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(Clinical Chemistry. 2001;47:1430-1436.)
© 2001 American Association for Clinical Chemistry, Inc.

Articles

Plasma Homocysteine Is Not Subject to Seasonal Variation

Michelle C. McKinley1, J.J. Strain1, Joseph McPartlin2, John M. Scott3 and Helene McNulty1a

1 Northern Ireland Centre for Diet and Health (NICHE), University of Ulster, Coleraine BT52 1SA, Northern Ireland. Departments of
2 Clinical Medicine and
3 Biochemistry, Trinity College, Dublin 2, Republic of Ireland.

aAuthor for correspondence. Fax 44-028-7032-4965; e-mail H.McNulty{at}ulst.ac.uk.


   Abstract
Top
 Abstract
Introduction
 Subjects and Methods
 Results
 Discussion
 References
 
Background: Studies investigating the relationship between plasma total homocysteine (tHcy) and vascular disease usually rely on a single measurement. Little information is available, however, on the seasonal variability of plasma tHcy. The aim of this study was to investigate the seasonal variation in fasting plasma tHcy and related B-vitamin intake and status in a group of people who did not consume fortified foods or take B-vitamin supplements.

Methods: In this longitudinal study, a group of 22 healthy people were followed for 1 year. A fasting blood sample and dietary information were collected from each individual every 3 months, i.e., at the end of each season.

Results: There was no significant seasonal variation in plasma tHcy or in B-vitamin intake or status with the exception of red cell folate (significantly lower in spring compared with autumn or winter) and serum folate (significantly lower in spring compared with the other seasons). Although the between-person variation in plasma tHcy was high (47%), the within-person variation was low (11%). This low variation, combined with the low methodologic imprecision of 3.8%, yielded a high reliability coefficient for plasma tHcy (0.97).

Conclusions: Although there was a small seasonal variation in folate status, there was no corresponding seasonal variation in plasma tHcy. The high reliability coefficient for plasma tHcy suggests that a single measurement is reflective of an individual’s average plasma tHcy concentration, thus indicating its usefulness as a potential predictor of disease. This, however, needs to be confirmed in different subgroups of the population.


   Introduction
Top
 Abstract
 Introduction
Subjects and Methods
 Results
 Discussion
 References
 
Prospective and case-control studies indicate that mild increases in total plasma homocysteine (tHcy) 1 are associated with an increased risk of cerebrovascular, peripheral vascular, and cardiovascular disease (1). The relationship between tHcy and vascular disease is graded, indicating that even small increases in plasma tHcy may pose a risk for vascular disease (2)(3)(4). Hcy metabolism is dependent on four B vitamins: folate (5), vitamin B12 (5), vitamin B6 (6), and riboflavin (vitamin B2) (7). Whereas the potential role of riboflavin as a determinant of tHcy has been investigated in just one human study (8), many studies have shown an inverse relationship between tHcy and the intake and status of vitamin B6 (9)(10)(11), vitamin B12 (10)(12), and folate (10)(12). The intake of these B vitamins, in particular folate, can vary from season to season because of variations in the price and availability of food (13). A potential consequence of this seasonal variation is that it could also affect tHcy concentrations because, even at low doses, the B vitamins can significantly affect tHcy concentrations (14)(15)(16)(17). Little information, however, is available on the seasonal variation of B-vitamin status or plasma tHcy.

Studies investigating the relationship between plasma tHcy and vascular disease, as well as intervention studies investigating the lowering of plasma tHcy, usually rely on a single measurement of plasma tHcy (18). Although much is known about the short-term variability of plasma tHcy (19)(20)(21)(22)(23), to our knowledge, only two studies have looked at the long-term variation of plasma tHcy (20)(24). One of these studies (20) involved small numbers (n = 9), with sampling of subjects at only one time point (30 months) compared with baseline. It therefore could not be considered a seasonal study. The second study, by Clarke et al. (24), was an investigation of seasonal variation in tHcy in an elderly sample, followed at 2-month intervals for 14 months. However, the study did not exclude vitamin users or consumers of fortified food, either of which could mask any natural seasonal variation. Because intervention studies frequently span more than one season, any natural seasonal or long-term variation in plasma tHcy concentrations could confound, mask, or bias the results of a long-term study. The aim of this study was to investigate the seasonal variation in B-vitamin intake and status and plasma tHcy concentrations in a group of people who did not consume fortified foods or take B-vitamin supplements.


