Clinical Science (1991) 80,277-280
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Editorial Review
Stable isotopes in clinical research: safety reaffirmed PETER J. H. JONES AND STANLEY T. LEATHERDALE Division of Human Nutrition, University of British Columbia, Vancouver, British Columbia, Canada
INTRODUCTION
DEUTERIUM
The use of non-radioactive stable-isotope tracers in biomedical experimentation and diagnosis is generally considered ethically acceptable in humans at all Me-cycle stages. These isotopes have therefore emerged as tracers of choice in metabolic studies, despite the cost and labour-intensive associated methodologies. Applications and related methods have been the focus of previous thorough reviews [ 1-41. Although stable isotopes emit no potentially harmful ionizing radiation, mass differences exist between the tracer and predominant elemental form. Such differences cause stable isotopes to exhibit isotope effects in biological systems when present in excess of natural abundance [5]. Isotope effects may have an adverse impact at the cellular or whole-organism level owing to the complex interrelationships between enzymic reactions proceeding in vivo. Although isotope effects exist for all stable isotopes, the most significant would be expected when compounds are labelled with deuterium (D), because of the greater mass discriminationbetween D and protium. Safety limits for stable isotopes, addressed in previous reports [5, 61, remain to be established. The increasing clinical use of stable isotopes in clinical research, and the documentation of side-effects at lower levels of use than previously reported [7], just@ further investigation of thresholds for stable-isotope toxicity in humans. The natural abundance and isotopic enrichment likely to be reached in typical biomedical tracer applications for some stable isotopes are listed in Table 1. The purpose of this review is to establish minimal levels of stable-isotope usage associated with toxicity or side-effectsin relation to those currently used in human biomedical research. The following terminology will be used for isotopes of hydrogen: H or protium for 'H, and D or deuterium for 2H.
Nature and mechanism of toxicity
Correspondence: Dr Peter J. H. Jones, Division of Human Nutrition, School of Family and Nutritional Sciences, 2205 East Mall, University of British Columbia, Vancouver, B.C., Canada V6T 1W5.
Mammalian cells are accustomed to molecules containing D at natural abundance levels. However, tissues containing high concentrations of D exhibit a multitude of D effects. These include impaired protein and nucleic acid synthesis, altered conformation and stability of biopolymers, altered rates of enzymic reactions due to the deuteration of either the enzyme or substrate, impaired cell division and morphological changes [8,9]. While some toxic manifestations of deuteration are reversible, high cmcentrations prove lethal. No unified explanation of the ultimate cause of death from deuteration is available as D 2 0 toxicity induces a systemic disorder [8, lo]. Numerous disturbances, including renal functional impairment, central nervous system disorder, cardiac abnormalities, enzymic differences, and hormonal and glucose metabolism imbalances, may contribute to D 2 0toxicity [ll].The inability of mice and rats to reproduce at sub-lethal levels of deuteration [12, 131, and the suppression of haematopoiesis resulting in anaemia and immunosuppression [14, 151, have been attributed to the ability of D to interfere with DNA synthesis and mitosis, causing disturbed cell proweration and increased cell death. The sigtllficantreplacement of H by D in living systems after administrationof D 2 0leads to a defined deuteration sequence [15]. Initial events in this sequence are caused Table 1. Natural abundance and isotopic enrichment attained in humans for some common stable isotopes during typical biomedical tracer applications Elemental isotope ZH I3H I5N '80
Natural abundance
(YO) 0.015 1.111 0.360 0.200
Tissue enrichment (mg/kg body wt.)
