethesda Conference proposed hyperhomocysteinaemia as one of the new cardiovascular risk factors [3, 4].

Inasmuch as it involves a potentially modifiable and correctable risk factor, this hypothesis is of great health care importance since it opens the door to a new form of intervention against cardiovascular diseases, such as dietary supplementation with group B vitamins and folic acid.

Homocysteine is a sulphur-containing amino acid derived from methionine, present in proteins of animal origin ingested as part of dietary intake. It is produced by intracellular demethylation of methionine and then exported to the plasma, where it circulates, mainly in its oxidised form.

Homocysteine is metabolised to methionine via remethylation or to cysteine via transulphuration at the hepatic level. During vitamin B₆-dependent transulphuration, homocysteine is irreversibly catabolised into cysteine, thanks to the action of cystathionine-β-synthetase enzyme, in the presence of serine. Most of the homocysteine is remethylated, regenerating methionine, mainly due to the action of methionine synthetase, the enzyme that depends on the action of methylcobalamine (vitamin B₁₂) as a co-factor and folate, in the form of 5-methyl-tetrahydrofolate, as a methyl group donor.

The MTHFR C677T mutation is the most frequent cause of moderate hyperhomocysteinaemia due to genetic factors. The TT genotype has been found to have a direct effect on homocysteine concentration, with MTHFR enzymatic activity being reduced by as much as 50% in individuals with this mutation [5]. This polymorphism is present in its homozygous form in 5%-18% of the population [6].

The effect of the TT homozygote genotype on homocysteine concentration is not observed, however, when folic acid levels are elevated [7]. This is due to the fact that, despite the reduction in its enzymatic activity, MTHFR has sufficient 5-10 methyltetrahydrofolate substrate to generate the 5-methyltetrahydrofolate necessary for remethylation of homocysteine.

When these reactions are altered, homocysteine accumulates and is excreted into the blood, causing hyperhomocysteinaemia [8], which, according to a number of epidemiological studies, is associated with an increase in the probability of suffering from CVDs [9‐15].

Hyperhomocysteinaemia is defined as elevated concentration of fasting plasma total homocysteine.

As is the case with other parameters, such as arterial hypertension, no threshold plasma homocysteine value has been established, above which the risk of suffering from a vascular disease is increased [16, 17]. In most studies, this threshold is obtained arbitrarily, with values above the mean found in the control group plus two standard deviations [17‐20], or above the 95th percentile, being used to define hyperhomocysteinaemia [16, 17, 20‐22].

In Spain, Pijoan et al. constructed a reference interval (5-15 μmol/l) based on a sample of 396 apparently healthy individuals [23].

Plasma homocysteine levels increase with weight, height, body mass index, blood pressure, systolic and diastolic pressure, age, male gender and MHTFR 677TT genotype. In contrast, they decrease with high plasma folate and intraerythrocyte, vitamin B₆ and vitamin B₁₂ concentrations.

The established drug treatments for hyperhomocysteinaemia are vitamin supplements consisting of folate, vitamin B₆, vitamin B₁₂ or any combination of the three [24].

In the healthy paediatric population, homocysteine levels are influenced more by biochemical than by genetic factors, due to the high folate reserves present during childhood. No significant differences in homocysteine levels were observed between boys and girls until postpubertal age, when these rise in males [25].

Arterosclerosis begins at early ages of life [26, 27] and environmental factors in childhood are known to be linked to the appearance of cardiovascular disease at adult age [28].

Determination of plasma homocysteine in children could have a predictive value vis-à-vis adult vascular risk [29].

For a level of precision of 3%, an estimated prevalence of 5% and a confidence level of 95%, a minimum sample size of 202 was needed. Assuming lost of 20%, a total of 243 subjects was needed. (EPI-INFO 2002; version 6).

The study included subjects of both sexes, who were aged over 13 years, attended schools in the Madrid Region and produced informed consent. The following were subjects excluded from the study: those whose parents failed to sign the authorisation to participate; those who were authorised but were nevertheless unwilling to participate; and those who presented with some type of acute or chronic illness that might affect the variables of interest.

In each case, data were collected on age, gender, physical activity, blood pressure and anthropometric variables.

