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A relationship between vitamin B12, folic acid, ascorbic acid, and mercury uptake and methylation
Life Sciences, Vol. 47, pp. 167-173 Printed in the U.S.A.
Pergamon Press
A RELATIONSHIP B ~ E E N VITAMIN B12, FOLIC ACID, ASCORBIC ACID, AND MERCURY UPTAKE AND METHYLATION
Nancy E. Zorn John T. Smith Department of Nutrition and Food Sciences College of Human Ecology and Agricultural Experiment Station The University of Tennessee, Knoxville, TN 37996-1900 (Received in final form May 7, 1990) Summary Ingestion of megadoses of certain vitamins appears to influence the in vivo methylation of mercuric chloride in guinea pigs. The addition of megadoses of vitamin B]2 fed either singularly or in combination with folic acid resulted in increased methylmercury concentrations in the liver. Moreover, percent methylmercury levels were significantly increased with B12 treatment in the liver (B]2 only and B12/folic acid) and brain (B12/vitamin C). Incorporation of high levels of folic acid into the dietary regime also increased the methylmercury concentration particularly in the liver and hair tissues. The addition of vitamin C in the diet, particularly in combination with B12 (brain) or folic acid (muscle) resulted in increased methylmercury levels in these tissues and percent methylmercury values with B12 in the muscle and brain tissue. Organic mercury compounds are one of the important environmental pollutants in our biosphere and much research has been conducted to explore the toxicity of this metal. Mass poisonings have been reported in which methylmercury has been implicated. The first reported instance took place in the 1950's in Minamata, Japan over a period of seven years. People were poisoned as a result of consuming fish which had acquired high concentrations of methylmercury due to contamination of local waters with industrial waste (1, 2). In addition, methylmercury and other alkylmercury compounds have been widely used in agriculture as antifungal agents in the storage of seeds. This has lead to several outbreaks of poisoning from the use of treated seeds for food (3, 4). A more recent concern has been the level of mercury exposure as a result of dental amalgams (5, 6, 7). Despite these events, mercury is still widely used in the production of fungicides, insecticides, paints, and electrical equipment. Many industrial wastes which contain mercury are discarded in oceans, rivers, lakes, and landfills (8, 9). Thus, the general public continues to be exposed to mercury from a number of rather widespread sources. A number of mechanisms have been proposed for metabolism of inorganic mercury in vivo. Early research has shown that rats injected with mercuric salts will excrete a small portion as volatile mercury, thus, suggesting that these animals are able to convert inorganic mercury either by a methylation reaction or reduction to the metallic form (10). Additional research done by Ogata and associates (11, 12) has demonstrated that elemental mercury can be oxidized to produce the mercurous ion (Hg+), and the mercuric ion (Hg++). Another metabolic fate of inorganic mercury compounds is the biomethylation of mercuric ions via methyl B12 which has been shown to occur in certain bacteria (13, 14). Methylmercury compounds pass easily through the blood-brain barrier and the placenta and are more likely to target the nervous system, testes, and the developing embryo/fetus (15). Additionally, the toxicity of short-chain alkylmercury compounds, such as methylmercury, is accentuated due to their persistence in mammals. 0024-3205/90 $3.00 +.00 Copyright (c) 1990 Pergamon Press plc
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The vitamin supplement business has blossomed into a multibillion dollar industry due to the large-scale consumption of these products. It has been estimated that 40% to 50% of the adult population, both active and sedentary, consume self-prescribed vitamin supplements (16). Recognizing that the biological formation of methyimercury requires the coenzyme methylcobalamine (vitamin B12) (13) and that the regeneration of methyl-cobalamine would require one of the forms of tetrahydrofolate (folic acid) (17), it appears conceivable that individuals who consume megadoses of B]2 and folic acid would increase their potential to form methylmercury. Additionally, an investigation has shown that ingestion of megadoses of vitamin C with other vitamins permits increases in the level of metabolically active cobalamin (vitamin B12) as well as other vitamins (18). The purpose of this investigation was to examine if there is a relationship between consumption of megavitamins and mercury retention and metabolism. Methods Animals and Dietary Re.qime: A pilot study was initially conducted to determine whether rats or guinea pigs would be best suited for this study (19). It was determined that the guinea pig will accumulate more folic acid and vitamin B]2 than the rat with a dietary excess. Eight groups of five male guinea pigs each weighing approximately 250 grams were purchased from Hilltop Animal Company and were fed Purina Guinea Pig Chow 5025 and megadoses of vitamin B]2, folate, and/or vitamin C. The vitamins were weighed and subsequently triturated with sucrose to a total weight of 500 g and yielded a final concentration of 4 mg folic acid, 50 #g B]2 and 1.8 g ascorbic acid per 4 g of triturate. This was then added to ground guinea pig chow at a concentration of 4g/100g. The control group consisted of animals fed ad libitum chow ground guinea pig containing 4g of sucrose per 100 g. One group of animals was fed a diet supplemented with megadoses of vitamin B]2, folic acid, and vitamin C. This group was referred to as the all vitamin treatment animals. Three groups of animals were provided diets which included megadoses of either B12 and folic acid, B]2 and vitamin C, or folic acid and vitamin C. These groups served as the combination vitamin treated animals and were referred to according to their respective vitamin blends. Finally, three groups of guinea pigs were given diets supplemented with the individual vitamins. These groups were called by the respective vitamin treatment, i.e. B]2 only, folic acid only, vitamin C only. All diets were fed ad libitum for a total of eight weeks. The animals were maintained on a 12-hour light dark cycle in a controlled temperature environment. Distilled water was freely available to all animals. Animals were weighed weekly to assess comparable weight gain and food consumption with the different dietary regimes. At the end of the dietary period, each animal was given 0.6 mg HgCI2/kg body weight as a subcutaneous injection in isotonic saline every other day for nine doses (20). Animals were observed throughout this treatment for overt signs of mercury toxicity. Twenty-four hours after the last injection, the animals were sacrificed and their liver, heart, brain, lungs, spleen, kidneys, semitendinosus muscle, and hair were removed and frozen at -80°C for subsequent mercury determinations. All tissues were examined for toxicity effects of mercury. In addition, the sites of injection were inspected for any indication of toxicity. Upon thawing a 25% (w/v) homogenate was prepared for each tissue using 1.5 M potassium hydroxide. Mercury Analysis: All reagents were analytical grade and specially selected for their low mercury content. Glassware was cleaned with nitric acid (4 M) and rinsed with demineralized water. All solutions were prepared using demineralized water only. Mercury determinations were carried out by cold-vapor atomic absorption spectrometry (AAS). Absorption was measured at 253.7 nm with a Perkin-Elmer Model 3030 atomic absorption spectrophotometer equipped with a mercury, hollow cathode lamp. A cold-vapor apparatus was laboratory assembled according to directions provided by a Perkin-EImer representative (personal communication) with an absorption cell (100 mm X 20 mm i.d.) used for the AAS measurements. The gas was first passed through an absorption cell (50 mm X 20 mm) containing potassium persulfate (Baker Chemical Company, Phillipsburg, N.J., Analytical Grade) which dried the material. After each measurement, the mercury was adsorbed in a charcoal absorption tube (65 mm X 35 mm i.d.) which was connected to the apparatus with a bypass arrangement. The system was closed by means of a peristalic pump.