(mg/kg body wt.) 15 2000 110 1300
95 2015 120 1480
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by characteristics of D 2 0 as a solvent which differ from those of H,O. Such isotope effects result from the higher viscosity, decreased solubility of electrolytes, and decreased self-ionization of D,O in solution. Subsequently, D replaces hydrogen atoms in labile linkages to oxygen, sulphur and nitrogen, while hydrogen-carbon bonds are little affected. Later, D,O is included in biosyntheses of organic compounds produced containing D in stable bonds to carbon, causing constitutional isotope effects. Deuterated molecules differ from those containing protium in several ways. Not only do equilibrium properties of D-containing compounds frequently vary from those of the corresponding hydrogen compounds, but the rate at which D-containing compounds undergo chemical reaction may differ as well. For example, 2-deuteropropan01 is oxidized at one-sixth the rate of ordinary propanol by chromic acid at 40°C [16]. Various factors contribute to the alteration in reactivity of bonds to D as compared with bonds to protium, but of these the most important is the smaller zero-point energy difference for many D-associated bonds, compared with those of protium. This difference is reflected in slightly, but distinctly, more stable bonds containing D [16]. The overall effect of deuteration appears to be a depression of tissue metabolism due to lower reaction rates of deuterated compounds in vivo. Reaction rates that apparently increase after deuteration may be the consequence of a depression of inhibitory processes [ 171. This manifestation of deuteration is defined as a kinetic isotope effect and occurs because the rate of a chemical reaction is sensitive to atomic mass at a particular position in one of the reacting species [4]. The toxicity of D is a function of both dose and duration of administration. Difference in survival time of animals is related to the rate at which deuteration to toxic concentrations occurs and not to the absolute increase in D level [13]. It has been demonstrated that the lethal level of deuteration of larger mammals is similar to that of smaller mammals despite up to a 500-fold difference in weight [18].Thus in mammals the magnitude of the effect of D is likely to be independent of the size of the organism. Beyond the concern as to the effects of acute dosing with D, consideration must also be given to the duration of administration. D obtained by the body from D 2 0 is readily incorporated from body water into molecular positions of tissue components that are metabolically stable. Thus body enrichment will continue to increase with a steady intake of D, although upon cessation of isotope administration body concentrations will gradually decline to baseline levels normally observed. The accumulation of D 2 0 in body fluid and its clearance are rapid, whereas the flux of D bound within organic molecules is relatively slow and varies with the tissue under study [ 151. Threshold for toxicity in animals A graded dose-response has been observed in deuteration experiments in animals. Early ‘classic’ studies show-
ing deleterious effects of D in biological systems were carried out in lower life forms. Tadpoles, worms, small fish and some protozoa succumbed within 1-3 h when placed in 92% D,O [ 191. In succeeding years, the toxicity of D was demonstrated in mice, rats and dogs, where body water deuteration of 30-40% was observed to be lethal [13,17, 18,201, although the cause of death was not defined. When animals were given doses of D,O, rapidly raising the levels of D in the plasma to 40%, body temperatures dropped immediately and death occurred within a short time [20]. Czajka et al. [18]examined the physiological effects of deuteration in two dogs. One dog died after an acute dose resulting in a 33-35% concentration of D,O in body fluids. The second dog survived at a 20% level of deuteration, but exhibited similar signs to those observed in the acutely dosed animal. Signs included lymphopenia and an increase in polymorphonuclear leucocyte numbers, hypoglycaemia related to the degree of deuteration, electrocardiographic changes indicative of myocardial abnormality, and generalized muscle weakness. The survival of the dog deuterated at 20% is consistent with findings of other studies, where deuteration in animals below the lethal threshold can be achieved which is compatible with life, but at which definite physiological manifestations become evident. For example, mice and rats were observed to be asymptomatic at D levels of 20-25%, with characteristic toxic symptomology appearing at higher levels of deuteration [17]. Similarly, a condition of one-fifth saturation with D,O in mice was observed to be associated with deleterious effects, including decreased body temperature and hyperexcitability, although compatible with an apparent state of health [20]. Also, mice maintained on 25% D 2 0 in drinking water for up to 280 days showed no effect of concentrations of D in water of 18-20% on body weight or longevity. When the D content of drinking water was raised to 25-3070, fetal viability ceased [ 121. D in body fluids at a level of below 20% is generally non-lethal in animals, but is accompanied by variable physiological manifestations. Concentrations of D in serum water of about 16% did not influence the body weight or lifespan of mice [13], nor did they affect the sensitive proliferative activity of renewing cells in the bone marrow or in the small intestine [21, 221. Below concentrations of D in body water of 15%, harmful effects in mammals have not been detected and levels of deuteration of at least 15% must be maintained by continual dosage in animal models before adverse effects become evident [8, 93. Threshold for side-effects in human studies Evidence from animal studies has precluded trials using large quantities of D in human subjects. However, D 2 0 has been given to humans at lesser doses in many kinetic studies designed to measure metabolite elimination, biosynthesis or degradation. No evidence of toxicity has been reported. Certain temporary side-effects have been noted at moderate amounts employed, which permit