   Subjects and Methods
Top
 Abstract
 Introduction
 Subjects and Methods
Results
 Discussion
 References
 
subject recruitment
Ethical approval was granted by the Research Ethical Committee of the University of Ulster, and subjects gave written, informed consent. Subjects 18–60 years of age were recruited from the staff and students at the University of Ulster, Coleraine. All potential subjects were interviewed, using a short medical questionnaire, regarding general health and use of drugs and supplements. Subjects who were suffering from gastrointestinal disease or hematologic disorders, taking any supplements containing B vitamins, consuming fortified foods, or who had a history of vascular, hepatic, or renal disease were excluded from the study.

study design
A 20-mL fasting blood sample was taken from each subject at the end of each season, i.e., May (spring), August (summer), November (autumn), and February (winter), respectively. At the same time as blood collection, a trained nutritionist (M.M.) collected dietary information. Each volunteer gave four 20-mL fasting blood samples, one representing each season of the year. Three tubes were collected from each subject: one 8-mL EDTA tube for plasma and red blood cell extraction; one 4-mL EDTA tube for preparation of red cell lysate and measurement of packed cell volume; and one 8-mL serum-separation tube for serum extraction.

The EDTA tube for measurement of tHcy, vitamin B6 status [erythrocyte aspartate aminotransferase activation coefficient (EASTAC)], and riboflavin status [erythrocyte glutathione reductase activation coefficient (EGRAC)] was wrapped in foil and placed on ice immediately after collection and centrifuged within 1 h of collection. This EDTA tube was centrifuged at 719g for 15 min to separate plasma and red blood cells. After centrifugation, the plasma layer was removed and stored. The remaining red blood cells were washed three times with phosphate-buffered saline. The saline and buffy layers were removed after each centrifugation, and the resulting washed red cells were stored.

A red cell folate lysate was prepared from the 4-mL EDTA tube by diluting blood 1 in 10 with a freshly prepared solution of 10 g/L ascorbic acid. The packed cell volume (required for the calculation of red cell folate concentration) was measured in the remaining whole blood in an automated Coulter Counter (Causeway Health and Social Services Trust Laboratories, Coleraine, Northern Ireland).

Serum-separation tubes were centrifuged at 719g, and the serum layer was removed. All preparations were stored at -70 °C for batch analysis at the end of the study. For each analyte, the interassay CV was as follows: plasma tHcy, 3.8%; red cell folate, 5.8%; serum folate, 8.1%; serum vitamin B12, 9.1%; EGRAC, 5.4%; and EASTAC, 1.8%.

biochemical measurements
Plasma tHcy was measured by fluorescent polarized immunoassay (25); red cell folate (26), serum folate (26), and serum vitamin B12 (27) were measured by microbiological assay. EASTAC (28) and EGRAC (29) were measured by enzyme assay on the Cobas Fara centrifugal analyzer (Roche Diagnostics). For all assays, samples were analyzed blind in duplicate (except for EGRAC where triplicate samples were measured) and within 6 months after sampling. Quality control was provided by repeated analysis of stored batches of pooled erythrocytes (for EASTAC and EGRAC), plasma (tHcy), serum (vitamin B12, folate), or red cell lysates (red cell folate) covering a wide range of values. For folate and vitamin B12, the control material corresponded to the deficiency cutoff, three times this value, and six times this value; for homocysteine, Nexo et al. (25) give a full description of assay performance; for EASTAC and EGRAC, the analyte concentrations in control material were calculated at the low, medium, and upper ends of the reference intervals.

dietary intake and anthropometric data
At the end of each season, dietary information was collected using a diet history interview covering usual dietary intake for the past 3 months. This information was cross-checked with a food frequency questionnaire specifically designed to focus on sources of the B vitamins. The diet history method involved an open-ended interview lasting ~1 h. During the interview, a trained nutritionist (M.M.) asked questions about general dietary habits, usual meal and snack pattern, the place of food consumption, foods typically consumed during the week and at weekends, methods of food preparation, and sizes of portions. The food frequency questionnaire was completed by subjects before the diet history interview and was used to cross-check the information provided during the diet history interview. Any discrepancy between the diet history and the information recorded in the food frequency questionnaire was discussed and clarified at the interview.

