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Technical Briefs |
1
Department of Clinical Biochemistry, Hvidovre Hospital, DK-2650 Hvidovre, Denmark, and
2
Department of Clinical Biochemistry, Aalborg Hospital, DK-9100 Aalborg, Denmark;
a author for correspondence: fax 45 3675 0977, e-mail hcy{at}forum.dk
Increased concentrations of the amino acid homocysteine—hyperhomocysteinemia—are correlated with atherosclerotic and thrombotic diseases (1)(2). High concentrations can be lowered by diet and dietary supplements with vitamins B12, folate, and pyridoxine. Homocysteine is produced in the metabolism of the essential amino acid methionine and is converted by cystathionine ß-synthase to cystathionine and by methionine synthase back to methionine. These enzymatic reactions are dependent on sufficient concentrations of the vitamins B12, folate, and pyridoxine.
Knowledge of biological, postprandial, and orthostatic variations are important in judging significant changes in results and error sources in blood sampling conditions (3), and several studies on the biological variation of plasma homocysteine have been published (4)(5)(6).
Fasting blood samples traditionally have been recommended for plasma homocysteine measurement because postprandial changes produce a modest decrease in the first hours and an increase after 8 h (7)(8).
Orthostatic changes can also be important in the monitoring of homocysteine in patients with atherosclerotic and thrombotic diseases. Because most homocysteine is bound to albumin, the decrease with supine posture is expected to be 5–10%.
In this study we examined the day-to-day, postprandial, and orthostatic variations of plasma total homocysteine.
Blood samples were obtained from 19 healthy hospital employees (11 women and 8 men) ages 19–60 years (median age, 44 years). None of these individuals was or became pregnant or had medical diseases. The intake of oral contraceptives, intermittent asthma and allergy medicines, nonsteroidal antiinflammatory drugs, acetaminophen, and multivitamins was allowed during the study. Multivitamins were taken on a regular basis by four persons. Blood hemoglobin, erythrocyte folate, and serum cobalamin concentrations in all subjects were within the reference intervals.
Participants provided fasting blood samples after arrival for work (0800 to 1000) and nonfasting samples after lunch (1215 to 1500). Samples were collected daily for 5 days from subjects in an upright position. In addition, samples for plasma homocysteine, serum albumin, and serum sodium were obtained from nine participants before and after 30 min of supine rest.
Samples were collected in K3EDTA, and all plasmas were separated within 30 min (except four samples used for biological variation that were separated at 35, 50, 60, and 60 min). After separation, the samples were frozen at -20 °C and kept frozen until analysis. The samples were not kept on ice before separation to reflect routine sampling conditions. Samples were assayed in singlicate, and all samples from the same person were assayed in one batch.
The study was conducted in accordance with the Helsinki Declaration of 1975 and was approved by the Regional Scientific Ethics Committee. All participants gave informed consent.
The method used to determine total plasma homocysteine was HPLC and has been described previously (9). The within-run analytical CV was 4.9%, and the between-run CV was 4.5%. The material used for internal quality control was plasma from a healthy donor. An external quality-assurance program with participants from all of Scandinavia (10) has shown results with maximum differences of 6% from the mean value.
Sodium and albumin in serum were determined by the dry-chemistry methods on a Vitros system (Johnson & Johnson). The analytical CVs were 1% for sodium and 1.5% for albumin (total). The samples were analyzed using the same batch of slides.
The formulas used in the calculation of biological, postprandial, and
orthostatic CVs are given below:
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where CVB is the coefficient of biological variation, CVI is the coefficient of within-person biological variation, CVG is the coefficient of between-person (group) variation, CVA is the coefficient of analytical variation, and CVT is the coefficient of total variation. The CVIs were calculated according to the mean-square successive difference method, which is less sensitive to trends than the more usual variance method (11).
Thus, the variance of fasting to postprandial values was calculated as:
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This can also be regarded as the within-day or ~6 h variation.
The variance of the orthostatic values was calculated as:
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The variance of within-person day-to-day values was calculated as:
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The median CVI and the CVI at the 25th and 75th percentiles were calculated because of the heterogeneity of variance.