Safety of stable isotopes estimation of threshold levels for D intake that avoid any adverse effect. Side-effects in humans have been documented, with single oral doses of less than 250 g of D 2 0 resulting in severe, but transitory, episodes of vertigo [7].From 140 to 250g of 99.5% D 2 0 were consumed orally by study subjects depending on amounts of D required to elevate the body water D content to 0.5%. At least two subjects out of the five studied reported transitory vertigo, beginning within 30 min of the initial D 2 0 ingestion and lasting several hours. This side-effect was attributed possibly to a perturbation of the specific gravity of vestibular fluid. In addition, one subject reported a transitory increase of his gradually developing presbyopia. The same study cites a personal communication reporting vertigo at lower levels of D,O administration. Vertigo accompanied by nausea was reported to have resulted from the consumption of a quantity of D20 required to dose body water at 0.2% [7]. Not in any case was there mention of negative health effects other than vertigo. In a single subject given D 2 0 to maintain body water D enrichment at 0.5%over 130 days, neither acute side-effects nor subsequent toxic symptoms were noted [23]. At lower levels, doses of D 2 0 resulting in body water enrichments of 0.05% [24], 0.07% [25]and 0.12% (P. J. H. Jones et al., unpublished work) have produced no reported negative physiological effects in over 30 male subjects tested, with a single exception. One subject reported dizziness which restricted ambulatory function for 3 h after a dose of 30 g of 99.8 atom o/' excess D20, enriching body water to about 0.07% D. Generally, subjects report D 2 0as possessing a sweet pleasant taste compared with H,O, but notice no ill effects after oral consumption. Other studies using similar or lower concentrations of D report no ill effects after administration [26,27]. From these observations it is suggested that the threshold for noticeable side-effectsexists at some dosage between 70 and 140 g of 100% D,O, although subjects given the latter dose gradually over the evening period report a lessening or absence of vertigo effects [7]. Although such side-effects are discomforting to research subjects, absence of follow-up toxicity in animals and humans given D over prolonged time intervals at these levels weighs against the possibility of long-term deleterious action. Possible therapeutic applications of D Therapeutic applications of D have been examined. Anti-neoplastic actions of D, due to its influence on mitosis, have been successfully exploited with inhibition of tumour proliferation in mice at a 16% level of deuteration without toxic side-effects [28]. Giving D to mice has been associated with lessened susceptibility to lethal doses of whole-body y-irradiation, possibly due to a protective effect on pluripotential stem cells [22].In addition, deuteration of cytotoxic drugs has been shown to be effective in reducing formation of teratogenic compounds in vivo 1291. Such findings offer the possibility that
279
deuteration of body water and metabolite compartments may be of some potential health benefit. TOXICITY WITH OTHER STABLE ISOTOPES Less information is available concerning toxic biological effects of 13C, 15N, I 8 0 and other elemental stable isotopes compared with D, although all have been utilized as tracers in a myriad of clinical studies. The paucity of knowledge concerning toxicity of heavier isotopes may reflect the general perception that biological consequences of compounds labelled with these higher elements will be minimized because of smaller mass differences, and almost identical physical and chemical properties, between the stable isotope and predominating elemental forms. No investigations exist dealing with the toxicity of the stable isotopes of nitrogen or sulphur. Biological consequences of the substantial replacement of body carbon and oxygen with their respective stable isotopes have been studied. Replacement of up to 60% of body oxygen by l 8 0 revealed a total absence of adverse physiological or biochemical consequences[30]. Here, l8O replacement resulted from three generations of mice existing in an atmosphere of 90% 1 8 0 2 with the third generation consuming 90% H2180. A similar lack of effect noted in baboons breathing H,180 indicates the apparent innocuous nature of this stable isotope [29]. The natural abundance of 13C (1.1'/0) is substantially higher than that of many other stable isotopes used as tracers; thus the relative I3C enrichment over background generated in metabolic studies is generally small compared with relative enrichments associated with other stable isotopes. Toxicity of 13C has been examined in animals given amounts far in excess of those employed in research. Sixty per cent body enrichment with I3C was achieved in mice over a period of over 200 days, without any detrimental effect [31].The lack of teratogenicity or embryotoxicity of 13C has also been demonstrated. Preimplantation embryos enriched with 15-20% 13C, concentrations tenfold higher than usually achieved in clinical use, developed with no detectable alterations [32], as did mouse limb-buds with DNA fractions containing up to 60% 13C [29]. Thus, although the effect of even higher tissue enrichment is uncertain, the small requirements of 13Cas a tracer in most clinical studies, particularly relative to its naturally high abundance, precludes any discernible risk of toxicity. SUMMARY
Approaching half a century of stable-isotope usage in human metabolic studies has been without documented significant adverse effect. Side-effects with acute D dosing are transitory with no demonstrated evidence of permanent deleterious action. The threshold of D toxicity has been defined in animals and is far in excess of concentrations conceivably used in human studies. The possibility that D may have additional beneficial pharmacological applications cannot be excluded.
