Review Themed Issue: Radioisotopic Bioanalysis

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A historical perspective on radioisotopic tracers in metabolism and biochemistry

Radioisotopes are used routinely in the modern laboratory to trace and quantify a myriad of biochemical processes. The technique has a captivating history peppered with groundbreaking science and with more than its share of Nobel Prizes. The discovery of radioactivity at the end of the 19th century paved the way to understanding atomic structure and quickly led to the use of radioisotopes to trace the fate of molecules as they flowed through complex organic life. The 1940s saw the first radiotracer studies using homemade instrumentation and analytical techniques such as paper chromatography. This article follows the history of radioisotopic tracers from meager beginnings, through to the most recent applications. The author hopes that those researchers involved in radioisotopic tracer studies today will pause to remember the origins of the technique and those who pioneered this fascinating science.

“You have to know the past to understand the present.”– Carl Sagan The development of radioisotopic tracer techniques has been pivotal to our understanding of a wide range of biological functions, from routine quantitative measurement, the study of xenobiotic and constitutive metabolic pathways, to metabolic flux and DNA binding. Without a hint of hyperbole, the advent of radioisotopic tracers has been called ‘the most important scientific tool invented since the microscope’ [1] . For most biochemistry and metabolism laboratories and certainly anywhere conducting absorption, distribution, metabolism and excretion (ADME) studies in laboratory animals or humans, the use of radioisotopes is taken virtually for granted. It is, however, worth pausing from our daily radioisotopic routine to consider the origins of the technique and how the associated technology grew around it. The first reference I could find to the term ‘radioisotopic tracer’ in the current context was by Frederick Soddy in his 1909 book on radium [2] . The reference does not refer to any biological application; he suggests using radon gas (discovered just 9 years earlier) as a tracer to follow pipelines and leakages,

10.4155/BIO.14.286 © 2015 Future Science Ltd

Graham Lappin School of Pharmacy, University of Lincoln, LN6 7TS, UK [email protected]

although the underlining principles he suggested were very similar as those quoted by Melvin Calvin for the elucidation of the photosynthetic pathway (see below). Soddy’s 1909 book is enlightening in both what was known at that time and, perhaps more so, in what still remained to be discovered. Soddy mentions, for example, his suspicion that α-particles are the same as helium nuclei, although this was not verified until 1932 when James Chadwick discovered the neutron [3] . The story of radioactivity is inextricably molded with the elucidation of atomic structure and it is here that we will begin with the earliest origins. The beginnings The story of radioactivity starts with Wilhelm Röntgen (1845–1923), the discoverer of x-rays [4] , followed just 2 years later with JJ Thompson’s discovery of the electron [5] . Both Röntgen and Thompson produced their ‘rays’ artificially but this was quickly followed by the serendipitous discovery of naturally occurring radioemissions by Henri Becquerel in 1897 [6] . Becquerel thought that certain minerals might be able to absorb visible light then emit the newly discovered

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Key terms ADME: Absorption, distribution, metabolism and excretion. ADME data can be gathered in a variety of ways and one commonly applied approach is to administer a radiolabeled drug (typically 14C) to laboratory animals or humans, followed by the radioanalysis of excreta (and tissues in animal studies) to determine the distribution, metabolic pathway and rates and routes of excretion. Radioisotopes: Elements are defined by the number of protons in the nucleus, whilst isotopes are elements with varying numbers of neutrons. As a general rule, as the number of protons and neutrons in the nucleus becomes uneven, the nucleus becomes unstable and undergoes radioactive decay. Becquerels, Curies and dpm: The basic unit of radioactive decay, based upon the number of atomic disintegrations per unit time, depicted as either dps or dpm (for disintegrations per second and disintegrations per minute, respectively). One Becquerel is equal to 1 dps and 1 Curie is equal to 3.7 × 1010 dps. Carbon-14 (14C): Using the modern terminology, 14 carbon-14 is depicted as 12 C , the superscript being the mass number and the subscript being the atomic number. By convention, when biological tracers are used, the atomic number is omitted, with the mass number identifying the specific isotope (i.e., 14C). The earliest of publications, however, used C14, with the mass number following the symbol for the element. The precise point where the convention for the nomenclature changed so that the mass number preceded the symbol for the element is unclear. PET imaging: Imaging technique where positronemitting isotopes are used to label tracers which are then introduced into humans or animals. The existence of the positron was predicted in 1928 from an equation by Paul Dirac (Nobel Prize in 1933). The existence of the positron was confirmed by 1931. Half-life (t½): Time taken for half a population of atoms to decay. Radioactive decay is exponential, in that for each half-life, half the population decays but it can never in theory reach zero. Grignard reagents: Organometallic-based reagents developed by Victor Grignard (Nobel Prize 1912) important in synthetic chemistry for the formation of carbon–carbon bonds and hence widely used in the chemical synthesis of 14 C compounds.