The index of individuality = CVI/CVG (3).
The reference change value = 1.96 x 21/2 x (CVI2 + CVA2)1/2, showing the change that can be explained by analytical and biological variation covering 95% of the changes (probability level, 95%; probability of false alarm, 0.05) (12).
The heterogeneity of variance was tested by calculating whether the CVI values were all estimates of the same true variance (EVV), using the formula in Ref. (12).
Two-way ANOVA with replication was used to determine whether there was a systematic difference between fasting and postprandial values.
The mean fasting and postprandial plasma homocysteine values did not differ through the five weekdays (no trend over the week). The mean plasma homocysteine values for women were significantly lower than for men (8.7 vs 10.0 µmol/L; P = 0.004, two-tailed unpaired t-test).
The observed changes in homocysteine concentrations and calculated
values are listed in Table 1
. As can be seen in Table 1
and Fig. 1
, the plasma homocysteine decreases after 30 min of supine rest
were larger than can be explained by protein binding.
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Our values for within-person biological variation were comparable to the CVI values obtained by others: 7.03% (weekly sampling) (4), 9.4% (sampling at 2-week intervals) (5), and 9% (sampling at 2-month interval) (6). There was no or very little seasonal variation in plasma homocysteine (6). Cobbaert et al. (5) observed CVI values between 0.0% and 26.1%, which are values very close to our CVI range. Guttormsen et al. (7) observed a day-to-day maximum change in plasma homocysteine, including postprandial values, between 15% and 39% (analytical imprecision was 2–4%). Santhosh-Kumar et al. (13) measured serum homocysteine 3 weeks apart and found that the second values were between 46% and 165% of the first values (analytical imprecision was 8%).
All blood samples for biological variation in our study were drawn with the subjects in an upright position so that orthostatic variation would not affect the result. Physical exertion before blood sampling was not standardized, but in general sampling was performed in a relaxed condition. In a recent publication, there was a slight (1.2 µmol/L, 11.5%) increase immediately after acute exercise on plasma homocysteine (14).
Postprandial values taken from 1215 to 1500 did not differ
significantly from fasting values taken in the morning from 0800 to
1000. The meals eaten were traditional, i.e., cereals in the morning
and more protein-rich food at noon. It does not seem to be necessary to
collect a homocysteine sample in a fasting condition, although the
first hours after breakfast were not investigated in this study. The
postprandial blood sample was collected 30 min to 3 h after lunch
and 4–7 h after breakfast. Ubbink et al. (8) observed a
mean decrease 2 h after breakfast of 0.57 µmol/L and 4 h after
breakfast of 0.72 µmol/L, with a maximum decrease of ~1.8 µmol/L.
These values are also small compared with the random unavoidable
day-to-day variation (see Table 1
).
The surprisingly large orthostatic variation might suggest a function of homocysteine in the vasomotor response, which is triggered by the centralization of the blood flow at supine rest. A connection between homocysteine and nitric oxide, a potent vasodilator, that might explain this variation has been found (15). Whatever the cause, the large orthostatic variation has practical implications in monitoring of patients, who may go from bedridden to non-bedridden and later, to outpatient status. The mean value of plasma homocysteine is ~30% lower at day 1 compared with day 3 in acute myocardial infarction (16). This might be explained by orthostatic changes instead of acute-phase reaction.
Because the index of individuality is 0.4 (0.6 for women), the population-based reference intervals are not very useful in monitoring individual patients and as screening procedure to pick up illness in a particular individual (3)(12). Persons with homocysteine values in the upper part of the reference interval have a higher risk of atherosclerotic events than persons with homocysteine values in the lower part (1), making the reference interval even less useful. Risk calculation at a certain concentration of plasma homocysteine and assessment of significant response to treatment are the most important features.
