`
`"H mm 1
`
`MARCH 1975
`
`Volume 64 Number 3
`
`Coden:
`
`JPMSAE 64(3) 867-534 (1975)
`
`
`
`A puincation of the American Pharmaceutical Association
`
`rm:u.
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`This mate-rialh‘asmfiiefi
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`CON1018
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`

`

`Journal of
`
`Pharmaceutical
`
`Sciences
`
`MARCH 1975
`
`VOLUME 64 NUMBER 3
`
`acme N LM and may be
`
`iTHE ECONOMICS OF PUBLICATION
`A person does not need to venture further than the local supermarket to
`verify that the price of everything has been climbing at a fantastic rate. The
`economists tell us we have been caught in an “inflation spiral.”
`Well, as bad as the situation may be in the case of food prices and other
`commodities sold in the supermarket, printers and printing companies have
`been increasing prices to their clients at an even faster pace. This is the re-
`sult of all the customary factors, including labor, taxes, equipment, utilities,
`and so on, but it is especially due to one other; namely, the cost of paper
`stock.
`Anyone even remotely connected with the production of printed material
`has probably had the shocking experience during the past year or so of being
`told that the price of paper was increasing on a monthly or even a weekly
`basis. The overall result was that comparable paper stock ended up costing
`anywhere from double to four times as much as it did about a year earlier.
`And to add distressing insult to injury, paper supplies for a time were run-
`ning so short that the customer was told to feel fortunate in being able to get
`that supply of paper even at several times what it had cost just a year before.
`The combination of month-to-month paper shortages and week-to-week price
`increases explains why J. Pharm. Sci. has been printed on several different
`types of paper stock over the past eighteen months.
`We are devoting our comments this month to a brief discussion of publica-
`tion costs primarily to inform the readership of the fact that the process of
`producing J. Pharm. Sci. each month and putting it into the hands of our
`readers is a costly undertaking; and it is rapidly becoming more costly.
`Many people assume that income from publications represents a profita-
`ble activity for an association or society. For some few organizations this may
`be the case, but for most it is not. Specifically, it has been the policy of the
`American Pharmaceutical Association, as publisher of J. Pharm. Sci.—-and
`various other professional, technical, and scientific publications—to produce
`and make available such publications on essentially a self-sustaining basis; in
`fact, rates to members have often been set at a particularly low level requir-
`ing that they be subsidized from membership dues.
`All of this is rather evident from an inspection of the APhA financial
`statements. Although these statements are released and distributed each -
`year—as in the case of most financial reports—they make awfully dull read-
`ing, and few people give them even a cursory examination.
`But if they did read these reports, they would note that J. Pharm. Sci. in-
`come has been growing modestly from $244,000 in 1972, to $260,000 in 1973,
`to $290,000 in 1974, and to $288,000 in 1975 (all figures rounded and latter
`figures estimated).
`income
`Simultaneously, however, during this same period of modest
`growth, direct expenses attributable to production of J. Pharm. Sci. have
`grown by leaps and bounds from $265,000 in 1972, to $293,000 in 1973, to
`$322,000 in 1974, and to $370,000 in 1975.
`Without going into any great detail, two things are evident from these fig-
`ures; namely:
`(a) As the first observation, the APhA member subscriber pays only a
`small additional premium to receive this Journal, but a substantial propor-
`tion of his or her membership dues is allocated to support the Journal. At the
`current rate of total income resulting from subscription charges, page charges,
`advertising revenue, and reprint sales, APhA is providing a very substan-
`tial subsidy from membership dues—for 1975 this will amount to approxi-
`mately $82,000—in order to make J. Pharm. Sci. available.
`(b) As the second observation, we may look at how this “cost/benefit
`ratio” translates to each individual Journal recipient, whether the recipi-
`ent is a member subscriber, nonmember subscriber, or institutional sub-
`scriber. With an overall circulation of approximately 12,000 and an antici-
`pated 1975 production cost of $370,000, it is evident that the proportionate
`cost of simply providing the average subscriber with the Journal for a peri-
`od of one year is running just under $31—and this figure of $31 is extreme-
`ly conservative since it does not include any overhead and similar types of
`occupancy expenses.
`Every clergyman tells his congregation something to the effect that: “I do
`not wish to talk about money, but .