Vol. 47, No. 2, 1990
Vitam±ns Effect on Mercury Metabollsm
The method used in this research was adapted from a number of sources (21, 22, 23) and allowed for selective reduction of inorganic mercury and methylmercury using the same sample. Stannous chloride was used for the reduction of inorganic mercury while a stannous-cadmium chloride mixture was necessary for methylmercury determinations. Mercuric chloride and methylmercury standards were used to evaluate the effectiveness of the stannous chloride and stannous-cadmium chloride reduction specificity. A known quantity of either mercuric chloride or methylmercury was analyzed using the specific reduction agent listed above. Recovery of these standards was 99-100% for inorganic mercury and 97-99% for methylmercury. Thus, the validity of this methodology was established. In addition, previous research done with gas chromatography and direct current plasma atomic emission spectrometry to examine this methodology (22, 24) has demonstrated the reliability/specificity of this type of reduction. These two measurements were then added together to derive total mercury concentrations. At the beginning of each run, inorganic mercury standards (0.4 #g Hg/ml) were run by adding 1 ml of 10% stannous chloride solution and absorption values read. Also, absorption was measured for the methylmercury standard (0.5 ~g CH3Hg/ml) by following the procedure listed below for methylmercury determination. Representative samples of each tissue homogenate were pipetted to Biological Oxygen Demand (BOD) bottles: 5 ml of liver and muscle; 2.5 ml of brain, lungs, and heart plus 2.5 ml of 1.5 M KOH; 1.0 ml of kidney and spleen plus 4.0 ml of 1.5 M KOH; 1 ml of blood plus 5 ml of 1.5 M KOH; and 0.25 g weighed hair sample plus 5.0 ml of 1.5 M KOH. Then, 1 ml of 1% cysteine and 1.24 ml of 12 N sodium hydroxide were added to each reaction vessel. At all times, a total of 22.4 meq of hydrogen ions was maintained in each sample solution. The samples were digested in a water-bath at 85°C for five minutes. After digestion, the samples were immediately placed in an ice bath and 12.76 ml of 1% sodium chloride were added to each bottle. Total reaction volume was 20 ml and all samples were maintained on ice until AAS was performed. Each B.O.D. bottle containing the digested sample was placed in an ice water bath under the aerator tube assembly. The following were added in sequence; 1-2 drops Antifoam A Emulsion (Sigma Chemical Company, St. Louis, MO), 10 ml of 8 M sulfuric acid, 1 ml of 10% tin (11) chloride solution, 20 ml of 45% sodium hydroxide, and 50 ml of demineralized water. The aerator was immediately attached, the pump turned on, and the air flow continued through the reaction vessel for exactly one minute. At this time the absorption for inorganic mercury was read. The aerator tube was immediately removed from the sample and the air flow switched to remove the mercury through the activated charcoal chamber. The pump was allowed to run for a maximum of one minute or until absorption was zeroed, at which time the air flow was stopped. Exactly two minutes after the first reduction process was begun, 10 ml of 8 M sulfuric acid, 2 ml of tin (11)chloride - cadmium chloride reagent, and 20 ml of 45% sodium hydroxide were added sequentially to the sample. The aerator tube was attached immediately and the pump activated. The reaction was allowed to occur until a maximum absorption for methylmercury was obtained and recorded. At the end of the run, the air flow was again diverted through the charcoal absorption tube to remove all mercury. Statistical Analysis: All data were evaluated by Analysis of Variance (ANOVA) performed with SAS (SAS Program) using the General Linear Model (GLM) procedures. Differences between groups for each tissue were determined by Duncan's multiple-range test (25). Resu~s All animals were healthy upon sacrifice and tissues were examined for mercury toxicity with no evidence of damage observed in any group. Additionally, there was no indication of inflammation, sloughing, or granulomas at the site of injection. Weight data showed comparable weight gain within the various groups, however, a slight weight loss was observed for all animals after mercury injections were begun (26).