x-rays (known as Röntgen rays at the time) by phosphorescence. He examined many minerals with no success until he tried uranium salts, whereupon he detected the Röntgen rays that he sought. When he reran his experiments, however, he found that uranium salts emitted the rays spontaneously in the absence of light, thus spectacularly demonstrating the importance of including experimental controls. The story goes that Becquerel was not much interested in the discovery at the time and handed it on to a slightly troublesome student looking for a PhD project. That student was Marie Sklodowska, who later married

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Pierre Curie, thus forming one of the most famous scientific partnerships in history – Pierre and Marie Curie. It is worth remembering that Marie Curie lived at a time when women were second-class citizens and a woman scientist was an anathema. Indeed the reason she moved to Paris in the first place was that the Sorbonne was one of the very few universities to accept women students, although admittedly she was also getting into political trouble in her native Poland at that time. Marie, along with her husband Pierre, shared the Nobel Prize with Becquerel in 1903. Following Pierre’s death 3 years later, after he fell under a horse-drawn cart, Marie continued her work and was awarded a second Nobel Prize in 1911. The Curie legacy does not end there as her daughter, Irène Joliot-Curie, went on to win the 1935 Nobel Prize, together with her husband Frédéric, in recognition of the synthesis of new radioactive elements. Although the story of the Curies is a fascinating one, it has little direct relevance to the story told here. I will therefore just mention that it was Marie Curie herself that first coined the term ‘radioactivity’ and the units of radioactivity today are either Becquerels (Bq) or Curies (Ci) and leave the tale of the Curies there. By the 1940s the structure of the atom was understood and researchers were searching for new radioactive isotopes. The two most widely used radioisotopic tracers in laboratory biochemical research today are tritium (3H) and carbon-14 (14C). Ernest Rutherford, M. L. Oliphant and Paul Harteck first made tritium in 1934 by the collision of deuterium atoms [7] . That initial making of tritium had little to do with thoughts of biochemistry, but was all part of investigating the inner workings of the atom. The existence of 14C was predicted before it was discovered in 1940 by Martin Kamen and Samuel Ruben whilst working with the Nobel Laureate Ernest O. Lawrence at the Berkeley Radiation Laboratory [8] . Unlike tritium, the search for 14C was motivated by the idea that it could be used as an organic radioisotopic tracer to elucidate biochemical pathways. In fact, 11C, a positronemitting isotope of carbon, was also discovered by Kamen prior to 14C. He used 11C to show that water was the source of molecular oxygen in photosynthesis and not CO2  [9] . It is one of those twists of history that this very first biological radioisotopic tracer study, published in 1939, was conducted not with what are now routine radioisotopes but with 11C, used today almost exclusively in PET imaging. The problem with 11C for Kamen, however, was that it has a very short half-life of only 20.3 min, which prompted the search for 14C, an isotope of carbon with a half-life more amenable to biochemical experiments (the half-life of 14C is 5730 ± 40 years).