`.
`. .".Similarly, we would also prefer to
`avoid doing so. However, we feel that these facts and figures will be revealing
`to our readers, and especially to our APhA member subscribers by acquaint-
`ing them more fully. with one of their membership benefits.
`We also feel that these facts and figures will provide a better under-
`standing of why all associations, societies, and commercial publishers are se-
`riously reexamining their publication programs and in many cases are insti-
`tuting or at least considering radical changes in them. If the publishers of
`Life, Look, and the Saturday Evening Post had discerned the winds of
`change early enough and taken action in time to adjust to them, their demise
`may have been avoided, and these once-popular mass-circulation weekly
`magazines would probably have survived. By the same token, current trends
`and changing circumstances make it appear that technical and scientific
`periodicals will need to undergo some major changes in coming years if they
`are to survive and prosper.
`—EGF
`2
`
`MARY H. FERGUSON
`Editor
`'
`
`L. LUAN CORRIGAN
`Assistant Editor
`
`SHELLY ELLIOTT
`Production Editor
`
`CHRISTINE L. BAILEY
`Copy Editor
`
`EDWARD G. FELDMANN
`Contributing Editor
`
`SAMUEL W. GOLDSTEIN
`Contributing Editor
`
`LELAND J. ARNEY
`Publications Director
`
`EDITORIAL ADVISORY BOARD
`
`LYNN R. BRADY
`
`GERHARD LEVY
`
`WILLIAM O. FOYE
`
`CARL J. LINTNER, JR.
`
`HARRY B. KOSTENBAUDER
`
`G. VICTOR ROSSI
`
`The Journal of Pharmaceutical Sciences is published
`monthly by the American Pharmaceutical Association
`at 2215 Constitution Ave., N.W., Washington, DC
`20037. Second-class postage paid at Washington, DC,
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`Claims—Missing numbers will not be supplied if dues
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`© Copyright 1975, American Pharmaceutical Associa-
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`20037; all rights reserved.
`
`This material was copied
`
`2
`
`

`

`Journal of
`Pharmaceutical
`Sciences
`
`MARCH 1975
`VOLUME 64 NUMBER 3
`
`REVIEW ARTICLE
`
`Studies with Deuterated Drugs
`
`MARTIN I. BLAKE *=, HENRY L. CRESPI *, and JOSEPH J. KATZ
`
`Keyphrases Drugs, deuterated-deuterium
`biological and iso-
`tope effects, toxicity and pharmacological and biological effects of
`deuterium oxide, heavy water as a drug, various deuterated drugs,
`applications, review 0 Deuterated drugs-barbiturates, hormones,
`anesthetics, antibiotics, alkaloids, amino acids, sympathomimetic
`amines, applications, review 0 Heavy water-carcinolytic agent,
`measure of total body water, and neutron radiography, review
`0 Deuterium oxide-taxicity and pharmacological and biological
`effects, review
`
`CONTENTS
`Introduction .....................
`................... 367
`.................. .368
`Biological Effects of Deuterium ...
`Deuterium Isotope Effects ............................
`.368
`General Toxicity of Deuterium Oxide ................... .369
`Pharmacological and Biological Effects of
`Deuterium Oxide ................................
`.371
`Classification of Deuterated Drugs ..................
`Heavy Water as a Drug.. ............................
`Heavy Water as a Carcinolytic Agent ................
`Heavy Water as Measure of Total
`Bodywater ......................................
`374
`Heavy Water in Neutron Radiography .................. .374
`Rarbiturates ........................................... 375
`Hormones and Related Compounds ......................
`.376
`Anesthetics ..................................
`Antibiotics ..................................
`Alkaloids .............................................. 379
`Amino Acids ..........................................
`.381
`Sympathomimetic Amines ..............................
`.382
`Miscellaneous Deuterated Compounds of Biological
`Importance .......................................... 384
`Bioanalytical Applications. .............................
`.385
`Methods ............................................. 385
`Applications Involving Deuterated Drugs. ............... .386
`
`Conclusion ............................................