The study population totalled 317 students, 50% aged 13 years, 35% aged 14 years and 15% aged 15 or more years; 54% were females and 46% males. The mean age of the study subjects was 13.69 years, 95% CI (13.60-13.78), and their anthropometric characteristics corresponded to the expected values for their age.

Table 1 shows the serum or plasma values for the principal biochemical variables and possible hormonal factors implicated in cardiovascular disease. Mean concentrations were as follows: serum folate, 7.83 nM, 95% CI (7.42-8.23) nM; vitamin B₁₂, 503 pM, 95% CI (478-528) pM; and homocysteine, 4.92 μM, 95% CI (4.50-5.35) μM.

Taking the classic values as reference, prevalence of hyperhomocysteinaemia in the study population was 1.26% for 15 μmol/L, and 2.52% for 12 μmol/L. Table 2 defines different cut-off points for hyperhomocysteinaemia in children.

When analysing MTHFR C677T genotype, normal homozygous CC in the methylenetetrahydrofolate reductase enzyme was present in 37.8%, heterozygous CT genotype was present in 47.3%, and TT genotype, corresponding to the homozygous mutation, was present in 14.9% of cases.

Table 3 shows the prevalence of potential cardiovascular risk factors and variables implicated in homocysteine metabolism. Of the total study population, 23.8% displayed deficient serum folate levels (≤ 5.3 nmol/l) and 24.6% displayed deficient vitamin B₁₂ levels (≤ 336 pmol/l). Overweight, elevated glucose levels, deficient vitamin B₁₂ and HDL cholesterol values were greater in boys, whilst girls showed greater sedentariness and hypercholesterolaemia. Prevalence of occasional tobacco and alcohol use increased with age. Folate deficit was significantly greater among subjects that displayed total (homozygote TT) mutation of the MTHFR C677T genotype (Table 3).

In order to assess analyze the influence of vitamin and metabolic factors in homocysteine levels in this population group, hyperhomocysteinemia was defined as percentile 90^th. Crude analysis showed how prevalence of hyperhomocysteinaemia increases with male gender (32.4% vs. 18.0% in female), age (21.0%, 22.3%, 40.4% for 13, 14 and >14, respectively), mutation of the MTHFR C677T genotype (15.1%, 24.8%, 46.8% for CC, CT and TT genotype, respectively), HDL cholesterol (22,7% vs. 37.2% for normal and low levels, respectively) and deficient folate values (19.4 vs. 43.2% for normal and low levels, respectively). These independent variables, as well of other of interest as vitamin B₁₂, were included in a multivariate logistic regression model, obtaining adjusted Odds Ratios for the principal study factors (Table 4).

Deficient HDL cholesterol and serum folate levels registered adjusted Odds Ratios of OR = 2.786, 95% CI (1.089-7.126) and OR = 5.140, 95% CI (2.347-11.256) respectively. The MTHFR C677T mutant genotype raised the risk of hyperhomocysteinaemia (CC→CT: OR = 2.362; 95% CI (1.107-5.042) CC→TT: OR = 6.124, 95% CI (2.301-16.303))

After plausible biologically interactions have been ruled out, the multiplicative nature of the association among several risk factors in the sample can be observed, namely, prevalence of hyperhomocysteinaemia increases in accordance with the number of risk factors (FR) that accumulate.

From Table 5, it will be seen how the prevalence of hyperhomocysteinaemia rises when deficient folate and HDL cholesterol values and mutant genotype coincide. Shown in Figure 1 is the cumulative effect of the most important risk factors, i.e., genotype, folate deficit and HDL cholesterol deficit. An indiviual with CT genotype and deficient folate and cHDL is almost 34 times more likely to suffer from hyperhomocysteinaemia; and if that same individual has these deficits plus the TT genotype, the likelihood of his/her having high serum homocysteine levels is then 87 times higher.

This study suffers from the limitations inherent in all cross-sectional studies, in that it does not allow for causal or temporal relationships among variables to be established, thus rendering it necessary for subsequent analytical epidemiological studies to be conducted in order to confirm the results and hypotheses generated. Furthermore, some confounding variables may not have not been included in this study, e.g., methionine intake, creatinine levels, or polymorphisms of the nicotinamide-methyltransferase enzyme (chromosome 11q23), which have recently been linked to plasma homocysteine levels [33].