A trained nutritionist (M.M.) quantified portion sizes, using published food portion sizes (30), and calculated nutrient intakes, using the nutrient analysis program Comp-Eat (Lifeline; Nutritional Services Ltd.). Body mass index was calculated using height (m) and weight (kg) measurements. Basal metabolic rate (BMR) was calculated using formulas of Schofield (31) based on sex, height, and weight for age groups 18–30 and 30–60 years. The ratio of BMR to energy intake (obtained from the diet history) was calculated on an individual basis to identify likely underreporters of food intake according to the statistically derived cutoff limits of Goldberg et al. (32).

statistical analysis
All statistical analyses were performed using the Statistics Package for the Social Sciences (SPSS) computer software package. Seasonal differences were examined using repeated-measures ANOVA with the least significant difference test. P <0.05 was considered significant. The within-person (intraindividual) variation (CVw) in plasma tHcy was assessed by initially calculating the mean and SD (CV = 100 x SD/mean) for the four plasma tHcy measurements (spring, summer, autumn, and winter) on an individual basis. From these data, the mean CVw and the between-person (interindividual) variance (CVb) for all 22 subjects were then calculated. The total population variance (CVtotal) was calculated using the following equation:

where CVm is the between-run methodologic variance of the immunoassay for plasma tHcy.

The reliability coefficient (R) was calculated as the ratio of between-person variance (CVb) to total observed population variance (CVtotal).


   Results
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
Discussion
 References
 
seasonal variation in dietary intake
A total of 22 individuals 23–55 years of age (mean age, 33.4 ± 11.9 years) completed the longitudinal study; the group comprised 8 males (mean age, 32.6 ± 11.5 years) and 14 females (mean age, 33.9 ± 12.5 years). Dietary intake of energy and B vitamins was compared across the four seasons (Table 1 ). A ratio of energy intake to BMR of <1.14 was used to identify individuals who were likely to have underreported usual dietary intake (32). With this cutoff, two underreporters were identified in the summer and autumn, respectively, and one underreporter was identified in the winter and spring. These three underreporters were excluded from the dietary analysis shown in Table 1 . Dietary intakes of folate, vitamin B6, vitamin B12, and riboflavin below the United Kingdom reference nutrient intake (RNI) (33) were found in 27.3%, 9.2%, 10.2%, and 20.5% (mean of four seasons) of the group, respectively. There was no significant seasonal variation in the percentage of individuals below the RNI across the seasons. The dietary intake of all subjects was above the lower RNI (32). Dietary intakes of folate, vitamin B6, vitamin B12, and riboflavin below the United States recommended daily amount (RDA) (34) were found in 95.0%, 9.2%, 77.3%, and 20.5% (mean of four seasons) of the group, respectively. There was no significant seasonal variation in the percentage of individuals below the RDA across the seasons. Significant seasonal variation was not observed in the dietary intake of the B vitamins, nor was dietary intake of the B vitamins significantly correlated with plasma tHcy in any season (data not shown).


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  		Table 1. Seasonal variation in energy and B-vitamin intake1 of 19 healthy people (7 males, 12 females; ages, 23–55 years) assessed using a combined diet history and food frequency questionnaire method.