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For isotopes other than D, evidence of observed toxicity remains t o b e produced even at dosages far in excess of the range used in metabolic studies. Absence of adverse effect may be attributable t o small mass differences and the similar properties of tracer and predominantly abundant isotope. Absolute determination of stable isotope toxicity in humans is rendered impossible by ethical considerations. Also, the precision of extrapolating toxicity thresholds from animal studies remains unknown. However, should perturbation of the delicate homoeostatic characteristic of living organisms occur with use of stable isotopes, it is almost undoubtedly at some level of administration greatly in excess of those administered currently in biomedical research.
ACKNOWLEDGMENTS
We acknowledge the skilled assistance of Rhonda Ellwyn in the preparation of the manuscript. O u r own work reported here was supported by grants from British Columbia Health C a r e Research and the B.C. and Yukon Heart Foundations.
REFERENCES 1. Klein, P.D. & Klein, E.R. Stable isotopes in biomedical
research. Spectra 1982; 8,9- 12. 2. Bier, D.M. The use of stable isotopes in metabolic investigation. Baillieres Clin. Endocrinol. Metab. 1987; 1, 817-34. 3. Wolfe, R.R. Tracers in metabolic research: radioisotope and stable isotope/mass spectrometry methods. New York: Alan R. Liss, Inc., 1984. 4. Hayes, J.M. Fractionation, et ul.: an introduction to isotopic measurements and terminology. Spectra 1982; 8,3-9. 5. Klein, P.D. & Klein, E.R. Stable isotopes: origins and safety. J. Clin. Pharmacol. 1986; 26,378-82. 6. Thompson, G.N., Pacy, P.J., Ford, G.C. & Halliday, D. Practical considerations in the use of stable isotope labelled compounds as tracers in clinical studies. Biomed. Environ. Mass Spectrom. 1989; 18,321-7. 7. Taylor, C.B., Mikkelson, B., Anderson, J.A. & Forman, D.T. Human serum cholesterol synthesis measured with the deuterium label. Arch. Pathol. 1966; 81,213-31. 8. Thomson, J.F. Biological effects of deuterium. New York: Pergamon Press, 1963. 9. Katz, J.J. & Crespi, H.L. Isotope effects in biological systems. In: Collins, C.J. & Bowman, N.S., eds. Isotope effects in chemical reactions. New York: Van Nostrand Reinhold Co., 1971: 286-363. 10. Bachner, P., McKay, D.G. & Rittenberg, D. The pathologic anatomy of deuterium intoxication. Proc. Natl. Acad. Sci. U.S.A. 1964; 51,464-71. 11. Blake, M.I., Crespi, H.L. & Katz, J.J. Studies with deuterated drugs. J. Pharm. Sci. 1975; 64,367-91. 12. Czajka, D.M. & Finkel, A.J. Effect of deuterium oxide on the reproducive potential of mice. Ann. N.Y. Acad. Sci. 1960; 84,770-9.