In Spain, Gutiérrez-Revilla et al. (n = 83), Vilaseca et al. (n = 195) and Mainou et al. (n = 80) have respectively published cross-sectional studies in children and adolescents. The first two targeted the population aged 0 to 18 years, and the third the population aged 5 to 18 years. Ours is the first study conducted in Spain and focused on the adolescent population to have a substantial sample size.

In Western countries, vitamin deficiencies are rather infrequent, but in populations with important vitamin deficiencies, plasma homocysteine levels rise until reaching age-adjusted hyperhomocysteinaemia prevalences of 73% in men and 41% in women, as shown by a recent study conducted in Iran [34].

The distribution of the various polymorphisms of the MTHFR C677T enzyme in our sample was very similar to that observed in other studies [35, 36], i.e., Hiraoka, with 32.9%, 51.6% and 15.5%, in CC, CT and TT respectively [37], or Gutiérrez-Revilla et al., with 42.2%, 41% and 16.9%, in CC, CT and TT respectively [25]. The cardiovascular risk factors studied showed no significant variation according to MTHFR genotype, which only affects folate and serum homocysteine levels.

The mean homocysteine values obtained by us are very similar to those found in children and adolescents in Norway and the USA, namely, 5.25 and 5 μmol/l, respectively [38, 39]. There is no international consensus regarding the establishment of a cut-off point for defining hyperhomocysteinaemia, and there are still fewer data relating to the adolescent population. Prevalence of hyperhomocysteinaemia in the study population was 1.26% for 15 μmol/L and 2.52 for 12 μmol/L, results very similar to those recently obtained in young adults aged 20-22 years in Tokyo (1.18% for > 15 μmol/L) [37]. Taking into account other international studies, which use percentiles for determining other magnitudes in childhood and adolescence, the 90^th percentile is considered a good cut-off point for defining hyperhomocysteinaemia in developed countries [29, 40, 41]. In our study, this cut-off allowed us to show a statistically significant increase of plasma homocysteine levels depending on the study variables. In line with other studies [5, 23, 42‐46], prevalence of hyperhomocysteinaemia was seen to increase in our study with the MTHFR C677T mutant allele (2- and 6-fold for partial and total C→T mutation, respectively), serum folate deficit (5-fold) and cHDL deficit (2.7-fold). No significant association was found with vitamin B₁₂ after adjusting by age, gender and CV risk factors.

It has been observed that the coexistence of several risk factors, none of which is necessarily elevated to an extraordinary degree, in any given individual can actually be more dangerous than the existence of a single factor, regardless of its magnitude [44, 47]. In our study, as previously described in Guo et al [48], prevalence of hyperhomocysteinaemia increases when risk factors accumulate. The probability of being hyperhomocysteinemic is 5 times higher with deficit serum folate levels than it is with normal values. If, the person concerned also happens to be a carrier of the MTHFR TT polymorphism, this risk is then multiplied by 30 because his/her ability to metabolise homocysteine is very reduced; and if, in addition to this, such a person has HDL cholesterol levels below 35 mg/dl, the likelihood of his/her being hyperhomocysteinemic is multiplied by 90. These results suggest that the higher decile is an appropriate cut-off to define homocysteinemia in adolescents.

While many national and international studies link deficit serum folate levels to hyperhomocysteinaemia and suggest dietary supplementation as a preventive strategy for reducing cardiovascular risk [48‐53], other authors contend that, as hyperhomocysteinaemia has not yet been shown to be an independent risk factor, dietary supplementation is not justified [54].

We thus feel that we are confronted by a still unresolved research question, and trust that our study may serve as a further contribution towards finding an answer in this field of investigation

A good definition of hyperhomocysteinaemia in adolescents is the 90^th percentile, equivalent to 8.23 μmol/L.

Risk factors for hyperhomocysteinaemia are cHDL and folate deficiency, and the MTHFR C677T mutant genotype. Coexistence of all three factors increases the risk of suffering from hyperhomocysteinaemia 87-fold.