- Email: [email protected]
Zinc supplementation does not alter plasma homocysteine, vitamin B12 and red blood cell folate concentrations in French elderly subjects
ARTICLE IN PRESS
Journal of Trace Elements in Medicine and Biology 23 (2009) 15–20 www.elsevier.de/jtemb
CLINICAL STUDIES
Zinc supplementation does not alter plasma homocysteine, vitamin B12 and red blood cell folate concentrations in French elderly subjects Ve´ronique Ducrosa,b,,1, Maud Andriollo-Sanchezb,c,1, Josiane Arnauda,b,1, Nathalie Meunierd, Franc¸ois Laportea,b,c, Isabelle Hininger-Favierb,c,1, Charles Coudrayd, Monique Ferrye, Anne-Marie Rousselb,c,1 a
De´partement de Biologie Inte´gre´e, CHU de Grenoble, Grenoble F-38043, France Inserm, U884, Grenoble F-38000, France c Universite´ Joseph Fourier, Grenoble F-38000, France d Unite´ Maladies Me´taboliques et Micro-nutriments, INRA, Clermont-Ferrand/Theix F-63122, France e Service de Ge´riatrie, Centre Hospitalier de Valence, Valence F-26000, France b
Received 20 December 2007; accepted 4 August 2008
Abstract Background/aims: In ageing, low folates and vitamin B12 status are frequent and can explain the increase of plasma homocysteine level. Zinc is involved in the folates and vitamin B12 metabolism with opposite actions. The aim of this study was to investigate the effects of zinc supplementation on homocysteine and vitamin B12 plasma levels as well as red blood cell folate level in French ageing subjects participating in the ZENITH study. Methods: Apparently healthy middle-aged (55–70 years) and free-living older (70–85 years) subjects were enrolled. They were randomly allocated to three groups: 0, 15 or 30 mg Zn per day for 6 months as zinc gluconate in addition to their usual dietary intake. Results: At baseline, plasma homocysteine levels (15.273.5 mmol/L) in older people were higher than in the middleaged subjects (12.772.7 mmol/L) and was negatively correlated with vitamin B12 values (p ¼ 0.0036, r ¼ 0.215) and with RBC folate levels (po0.0001, r ¼ 0.30). These results are in agreement with previous data. However, we found no correlation between the biomarkers of zinc status and homocysteine, vitamin B12 or folate levels at baseline. Moreover, 6-month zinc supplementation did not modify homocysteine, vitamin B12 and RBC folate values in either of the groups. Conclusions: Zinc supplementation at moderate doses do not lead to deleterious effect on folate or vitamin B12 status in ageing healthy free-living people, but does not have any beneficial effects on homocysteine metabolism either. r 2008 Elsevier GmbH. All rights reserved. Keywords: Zinc supplementation; Homocysteine; Vitamin B12; Folates; Ageing
Corresponding author at: De´partement de Biologie Inte´gre´e, CHU
de Grenoble, BP 217, 38043 Grenoble, France. Tel.: +33 4 76 76 92 75; fax: +33 4 76 76 56 64. E-mail address: [email protected] (V. Ducros). 1 SFERETE’s member. 0946-672X/$ - see front matter r 2008 Elsevier GmbH. All rights reserved. doi:10.1016/j.jtemb.2008.08.003
Introduction The balance amongst micro-nutriments is crucial in the elderly and interactions between trace elements and vitamins, possibly occurring in single supplementation,
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V. Ducros et al. / Journal of Trace Elements in Medicine and Biology 23 (2009) 15–20
have to be considered with caution. It is well documented that folates and vitamin B12 deficiencies result in increased homocysteine levels which are, in ageing people, important risk factors of cardiovascular disease, cognitive decline and mortality [1,2]. Zinc (Zn) deficiency has been reported to increase with age [3,4]. The ZENITH (Zinc Effect in Nutrient/nutrient Interactions and Trends on Health and ageing) study was built to evaluate the effects of zinc on health indices in middleaged and older people. The aim and design of the ZENITH study have been described elsewhere [5,6]. Zinc treatment did not alter folate status despite at baseline a moderate positive correlation between red blood cells (RBC) folate and RBC zinc levels (r ¼ 0.11, po0.05) was noted [7]. There is much evidence of interactions between zinc and group B vitamins. Marginal or low Zn status has been reported to modify vitamin B12 metabolism and decreases absorption of food folates because the intestinal brush-border pteroylglutamate hydrolase, which is responsible for cleaving dietary folate prior to absorption, is zinc dependent [8,9]. Therefore, it could be hypothesized that zinc can participate in the regulation of the homocysteine pathway. The aim of the present work was to evaluate the effect of zinc supplementation on homocysteine and on the two co-factors of its metabolism (vitamin B12 and folate) in middle-aged and older French subjects of the ZENITH study.
Subjects and methods
Subjects Ninety-five volunteers (48 men and 47 women), aged 55–70 years, were recruited in Clermont-Ferrand (Theix, France) and 89 volunteers (46 men and 43 women), aged 70–87 years, were involved in Grenoble (France). The general recruitment, screening and enrolment of these subjects together with information and ethical consideration have been described elsewhere [6]. Fully informed written consent was required before taking part in the study. Based on the screening data, participants had to fulfill the following requirements: smoking less than 10 g/d of tobacco, alcohol consumption less than 30 g/d of alcohol for men and less than 20 g/d for women, less than 3 drugs/d for 55–70 years and less than 4 drugs/d for 70–85 years, no medical treatment which could affect Zn status (antidepressants, laxatives or hormone replacement therapy), no nutritional supplements, no chronic diseases such as diabetes, hypertension, cancer, no cognitive impairment (minimental state evaluation score greater than 23), nor depression (geriatric depression scale score less than 6), and a body mass index between 20 and 30 kg/m2. For participants satisfying the above criteria, a biochemical profile was performed, which included a full blood profile, including kidney and liver function evaluation. On the basis of these data, volunteers were excluded if they suffered from renal insufficiency, had bad hepatic performance, evidence of malabsorption or inflammatory chronic pathologies, and were included if their serology for HIV and hepatitis C viruses was negative.