13. Katz, J.J., Crespi, H.L., Czajka, D.M. & Frinkel, A.J. Course of deuteration and some physiological effects of deuterium in mice. Am. J. Physiol. 1962; 203,907-13. 14. Laissue, J.A. & Stoner, R.D. Deuterium isotope effects on lymphoid tissues and humoral antibody responses in mice. Virchows Arch. 1979; 383,149-66. 15. Adams, W.H. & Adams, D.G. Effects of deuteration on hematopoiesis in the mouse. J. Pharmacol. Exp. Ther. 1988; 244,633-9. 16. Katz. J.J. Chemical and biological studies with deuterium. Am. Sci. 1969; 48,544-80. 17. Katz, J.J., Crespi, H.L., Hasterlik, R.J., Thomson, J.F. & Finkel, A.J. Some observations on biological effects of deuterium, with special reference to effects on neoplastic processes. J. Natl. Cancer Inst. 1957; 18,641-58. 18. Czajka, D.M., Finkel, A.J., Fischer, C.S. & Katz, J.J. Physiological effects of deuterium on dogs. Am. J. Physiol. 1961; 201,357-62. 19. Taylor, H.S., Swingle, W.W., Eyring, H. & Frost, A.A. The effect of water containing the isotope of hydrogen upon freshwater organisms. J. Chem. Phys. 1933; 1,751. 20. Barbour, H.G. The basis of the pharmacological action of heavy water in mammals. Yale J. Biol. Med. 1937; 9, 551-65. 21. Hodel, A,, Gebbers, J.-O., Cottier, H. & Laissue, J.A. Effects of prolonged moderate body deuteration on proliferative activity in major cell renewal systems in mice. Life Sci. 1983; 30, 1987-96. 22. Laissue, J.A., Altermatt, H.J., Bally, E. & Gebbers, J.-0. Protection of mice from whole body gamma irradiation by deuteration of drinking water: hematological findings. Exp. Hematol. 1987; 15, 177-80. 23. Steinberg, D., Mize, C.E., Avigan, J., Fales, H.M., Eldjarn, L., Try, K., Stokke, 0. & Refsum, S. Studies on the metabolic error in Refsum’s disease. J. Clin. Invest. 1967; 46,3 13-22. 24. Jones, P.J.H., Scanu, A.M. & Schoeller, D.A. Plasma cholesterol synthesis using deuterated water in humans: effects of short-term food restriction. J. Lab. Clin. Med. 1988; 111,627-33. 25. Jones, P.J.H. & Schoeller, D.A. Evidence for diurnal periodicity in human cholesterol synthesis. J. Lipid Res. 1990; 31,667-73. 26. Schoeller, D.A., Ravussin, E., Schutz, Y., Acheson, K.J., Baertschi, P. & Jequier, E. Energy expenditure by doubly labelled water: validation in humans and proposed calculations. Am. J. Physiol. 1986; 250, R823-30. 27 Luksaki, H.C. & Johnson, P.E. A simple inexpensive method of determining total body water using a tracer dose of D,O and infrared absorption of biological fluids. Am. J. Clin. Nutr. 1984; 41,363-70. 28. Altermatt, H.J., Gebbers, J.-0. & Laissue, J.A. Heavy water delays growth of human carcinoma in nude mice. Cancer 1988; 62,462-6. 29. Spielmann, H. & Nau, H. Embryotoxicity of stable isotopes and use of stable isotopes in studies of teratogenic mechanisms. J. Clin. Pharmacol. 1986; 26,474-80. 30. Wolf, D., Cohen, H., Meshorer, A., Wasserman, 1. & Samual, D. The effect of I8O on the growth and reproduction of mice. In: Klein, E.R. & Klein, P.D., eds. Stable isotopes: proceedings of the Third International Conference. New York: Academic Press, 1978: 353-60. 31. Gregg, C.T., Hutson, J.Y., Prine, J.R., Ott, D.G. & Furchner, J.E. Substantial replacement of mammalian body carbon with carbon-13. Life Sci. 1973; 13,775-82. 32. Gregg, C.T. Some applications of stable isotopes in clinical pharmacology.Eur. J. Clin. Pharmacol. 1974; 7,315-9.
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Editorial Review
Stable isotopes in clinical research: safety reaffirmed PETER J. H. JONES AND STANLEY T. LEATHERDALE Division of Human Nutrition, University of British Columbia, Vancouver, British Columbia, Canada
INTRODUCTION
DEUTERIUM
The use of non-radioactive stable-isotope tracers in biomedical experimentation and diagnosis is generally considered ethically acceptable in humans at all Me-cycle stages. These isotopes have therefore emerged as tracers of choice in metabolic studies, despite the cost and labour-intensive associated methodologies. Applications and related methods have been the focus of previous thorough reviews [ 1-41. Although stable isotopes emit no potentially harmful ionizing radiation, mass differences exist between the tracer and predominant elemental form. Such differences cause stable isotopes to exhibit isotope effects in biological systems when present in excess of natural abundance [5]. Isotope effects may have an adverse impact at the cellular or whole-organism level owing to the complex interrelationships between enzymic reactions proceeding in vivo. Although isotope effects exist for all stable isotopes, the most significant would be expected when compounds are labelled with deuterium (D), because of the greater mass discriminationbetween D and protium. Safety limits for stable isotopes, addressed in previous reports [5, 61, remain to be established. The increasing clinical use of stable isotopes in clinical research, and the documentation of side-effects at lower levels of use than previously reported [7], just@ further investigation of thresholds for stable-isotope toxicity in humans. The natural abundance and isotopic enrichment likely to be reached in typical biomedical tracer applications for some stable isotopes are listed in Table 1. The purpose of this review is to establish minimal levels of stable-isotope usage associated with toxicity or side-effectsin relation to those currently used in human biomedical research. The following terminology will be used for isotopes of hydrogen: H or protium for 'H, and D or deuterium for 2H.