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As indicated in the methods section, a total of nine tissues were analyzed from each animal. However, due to the limitations of this publication, only the results of four tissues will be reported here. The data presented in Table I show the micrograms of inorganic and methylmercury per gram of wet liver in guinea pigs fed the various vitamin regimes. The individual vitamin treated groups had significantly higher inorganic and methylmercury concentrations than controls and all other treated groups. The B]2/folic acid group had more (p<0.05) inorganic mercury than the all vitamin and B12/vitamin C groups. Of importance, are the data showing that the B12/folic acid group (.67 #g) had a significantly higher methylmercury concentration than the control (.37 #g), all vitamins (.25 #g), B12/vitamin C (.27 ~,g), and folic acid/vitamin C (.38 #g) groups. TABLE I Mercury Levels in the Liver Tissue of Guinea Pigs with Different Vitamin Dietary Regimes #.q HCl/.q wet tissue Treatment
Inorganic Hg
Methyl Hg
Controls All Vitamins B12/Folic Acid B12/Vitamin C Folic Acid/Vitamin C B/2 Only Folic Acid Only Vitamin C Only
0.78 0.52 1.05 0.61 0.78 1.58 1.60 1.72
0.37 0.25 0.67 0.27 0.38 1.16 1.22 1.08
± 0.07.a'b ÷ 0.04 o ± 0.11 b 0.05 . 0.05 a ' ° ± 0.07 c ± 0.32 c + 0.24 c
± ± + ± ± ± ± +
0.05 a 0.01 a 0.04 D 0.02 a 0.03 a 0.18 c 0.12 c 0.08 c
Values represent the average ± S.E.M. Data showing a common superscript letter in a column are not significantly different (p>0.05). The brain was an important tissue to investigate in this study since only the methylated form of mercury is able to cross the blood-brain barrier. However, as shown in Table II, there were no significant differences in the concentration of inorganic and methylmercury between treatments. TABLE II Mercury Levels in the Brain Tissue of Guinea Pigs with Different Vitamin Dietary Regimes ~,Cl H.q/.q wet tissue Treatment
Inorganic Hg
Methyl Hg
Controls All Vitamins B12/Folic Acid B]2Nitamin C FoUc Acid/Vitamin C B12 Only Folic Acid Only Vitamin C Only
0.30 0.19 0.16 0.20 0.25 0.27 0.30 0.23
0.24 0.18 0.15 0.29 0.18 0.19 0.18 0.19
± 0.04 a ± 0.02 a 0.01 a ± 0.04 a ± 0.04 a _+ 0.02 a ± 0.02 a ± 0.01 a
± 0.04 a _+ 0.02 a ± 0.01 a ± 0.07 a ± 0.04 a 0.01 a _+ 0.01 a ± 0.01 a
Values represent the average ± S.E.M. Data showing a common superscript letter in a column are not significantly different (p>0.05).
Vol. 47, No. 2, 1990
Vitamins Effect on Mercury Metabolism
Table III shows the data for muscle tissue. Of particular interest in this tissue is the folic acid/vitamin group which displayed the highest (p<0.05) concentrations of both forms of mercury. TABLE III Mercury Levels in the Muscle Tissue of Guinea Pigs with Different Vitamin Dietary Regimes
~,.q H.q/.q wet tissue Treatment
Inorganic Hg
Methyl Hg
Controls All Vitamins B12/Folic Acid B12Nitamin C Folic AcidNitamin C B12 Only Folic Acid Only Vitamin C Only
0.21 0.10 0.12 0.27 1.33 0.18 0.15 0.16
0.18 0.13 0.19 0.31 0.83 0.14 0.10 0.07
± ± ± ±
0.06 a 0.01 a 0.01 a 0.15 a 0.71D ± 0.02 a 0.02 a ± 0.01 a
± ± + ± + ±
0.03 a 0.03 a 0.02 a 0.10 a 0.37 b 0.01 a 0.02 a ~ 0.01 a
Values represent the average ± S.E.M. Data showing a common superscript letter in a column are not significantly different (p>0.05). In hair tissue (Table IV), inorganic mercury concentration did not differ among the groups. Both the folic acid group and vitamin C group had methyl and total mercury concentrations which were significantly higher than controls. Of interest also was the fact that the folate group had significantly more methylmercury than the all vitamin and B12/folic acid groups. TABLE IV Mercury Levels in the Hair Tissue of Guinea Pigs with Different Vitamin Dietary Regimes
~,.q H.q/.q wet tissue Treatment
Inorganic Hg
Methyl Hg
Controls All Vitamins B]2/Folic Acid B]2 Only Folic Acid Only Vitamin C Only
0.80 0.73 0.87 0.95 0.95 0.95
0.65 0.70 0.78 1.27 1.84 1.47
± 0.07 a ± 0.12 a ± 0.09 a 0.14 a ± 0.09 a ± 0.15 a
± 0.06 a, 0.09a'.o 0 04 a ' ° ÷ 0128a'b'c ± 0.62 c ± 0.39 ° ' c
Values represent the average ± S.E.M. Data showing a common superscript letter in a column are not significantly different (p>0.05). The percent methylmercury (of total mercury) values for the four tissues are presented in Table V. In the liver, the B]2/folic acid and individual vitamin treated groups
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TABLE V Percent 1 Methylmercury Values in Various Tissues of Guinea Pigs with Different Vitamin Dietary Regimes
Treatment
Liver
Brain
Tissue Muscle
Controls All Vitamins B]2/Folic Acid B]2/Vitamin C FoliCB12 OnlyACidNitaminC
a2 31 a,b 33 b,c 40 a 31 a,b 3341c
a,b 44 49 a 49 59 ca a,b 4242a,b
a,b,c 49 a,b,c 53 b 61 b,c 57 a,b,c 4550a,b,d
47a -56a,b
Folic Acid Only Vitamin C Only
44 c 39 b,c
b 4437a,b
a,d 3140d
60b ' 57a, o
Hair 45a" 49a,D
1Percent methylmercury determined as a portion of total mercury concentration present in tissue. 2Data showing a common superscript letter in a column are not significantly different (p>0.05). exhibited significantly higher values than the controls, while in brain tissue, only the B/2/vitamin C group had an elevated (p<0.05) percent methylmercury level compared to all other groups. In muscle and blood tissue only, the vitamin C only group had significantly lower percent methylmercury levels than controls. In hair tissue, the folic acid group had significantly higher percent methylmercury than the controls. Discussion It was the purpose of this investigation to determine if the ingestion of megadoses of vitamin B]2, folic acid or vitamin C would increase the methylation of inorganic mercury and/or alter the metabolism of subcutaneous doses of inorganic mercury. The data presented in Table I support the concept that megadoses of these vitamins will increase the methylation of inorganic mercury. Feeding diets supplemented with megadoses of B12/folic