Design of the study Biochemical measurements The present study reports a secondary analysis performed on ZENITH volunteers for whom plasma homocysteine was available. Subjects were middle-aged (55–70-year-old) and older (470-year-old) people recruited in France (Clermont-Ferrand/Theix and Grenoble). In both centers, subjects were divided in three groups receiving a placebo (lactose), or 15 mg Zn/d or 30 mg Zn/d for 6 months given as gluconate supplied by E-Pharma (Creapharm, Gannat, France). These Zn doses correspond to once or twice the zinc requirement [10] and deleterious effects on copper status have been reported at dose higher than 45 mg/d [11]. Zn capsules were distributed to the subjects at the beginning of the trial and at 3 months. At 3 and 6 months, the subjects were asked to return any remaining capsules, and the degree of apparent compliance was estimated from the number of delivered and returned capsules. Compliance, expressed as a proportion of the intended supplements consumed during zinc supplementation, did not differ among the groups; the mean compliance was around 98% in both groups.
All blood samples were collected after a 12-h overnight fast. Urine samples were taken in the morning after elimination of the first stream. For zinc determination, blood was collected using trace element-free Vacutainers tubes (Becton Dickinson, Pont de Claix, France); urine samples were acidified with pure hydrochloric acid (final acid concentration: 1%) and then stored at 20 1C until analysis. Urinary creatinine concentrations were measured by a modified Jaffe method (Metras Creatinine assay, Quidel Corporation, CA). Serum, RBC and urinary zinc levels were carried out by flame atomic spectrometry technique using a Perkin-Elmer 560 (Norwalk, Connecticut, USA) [12]. Homocysteine and vitamin B12 were measured on an EDTA plasma sample stored at 80 1C and RBC folates were determined on an aliquot of whole blood EDTA. Plasma homocysteine and vitamin B12 as well as RBC folates were measured by a competitive immunoassay with direct chemiluminescence detection on an automatised immunoanalyzer (ADVIA Centaurs, Siemens
ARTICLE IN PRESS V. Ducros et al. / Journal of Trace Elements in Medicine and Biology 23 (2009) 15–20
Medical Solutions Diagnostics, Puteaux, France). A serum-based standard reference materials (SRM 1955), used for validation of homocysteine levels, was tested to validate the reproducibility and the accuracy of the homocysteine determinations. CV inter-runs (reproducibility) was 7.7% for values at 3.98 mmol/L (level I) and 6.3% for values at 17.7 mmol/L (level III). The accuracy was 88.276.9% (n ¼ 3) at 3.98 mmol/L and 97.776.2% (n ¼ 3) at 17.7 mmol/L. Quality controls were assessed in every series of samples to check the reproducibility and accuracy of the measurements. We used Seronorms (Sero, Billingstad, Norway) and Utak controls (Utak Labs Inc., Valencia, CA) internal quality controls for zinc; QC cardiac markers 1,2,3 (Siemens) for homocysteine; QC ligand plus 1,2,3 (Siemens) for vitamin B12, and Lyphochek whole blood control (Bio-Rad, Marne-la-Coquette, France) for folates. For homocysteine, CV inter-runs (reproducibility) was 5.2% for values at 8.8 mmol/L and 4.2% for values at 19.7 mmol/L. The accuracy was 102.575.4% (n ¼ 6) at 8.8 mmol/L and 103.574.3% (n ¼ 6) at 19.7 mmol/L. For vitamin B12, CV inter-runs (reproducibility) were 3.8% for values at 617 pg/mL and 10.9% for values at 323 pg/mL. The accuracy was 97.773.7% (n ¼ 6) at 617 pg/mL and 96.80710.6% (n ¼ 6) at 323 pg/mL. For RBC folates, CV inter-runs were 11.5% for values at 4.8 ng/mL and 8.3% for values at 17.2 ng/mL. The accuracy was 99.0711.4% (n ¼ 13) at 4.8 ng/mL and 99.178.2% (n ¼ 15) at 17.2 ng/mL. Precision values for zinc have already been given [13]. All determinations of each parameter were done in the same laboratory, at the same time, and in random order.
Statistical analysis Data are presented as mean7standard deviation for homocysteine values and biomarkers of the Zn status or median7interquartile (IQ) for vitamin B12 and folate
17
values depending on the normal distribution or not of the parameter. Vitamin B12 and RBC folate values were log-transformed to approach Gaussian distribution. At baseline, statistical analysis between the two groups of elderly people was based on a Student t-test; in each group, gender influence was tested. The effect of 6 month zinc supplementation was analysed by analysis of variance (ANOVA) with repeated measurements including (centre, sex and zinc treatment) and was performed using Statview software version 5.0. Pearson’s correlations were performed to explore relationships between plasma homocysteine and RBC folates or plasma vitamin B12 or parameters of zinc status. The limit of statistical significance was set at po0.05.