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A historical perspective on radioisotopic tracers in metabolism & biochemistry

Following the discovery of 14C, the US government became involved in World War II and Kamen and Ruben’s careers took divergent paths. Ruben researched mechanisms of action for poisonous gases and died in 1943 in a tragic laboratory accident involving phosgene. Kamen, an immigrant from Canada, came under investigation for his left-leaning political views and lost his job with the Manhattan Project in July 1944. To pay the bills, he took a job in the shipyards near to Berkeley. Seven years later, he would face accusations in front of the House Un-American Activities Committee and lose his passport. He eventually cleared his name in the 1950s, sued the newspapers for libel and was given the Enrico Fermi Award in 1995. He died aged 89 in 2002. The first biomedical radioisotopic tracer studies Prior to the availability of radioisotopes, advances in metabolic research (constitutive and xenobiotic) were severely constrained. In what is one of the more bizarre examples of how compounds were traced in the absence of radioisotopes, in 1917 the biological fate of strychnine was investigated using a ‘fasted tree frog assay’ [10] . Strychnine, an alkaloid derived from Strychnos nux vomica, was used medicinally at the time in cathartic pills. There was therefore interest in the metabolic fate of strychnine, which was investigated by administration of cathartic pills and collection of urine over time. The presence of strychnine in the urine was assayed by exposure to fasted tree frogs that were, apparently, highly susceptible to its toxicity. The number of dead frogs was then correlated with the amount of strychnine excreted. The chemical structure of strychnine proved difficult to elucidate and it was not synthesized until 1954, leading to a Nobel Prize for Robert Woodward in 1965. It was only in the 1960s that the metabolic fate of strychnine was elucidated with the use of 3H and 14C labels [11,12] . Tritium was used in nuclear weapons, which both dominated and restricted its early use. Tritium was famously used, however, along with 14C in the elucidation of the photosynthetic pathway for which Melvin Calvin (1911–1997) won the Nobel prize in 1961 [13] . Calvin worked at the University of California in Berkeley and therefore was able to access to the newly discovered 14C isotope. Calvin summarized the usefulness of radiotracers in his Nobel lecture [14] : “One of the principal difficulties in such an investigation as this, in which the machinery which converts the CO2 , to carbohydrate and the substrate upon which it operates are made with the same atoms, namely,

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carbon and its near relatives is that ordinary analytical methods will not allow us to distinguish easily between the machinery and its substrate. However, the discovery of the long-lived isotope of carbon, carbon-14, by Samuel Ruben and Martin Kamen in 1940 provided the ideal tool for the tracing of the route along which carbon dioxide travels on its way to carbohydrate…” Calvin fed 14CO2 and 3H 2O to algal cultures, then dropped the algae into alcohol at specified times, from a few seconds to many minutes. He then separated metabolites extracted from the algae using what was then a state-of-the-art method: 2D paper chromatography. The paper chromatographs were exposed to photographic film, which revealed the presence of intermediates in the photosynthetic pathway as they incorporated the radioisotope over time. One of the greatest challenges was in the identification of the various metabolites, something that modern LC–MS could have accomplished in a fraction of the time. Although the work of Calvin was undoubtedly a milestone in the use of radioisotopic tracers that laid the foundations of many studies to come, his work was not the first to use 14C to trace a metabolic pathway. Perhaps surprisingly, the earliest publication where 14 C was used as a radiotracer was in 1948 where the metabolism of a xenobiotic (dibenzanthracene) was followed in the mouse [15] . The study was performed at the University of California, Berkeley (the same institution where Calvin worked). The results look remarkably like any modern ADME study, reporting quantitative distribution in tissues and rates and routes of excretion in urine, faeces and expired air. Biliary elimination was studied along with the effects of different routes of administration. The authors reported a mean of 98.8% recovery, an achievement that many a laboratory would be proud of today and they did this using a homemade liquid scintillation counter (see below). Of course at that time, the researchers did not have a catalog of 14C-labeled compounds and they had to synthesize their own radiolabeled test substances. Papers were published around this time, for example, by Dauben et al. in 1947 [16] on synthetic methods for 14C incorporation, principally using Grignard reagents. The metabolism of 14C-methadone in the rat was reported in 1949 [17] and the metabolism of carcinogenic 14C-2-acetylamonofluorene in the rat was first studied in 1950 [18] . Although perhaps not entirely routine by that time, the use of radioisotopic tracers in ADME studies was certainly established by the early to mid-1950s. By 1955 the Atomic Energy Commission at Oak Ridge (Tennessee, USA) was making 64,000 shipments of radioactive materials per year to hospitals and research laboratories [1] .