acid, or B12, folic acid or vitamin C resulted in an 81 to 230% increase in methylmercury in the liver tissue as compared to control. There were no significant differences in MeHg content between groups supplemented with B]2, folic acid or vitamin C. The significant increase above control obtained with the B12/folic acid combination was significantly lower than obtained with the individual vitamins. It could be argued that since the liver values for both inorganic mercury and methylmercury for rats fed these three vitamin supplemented diets are high and not significantly different that these values represent a systematic error. In defense, it should be noted that supplementation with B]2, folic acid and vitamin C did not result in significant increases in methyl or inorganic mercury in muscle or brain tissue nor inorganic concentration in hair tissue. Only folic acid or vitamin C supplementation increased the methyl mercury concentration of hair. Therefore, a systematic measurement error seems an unlikely explanation for these data. Since the methylmercury concentration in the hair is a reflection of the body burden of mercury over time (26) the positive relationship observed between the high levels of methyl mercury found in the liver and the hair of the guinea pigs fed the individual vitamin supplements could be used as additional proof that megadoses of these vitamins increased the body burden of methylmercury. If, however, these data from liver and hair reflect the body burden of methylmercury then the consistency of the data from the brain and the assumption that only methylmercury passes the blood brain barrier (15) would allow the assumption that the brain has a limited capacity to take up methylmercury. Once, however, the methylmercury is taken up by the brain it appears that with exception of mice fed a diet supplemented with B12 and vitamin C that the methylmercury is demethylated to give a ratio of 1.2:1 inorganic to methylmercury. The combination of
Vol. 47, No. 2, 1990
Vitamins Effect on Mercury Metabolism
B12/vitamin C supplementation appeared to suppress demethylation since the inorganic:methyl mercury ratio was 0.7:1. Thenen (27) has shown vitamin C ingestion raised the B]2 levels in B12 deficient rats. It is conceivable then that the B]2/vitamin C supplementation suppressed the formation of inorganic mercury in the brain by keeping B12 high to remethylate inorganic mercury. Therefore, although the total concentration of mercury in the brain is not elevated in guinea pigs fed diets supplemented with B]2/vitamin C, the fact that a significantly greater percentage is in the form of methylmercury could result in an increase in neurotoxicity. No explanation has been offered for the large increase in mercury both inorganic and methyl in the muscle tissue of guinea pigs fed the diet supplemented with folic acid/vitamin C. Since the values were suspect, they were repeated several times. It is believed, therefore, that the values are real but no explanation can be offered. In conclusion, the methylation of inorganic mercury in vivo can be increased with the ingestion of megadoses of vitamin B]2, folic acid, or vitamin C. Therefore, individuals who take large amounts of these nutritional supplements and who are exposed to mercury sources could have increased methylmercury formation with subsequent neurological damage, especially with high combinations of vitamin B/2 and vitamin C. Additionally, dental professionals should become aware of this potential. Further research is required to elucidate these effects and the possible mechanisms which are involved in the methylation of mercury in vivo. References 1. M. KUTSUMA, Minamata Dis., Kutama University Press, Japan (1968). 2. R.P. JUNGHANS, Environ. Res. 3_1:1-31 (1983). 3. F. BAKIR, S.F. DAMLUJI, L. AMIN-ZAKI, M. MURTADHA, A. KHALIDI, N.Y. AL-RAWl, S. TIKRITI, H.I. DHAHIR, T.W. CLARKSON, J.C. SMITH and R.A. DOHERTY, Science 18_!:230241 (1973). 4. P.L. SCHULLER, Cadmium, Lead, Mercury and Methyimercury Compounds, Food and Horticulture Organization of the United Nations (1976). 5. C. NALEWAY, R. SAKAGUCHI, E. MITCHELL, T. MULLER, W.A. AYER and J.J. HEFFERREN, JADA 11.__[1:37-42(1985). 6. S.B. CHANG, C. SlEW and S.E. GRUNINGER, J. Anal. Toxicol. 11--137-139 (1987). 7. M.J. VIMY and F.L. LORSCHEIDER, J. Dent. Res. 64:1069-1071 (1985). 8. B. LOFORTH, Ecol. Res. Comm. Bull. _4:5-44 (1970). 9. M. BERLIN, Effects and Dose-Response Relationships of Toxic Metals, G. F. Nordber, pp. 235-245, Elsevier Scientific, Amsterdam (1976). 10. T. CLARKSON and A. ROTHSTEIN, Health Phys. 10:1115-1121 (1964). 11. M. OGATA, M. IKEDA and Y. SUGATA, Environ. Health 34:218-221 (1979). 12. M OGATA, K. KENMOTSU, N. HIROTA, T. MEGURA and H. AIKOH, Arch. Environ. Health 4_.22:26-30(1987). 13. W.P. RIDLEY, L.J. DIZIKES and J.M. WOOD, Sci. 19"7:329-3,32 (1977). 14. C.T. WALSH, M.D. DISTEFANO, M.J. MOORE, L.M. SHEWCHUK and G.L. VERDINE, FASEB J. 2:124-130 (1988). 15. N.K. MOI-rET, C.M. SHAW and T.M BURBACHER, Environ. Health Perspect 63:133-40 (1985). 16. V. ARONSON, Physician Sportsmed. 1__4:209-212(1986). 17. I. CHANARIN, R. DEACON, M. LUMB, M. MUIR and J. PERRY, Blood 6__66:479-489(1985). 18. H. BAKER, L. PAULING and O. FRANK, Nutr. Reports Internatl. 2:3:669-677 (1981). 19. J. T. SMITH and N. Z. WATSON, Fed. Proc. 4,5:822 (1986). 20. M. CIKRT, L. MAGOS and R.T. SNOWDEN, Toxicol. Lett. 2_0:189-194 (1984). 21. L. MAGOS, Analyst 9_66:847-853(1971). 22. T. GOVANOLI-JAKUBCZAK, M.R. GREENWOOD, J.C. SMITH and T.W. CLARKSON, Clin. Chem. 20:222-229 (1974). 23. J.L. KACPRZAK and R. CHVOJKA, JAOAC 5.99:153-157(1976). 24. L.H,J. LAJUNEN and A. KINNUNEN, at. Spectroscopy 6:49-52 (1985). 25. D.B. DUNCAN, Biometrics 1._!:1-42 (1955). 26. N.E. ZORN and J.T. SMITH, Biochem. Arch. 5:141-146 (1989). 27. S.W. THENEN, J. Nutr. 11__9:1107-1114(1989). 28. L. TOLLEFSON and F. CORDLE, Environ. Health Perspect. 6_.88:203-208(1986).