seasonal variation in b-vitamin status and plasma tHcy
The seasonal variations in biochemical status of the B vitamins and plasma tHcy throughout the four seasons are shown in Table 2 . There was no significant seasonal variation in the status of vitamin B6 (EASTAC), serum vitamin B12, or riboflavin (EGRAC). The status of both serum folate and red cell folate showed some seasonal variation. Serum folate status was significantly lower in spring compared with the other three seasons (P = 0.006, 0.027, and 0.029 for summer, autumn, and winter, respectively). Red cell folate status was significantly lower in spring compared with autumn (P = 0.041) or winter (P = 0.026). The most profound seasonal difference in both serum folate and red cell folate status occurred between winter and spring: serum folate, 14.48 ± 7.59 and 10.83 ± 3.90 nmol/L, respectively (difference of 3.65 nmol/L); red cell folate, 817.9 ± 318.6 and 724.4 ± 312.9 nmol/L, respectively (difference of 93.5 nmol/L). Despite the seasonal variation in serum folate and red cell folate concentrations, plasma tHcy concentrations did not show any significant variation. Table 3 shows the variability in measurements of plasma tHcy and the relevant B vitamins. The methodologic CV for each analyte was calculated by averaging the CVs obtained for low, medium, and high control material. Reliability coefficients were high for all measurements, ranging from 0.94 (serum folate) to 0.99 (EASTAC).


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  		Table 2. Seasonal variation in plasma tHcy and B-vitamin status of 22 healthy people (8 males, 14 females) 23–55 years of age.


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  		Table 3. Variability in plasma tHcy, red cell folate, serum folate, serum vitamin B12, EGRAC, and EASTAC.

Plasma tHcy failed to show any correlation with EASTAC or EGRAC, but was significantly correlated with serum folate, red cell folate, and serum vitamin B12. For serum folate, the correlations with plasma tHcy in spring, summer, autumn, and winter were r = -0.43 (P = 0.042), r = -0.462 (P = 0.003), r = -0.433 (P = 0.044), and r = -0.588 (P = 0.004), respectively. For red cell folate, the correlations with plasma tHcy in spring, summer, autumn, and winter were r = -0.502 (P = 0.012), r = -0.531 (P = 0.011), r = -0.561 (P = 0.007), and r = -0.513 (P = 0.015), respectively. For serum vitamin B12, the correlations with plasma tHcy in spring, summer, autumn, and winter were r = -0.444 (P = 0.038), r = -0.310 (P = 0.040), r = -0.332 (P = 0.031), and r = -0.491 (P = 0.021), respectively.


   Discussion
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
References
 
The aim of this study was to investigate the extent of seasonal variation in the intake and status of relevant B vitamins and plasma tHcy. We found that, whereas folate status showed a small but significant seasonal variation, there was no corresponding significant variation in tHcy or in the status of the other relevant B vitamins. This suggests that the change in red cell and serum folate status observed across the seasons in this study, although statistically significant, was not of biological significance. Unlike a previous seasonal study (24), this study excluded individuals who consumed fortified foods or took B-vitamin supplements. This is because regular consumption of fortified foods or supplements would be expected to ensure a high status of relevant B vitamins throughout the year, which may in turn mask any changes in status arising from a seasonal variation in intake of foods naturally rich in these nutrients. Blood samples and dietary information were collected at the end of each season to ensure consistency and because red cell folate is a retrospective index that reflects folate status during the previous 3 months, i.e., closely reflecting the life span of the red blood cell. Collecting blood samples mid-season would not have given a true representation of folate status for that particular season.

Wickham et al. (13) were the first to report a seasonal variation in folate status, but this study used a cross-sectional design (i.e., a different group of people was sampled during each season of the year), which is an unsuitable way of examining seasonal patterns. A longitudinal design, such as that used in the current study, represents a more appropriate way of addressing the issue of seasonality. Consistent with the results of the present study, Wickham et al. (13) found lower serum folate concentrations during March to June vs November to February (mean difference between seasons, 4.76 nmol/L compared with 3.65 nmol/L in the present study). However, a corresponding change in red cell folate concentrations was not detected until the following season, which the authors interpreted as being attributable to a time lag between diminished folate intake and the 4-month turnover of red blood cells. The authors did not specify, however, at what point during the season blood samples were taken or whether they were taken consistently at the same time point during each season. The lack of red cell folate response in the study by Wickham et al. (13) may simply have been attributable to inconsistent or inappropriate timing of the blood sampling compared with serum folate, which would tend to show a more immediate response to a recent change in dietary intake. More recently, Bates et al. (35) found evidence of a moderate seasonal variation in plasma pyridoxal phosphate (vitamin B6) concentrations, with the lowest values occurring in winter (January to March) and the highest in summer (July to September). They did not, however, find any such variation in pyridoxic acid or vitamin B6 intake, nor did they report how the variation in pyridoxal phosphate affected tHcy concentrations. We did not observe any variation in either vitamin B6 intake or in EASTAC concentrations, a functional indicator of vitamin B6 status, in the present study.