Nature and mechanism of toxicity
Correspondence: Dr Peter J. H. Jones, Division of Human Nutrition, School of Family and Nutritional Sciences, 2205 East Mall, University of British Columbia, Vancouver, B.C., Canada V6T 1W5.
Mammalian cells are accustomed to molecules containing D at natural abundance levels. However, tissues containing high concentrations of D exhibit a multitude of D effects. These include impaired protein and nucleic acid synthesis, altered conformation and stability of biopolymers, altered rates of enzymic reactions due to the deuteration of either the enzyme or substrate, impaired cell division and morphological changes [8,9]. While some toxic manifestations of deuteration are reversible, high cmcentrations prove lethal. No unified explanation of the ultimate cause of death from deuteration is available as D 2 0 toxicity induces a systemic disorder [8, lo]. Numerous disturbances, including renal functional impairment, central nervous system disorder, cardiac abnormalities, enzymic differences, and hormonal and glucose metabolism imbalances, may contribute to D 2 0toxicity [ll].The inability of mice and rats to reproduce at sub-lethal levels of deuteration [12, 131, and the suppression of haematopoiesis resulting in anaemia and immunosuppression [14, 151, have been attributed to the ability of D to interfere with DNA synthesis and mitosis, causing disturbed cell proweration and increased cell death. The sigtllficantreplacement of H by D in living systems after administrationof D 2 0leads to a defined deuteration sequence [15]. Initial events in this sequence are caused Table 1. Natural abundance and isotopic enrichment attained in humans for some common stable isotopes during typical biomedical tracer applications Elemental isotope ZH I3H I5N '80
Natural abundance
(YO) 0.015 1.111 0.360 0.200
Tissue enrichment (mg/kg body wt.)
(mg/kg body wt.) 15 2000 110 1300
95 2015 120 1480
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P. J. H. Jones and S. T. Leatherdale
by characteristics of D 2 0 as a solvent which differ from those of H,O. Such isotope effects result from the higher viscosity, decreased solubility of electrolytes, and decreased self-ionization of D,O in solution. Subsequently, D replaces hydrogen atoms in labile linkages to oxygen, sulphur and nitrogen, while hydrogen-carbon bonds are little affected. Later, D,O is included in biosyntheses of organic compounds produced containing D in stable bonds to carbon, causing constitutional isotope effects. Deuterated molecules differ from those containing protium in several ways. Not only do equilibrium properties of D-containing compounds frequently vary from those of the corresponding hydrogen compounds, but the rate at which D-containing compounds undergo chemical reaction may differ as well. For example, 2-deuteropropan01 is oxidized at one-sixth the rate of ordinary propanol by chromic acid at 40°C [16]. Various factors contribute to the alteration in reactivity of bonds to D as compared with bonds to protium, but of these the most important is the smaller zero-point energy difference for many D-associated bonds, compared with those of protium. This difference is reflected in slightly, but distinctly, more stable bonds containing D [16]. The overall effect of deuteration appears to be a depression of tissue metabolism due to lower reaction rates of deuterated compounds in vivo. Reaction rates that apparently increase after deuteration may be the consequence of a depression of inhibitory processes [ 171. This manifestation of deuteration is defined as a kinetic isotope effect and occurs because the rate of a chemical reaction is sensitive to atomic mass at a particular position in one of the reacting species [4]. The toxicity of D is a function of both dose and duration of administration. Difference in survival time of animals is related to the rate at which deuteration to toxic concentrations occurs and not to the absolute increase in D level [13]. It has been demonstrated that the lethal level of deuteration of larger mammals is similar to that of smaller mammals despite up to a 500-fold difference in weight [18].Thus in mammals the magnitude of the effect of D is likely to be independent of the size of the organism. Beyond the concern as to the effects of acute dosing with D, consideration must also be given to the duration of administration. D obtained by the body from D 2 0 is readily incorporated from body water into molecular positions of tissue components that are metabolically stable. Thus body enrichment will continue to increase with a steady intake of D, although upon cessation of isotope administration body concentrations will gradually decline to baseline levels normally observed. The accumulation of D 2 0 in body fluid and its clearance are rapid, whereas the flux of D bound within organic molecules is relatively slow and varies with the tissue under study [ 151. Threshold for toxicity in animals A graded dose-response has been observed in deuteration experiments in animals. Early ‘classic’ studies show-