`388
`References ............................................. 388
`
`INTRODUCTION
`A drug may be defined as an agent used in the di-
`agnosis, cure, mitigation, treatment, or prevention of
`disease in humans or animals. Included in this cate-
`gory are articles (other than food) used to affect the
`structure or any function of the body of humans or
`animals. This definition distinguishes a drug from a
`chemical. A drug that contains a radioactive nuclide
`in the molecule is known as a radiopharmaceutical,
`and several dozens of these are useful clinical agents.
`At the present time, there are no drugs on the mar-
`ket that contain deuterium in the molecule or, for
`that matter, are enriched in any other stable isotope.
`Numerous deuterated drugs have been obtained by
`organic synthesis or by biosynthesis. Several of these
`substances are completely deuterated, some are high-
`ly deuterated, and others are partially deuterated,
`but the majority are deuterated only in specific mo-
`lecular positions.
`Deuterated drugs have proved useful in studying
`isotope effects, in permitting a better understanding
`of the mechanism of drug action, and in elucidating
`metabolic and biosynthetic pathways. Some are sim-
`ply laboratory curiosities awaiting future study.
`Heavy water (2H20) itself has been examined for
`therapeutic applications. This review is concerned
`with the laboratory application of both heavy water
`and deuterated drugs.
`
`Val. 64, No. 3, March 1975 I367
`
`3
`
`

`

`Table I-Comparison of Some Physical Properties of Water
`and Deuterium Oxidea
`
`Property
`
`Melting point
`Boiling point
`Density, d4z0, g/ml
`Temperature of maximum density
`Viscosity, centipoise, 20'
`Surface tension, dynes/cm, 25 O
`Dielectric constant, 25"
`Heat of formation, cal/mole,
`liquid
`Free energy of formation, cal/
`mole, liquid
`Entropy, eu/mole, liquid
`Heat of fusion, cal/mole
`Heat of vaporization, cal/mole,
`25 O
`Zero-point energy, cal/mole
`
`~~
`
`a See Ref. 16.
`
`Deute-
`rium
`Oxide
`
`3.81'
`101.72'
`1.1056
`11.23"
`1 . 2 5
`71.93
`78.93
`-70,410
`
`-58,200
`18.19
`1,515
`10,846
`
`Water
`
`0.00"
`100.000
`0.9982
`3 . 9 8 "
`1.005
`71.97
`78.54
`- 68,320
`
`-56,690
`16.75
`1,436
`10,515
`
`13,219
`
`9,664
`
`Biological Effects of Deuterium-Deuterium
`(2H, D) is the rare, stable heavy isotope of hydrogen
`(lH). It was discovered (1, 2) in 1932 and was isolated
`in a high state of purity (3) shortly thereafter. Deute-
`rium occurs in nature to the extent of about 1 part in
`6400 and is obtained primarily by isolation of deute-
`rium oxide from ordinary water; its concentration
`ranges from 0.0156% in sea water to 0.0139% in fresh
`water. Heavy water is the simplest deuterium-con-
`taining compound and differs from ordinary water in
`many of its properties (Table I). It can be reasonably
`expected that the mass differences associated with
`the replacement of deuterium by hydrogen in a mole-
`cule will distinctly affect its physical and chemical
`properties. Such mass differences can also be expect-
`ed to produce changes in the biological behavior of
`deuterium compounds.
`Extensive deuterium isotope studies were preclud-
`ed in the 1930's and early 1940's because of the great
`scarcity of heavy water. Since heavy water is very
`suitable as a moderator in certain types of nuclear re-
`actors, efficient methods have been developed to iso-
`late heavy water from natural sources. Hence, deute-
`rium (as heavy water) has become a tonnage, high
`purity, industrial chemical available at low to moder-
`ate cost. The ready availability of heavy water to the
`scientific community as a result of advances in nucle-
`ar technology has resulted in a burgeoning interest in
`the biological effects of deuterium.
`The biological implications of deuterium substitu-
`tion were recognized almost immediately after its dis-
`covery. It soon became apparent that extensive re-
`placement of hydrogen by deuterium in biological
`systems could produce deleterious effects. The early
`literature for 1932-1950 has been reviewed thorough-
`ly (4). Much of the early work, however, is contradic-
`tory and of questionable value, primarily because of
`poor experimental design forced by inadequate and
`impure supplies of heavy water and because of the
`absence of suitable analytical techniques for measur-
`ing accurately the deuterium content of biological
`systems.