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Pergamon Press
A RELATIONSHIP B ~ E E N VITAMIN B12, FOLIC ACID, ASCORBIC ACID, AND MERCURY UPTAKE AND METHYLATION
Nancy E. Zorn John T. Smith Department of Nutrition and Food Sciences College of Human Ecology and Agricultural Experiment Station The University of Tennessee, Knoxville, TN 37996-1900 (Received in final form May 7, 1990) Summary Ingestion of megadoses of certain vitamins appears to influence the in vivo methylation of mercuric chloride in guinea pigs. The addition of megadoses of vitamin B]2 fed either singularly or in combination with folic acid resulted in increased methylmercury concentrations in the liver. Moreover, percent methylmercury levels were significantly increased with B12 treatment in the liver (B]2 only and B12/folic acid) and brain (B12/vitamin C). Incorporation of high levels of folic acid into the dietary regime also increased the methylmercury concentration particularly in the liver and hair tissues. The addition of vitamin C in the diet, particularly in combination with B12 (brain) or folic acid (muscle) resulted in increased methylmercury levels in these tissues and percent methylmercury values with B12 in the muscle and brain tissue. Organic mercury compounds are one of the important environmental pollutants in our biosphere and much research has been conducted to explore the toxicity of this metal. Mass poisonings have been reported in which methylmercury has been implicated. The first reported instance took place in the 1950's in Minamata, Japan over a period of seven years. People were poisoned as a result of consuming fish which had acquired high concentrations of methylmercury due to contamination of local waters with industrial waste (1, 2). In addition, methylmercury and other alkylmercury compounds have been widely used in agriculture as antifungal agents in the storage of seeds. This has lead to several outbreaks of poisoning from the use of treated seeds for food (3, 4). A more recent concern has been the level of mercury exposure as a result of dental amalgams (5, 6, 7). Despite these events, mercury is still widely used in the production of fungicides, insecticides, paints, and electrical equipment. Many industrial wastes which contain mercury are discarded in oceans, rivers, lakes, and landfills (8, 9). Thus, the general public continues to be exposed to mercury from a number of rather widespread sources. A number of mechanisms have been proposed for metabolism of inorganic mercury in vivo. Early research has shown that rats injected with mercuric salts will excrete a small portion as volatile mercury, thus, suggesting that these animals are able to convert inorganic mercury either by a methylation reaction or reduction to the metallic form (10). Additional research done by Ogata and associates (11, 12) has demonstrated that elemental mercury can be oxidized to produce the mercurous ion (Hg+), and the mercuric ion (Hg++). Another metabolic fate of inorganic mercury compounds is the biomethylation of mercuric ions via methyl B12 which has been shown to occur in certain bacteria (13, 14). Methylmercury compounds pass easily through the blood-brain barrier and the placenta and are more likely to target the nervous system, testes, and the developing embryo/fetus (15). Additionally, the toxicity of short-chain alkylmercury compounds, such as methylmercury, is accentuated due to their persistence in mammals. 0024-3205/90 $3.00 +.00 Copyright (c) 1990 Pergamon Press plc
168
Vitamins
Effect
on M e r c u r y
Metabolism
Vol.
47, No.
2, 1990
The vitamin supplement business has blossomed into a multibillion dollar industry due to the large-scale consumption of these products. It has been estimated that 40% to 50% of the adult population, both active and sedentary, consume self-prescribed vitamin supplements (16). Recognizing that the biological formation of methyimercury requires the coenzyme methylcobalamine (vitamin B12) (13) and that the regeneration of methyl-cobalamine would require one of the forms of tetrahydrofolate (folic acid) (17), it appears conceivable that individuals who consume megadoses of B]2 and folic acid would increase their potential to form methylmercury. Additionally, an investigation has shown that ingestion of megadoses of vitamin C with other vitamins permits increases in the level of metabolically active cobalamin (vitamin B12) as well as other vitamins (18). The purpose of this investigation was to examine if there is a relationship between consumption of megavitamins and mercury retention and metabolism. Methods Animals and Dietary Re.qime: A pilot study was initially conducted to determine whether rats or guinea pigs would be best suited for this study (19). It was determined that the guinea pig will accumulate more folic acid and vitamin B]2 than the rat with a dietary excess. Eight groups of five male guinea pigs each weighing approximately 250 grams were purchased from Hilltop Animal Company and were fed Purina Guinea Pig Chow 5025 and megadoses of vitamin B]2, folate, and/or vitamin C. The vitamins were weighed and subsequently triturated with sucrose to a total weight of 500 g and yielded a final concentration of 4 mg folic acid, 50 #g B]2 and 1.8 g ascorbic acid per 4 g of triturate. This was then added to ground guinea pig chow at a concentration of 4g/100g. The control group consisted of animals fed ad libitum chow ground guinea pig containing 4g of sucrose per 100 g. One group of animals was fed a diet supplemented with megadoses of vitamin B]2, folic acid, and vitamin C. This group was referred to as the all vitamin treatment animals. Three groups of animals were provided diets which included megadoses of either B12 and folic acid, B]2 and vitamin C, or folic acid and vitamin C. These groups served as the combination vitamin treated animals and were referred to according to their respective vitamin blends. Finally, three groups of guinea pigs were given diets supplemented with the individual vitamins. These groups were called by the respective vitamin treatment, i.e. B]2 only, folic acid only, vitamin C only. All diets were fed ad libitum for a total of eight weeks. The animals were maintained on a 12-hour light dark cycle in a controlled temperature environment. Distilled water was freely available to all animals. Animals were weighed weekly to assess comparable weight gain and food consumption with the different dietary regimes. At the end of the dietary period, each animal was given 0.6 mg HgCI2/kg body weight as a subcutaneous injection in isotonic saline every other day for nine doses (20). Animals were observed throughout this treatment for overt signs of mercury toxicity. Twenty-four hours after the last injection, the animals were sacrificed and their liver, heart, brain, lungs, spleen, kidneys, semitendinosus muscle, and hair were removed and frozen at -80°C for subsequent mercury determinations. All tissues were examined for toxicity effects of mercury. In addition, the sites of injection were inspected for any indication of toxicity. Upon thawing a 25% (w/v) homogenate was prepared for each tissue using 1.5 M potassium hydroxide. Mercury Analysis: All reagents were analytical grade and specially selected for their low mercury content. Glassware was cleaned with nitric acid (4 M) and rinsed with demineralized water. All solutions were prepared using demineralized water only. Mercury determinations were carried out by cold-vapor atomic absorption spectrometry (AAS). Absorption was measured at 253.7 nm with a Perkin-Elmer Model 3030 atomic absorption spectrophotometer equipped with a mercury, hollow cathode lamp. A cold-vapor apparatus was laboratory assembled according to directions provided by a Perkin-EImer representative (personal communication) with an absorption cell (100 mm X 20 mm i.d.) used for the AAS measurements. The gas was first passed through an absorption cell (50 mm X 20 mm) containing potassium persulfate (Baker Chemical Company, Phillipsburg, N.J., Analytical Grade) which dried the material. After each measurement, the mercury was adsorbed in a charcoal absorption tube (65 mm X 35 mm i.d.) which was connected to the apparatus with a bypass arrangement. The system was closed by means of a peristalic pump.