ing deleterious effects of D in biological systems were carried out in lower life forms. Tadpoles, worms, small fish and some protozoa succumbed within 1-3 h when placed in 92% D,O [ 191. In succeeding years, the toxicity of D was demonstrated in mice, rats and dogs, where body water deuteration of 30-40% was observed to be lethal [13,17, 18,201, although the cause of death was not defined. When animals were given doses of D,O, rapidly raising the levels of D in the plasma to 40%, body temperatures dropped immediately and death occurred within a short time [20]. Czajka et al. [18]examined the physiological effects of deuteration in two dogs. One dog died after an acute dose resulting in a 33-35% concentration of D,O in body fluids. The second dog survived at a 20% level of deuteration, but exhibited similar signs to those observed in the acutely dosed animal. Signs included lymphopenia and an increase in polymorphonuclear leucocyte numbers, hypoglycaemia related to the degree of deuteration, electrocardiographic changes indicative of myocardial abnormality, and generalized muscle weakness. The survival of the dog deuterated at 20% is consistent with findings of other studies, where deuteration in animals below the lethal threshold can be achieved which is compatible with life, but at which definite physiological manifestations become evident. For example, mice and rats were observed to be asymptomatic at D levels of 20-25%, with characteristic toxic symptomology appearing at higher levels of deuteration [17]. Similarly, a condition of one-fifth saturation with D,O in mice was observed to be associated with deleterious effects, including decreased body temperature and hyperexcitability, although compatible with an apparent state of health [20]. Also, mice maintained on 25% D 2 0 in drinking water for up to 280 days showed no effect of concentrations of D in water of 18-20% on body weight or longevity. When the D content of drinking water was raised to 25-3070, fetal viability ceased [ 121. D in body fluids at a level of below 20% is generally non-lethal in animals, but is accompanied by variable physiological manifestations. Concentrations of D in serum water of about 16% did not influence the body weight or lifespan of mice [13], nor did they affect the sensitive proliferative activity of renewing cells in the bone marrow or in the small intestine [21, 221. Below concentrations of D in body water of 15%, harmful effects in mammals have not been detected and levels of deuteration of at least 15% must be maintained by continual dosage in animal models before adverse effects become evident [8, 93. Threshold for side-effects in human studies Evidence from animal studies has precluded trials using large quantities of D in human subjects. However, D 2 0 has been given to humans at lesser doses in many kinetic studies designed to measure metabolite elimination, biosynthesis or degradation. No evidence of toxicity has been reported. Certain temporary side-effects have been noted at moderate amounts employed, which permit
Safety of stable isotopes estimation of threshold levels for D intake that avoid any adverse effect. Side-effects in humans have been documented, with single oral doses of less than 250 g of D 2 0 resulting in severe, but transitory, episodes of vertigo [7].From 140 to 250g of 99.5% D 2 0 were consumed orally by study subjects depending on amounts of D required to elevate the body water D content to 0.5%. At least two subjects out of the five studied reported transitory vertigo, beginning within 30 min of the initial D 2 0 ingestion and lasting several hours. This side-effect was attributed possibly to a perturbation of the specific gravity of vestibular fluid. In addition, one subject reported a transitory increase of his gradually developing presbyopia. The same study cites a personal communication reporting vertigo at lower levels of D,O administration. Vertigo accompanied by nausea was reported to have resulted from the consumption of a quantity of D20 required to dose body water at 0.2% [7]. Not in any case was there mention of negative health effects other than vertigo. In a single subject given D 2 0 to maintain body water D enrichment at 0.5%over 130 days, neither acute side-effects nor subsequent toxic symptoms were noted [23]. At lower levels, doses of D 2 0 resulting in body water enrichments of 0.05% [24], 0.07% [25]and 0.12% (P. J. H. Jones et al., unpublished work) have produced no reported negative physiological effects in over 30 male subjects tested, with a single exception. One subject reported dizziness which restricted ambulatory function for 3 h after a dose of 30 g of 99.8 atom o/' excess D20, enriching body water to about 0.07% D. Generally, subjects report D 2 0as possessing a sweet pleasant taste compared with H,O, but notice no ill effects after oral consumption. Other studies using similar or lower concentrations of D report no ill effects after administration [26,27]. From these observations it is suggested that the threshold for noticeable side-effectsexists at some dosage between 70 and 140 g of 100% D,O, although