`A conference under the auspices of the New York
`
`368 /Journal of Pharmaceutical Sciences
`
`Academy of Sciences in 1960 (5) did much to revive
`and stimulate interest in studies on the biological ef-
`fects of deuterium. Many of the relevant papers in
`that monograph will be referred to later in this re-
`view. In the same year, Katz published reports (6, 7)
`that summarized both the early studies in this field
`and also included new research undertaken during
`the 1950's, primarily a t Argonne National Laborato-
`ry * The physiological effects of deuterium were the
`subject of a comprehensive monograph (8) in 1963.
`Flaumenhaft et al. (9) reviewed the biological effects
`of deuterium and focused their comments on isotope
`effects on the cellular level. They noted specifically
`the isotope effects on the growth of microorganisms
`and higher plants in deuterated media and the cytol-
`ogy of deuterated cells.
`Katz, Crespi, Blake, and coworkers (10-15) pub-
`lished a series of articles covering various aspects of
`deuterium isotope effect studies, with special empha-
`sis on applications related to problems of a chemical
`and biochemical nature. The culturing of various mi-
`croorganisms in deuterated media, the effect of deu-
`teration on the stability of certain proteins and nu-
`cleic acids, and isotope effects in the metabolism of
`deuterated glucose and mannose by ascites tumor
`cells were reviewed (10).
`Another review (11) covered the effect of deuteri-
`um substitution on the conformation of certain bio-
`polymers and deuterium isotope effects in carbohy-
`drate metabolism and in the potentiation of tumor
`chemotherapy. A third paper (12) described the culti-
`vation of deuterated organisms and their utilization
`as a practical source of 2H and 1H-2H compounds
`that are very useful for PMR spectroscopy of bio-
`polymers; it also described the utility of deuterated
`organisms for following the path of hydrogen in living
`organisms.
`In 1965, Katz (13) presented a comprehensive re-
`view of deuterium isotope effects on living organisms
`and biopolymers and illustrated the power of these
`methods by a discussion of biogenesis of chlorophyll.
`In a more recent review, the application of NMR
`studies to the biosynthesis of the important plant
`constituents bacteriochlorophyll, chlorophylls a and
`b, and the clavine alkaloids was discussed (14). The
`most recent and perhaps the most comprehensive re-
`view of deuterium isotope effects on biological sys-
`tems was published in 1971 (15). In the present re-
`view, principal emphasis will be directed to deuterat-
`ed drugs and their application.
`basis for the
`Deuterium Isotope Effects-The
`frequently profound chemical and biological conse-
`quences of deuteration merits some discussion. The
`kinetic isotope effect on the rates of chemical reac-
`tion resulting from substitution of deuterium for hy-
`drogen, which must certainly be implicated in the bi-
`ological effects of deuterium, has received thorough
`theoretical treatment (16-20). The difference in mass
`between deuterium and hydrogen causes the vibra-
`tional frequencies of carbon, oxygen, and nitrogen
`bonds to deuterium to have lower frequencies than
`corresponding bonds to hydrogen. As a result, the
`
`4
`
`

`

`Natural
`Abundance
`Isotope
`
`'H
`'H
`
`Heavy
`Isotope
`
`2H
`3H
`'3C
`1 4 C
`"N
`1 8 0
`3 5 s
`
`Table 11-Estimated Maximum Possible Rate Constant
`Ratios at 25" for Various Stable Isotopes
`
`chemical bonds involving 2H will generally be more
`stable than those of 'H.
`It has been calculated that the zero-point energy
`(lowest ground-state vibrational level) for many
`bonds to deuterium is about 1.2-1.5 kcal/mole small-
`er than for bonds to hydrogen. The zero-point energy
`of a bond undergoing chemical reaction reflects the
`ease with which the molecule is activated from a
`ground state to the transition state required for bond
`scission to occur. The more stable deuterium bond
`requires a greater energy of activation to achieve the
`transition state; as a consequence, the rate of reaction
`Rate constant for the light isotope is the numerator.
`involving a bond to deuterium is generally slower
`than that involving a bond to hydrogen. Thus, substi-
`clear. Isotope effects produced by hydrogen atoms
`tution of deuterium for hydrogen in a chemical bond
`that exchange very slowly (if, in fact, they exchange
`can affect significantly the rate of bond cleavage and
`at all) with water, as is the case for most carbon-hy-
`exert marked effects on the relative rates of chemical
`drogen bonds, must be distinguished from those in
`reactions.