Results The mean plasma homocysteine, median RBC folates or vitamin B12 concentrations at baseline are shown by age group and gender in Table 1. RBC folates are lower in the older group (po0.05) with a more significant decrease in the men group compared with the women, as previously described for the entire study [7]. Homocysteine levels were significantly different between the two age groups getting higher with age. Although the difference in plasma homocysteine levels between genders was more pronounced in the middle-aged group, the homocysteine levels were unaffected by gender (p40.05). A significantly positive correlation was found between homocysteine levels and age (po0.0001, r ¼ 0.358) which was stronger among women (po0.0001, r ¼ 0.433). Vitamin B12 levels were not significantly different among gender or age groups. If the biological vitamin B12 deficiency threshold was set less than 200 pg/mL [14], 4 men in Grenoble (4.5%) and 2 men plus 4 women in Theix (6.3%) can be considered at risk from vitamin B12 deficiency at
Table 1. Plasma homocysteine (mean7SD), vitamin B12 (median7IQ), RBC folates (median7IQ) values and biomarkers of Zn status (mean7SD) at baseline in French middle-aged and older subjects 55–70 years
RBC folate (ng/mL) Vitamin B12 (pg/mL) Homocysteine (mmol/L) Serum zinc (mmol/L) RBC zinc (mmol/L) Urinary zinc (mmol/ mmol creatinine)
470 years
Females (n ¼ 47)
Males (n ¼ 48)
All (n ¼ 95)
Females (n ¼ 43)
Males (n ¼ 46)
387789 3537205 12.272.4 12.971.6 212758 0.6570.38
373790 3357117 13.273.0 12.971.6 235765 0.6770.30
378787 3407156 12.772.7 12.971.6 224762 0.6670.34
384793a 3667194 15.173.6*** 13.171.5 190753 1.0570.68***
3187102** 3427149 15.373.4** 12.871.4 188747*** 0.9170.60*
*po0.05, **po0.01, ***po0.001, mean or median values significantly different from those of middle-aged subjects. a,b Effect of gender: medians not sharing a common letter are significantly different (po0.01).
All (n ¼ 89) b
3597107* 3537169 15.273.5*** 12.971.4 189749*** 0.9870.64***
ARTICLE IN PRESS
o0.001
NS
o0.05 363785 3577163 373761 3787123 385769 3617107 RBC folate (ng/mL) Before treatment After 6 months
3747175 3747129
3667110 320785
3167118 3377117
NS 3117247 3367198 3657159 3077159 3247120 3387137 3467176 3447151 Vitamin B12 (pg/mL) Before treatment After 6 months
3547176 3287213
3547114 3097169
15.173.7 14.774.7 14.973.3 14.673.8 13.272.8 13.072.7 12.172.6 12.973.4
12.872.8 12.873.3
15.573.4 15.774.4
30 29 30 31 32 32
15 mg Zn
NS ¼ Non significant. a Differences during supplementation was tested by ANOVA with repeated measurements to explore the effect of age, sex, time and zinc treatment.
o0.05
NS
NS NS
NS o0.0001
Age effect 30 mg Zn 15 mg Zn Placebo Placebo
30 mg Zn
470 years 55–70 years
P-valuea
Time effect
NS
Dose effect
NS
Sex effect
V. Ducros et al. / Journal of Trace Elements in Medicine and Biology 23 (2009) 15–20
N Homocysteine (mmol/L) Before treatment After 6 months
Table 2. subjects
Homocysteine (mean7SD), vitamin B12 (median7IQ), and RBC folates (median7IQ) before and after 6 month treatment in French middle-aged and older French
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baseline. Despite remaining in the reference range [15], zinc status decreased with age (Table 1) as evidenced by a significant decrease in RBC zinc in older men (po0.001). The increase in urinary zinc concentrations found in both gender (po0.001) has been reported to reflect an increased muscle catabolism, renal disturbances or diabetes [16,17] and therefore may contribute to a decrease in Zn status observed in the older subjects. Negative correlations were found significant between homocysteine and RBC folate levels (po0.0001, r ¼ 0.30), and between homocysteine and vitamin B12 levels (p ¼ 0.0036, r ¼ 0.215). No correlation was found between the zinc status parameters (serum, RBC, or urinary Zn) and plasma homocysteine or vitamin B12. Six-month zinc supplementation had no effect on homocysteine levels neither on vitamin B12 levels and RBC folate levels as shown in Table 2. For plasma homocysteine, differences between age classes were observed with higher values in the older. For RBC folates, significant effect of age (o0.05) with lower values in the older and time effect (o0.05) were noted as well as a gender effect (o0.001). No interaction age by time, sex by time or zinc treatment by time was observed for the three parameters (homocysteine, vitamin B12 and RBC folate).
Discussion The relationship between zinc and homocysteine pathway is complex as zinc seems to be involved in the folates and vitamin B12 metabolism with opposite actions. In the ZENITH core study, we noted a significant correlation at baseline between RBC folates and RBC zinc despite the very low risk of both folate and zinc deficiencies in this population of middle-aged and older adults [13]. Therefore, we were interested in studying homocysteine and vitamin B12 levels before and after zinc supplementation on the French sub-group population as differences in vitamin intake and status between European countries have been reported [7]. As regards folates, interactions between zinc and folates have been reported several times at intestinal level although results are discrepant due to differences in folates and zinc status of the studied populations. In our laboratory, we demonstrated that zinc deficiency in pregnant rats decreases folate bioavailability [18] and on the contrary, high dose of folates have no incidence on zinc absorption in humans with normal zinc status [19]. In the same way, the intestinal absorption of a test dose of 25 mg Zn in human was not modified by 10 mg folic acid [20]. The decrease in the food-folate absorption in marginal or low zinc status is related to the intestinal brush-border zinc-dependent enzyme, pteroylglutamate hydrolase, which is responsible for cleaving
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dietary folates prior to absorption [8,9]. As regards vitamin B12 metabolism, data about zinc interactions are very scarce [21]. Dietary zinc deficiency in rats has been reported to increase hepatic methionine synthase activity [22], which in turn resulted in decreased-plasma homocysteine and folate concentrations without any modification of red blood cell folates. A decreased amount of hepatic 5-methyltetrahydrofolate was observed in zinc-deficient rats compared with pair-fed controls [22,23]. If we assume that altered metabolism of folate and homocysteine found in zinc-deficient rats may occur in humans, we may understand why, in this study, Zn supplementation did not affect plasma homocysteine and vitamin B12 levels nor RBC folates. The lack of effect of Zn supplementation on folate status was recently reported for the entire study [24]. Indeed the ZENITH population was not zinc deficient as previously reported [13]; and in the French sub-groups only 7.4% of the subjects of the middle-aged group and 5.6% in the older group, exhibited zinc plasma o10.7 mmol/L, the cutoff selected for defining zinc deficiency [25]. Despite the difference of geographical location and the difference of age, dietary habits at baseline in these two French healthy free-living people were not found very different on the basis of a self-report questionnaire [26]. Clermont-Ferrand and Grenoble are two towns located in mountain areas: Auvergne and Alpes, respectively. A wide national interventional study (SUVIMAX 1994–2002) in which zinc dietary intake was determined, showed no difference in zinc intake at baseline between these two areas (data not published). Plasma homocysteine levels were found adequate for the age groups revealing no real deficiency in vitamin B12 or folate as confirmed by the RBC folates and plasma vitamin B12 concentrations. Moreover, the highest intake of zinc (30 mg/d) in our study remained moderate despite higher than safety limits recommendations proposed by different countries. Our results show that homocysteine pathway remains normally regulated. Zinc supplementation did not involve any deleterious regulation mechanism such as an increase of homocysteine levels. Regardless the effects of zinc supplementation, this study allows us to obtain reference values for plasma homocysteine in function of age and sex. By this way, we confirm that plasma homocysteine levels increase with age and became similar for men and women after 70 years. Upper reference value for homocysteine has been set over 20 mmol/L in elderly aged more than 65 years in nonfolate supplemented population [27]. In our older group, where all the subjects are aged more than 65, 7/89 (7.9%) had plasma homocysteine level greater than 20 mmol/L at baseline with the highest value set at 25.2 mmol/L. Among these subjects, only one had low vitamin B12 level (190 pg/mL) which could be the cause of his mild hyperhomocysteimia and none had low-RBC folate level (o150 ng/mL).