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Vitam±ns Effect on Mercury Metabollsm
The method used in this research was adapted from a number of sources (21, 22, 23) and allowed for selective reduction of inorganic mercury and methylmercury using the same sample. Stannous chloride was used for the reduction of inorganic mercury while a stannous-cadmium chloride mixture was necessary for methylmercury determinations. Mercuric chloride and methylmercury standards were used to evaluate the effectiveness of the stannous chloride and stannous-cadmium chloride reduction specificity. A known quantity of either mercuric chloride or methylmercury was analyzed using the specific reduction agent listed above. Recovery of these standards was 99-100% for inorganic mercury and 97-99% for methylmercury. Thus, the validity of this methodology was established. In addition, previous research done with gas chromatography and direct current plasma atomic emission spectrometry to examine this methodology (22, 24) has demonstrated the reliability/specificity of this type of reduction. These two measurements were then added together to derive total mercury concentrations. At the beginning of each run, inorganic mercury standards (0.4 #g Hg/ml) were run by adding 1 ml of 10% stannous chloride solution and absorption values read. Also, absorption was measured for the methylmercury standard (0.5 ~g CH3Hg/ml) by following the procedure listed below for methylmercury determination. Representative samples of each tissue homogenate were pipetted to Biological Oxygen Demand (BOD) bottles: 5 ml of liver and muscle; 2.5 ml of brain, lungs, and heart plus 2.5 ml of 1.5 M KOH; 1.0 ml of kidney and spleen plus 4.0 ml of 1.5 M KOH; 1 ml of blood plus 5 ml of 1.5 M KOH; and 0.25 g weighed hair sample plus 5.0 ml of 1.5 M KOH. Then, 1 ml of 1% cysteine and 1.24 ml of 12 N sodium hydroxide were added to each reaction vessel. At all times, a total of 22.4 meq of hydrogen ions was maintained in each sample solution. The samples were digested in a water-bath at 85°C for five minutes. After digestion, the samples were immediately placed in an ice bath and 12.76 ml of 1% sodium chloride were added to each bottle. Total reaction volume was 20 ml and all samples were maintained on ice until AAS was performed. Each B.O.D. bottle containing the digested sample was placed in an ice water bath under the aerator tube assembly. The following were added in sequence; 1-2 drops Antifoam A Emulsion (Sigma Chemical Company, St. Louis, MO), 10 ml of 8 M sulfuric acid, 1 ml of 10% tin (11) chloride solution, 20 ml of 45% sodium hydroxide, and 50 ml of demineralized water. The aerator was immediately attached, the pump turned on, and the air flow continued through the reaction vessel for exactly one minute. At this time the absorption for inorganic mercury was read. The aerator tube was immediately removed from the sample and the air flow switched to remove the mercury through the activated charcoal chamber. The pump was allowed to run for a maximum of one minute or until absorption was zeroed, at which time the air flow was stopped. Exactly two minutes after the first reduction process was begun, 10 ml of 8 M sulfuric acid, 2 ml of tin (11)chloride - cadmium chloride reagent, and 20 ml of 45% sodium hydroxide were added sequentially to the sample. The aerator tube was attached immediately and the pump activated. The reaction was allowed to occur until a maximum absorption for methylmercury was obtained and recorded. At the end of the run, the air flow was again diverted through the charcoal absorption tube to remove all mercury. Statistical Analysis: All data were evaluated by Analysis of Variance (ANOVA) performed with SAS (SAS Program) using the General Linear Model (GLM) procedures. Differences between groups for each tissue were determined by Duncan's multiple-range test (25). Resu~s All animals were healthy upon sacrifice and tissues were examined for mercury toxicity with no evidence of damage observed in any group. Additionally, there was no indication of inflammation, sloughing, or granulomas at the site of injection. Weight data showed comparable weight gain within the various groups, however, a slight weight loss was observed for all animals after mercury injections were begun (26).