In conclusion, the mean day-to-day change in fasting plasma homocysteine was 15.2% (1.3 µmol/L), and the maximum change was 62.2% (4.4 µmol/L). The mean CVI was 13%, but there was heterogeneity of variance with CVI values from 0% to 25%. The postprandial values did not differ systematically from the fasting values; therefore, it does not appear critical that the patient be in a fasting state when plasma homocysteine is measured. The orthostatic changes after 30 min were up to 29.9% (3.5 µmol/L), with a mean of 19% (2.1 µmol/L), and correlated only weakly to the albumin change. This might indicate a function of plasma homocysteine in the vasomotor response, which needs further investigation.
Acknowledgments
This study was supported by The Medical Research Foundation of Northern Jutland County (Nordjyllands Laegevidenskabelige Forskningsfond). We thank Gerda Mikkelsen and Susanne Øberg for excellent technical assistance.
References
The following articles in journals at HighWire Press have cited this article:
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  L. R. Solomon Cobalamin-responsive disorders in the ambulatory care setting: unreliability of cobalamin, methylmalonic acid, and homocysteine testing Blood, February 1, 2005; 105(3): 978 - 985. [Abstract] [Full Text] [PDF]
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 P. Quadri, C. Fragiacomo, R. Pezzati, E. Zanda, G. Forloni, M. Tettamanti, and U. Lucca Homocysteine, folate, and vitamin B-12 in mild cognitive impairment, Alzheimer disease, and vascular dementia Am. J. Clinical Nutrition, July 1, 2004; 80(1): 114 - 122. [Abstract] [Full Text] [PDF]
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 A. de Bree, V. Ducros, L. I. Mennen, I. Quere, S. Hercberg, and P. Galan Influence of Centrifugation Temperature on the Plasma Total Homocysteine Concentration Clin. Chem., June 1, 2003; 49(6): 1026 - 1027. [Full Text] [PDF]
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M. R. Fokkema, M. F. Gilissen, J. J. van Doormaal, M. Volmer, I. P. Kema, and F. A.J. Muskiet Fasting vs Nonfasting Plasma Homocysteine Concentrations for Diagnosis of Hyperhomocysteinemia Clin. Chem., May 1, 2003; 49(5): 818 - 821. [Full Text] [PDF]
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 A. De Bree, W. M. M. Verschuren, D. Kromhout, L. A. J. Kluijtmans, and H. J. Blom Homocysteine Determinants and the Evidence to What Extent Homocysteine Determines the Risk of Coronary Heart Disease Pharmacol. Rev., December 1, 2002; 54(4): 599 - 618. [Abstract] [Full Text] [PDF]
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 E. Nurk, G. S. Tell, S. E. Vollset, O. Nygard, H. Refsum, and P. M. Ueland Plasma Total Homocysteine and Hospitalizations for Cardiovascular Disease: The Hordaland Homocysteine Study Arch Intern Med, June 24, 2002; 162(12): 1374 - 1381. [Abstract] [Full Text] [PDF]
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 T. B. Shea, E. Rogers, J. Auer, R. Berent, B. Eber, S. Seshadri, and P. A. Wolf Homocysteine and Dementia N. Engl. J. Med., June 20, 2002; 346(25): 2007 - 2008. [Full Text] [PDF]
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A. Voortman, A. Melse-Boonstra, J. M. Schulz, J. Burema, M. B. Katan, and P. Verhoef Optimal Time Interval between Repeated Blood Sampling for Measurements of Total Homocysteine in Healthy Individuals Clin. Chem., October 1, 2001; 47(10): 1839 - 1841. [Full Text] [PDF]
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 A. N. FRIEDMAN, A. G. BOSTOM, J. SELHUB, A. S. LEVEY, and I. H. ROSENBERG The Kidney and Homocysteine Metabolism J. Am. Soc. Nephrol., October 1, 2001; 12(10): 2181 - 2189. [Abstract] [Full Text] [PDF]
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M. C. McKinley, J.J. Strain, J. McPartlin, J. M. Scott, and H. McNulty Plasma Homocysteine Is Not Subject to Seasonal Variation Clin. Chem., August 1, 2001; 47(8): 1430 - 1436. [Abstract] [Full Text] [PDF]
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| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
), serum
albumin (
), and serum sodium
(
) after
30 min of supine rest (nine subjects).