19
Vitamin B12 levels were found in the adult reference range for almost all the subjects. No severe deficiency (o100 pg/ml) was determined in both groups but surprisingly lower vitamin B12 values (6.3%) were found in the middle-aged group compared to the older one (4.5%) if the cutoff for defining vitamin B12 deficiency was set less than 200 pg/ml. For RBC folate levels, a significant age effect (o0.05) was found showing lower values in older subjects which can be related to the lower intake of folate in this group of subjects found at baseline [7] and after 6 months of Zn supplementation [24]. A significant (po0.001) gender effect was also noted on RBC folate levels showing higher values for women (392768; 383780 ng/ml) compared to men (364789; 351796 ng/ml) before and after Zn supplementation, respectively. The slight but significant decrease at 6 months (time effect) could be related to a seasonal effect and consequently, a decrease in dietary folate intake.
Conclusions This study allows us to obtain reference values for plasma homocysteine in function of age and gender and shows that a single zinc supplementation at moderate doses (15 or 30 mg/d) for 6 months does not affect vitamin B12 or folate status and does not modify agerelated increase of plasma homocysteine levels in healthy free-living elderly. These data suggest that, when there is no deficiency in vitamin B12 and folates, homocysteine pathway remains normally regulated and not related to zinc status. Zinc supplementation at moderate doses can be used in elderly subjects without concern regarding an increase in homocysteine plasma levels.
Acknowledgement We gratefully acknowledge Mrs. Sandrine Berthelin for her technical help and all the subjects for their cooperation. Funding: ZENITH is supported by the European Commission ‘‘Quality of Life and Management of Living Resources’’, Fifth Framework Programme, Contact no. QLK1-CT-2001-00168.
References [1] Gonzalez S, Huerta JM, Fernandez S, Patterson AM, Lasheras C. Homocysteine increases the risk of mortality in elderly individuals. Br J Nutr 2007;97:1138–43. [2] Haan MN, Miller JW, Aiello AE, Whitmer RA, Jagust WJ, Mungas DM, et al. Homocysteine, B vitamins, and
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the incidence of dementia and cognitive impairment: results from the Sacramento Area Latino Study on Aging. Am J Clin Nutr 2007;85:511–7. Hotz C, Peerson JM, Brown KH. Suggested lower cutoffs of serum zinc concentrations for assessing zinc status: reanalysis of the second National Health and Nutrition Examination Survey data (1976–1980). Am J Clin Nutr 2003;78:756–64. Mariani E, Cornacchiola V, Polidori MC, Mangialasche F, Malavolta M, Cecchetti R, et al. Antioxidant enzyme activities in healthy old subjects: influence of age, gender and zinc status. Biogerontology 2006;7:391–8. Coudray C, O’Connor JM, Maiani G, Cashman KD, Simpson EEA, Secker DL, et al. Introduction to the ZENITH study and summary of baseline results. Eur J Clin Nutr 2005;59:S5–7. Polito A, Meunier N, Andriollo-Sanchez M, Catasta G, Azzini E, Simpson EEA, et al. Screening and recruitment procedure of late-middle aged and older subjects: the ZENITH study. Eur J Clin Nutr 2005;59:S8–12. Polito A, Intorre F, Andriollo-Sanchez M, Azzini E, Raguzzini A, Meunier N, et al. Estimation of intake and status of vitamin A, vitamin E and folate in older European adults: the ZENITH. Eur J Clin Nutr 2005;59:S42–7. Halsted Ch. The intestinal absorption of dietary folates in health and diseases. J Am Coll Nutr 1989;8:650–8. Milne DB, Canfield WK, Mahalko JR, Sandstead HH. Effect of oral folic acid supplements on zinc, copper and iron absorption and excretion. Am J Clin Nutr 1984;39: 535–9. Maret W, Sandstead HH. Zinc requirements and the risks and benefits of zinc supplementation. J Trace Elem Med Biol 2006;20:3–18. Prasad AS. Essentiality and toxicity of zinc. Scand J Work Environ Health 1993;19(Suppl. 1):134–6. Arnaud J, Bellanger J, Bienvenu F, Chappuis P, Favier A. Recommended method for assaying serum zinc with flame atomic absorption. Ann Biol Clin 1986;44: 77–87. Andriollo-Sanchez M, Hininger-Favier I, Meunier N, Toti E, Zaccaria M, Brandollini-Bunlon M, et al. Zinc intake and status in middle-aged and older European subjects: the ZENITH study. Eur J Clin Nutr 2005;59: S37–41.