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Vol. 47, No. 2, 1990
As indicated in the methods section, a total of nine tissues were analyzed from each animal. However, due to the limitations of this publication, only the results of four tissues will be reported here. The data presented in Table I show the micrograms of inorganic and methylmercury per gram of wet liver in guinea pigs fed the various vitamin regimes. The individual vitamin treated groups had significantly higher inorganic and methylmercury concentrations than controls and all other treated groups. The B]2/folic acid group had more (p<0.05) inorganic mercury than the all vitamin and B12/vitamin C groups. Of importance, are the data showing that the B12/folic acid group (.67 #g) had a significantly higher methylmercury concentration than the control (.37 #g), all vitamins (.25 #g), B12/vitamin C (.27 ~,g), and folic acid/vitamin C (.38 #g) groups. TABLE I Mercury Levels in the Liver Tissue of Guinea Pigs with Different Vitamin Dietary Regimes #.q HCl/.q wet tissue Treatment
Inorganic Hg
Methyl Hg
Controls All Vitamins B12/Folic Acid B12/Vitamin C Folic Acid/Vitamin C B/2 Only Folic Acid Only Vitamin C Only
0.78 0.52 1.05 0.61 0.78 1.58 1.60 1.72
0.37 0.25 0.67 0.27 0.38 1.16 1.22 1.08
± 0.07.a'b ÷ 0.04 o ± 0.11 b 0.05 . 0.05 a ' ° ± 0.07 c ± 0.32 c + 0.24 c
± ± + ± ± ± ± +
0.05 a 0.01 a 0.04 D 0.02 a 0.03 a 0.18 c 0.12 c 0.08 c
Values represent the average ± S.E.M. Data showing a common superscript letter in a column are not significantly different (p>0.05). The brain was an important tissue to investigate in this study since only the methylated form of mercury is able to cross the blood-brain barrier. However, as shown in Table II, there were no significant differences in the concentration of inorganic and methylmercury between treatments. TABLE II Mercury Levels in the Brain Tissue of Guinea Pigs with Different Vitamin Dietary Regimes ~,Cl H.q/.q wet tissue Treatment
Inorganic Hg
Methyl Hg
Controls All Vitamins B12/Folic Acid B]2Nitamin C FoUc Acid/Vitamin C B12 Only Folic Acid Only Vitamin C Only
0.30 0.19 0.16 0.20 0.25 0.27 0.30 0.23
0.24 0.18 0.15 0.29 0.18 0.19 0.18 0.19
± 0.04 a ± 0.02 a 0.01 a ± 0.04 a ± 0.04 a _+ 0.02 a ± 0.02 a ± 0.01 a
± 0.04 a _+ 0.02 a ± 0.01 a ± 0.07 a ± 0.04 a 0.01 a _+ 0.01 a ± 0.01 a
Values represent the average ± S.E.M. Data showing a common superscript letter in a column are not significantly different (p>0.05).
Vol. 47, No. 2, 1990
Vitamins Effect on Mercury Metabolism
Table III shows the data for muscle tissue. Of particular interest in this tissue is the folic acid/vitamin group which displayed the highest (p<0.05) concentrations of both forms of mercury. TABLE III Mercury Levels in the Muscle Tissue of Guinea Pigs with Different Vitamin Dietary Regimes
~,.q H.q/.q wet tissue Treatment
Inorganic Hg
Methyl Hg
Controls All Vitamins B12/Folic Acid B12Nitamin C Folic AcidNitamin C B12 Only Folic Acid Only Vitamin C Only
0.21 0.10 0.12 0.27 1.33 0.18 0.15 0.16
0.18 0.13 0.19 0.31 0.83 0.14 0.10 0.07
± ± ± ±
0.06 a 0.01 a 0.01 a 0.15 a 0.71D ± 0.02 a 0.02 a ± 0.01 a
± ± + ± + ±
0.03 a 0.03 a 0.02 a 0.10 a 0.37 b 0.01 a 0.02 a ~ 0.01 a
Values represent the average ± S.E.M. Data showing a common superscript letter in a column are not significantly different (p>0.05). In hair tissue (Table IV), inorganic mercury concentration did not differ among the groups. Both the folic acid group and vitamin C group had methyl and total mercury concentrations which were significantly higher than controls. Of interest also was the fact that the folate group had significantly more methylmercury than the all vitamin and B12/folic acid groups. TABLE IV Mercury Levels in the Hair Tissue of Guinea Pigs with Different Vitamin Dietary Regimes
~,.q H.q/.q wet tissue Treatment
Inorganic Hg
Methyl Hg
Controls All Vitamins B]2/Folic Acid B]2 Only Folic Acid Only Vitamin C Only
0.80 0.73 0.87 0.95 0.95 0.95
0.65 0.70 0.78 1.27 1.84 1.47
± 0.07 a ± 0.12 a ± 0.09 a 0.14 a ± 0.09 a ± 0.15 a
± 0.06 a, 0.09a'.o 0 04 a ' ° ÷ 0128a'b'c ± 0.62 c ± 0.39 ° ' c
Values represent the average ± S.E.M. Data showing a common superscript letter in a column are not significantly different (p>0.05). The percent methylmercury (of total mercury) values for the four tissues are presented in Table V. In the liver, the B]2/folic acid and individual vitamin treated groups
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Vol. 47, No. 2, 1990
TABLE V Percent 1 Methylmercury Values in Various Tissues of Guinea Pigs with Different Vitamin Dietary Regimes
Treatment
Liver
Brain
Tissue Muscle
Controls All Vitamins B]2/Folic Acid B]2/Vitamin C FoliCB12 OnlyACidNitaminC
a2 31 a,b 33 b,c 40 a 31 a,b 3341c
a,b 44 49 a 49 59 ca a,b 4242a,b
a,b,c 49 a,b,c 53 b 61 b,c 57 a,b,c 4550a,b,d
47a -56a,b
Folic Acid Only Vitamin C Only
44 c 39 b,c
b 4437a,b
a,d 3140d
60b ' 57a, o
Hair 45a" 49a,D
1Percent methylmercury determined as a portion of total mercury concentration present in tissue. 2Data showing a common superscript letter in a column are not significantly different (p>0.05). exhibited significantly higher values than the controls, while in brain tissue, only the B/2/vitamin C group had an elevated (p<0.05) percent methylmercury level compared to all other groups. In muscle and blood tissue only, the vitamin C only group had significantly lower percent methylmercury levels than controls. In hair tissue, the folic acid group had significantly higher percent methylmercury than the controls. Discussion It was the purpose of this investigation to determine if the ingestion of megadoses of vitamin B]2, folic acid or vitamin C would increase the methylation of inorganic mercury and/or alter the metabolism of subcutaneous doses of inorganic mercury. The data presented in Table I support the concept that megadoses of these vitamins will increase the methylation of inorganic mercury. Feeding diets supplemented with megadoses of B12/folic acid, or