`which exchange occurs very rapidly, as is generally
`Large isotope effects on reaction rates are apparent
`the case for hydrogen atoms bonded to oxygen, sul-
`where cleavage involves a bond to deuterium at the
`fur, or nitrogen. Compounds occurring in biological
`systems will generally have both types of exchange-
`reaction site. In such instances the effect is referred
`able hydrogen present. If the isotopic composition of
`to as a primary isotope effect and is usually expressed
`the solvent is altered, this will effect a corresponding
`in terms of the ratio of the specific rate constants
`k H I ~ D . Bigeleisen (21) calculated the maximum pos-
`change in the isotopic composition of the exchange-
`sible ratios in specific rate constants for a number of
`able hydrogen in the solute molecules. In living sys-
`stable and radioactive isotopes (Table 11). Wiberg
`tems, therefore, solvent isotope effects include con-
`(17) calculated that at 25' the maximum positive pri-
`tributions from both primary and secondary isotope
`mary kinetic isotope effect that can be expected in a
`effects.
`chemical reaction involving the breaking of bonds to
`The compositional or constitutional isotope effects
`hydrogen is in the range of 7-10 for C-H, N-H,
`have been defined (15) as those arising from alter-
`and 0-H uersus C-D, N-D,
`and 0-D,
`respec-
`ations in the isotopic composition of the nonex-
`tively. For a number of reasons, these maximum ra-
`changeable hydrogens in the molecule in which all
`tios are not realized; more generally, values of k ~ / l z ~
`hydrogen sites in nonexchangeable positions are oc-
`are in the range of 2-5.
`cupied by deuterium. Hybrid constitutional deuteri-
`An observable isotope effect will only be apparent,
`um isotope effects occur when both hydrogen and
`or C-D
`of course, where the breaking of a C-H
`deuterium are present in nonexchangeable positions.
`bond is involved in the rate-determining step. In cer-
`When the isotopic composition of living organisms is
`tain acid-base-catalyzed reactions, depending on the
`changed, as when animals are administered deuteri-
`mechanism, an inverse isotope effect may take place
`um oxide in the drinking water, the initial effects of
`(22). The pronounced effects on rates of chemical
`deuteration are attributable primarily to a solvent
`reactions make deuterium isotope studies a particu-
`isotope effect. When the deuterium oxide enters the
`larly useful tool for elucidating the mechanisms of
`metabolic processes of the organism and is used for
`many reactions of biochemical significance (8).
`the biosynthesis of compounds containing both hy-
`Deuterium atoms in nonexchangeable positions lo-
`drogen and deuterium, then, in addition to a solvent
`cated near, but not at, the reaction center can give
`isotope effect, hybrid constitutional isotope effects
`rise to secondary isotope effects. While deuterium
`are induced.
`secondary isotope effects are real and measurable,
`Finally, in an organism grown in an environment of
`they are usually much smaller than primary isotope
`deuterium free of any hydrogen, constitutional iso-
`effects. Belleau (23) indicated that the k ~ / k ~
`for sec-
`tope effects become predominant. In the deuteration
`ondary isotope effects falls in the range of 1.05-1.25.
`of living organisms, both water and deuterium oxide
`Secondary isotope effects, although small, may be
`are usually present and hybrid isotope effects are
`important in biological systems sensitive to kinetic
`most prominent. These hybrid isotope effects are
`effects. The physical organic chemist also makes a
`most commonly encountered in living organisms
`distinction between solvent isotope effects, where
`subjected to partial deuteration and are the most dif-
`only the isotopic composition of the medium has
`ficult isotope effects to interpret at the molecular
`been altered, and primary and secondary isotope ef-
`level.
`fects resulting from the replacement of hydrogen by
`General Toxicity of Deuterium Oxide-Since
`deuterium in carbon to hydrogen bonds of organic
`deuterium oxide resembles ordinary water so closely,
`it is natural to speculate on the effects of this appar-
`compounds.