[14] Snow CF. Laboratory diagnosis of vitamin B12 and folate deficiency. A guide for the primary care physician. Arch Intern Med 1999;159:1289–98. [15] Iyengar GV. Reevaluation of the trace element content in reference man. Radiat Phys Chem 1998;51:545–60. [16] Arnaud J, Chappuis P, Jaudon MC, Bellanger J. Nutritional biological markers of deficiencies of zinc, copper and selenium. Ann Biol Clin 1993;51:589–604. [17] Delves HT. Assessment of trace element status. Clin Endocrinol Metabol 1985;3:725–60. [18] Favier M, Faure P, Roussel AM, Coudray C, Blache D, Favier A. Zinc deficiency and dietary folate metabolism in pregnant rats. J Trace Elem Electrolytes Health Dis 1993;7:19–24. [19] Arnaud J, Favier A, Herrmann MA, Pilorget JJ. Effect of folic and folinic acids on zinc intestinal absorption. Ann Nutr Metab 1992;36:157–61. [20] Keating JN, Wada L, Stokstad ELR, King JC. Folic acid: effect on zinc absorption in humans and in the rat. Am J Clin Nutr 1987;46:835–9. [21] Wang R, Zhu X, Guo F, Zhang W, Jia Z. Influence of different dietary levels of zinc on performance, vitamin B12, and blood parameters in lambs. Int J Vitam Nutr Res 2006;76:353–8. [22] Tamura T, Kaiser LL, Watson JE, Halsted CH, Hurley LS, Stokstad EL. Increased methionine synthase activity in zincdeficient rat liver. Arch Biochem Biophys 1987;256:311–6. [23] Hong KH, Keen CL, Mizuno Y, Johnston KE, Tamura T. Effect of dietary zinc deficiency on homocysteine and folate metabolism in rats. J Nutr Biochem 2000;11:165–9. [24] Intorre F, Polito A, Andriollo-Sanchez M, Azzini E, Raguzzini A, Toti E, et al. Effect of zinc supplementation on vitamin status of middle-aged and older European adults: the ZENITH study. Eur J Clin Nutr 2007 [advance online publication]. [25] King JC. Assessment of zinc status. J Nutr 1990;120: 1474–9. [26] Simpson EEA, O’Connor, Livingstone MBE, Rae G, Stewart-Knox BJ, Andriollo-Sanchez M, et al. Health and lifestyle characteristics of older European adults: the ZENITH study. Eur J Clin Nutr 2005;59:S13–21. [27] Refsum H, Smith AD, Ueland PM, Nexo E, Clarke R, McPartlin J, et al. Facts and recommendations about total homocysteine determinations: an expert opinion. Clin Chem 2004;50:3–32.
Journal of Trace Elements in Medicine and Biology 23 (2009) 15–20 www.elsevier.de/jtemb
CLINICAL STUDIES
Zinc supplementation does not alter plasma homocysteine, vitamin B12 and red blood cell folate concentrations in French elderly subjects Ve´ronique Ducrosa,b,,1, Maud Andriollo-Sanchezb,c,1, Josiane Arnauda,b,1, Nathalie Meunierd, Franc¸ois Laportea,b,c, Isabelle Hininger-Favierb,c,1, Charles Coudrayd, Monique Ferrye, Anne-Marie Rousselb,c,1 a
De´partement de Biologie Inte´gre´e, CHU de Grenoble, Grenoble F-38043, France Inserm, U884, Grenoble F-38000, France c Universite´ Joseph Fourier, Grenoble F-38000, France d Unite´ Maladies Me´taboliques et Micro-nutriments, INRA, Clermont-Ferrand/Theix F-63122, France e Service de Ge´riatrie, Centre Hospitalier de Valence, Valence F-26000, France b
Received 20 December 2007; accepted 4 August 2008
Abstract Background/aims: In ageing, low folates and vitamin B12 status are frequent and can explain the increase of plasma homocysteine level. Zinc is involved in the folates and vitamin B12 metabolism with opposite actions. The aim of this study was to investigate the effects of zinc supplementation on homocysteine and vitamin B12 plasma levels as well as red blood cell folate level in French ageing subjects participating in the ZENITH study. Methods: Apparently healthy middle-aged (55–70 years) and free-living older (70–85 years) subjects were enrolled. They were randomly allocated to three groups: 0, 15 or 30 mg Zn per day for 6 months as zinc gluconate in addition to their usual dietary intake. Results: At baseline, plasma homocysteine levels (15.273.5 mmol/L) in older people were higher than in the middleaged subjects (12.772.7 mmol/L) and was negatively correlated with vitamin B12 values (p ¼ 0.0036, r ¼ 0.215) and with RBC folate levels (po0.0001, r ¼ 0.30). These results are in agreement with previous data. However, we found no correlation between the biomarkers of zinc status and homocysteine, vitamin B12 or folate levels at baseline. Moreover, 6-month zinc supplementation did not modify homocysteine, vitamin B12 and RBC folate values in either of the groups. Conclusions: Zinc supplementation at moderate doses do not lead to deleterious effect on folate or vitamin B12 status in ageing healthy free-living people, but does not have any beneficial effects on homocysteine metabolism either. r 2008 Elsevier GmbH. All rights reserved. Keywords: Zinc supplementation; Homocysteine; Vitamin B12; Folates; Ageing
Corresponding author at: De´partement de Biologie Inte´gre´e, CHU
de Grenoble, BP 217, 38043 Grenoble, France. Tel.: +33 4 76 76 92 75; fax: +33 4 76 76 56 64. E-mail address: [email protected] (V. Ducros). 1 SFERETE’s member. 0946-672X/$ - see front matter r 2008 Elsevier GmbH. All rights reserved. doi:10.1016/j.jtemb.2008.08.003
Introduction The balance amongst micro-nutriments is crucial in the elderly and interactions between trace elements and vitamins, possibly occurring in single supplementation,
ARTICLE IN PRESS 16
V. Ducros et al. / Journal of Trace Elements in Medicine and Biology 23 (2009) 15–20
have to be considered with caution. It is well documented that folates and vitamin B12 deficiencies result in increased homocysteine levels which are, in ageing people, important risk factors of cardiovascular disease, cognitive decline and mortality [1,2]. Zinc (Zn) deficiency has been reported to increase with age [3,4]. The ZENITH (Zinc Effect in Nutrient/nutrient Interactions and Trends on Health and ageing) study was built to evaluate the effects of zinc on health indices in middleaged and older people. The aim and design of the ZENITH study have been described elsewhere [5,6]. Zinc treatment did not alter folate status despite at baseline a moderate positive correlation between red blood cells (RBC) folate and RBC zinc levels (r ¼ 0.11, po0.05) was noted [7]. There is much evidence of interactions between zinc and group B vitamins. Marginal or low Zn status has been reported to modify vitamin B12 metabolism and decreases absorption of food folates because the intestinal brush-border pteroylglutamate hydrolase, which is responsible for cleaving dietary folate prior to absorption, is zinc dependent [8,9]. Therefore, it could be hypothesized that zinc can participate in the regulation of the homocysteine pathway. The aim of the present work was to evaluate the effect of zinc supplementation on homocysteine and on the two co-factors of its metabolism (vitamin B12 and folate) in middle-aged and older French subjects of the ZENITH study.