B12, folic acid or vitamin C resulted in an 81 to 230% increase in methylmercury in the liver tissue as compared to control. There were no significant differences in MeHg content between groups supplemented with B]2, folic acid or vitamin C. The significant increase above control obtained with the B12/folic acid combination was significantly lower than obtained with the individual vitamins. It could be argued that since the liver values for both inorganic mercury and methylmercury for rats fed these three vitamin supplemented diets are high and not significantly different that these values represent a systematic error. In defense, it should be noted that supplementation with B]2, folic acid and vitamin C did not result in significant increases in methyl or inorganic mercury in muscle or brain tissue nor inorganic concentration in hair tissue. Only folic acid or vitamin C supplementation increased the methyl mercury concentration of hair. Therefore, a systematic measurement error seems an unlikely explanation for these data. Since the methylmercury concentration in the hair is a reflection of the body burden of mercury over time (26) the positive relationship observed between the high levels of methyl mercury found in the liver and the hair of the guinea pigs fed the individual vitamin supplements could be used as additional proof that megadoses of these vitamins increased the body burden of methylmercury. If, however, these data from liver and hair reflect the body burden of methylmercury then the consistency of the data from the brain and the assumption that only methylmercury passes the blood brain barrier (15) would allow the assumption that the brain has a limited capacity to take up methylmercury. Once, however, the methylmercury is taken up by the brain it appears that with exception of mice fed a diet supplemented with B12 and vitamin C that the methylmercury is demethylated to give a ratio of 1.2:1 inorganic to methylmercury. The combination of
Vol. 47, No. 2, 1990
Vitamins Effect on Mercury Metabolism
B12/vitamin C supplementation appeared to suppress demethylation since the inorganic:methyl mercury ratio was 0.7:1. Thenen (27) has shown vitamin C ingestion raised the B]2 levels in B12 deficient rats. It is conceivable then that the B]2/vitamin C supplementation suppressed the formation of inorganic mercury in the brain by keeping B12 high to remethylate inorganic mercury. Therefore, although the total concentration of mercury in the brain is not elevated in guinea pigs fed diets supplemented with B]2/vitamin C, the fact that a significantly greater percentage is in the form of methylmercury could result in an increase in neurotoxicity. No explanation has been offered for the large increase in mercury both inorganic and methyl in the muscle tissue of guinea pigs fed the diet supplemented with folic acid/vitamin C. Since the values were suspect, they were repeated several times. It is believed, therefore, that the values are real but no explanation can be offered. In conclusion, the methylation of inorganic mercury in vivo can be increased with the ingestion of megadoses of vitamin B]2, folic acid, or vitamin C. Therefore, individuals who take large amounts of these nutritional supplements and who are exposed to mercury sources could have increased methylmercury formation with subsequent neurological damage, especially with high combinations of vitamin B/2 and vitamin C. Additionally, dental professionals should become aware of this potential. Further research is required to elucidate these effects and the possible mechanisms which are involved in the methylation of mercury in vivo. References 1. M. KUTSUMA, Minamata Dis., Kutama University Press, Japan (1968). 2. R.P. JUNGHANS, Environ. Res. 3_1:1-31 (1983). 3. F. BAKIR, S.F. DAMLUJI, L. AMIN-ZAKI, M. MURTADHA, A. KHALIDI, N.Y. AL-RAWl, S. TIKRITI, H.I. DHAHIR, T.W. CLARKSON, J.C. SMITH and R.A. DOHERTY, Science 18_!:230241 (1973). 4. P.L. SCHULLER, Cadmium, Lead, Mercury and Methyimercury Compounds, Food and Horticulture Organization of the United Nations (1976). 5. C. NALEWAY, R. SAKAGUCHI, E. MITCHELL, T. MULLER, W.A. AYER and J.J. HEFFERREN, JADA 11.__[1:37-42(1985). 6. S.B. CHANG, C. SlEW and S.E. GRUNINGER, J. Anal. Toxicol. 11--137-139 (1987). 7. M.J. VIMY and F.L. LORSCHEIDER, J. Dent. Res. 64:1069-1071 (1985). 8. B. LOFORTH, Ecol. Res. Comm. Bull. _4:5-44 (1970). 9. M. BERLIN, Effects and Dose-Response Relationships of Toxic Metals, G. F. Nordber, pp. 235-245, Elsevier Scientific, Amsterdam (1976). 10. T. CLARKSON and A. ROTHSTEIN, Health Phys. 10:1115-1121 (1964). 11. M. OGATA, M. IKEDA and Y. SUGATA, Environ. Health 34:218-221 (1979). 12. M OGATA, K. KENMOTSU, N. HIROTA, T. MEGURA and H. AIKOH, Arch. Environ. Health 4_.22:26-30(1987). 13. W.P. RIDLEY, L.J. DIZIKES and J.M. WOOD, Sci. 19"7:329-3,32 (1977). 14. C.T. WALSH, M.D. DISTEFANO, M.J. MOORE, L.M. SHEWCHUK and G.L. VERDINE, FASEB J. 2:124-130 (1988). 15. N.K. MOI-rET, C.M. SHAW and T.M BURBACHER, Environ. Health Perspect 63:133-40 (1985). 16. V. ARONSON, Physician Sportsmed. 1__4:209-212(1986). 17. I. CHANARIN, R. DEACON, M. LUMB, M. MUIR and J. PERRY, Blood 6__66:479-489(1985). 18. H. BAKER, L. PAULING and O. FRANK, Nutr. Reports Internatl. 2:3:669-677 (1981). 19. J. T. SMITH and N. Z. WATSON, Fed. Proc. 4,5:822 (1986). 20. M. CIKRT, L. MAGOS and R.T. SNOWDEN, Toxicol. Lett. 2_0:189-194 (1984). 21. L. MAGOS, Analyst 9_66:847-853(1971). 22. T. GOVANOLI-JAKUBCZAK, M.R. GREENWOOD, J.C. SMITH and T.W. CLARKSON, Clin. Chem. 20:222-229 (1974). 23. J.L. KACPRZAK and R. CHVOJKA, JAOAC 5.99:153-157(1976). 24. L.H,J. LAJUNEN and A. KINNUNEN, at. Spectroscopy 6:49-52 (1985). 25. D.B. DUNCAN, Biometrics 1._!:1-42 (1955). 26. N.E. ZORN and J.T. SMITH, Biochem. Arch. 5:141-146 (1989). 27. S.W. THENEN, J. Nutr. 11__9:1107-1114(1989). 28. L. TOLLEFSON and F. CORDLE, Environ. Health Perspect. 6_.88:203-208(1986).
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