`A recent review article (15) pointed out that in liv-
`ently small difference in chemical composition on the
`ing organisms and in many biochemical and physio-
`toxicological properties of the molecule. Concern over
`logical systems the distinction between solvent and
`the toxicity of heavy water to living organisms was
`primary and secondary isotope effects is not always
`first expressed (24) soon after its discovery. A mouse
`
`Rate
`Ratio-,
`k i / h
`
`18
`60
`1.25
`1 . 5
`1.14
`1 . 1 9
`1.05
`
`Vol. 64, No. 3, March 1975 I369
`
`5
`
`

`

`fed the equivalent of about 1 g of pure heavy water
`over a 3-hr period survived the ordeal but showed
`definite signs of intoxication. Lewis (24) noted that:
`“The more he [the mouse] drank of the heavy water
`the thirstier he became.” Unfortunately, the experi-
`ment had to be terminated because the limited sup-
`ply of heavy water was exhausted.
`An extensive series of studies an the physiological
`effects of deuterium oxide in mice was next conduct-
`ed over a 5-year period starting in 1934 (25). The
`acute lethal dose for deuterium oxide in mice was 5-7
`m1/10 g of body weight. This amount was observed to
`be the lethal dose even when the deuterium oxide
`was administered as a 50% solution in water. When
`“concentrated” heavy water was administered orally,
`a crisis was reached on about the 5th day, at which
`time the body water became about one-third replaced
`by deuterium oxide and death generally occurred
`around the 7th day. However, a mouse ingesting 40%
`D20 in its drinking water for a 2-month period
`showed no apparent ill effects other than retarded
`weight gain; mice whose body fluids reached 20%
`deuteration showed no noticeable harmful effects
`when the heavy water was removed from the diet.
`Barbour and Trace (26) fed mice pure (99.5%) deu-
`terium oxide at a dosage rate of 1 m1/10 g of body
`weight/day. This intake generally proved fatal in 7
`days, at which time the mice were deuterated to the
`extent of 40-50%l. Death in these mice was preceded
`by a characteristic sequence of toxic manifestations.
`More recently, Thomson (8) thoroughly reviewed
`the literature dealing with the physiological and toxi-
`cological effects of deuterium oxide in mammals.
`Katz (7) reported that mice and rats cannot long sur-
`vive replacement of more than one-third of their
`body water by deuterium oxide. At the 20% replace-
`ment level, rats become hyperexcitable and more ag-
`gressive than normal; when the plasma levels ap-
`proach 30% DZO, rats frequently convulse when han-
`dled. When the body fluids are a t the 35% deutera-
`tion level, death generally results. Thomson (27) also
`observed that rats drinking deuterium oxide died
`when about one-third of their body water was re-
`placed by deuterium oxide. He concluded that the
`toxic effects were attributable to the summation of a
`multitude of small changes affecting the rates of en-
`zymatic reactions in the body.
`Czajka and Finkel (28) maintained mice on 25%
`D2O in the drinking water for up to 280 days without
`adverse effect on body weight or longevity. In these
`mice, ingestion of 25% D2O caused incorporation in
`the body fluids of about 18-20% deuterium. When
`the deuterium content of the drinking water was
`raised to the 2530% level, fetal viability ceased. Katz
`et al. (29) found that the median survival times for
`mice deuterated to different deuterium levels ranged
`from 60 days for mice drinking 40% D20 to 12 days
`for mice drinking 75% D2O. Differences in survival
`time were related to the rates at which deuteration
`
`Percent deuterium oxide throughout this paper refers to atom percent D
`mobile equilibrium: HzO + D20 == 2 HOD.
`in the aqueous fluid. A mixture of water and deuterium oxide is related by a
`
`370 /Journal o j Pharmaceutical Sciences
`
`reached toxic levels and not to differences in deuteri-
`um levels per se. Most animals died when the body
`tissue fluid concentration rose to between 30 and 40%
`D2O.
`Czajka et al. (30) studied the toxic manifestations
`of deuterium in two dogs. One beagle was maintained
`at 20% D20 in the body fluids for 50 days, and the
`other was maintained at the toxic range of 33-35% for
`a brief period. Deuteration was effected by replace-
`ment of ordinary water with deuterium oxide in the
`food and drink. These dogs (approximately 10 kg)
`appear to be the largest animals subjected to exten-
`sive deuteration. A 30% concentration in the blood
`plasma appears to be close to the acute danger level
`for dogs, as it is for mice and rats. Thus, in intact
`mammals the magnitude of deuterium effect appears
`on the whole to be quite independent of the size of
`the organism.