Subjects and methods
Subjects Ninety-five volunteers (48 men and 47 women), aged 55–70 years, were recruited in Clermont-Ferrand (Theix, France) and 89 volunteers (46 men and 43 women), aged 70–87 years, were involved in Grenoble (France). The general recruitment, screening and enrolment of these subjects together with information and ethical consideration have been described elsewhere [6]. Fully informed written consent was required before taking part in the study. Based on the screening data, participants had to fulfill the following requirements: smoking less than 10 g/d of tobacco, alcohol consumption less than 30 g/d of alcohol for men and less than 20 g/d for women, less than 3 drugs/d for 55–70 years and less than 4 drugs/d for 70–85 years, no medical treatment which could affect Zn status (antidepressants, laxatives or hormone replacement therapy), no nutritional supplements, no chronic diseases such as diabetes, hypertension, cancer, no cognitive impairment (minimental state evaluation score greater than 23), nor depression (geriatric depression scale score less than 6), and a body mass index between 20 and 30 kg/m2. For participants satisfying the above criteria, a biochemical profile was performed, which included a full blood profile, including kidney and liver function evaluation. On the basis of these data, volunteers were excluded if they suffered from renal insufficiency, had bad hepatic performance, evidence of malabsorption or inflammatory chronic pathologies, and were included if their serology for HIV and hepatitis C viruses was negative.
Design of the study Biochemical measurements The present study reports a secondary analysis performed on ZENITH volunteers for whom plasma homocysteine was available. Subjects were middle-aged (55–70-year-old) and older (470-year-old) people recruited in France (Clermont-Ferrand/Theix and Grenoble). In both centers, subjects were divided in three groups receiving a placebo (lactose), or 15 mg Zn/d or 30 mg Zn/d for 6 months given as gluconate supplied by E-Pharma (Creapharm, Gannat, France). These Zn doses correspond to once or twice the zinc requirement [10] and deleterious effects on copper status have been reported at dose higher than 45 mg/d [11]. Zn capsules were distributed to the subjects at the beginning of the trial and at 3 months. At 3 and 6 months, the subjects were asked to return any remaining capsules, and the degree of apparent compliance was estimated from the number of delivered and returned capsules. Compliance, expressed as a proportion of the intended supplements consumed during zinc supplementation, did not differ among the groups; the mean compliance was around 98% in both groups.
All blood samples were collected after a 12-h overnight fast. Urine samples were taken in the morning after elimination of the first stream. For zinc determination, blood was collected using trace element-free Vacutainers tubes (Becton Dickinson, Pont de Claix, France); urine samples were acidified with pure hydrochloric acid (final acid concentration: 1%) and then stored at 20 1C until analysis. Urinary creatinine concentrations were measured by a modified Jaffe method (Metras Creatinine assay, Quidel Corporation, CA). Serum, RBC and urinary zinc levels were carried out by flame atomic spectrometry technique using a Perkin-Elmer 560 (Norwalk, Connecticut, USA) [12]. Homocysteine and vitamin B12 were measured on an EDTA plasma sample stored at 80 1C and RBC folates were determined on an aliquot of whole blood EDTA. Plasma homocysteine and vitamin B12 as well as RBC folates were measured by a competitive immunoassay with direct chemiluminescence detection on an automatised immunoanalyzer (ADVIA Centaurs, Siemens
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Medical Solutions Diagnostics, Puteaux, France). A serum-based standard reference materials (SRM 1955), used for validation of homocysteine levels, was tested to validate the reproducibility and the accuracy of the homocysteine determinations. CV inter-runs (reproducibility) was 7.7% for values at 3.98 mmol/L (level I) and 6.3% for values at 17.7 mmol/L (level III). The accuracy was 88.276.9% (n ¼ 3) at 3.98 mmol/L and 97.776.2% (n ¼ 3) at 17.7 mmol/L. Quality controls were assessed in every series of samples to check the reproducibility and accuracy of the measurements. We used Seronorms (Sero, Billingstad, Norway) and Utak controls (Utak Labs Inc., Valencia, CA) internal quality controls for zinc; QC cardiac markers 1,2,3 (Siemens) for homocysteine; QC ligand plus 1,2,3 (Siemens) for vitamin B12, and Lyphochek whole blood control (Bio-Rad, Marne-la-Coquette, France) for folates. For homocysteine, CV inter-runs (reproducibility) was 5.2% for values at 8.8 mmol/L and 4.2% for values at 19.7 mmol/L. The accuracy was 102.575.4% (n ¼ 6) at 8.8 mmol/L and 103.574.3% (n ¼ 6) at 19.7 mmol/L. For vitamin B12, CV inter-runs (reproducibility) were 3.8% for values at 617 pg/mL and 10.9% for values at 323 pg/mL. The accuracy was 97.773.7% (n ¼ 6) at 617 pg/mL and 96.80710.6% (n ¼ 6) at 323 pg/mL. For RBC folates, CV inter-runs were 11.5% for values at 4.8 ng/mL and 8.3% for values at 17.2 ng/mL. The accuracy was 99.0711.4% (n ¼ 13) at 4.8 ng/mL and 99.178.2% (n ¼ 15) at 17.2 ng/mL. Precision values for zinc have already been given [13]. All determinations of each parameter were done in the same laboratory, at the same time, and in random order.