One of the primary aims of this study was to examine the consequence of any seasonal variation in B-vitamin status on tHcy concentration, now considered an independent risk factor for vascular disease. In the current study we found no seasonal variation in plasma tHcy as reflected in the high reliability coefficient (R = 0.97). The reliability coefficient of plasma tHcy (R = 0.97) in this long-term study compares well with the long-term reliability coefficient (0.88) estimated by Clarke et al. (24) and with the short-term reliability coefficients (0.94 and 0.90, respectively) of Garg et al. (20) and Rossi et al. (22). The within-person variation (CVw) of 11% for plasma tHcy in this study, which was calculated from samples collected at 3-month intervals, compares well with the results of other studies using different sampling time frames: 13%, daily sampling for 5 days (19); 7.03%, weekly sampling (20); 8.1%, weekly sampling (21); 8.3%, weekly sampling (22); 9.4%, sampling every 2 weeks (23); and 9%, sampling at 2-month intervals (24). The between-person variation (CVb) for plasma tHcy in this study (47%) was, however, somewhat higher than those noted by other investigators: 21% (19), 33.5% (20), 28% (21), 26.3% (22), 23.9% (23), and 24% (24).

Not only is plasma tHcy considered an important risk factor for vascular disease, but it is also a sensitive functional indicator of, primarily, folate status (14)(17) and also of other relevant B vitamins in the face of optimal folate status (16). This study further demonstrates that plasma tHcy has a high reliability coefficient, which compared well with other, more traditional, measures of B-vitamin status (red cell folate, R = 0.98; serum folate, R = 0.94; serum vitamin B12, R = 0.97; EGRAC, R = 0.98; and EASTAC, R = 0.99). This points to the potential usefulness of plasma tHcy as a screening tool in clinical situations, where it could be used as a proxy for general B-vitamin status. Individual tests of relevant B vitamins could then be pursued if an individual was found to have a borderline or increased plasma tHcy concentration. Results of this study are encouraging because they show that a single measurement of plasma tHcy is reflective of an individual’s average plasma tHcy concentration. The present study, therefore, suggests that a single measurement of tHcy can be relied on in studies examining its relationship with risk of vascular disease [and other diseases in which altered folate metabolism is thought to be important (36)(37)], and also for monitoring response to Hcy-lowering interventions. This finding, however, needs to be confirmed in different age groups, in different geographic locations where seasonal variations might be more extreme, and importantly, in different disease states.

In the present study, there was no significant correlation between the dietary intake of the relevant B vitamins and either B-vitamin status or plasma tHcy (data not shown). This result is not entirely surprising; few studies in the literature have reported significant interactions between B-vitamin intake and measurements of B-vitamin status (38)(39)(40)(41), including plasma tHcy. When correlations are found, they are not consistent for all B vitamins studied, tend to be weak, and are mainly driven by supplement users (of which there were none in this study) (38)(39)(40)(41). The most plausible explanation for the lack of correlation between intake and status is that blood concentration may be influenced by factors other than dietary intake, such as nutrient bioavailability in the case of folate (42), and atropic gastritis and hypochlorhydria in the case of vitamin B12 (43).

In conclusion, results from this study show that there was a small seasonal variation in serum folate and red cell folate status but no seasonal variation in plasma tHcy concentrations in this group of individuals. The high reliability coefficient for plasma tHcy indicates that a single measurement will accurately characterize an individual’s average tHcy concentration, thus indicating its usefulness as a potential predictor of disease. This, however, needs to be confirmed in different subgroups of the population.


   Acknowledgments
 
This study was supported by European Union Project BMH 4983549 and Abbott Germany.


   Footnotes
 
1 Nonstandard abbreviations: tHcy, total homocysteine; EASTAC, erythrocyte aspartate aminotransferase activation coefficient; EGRAC, erythrocyte glutathione reductase activation coefficient; BMR, basal metabolic rate; RNI, reference nutrient intake; and RDA, recommended daily amount.


   References
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 

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