`In one report (31) on humans, no ill effects were
`noted over 4 months when the body water was re-
`placed with deuterium oxide to the extent of 0.5%. In
`mammals it appears that up to 15% deuterium can be
`tolerated, but severe toxic effects and possibly death
`may result at the 30% level and higher. Although
`toxic effects appear to correlate with the overall level
`of deuteration, deuterium probably elicits harmful
`effects in subtle ways even in low concentration.
`Incorporation in viuo of small amounts of deuteri-
`um into sensitive molecules such as enzymes, nucleic
`acids, and similar important substances possibly
`could have serious consequences. Knapp and Gaffney
`(32) pointed out that the small amounts of stable iso-
`topes involved in labeling experiments probably
`would not cause harmful effects. Nevertheless, they
`cautioned that it would be wise to label the molecules
`in molecular positions that are metabolically stable
`to minimize the extent of distribution throughout the
`body. The possible toxicity of stable isotopes at low
`concentrations clearly merits further study.
`The ultimate cause of death from deuteration is
`not clear. Numerous disturbances are observed in-
`cluding renal function impairment, central nervous
`system (CNS) disturbances, cardiac involvement, en-
`zymic interferences, hormonal imbalances, and glu-
`cose metabolism disturbances. All of these factors
`seem to be involved in the death of the animal, and
`no single factor appears to be the principal cause of
`death. Only a few studies have been directed toward
`finding ways to increase the tolerance of animals to
`the ravages of high concentrations of deuterium
`oxide in the body fluids. An extensive study (33) was
`conducted in which a number of hormones, vitamins,
`and vitamin mixtures were examined for possible
`beneficial effects on the ability of mice to survive
`toxic levels of deuteration. The mice were adminis-
`tered daily injections of hormones, vitamins, or vita-
`min mixtures while being maintained on 50 or 75%
`D20 in the drinking water, but the improvement in
`survival was marginal at best.
`Notwithstanding the damaging effects imposed on
`animals from high levels of deuteration, deuterium
`oxide must nevertheless be considered a remarkably
`nontoxic substance. Few, if any, components of the
`
`6
`
`

`

`body can be replaced to anywhere the same extent as
`can water by deuterium oxide without the gravest
`consequences.
`Pharmacological and Biological Effects of
`Deuterium Oxide-Morowitz
`and Brown (4) re-
`viewed the early physiological studies in this area,
`and Thomson (8) prepared a comprehensive summa-
`ry of the literature up to the year 1963.
`Heavy water is readily absorbed from the GI tract
`and rapidly equilibrates with the body fluids. There
`appears to be no selective excretion of heavy water by
`the kidney. Pure (99.8 atom% D) heavy water has
`a taste not too different from distilled water or deaer-
`ated water. It is readily administered to animals in
`their drinking water, and its palatability may be en-
`hanced by addition of traces of sodium chloride.
`Smith (34) suggested that mice are capable of discri-
`minating between deuterium oxide and water, and he
`attributed this ability to the formation of the more
`stable deuterium-oxygen bonds which may be in-
`volved in the interaction between the water molecule
`and “a gustatory receptor site.” This study was later
`challenged (35), since the data were based on only
`two mice (litter mates)2.
`By the selection of the appropriate deuterium
`oxide concentration in the drinking water, the equi-
`librium deuterium oxide concentration in the body
`fluids can be readily controlled. The course of deu-
`teration has been intensively studied in mice (26, 29,
`36) and rats (27). Figure 1 illustrates the-rate of deu-
`teration (as reflected by the deuterium content of the
`urine) as a function of time when varying concentra-
`tions of heavy water were administered to mice in
`their drinking water. An equilibrium state in the
`body fluids was reached by about the 10th day. With
`low concentrations of heavy water in the drinking
`water, the deuterium level in urine reached about
`90% that of the ingested water. Where the deuterium
`oxide content of the drinking water was in the 20-
`50% range, equilibrium urine values were approached
`more slowly and were only 7040% that of the drink-
`ing water. Dilution was due primarily to the fact that
`the food administered to the animal was of ordinary
`isotopic composition. Similar results