Statistical analysis Data are presented as mean7standard deviation for homocysteine values and biomarkers of the Zn status or median7interquartile (IQ) for vitamin B12 and folate
17
values depending on the normal distribution or not of the parameter. Vitamin B12 and RBC folate values were log-transformed to approach Gaussian distribution. At baseline, statistical analysis between the two groups of elderly people was based on a Student t-test; in each group, gender influence was tested. The effect of 6 month zinc supplementation was analysed by analysis of variance (ANOVA) with repeated measurements including (centre, sex and zinc treatment) and was performed using Statview software version 5.0. Pearson’s correlations were performed to explore relationships between plasma homocysteine and RBC folates or plasma vitamin B12 or parameters of zinc status. The limit of statistical significance was set at po0.05.
Results The mean plasma homocysteine, median RBC folates or vitamin B12 concentrations at baseline are shown by age group and gender in Table 1. RBC folates are lower in the older group (po0.05) with a more significant decrease in the men group compared with the women, as previously described for the entire study [7]. Homocysteine levels were significantly different between the two age groups getting higher with age. Although the difference in plasma homocysteine levels between genders was more pronounced in the middle-aged group, the homocysteine levels were unaffected by gender (p40.05). A significantly positive correlation was found between homocysteine levels and age (po0.0001, r ¼ 0.358) which was stronger among women (po0.0001, r ¼ 0.433). Vitamin B12 levels were not significantly different among gender or age groups. If the biological vitamin B12 deficiency threshold was set less than 200 pg/mL [14], 4 men in Grenoble (4.5%) and 2 men plus 4 women in Theix (6.3%) can be considered at risk from vitamin B12 deficiency at
Table 1. Plasma homocysteine (mean7SD), vitamin B12 (median7IQ), RBC folates (median7IQ) values and biomarkers of Zn status (mean7SD) at baseline in French middle-aged and older subjects 55–70 years
RBC folate (ng/mL) Vitamin B12 (pg/mL) Homocysteine (mmol/L) Serum zinc (mmol/L) RBC zinc (mmol/L) Urinary zinc (mmol/ mmol creatinine)
470 years
Females (n ¼ 47)
Males (n ¼ 48)
All (n ¼ 95)
Females (n ¼ 43)
Males (n ¼ 46)
387789 3537205 12.272.4 12.971.6 212758 0.6570.38
373790 3357117 13.273.0 12.971.6 235765 0.6770.30
378787 3407156 12.772.7 12.971.6 224762 0.6670.34
384793a 3667194 15.173.6*** 13.171.5 190753 1.0570.68***
3187102** 3427149 15.373.4** 12.871.4 188747*** 0.9170.60*
*po0.05, **po0.01, ***po0.001, mean or median values significantly different from those of middle-aged subjects. a,b Effect of gender: medians not sharing a common letter are significantly different (po0.01).
All (n ¼ 89) b
3597107* 3537169 15.273.5*** 12.971.4 189749*** 0.9870.64***
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o0.001
NS
o0.05 363785 3577163 373761 3787123 385769 3617107 RBC folate (ng/mL) Before treatment After 6 months
3747175 3747129
3667110 320785
3167118 3377117
NS 3117247 3367198 3657159 3077159 3247120 3387137 3467176 3447151 Vitamin B12 (pg/mL) Before treatment After 6 months
3547176 3287213
3547114 3097169
15.173.7 14.774.7 14.973.3 14.673.8 13.272.8 13.072.7 12.172.6 12.973.4
12.872.8 12.873.3
15.573.4 15.774.4
30 29 30 31 32 32
15 mg Zn
NS ¼ Non significant. a Differences during supplementation was tested by ANOVA with repeated measurements to explore the effect of age, sex, time and zinc treatment.
o0.05
NS
NS NS
NS o0.0001
Age effect 30 mg Zn 15 mg Zn Placebo Placebo
30 mg Zn
470 years 55–70 years
P-valuea
Time effect
NS
Dose effect
NS
Sex effect
V. Ducros et al. / Journal of Trace Elements in Medicine and Biology 23 (2009) 15–20
N Homocysteine (mmol/L) Before treatment After 6 months
Table 2. subjects
Homocysteine (mean7SD), vitamin B12 (median7IQ), and RBC folates (median7IQ) before and after 6 month treatment in French middle-aged and older French
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baseline. Despite remaining in the reference range [15], zinc status decreased with age (Table 1) as evidenced by a significant decrease in RBC zinc in older men (po0.001). The increase in urinary zinc concentrations found in both gender (po0.001) has been reported to reflect an increased muscle catabolism, renal disturbances or diabetes [16,17] and therefore may contribute to a decrease in Zn status observed in the older subjects. Negative correlations were found significant between homocysteine and RBC folate levels (po0.0001, r ¼ 0.30), and between homocysteine and vitamin B12 levels (p ¼ 0.0036, r ¼ 0.215). No correlation was found between the zinc status parameters (serum, RBC, or urinary Zn) and plasma homocysteine or vitamin B12. Six-month zinc supplementation had no effect on homocysteine levels neither on vitamin B12 levels and RBC folate levels as shown in Table 2. For plasma homocysteine, differences between age classes were observed with higher values in the older. For RBC folates, significant effect of age (o0.05) with lower values in the older and time effect (o0.05) were noted as well as a gender effect (o0.001). No interaction age by time, sex by time or zinc treatment by time was observed for the three parameters (homocysteine, vitamin B12 and RBC folate).