subjects given the latter dose gradually over the evening period report a lessening or absence of vertigo effects [7]. Although such side-effects are discomforting to research subjects, absence of follow-up toxicity in animals and humans given D over prolonged time intervals at these levels weighs against the possibility of long-term deleterious action. Possible therapeutic applications of D Therapeutic applications of D have been examined. Anti-neoplastic actions of D, due to its influence on mitosis, have been successfully exploited with inhibition of tumour proliferation in mice at a 16% level of deuteration without toxic side-effects [28]. Giving D to mice has been associated with lessened susceptibility to lethal doses of whole-body y-irradiation, possibly due to a protective effect on pluripotential stem cells [22].In addition, deuteration of cytotoxic drugs has been shown to be effective in reducing formation of teratogenic compounds in vivo 1291. Such findings offer the possibility that
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deuteration of body water and metabolite compartments may be of some potential health benefit. TOXICITY WITH OTHER STABLE ISOTOPES Less information is available concerning toxic biological effects of 13C, 15N, I 8 0 and other elemental stable isotopes compared with D, although all have been utilized as tracers in a myriad of clinical studies. The paucity of knowledge concerning toxicity of heavier isotopes may reflect the general perception that biological consequences of compounds labelled with these higher elements will be minimized because of smaller mass differences, and almost identical physical and chemical properties, between the stable isotope and predominating elemental forms. No investigations exist dealing with the toxicity of the stable isotopes of nitrogen or sulphur. Biological consequences of the substantial replacement of body carbon and oxygen with their respective stable isotopes have been studied. Replacement of up to 60% of body oxygen by l 8 0 revealed a total absence of adverse physiological or biochemical consequences[30]. Here, l8O replacement resulted from three generations of mice existing in an atmosphere of 90% 1 8 0 2 with the third generation consuming 90% H2180. A similar lack of effect noted in baboons breathing H,180 indicates the apparent innocuous nature of this stable isotope [29]. The natural abundance of 13C (1.1'/0) is substantially higher than that of many other stable isotopes used as tracers; thus the relative I3C enrichment over background generated in metabolic studies is generally small compared with relative enrichments associated with other stable isotopes. Toxicity of 13C has been examined in animals given amounts far in excess of those employed in research. Sixty per cent body enrichment with I3C was achieved in mice over a period of over 200 days, without any detrimental effect [31].The lack of teratogenicity or embryotoxicity of 13C has also been demonstrated. Preimplantation embryos enriched with 15-20% 13C, concentrations tenfold higher than usually achieved in clinical use, developed with no detectable alterations [32], as did mouse limb-buds with DNA fractions containing up to 60% 13C [29]. Thus, although the effect of even higher tissue enrichment is uncertain, the small requirements of 13Cas a tracer in most clinical studies, particularly relative to its naturally high abundance, precludes any discernible risk of toxicity. SUMMARY
Approaching half a century of stable-isotope usage in human metabolic studies has been without documented significant adverse effect. Side-effects with acute D dosing are transitory with no demonstrated evidence of permanent deleterious action. The threshold of D toxicity has been defined in animals and is far in excess of concentrations conceivably used in human studies. The possibility that D may have additional beneficial pharmacological applications cannot be excluded.
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P. J. H. Jones and S. T. Leatherdale
For isotopes other than D, evidence of observed toxicity remains t o b e produced even at dosages far in excess of the range used in metabolic studies. Absence of adverse effect may be attributable t o small mass differences and the similar properties of tracer and predominantly abundant isotope. Absolute determination of stable isotope toxicity in humans is rendered impossible by ethical considerations. Also, the precision of extrapolating toxicity thresholds from animal studies remains unknown. However, should perturbation of the delicate homoeostatic characteristic of living organisms occur with use of stable isotopes, it is almost undoubtedly at some level of administration greatly in excess of those administered currently in biomedical research.
ACKNOWLEDGMENTS
We acknowledge the skilled assistance of Rhonda Ellwyn in the preparation of the manuscript. O u r own work reported here was supported by grants from British Columbia Health C a r e Research and the B.C. and Yukon Heart Foundations.
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