Discussion The relationship between zinc and homocysteine pathway is complex as zinc seems to be involved in the folates and vitamin B12 metabolism with opposite actions. In the ZENITH core study, we noted a significant correlation at baseline between RBC folates and RBC zinc despite the very low risk of both folate and zinc deficiencies in this population of middle-aged and older adults [13]. Therefore, we were interested in studying homocysteine and vitamin B12 levels before and after zinc supplementation on the French sub-group population as differences in vitamin intake and status between European countries have been reported [7]. As regards folates, interactions between zinc and folates have been reported several times at intestinal level although results are discrepant due to differences in folates and zinc status of the studied populations. In our laboratory, we demonstrated that zinc deficiency in pregnant rats decreases folate bioavailability [18] and on the contrary, high dose of folates have no incidence on zinc absorption in humans with normal zinc status [19]. In the same way, the intestinal absorption of a test dose of 25 mg Zn in human was not modified by 10 mg folic acid [20]. The decrease in the food-folate absorption in marginal or low zinc status is related to the intestinal brush-border zinc-dependent enzyme, pteroylglutamate hydrolase, which is responsible for cleaving
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dietary folates prior to absorption [8,9]. As regards vitamin B12 metabolism, data about zinc interactions are very scarce [21]. Dietary zinc deficiency in rats has been reported to increase hepatic methionine synthase activity [22], which in turn resulted in decreased-plasma homocysteine and folate concentrations without any modification of red blood cell folates. A decreased amount of hepatic 5-methyltetrahydrofolate was observed in zinc-deficient rats compared with pair-fed controls [22,23]. If we assume that altered metabolism of folate and homocysteine found in zinc-deficient rats may occur in humans, we may understand why, in this study, Zn supplementation did not affect plasma homocysteine and vitamin B12 levels nor RBC folates. The lack of effect of Zn supplementation on folate status was recently reported for the entire study [24]. Indeed the ZENITH population was not zinc deficient as previously reported [13]; and in the French sub-groups only 7.4% of the subjects of the middle-aged group and 5.6% in the older group, exhibited zinc plasma o10.7 mmol/L, the cutoff selected for defining zinc deficiency [25]. Despite the difference of geographical location and the difference of age, dietary habits at baseline in these two French healthy free-living people were not found very different on the basis of a self-report questionnaire [26]. Clermont-Ferrand and Grenoble are two towns located in mountain areas: Auvergne and Alpes, respectively. A wide national interventional study (SUVIMAX 1994–2002) in which zinc dietary intake was determined, showed no difference in zinc intake at baseline between these two areas (data not published). Plasma homocysteine levels were found adequate for the age groups revealing no real deficiency in vitamin B12 or folate as confirmed by the RBC folates and plasma vitamin B12 concentrations. Moreover, the highest intake of zinc (30 mg/d) in our study remained moderate despite higher than safety limits recommendations proposed by different countries. Our results show that homocysteine pathway remains normally regulated. Zinc supplementation did not involve any deleterious regulation mechanism such as an increase of homocysteine levels. Regardless the effects of zinc supplementation, this study allows us to obtain reference values for plasma homocysteine in function of age and sex. By this way, we confirm that plasma homocysteine levels increase with age and became similar for men and women after 70 years. Upper reference value for homocysteine has been set over 20 mmol/L in elderly aged more than 65 years in nonfolate supplemented population [27]. In our older group, where all the subjects are aged more than 65, 7/89 (7.9%) had plasma homocysteine level greater than 20 mmol/L at baseline with the highest value set at 25.2 mmol/L. Among these subjects, only one had low vitamin B12 level (190 pg/mL) which could be the cause of his mild hyperhomocysteimia and none had low-RBC folate level (o150 ng/mL).
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Vitamin B12 levels were found in the adult reference range for almost all the subjects. No severe deficiency (o100 pg/ml) was determined in both groups but surprisingly lower vitamin B12 values (6.3%) were found in the middle-aged group compared to the older one (4.5%) if the cutoff for defining vitamin B12 deficiency was set less than 200 pg/ml. For RBC folate levels, a significant age effect (o0.05) was found showing lower values in older subjects which can be related to the lower intake of folate in this group of subjects found at baseline [7] and after 6 months of Zn supplementation [24]. A significant (po0.001) gender effect was also noted on RBC folate levels showing higher values for women (392768; 383780 ng/ml) compared to men (364789; 351796 ng/ml) before and after Zn supplementation, respectively. The slight but significant decrease at 6 months (time effect) could be related to a seasonal effect and consequently, a decrease in dietary folate intake.
Conclusions This study allows us to obtain reference values for plasma homocysteine in function of age and gender and shows that a single zinc supplementation at moderate doses (15 or 30 mg/d) for 6 months does not affect vitamin B12 or folate status and does not modify agerelated increase of plasma homocysteine levels in healthy free-living elderly. These data suggest that, when there is no deficiency in vitamin B12 and folates, homocysteine pathway remains normally regulated and not related to zinc status. Zinc supplementation at moderate doses can be used in elderly subjects without concern regarding an increase in homocysteine plasma levels.
Acknowledgement We gratefully acknowledge Mrs. Sandrine Berthelin for her technical help and all the subjects for their cooperation. Funding: ZENITH is supported by the European Commission ‘‘Quality of Life and Management of Living Resources’’, Fifth Framework Programme, Contact no. QLK1-CT-2001-00168.
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