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Key terms Specific activity: Amount of radioactivity per mole or mass of a substance. The specific activity is limited by the half-life of the isotope, the shorter the half-life the higher the specific activity. For example, the maximum specific activity for 14C (half-life 5730 years) is 62.4 mCi/ mmol whilst for tritium (half-life 12.32 years) the maximum specific activity is 29 Ci/mmol. Metabolic turnover: Concerns the continual exchange, or flux, of cellular components by degradation and synthesis. All proteins, for example, have finite lifetimes and therefore need to be continuously replaced by metabolism. Turnover time, a commonly used parameter, is defined as the time taken for a pool of a specific metabolite or component to be completely replaced.

Milestone radioisotopic tracer studies Selecting specific radioisotopic tracer studies as examples, even those of great historical interest, is very difficult, given the ubiquity of the technique. In addition, radioisotopic tracers have been used in conjunction with stable isotopes, the latter being outside the scope of this article. Nevertheless, I have tried to pick out a few ‘golden nuggets’ of particular historical significance, whilst recognizing the injustice to those I have left out. Undoubtedly, Calvin’s use of 14C and 3H in the elucidation of the photosynthetic pathway (mentioned above) represents one of the key milestones in the use of radioisotopic tracers. Interestingly, however, radioactive (and stable) tracers were not used by Hans Krebs in the initial elucidation of the citric acid cycle. The work, which led to a Nobel Prize for Krebs in 1953, was conducted in various forms of isolated tissues, which left many questions regarding the relevance of the results to the intact organism. Subsequent to Krebs’ original work, stable 13C and radioactive 14C tracers were therefore used to confirm the presence of the citric acid cycle in living species [19] . Watson and Crick revealed the structure of DNA in 1953 [20] , but prior to this there was much debate regarding whether DNA or a protein was the molecule of inheritance. DNA was, however, confirmed as the molecule of inheritance in 1952, probably to the great relief of Watson and Crick and the dismay of those searching for a protein such as Johannes Miescher. Confirmation came from Alfred Hershey and Martha Chase who labeled the DNA and protein in bacteriophage with phosphorus-32 (32P) and sulphur-35 (35S)  [21] . Both 32P and 35S were products of nuclear research at Oak Ridge in the late 1940s and early 1950s [22] with half-lives of 14.3 and 87.4 days, respectively. At the time, the genetic part of bacteriophage was known generically as ‘nucleoprotein’ but Hershey and Chase separately labeled the protein with 35S and the DNA with 32P enabling them to distinguish the

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two types of molecules. They infected bacteria with the phage and then examined the proportions of 35 S-protein versus 32P-DNA that entered the cell. Initial experiments were complicated by the difficulty of separating intact phage from infected bacteria but eventually they were able to show that the transfer of 32P-DNA predominated, confirming this as the molecule of inheritance. Staying with the theme of DNA, elucidation of the double helix opened up many questions about the inheritance mechanism. Such questions included, at how many sites did the replication process originate and in which direction did it proceed? A biochemist at Cold Spring Harbor (New York, USA) called John Cairns attempted to design a number of experiments to answer these questions but his initial attempts were considered too complicated to have any practical chance of success. He then hit upon the idea of growing bacteria in cultures fed with 3H-thymadine and using autoradiography. He exposed x-ray film to very carefully isolated DNA strands (now radioisotopically labeled with 3H) and then developed the film to observe the images of replicating DNA. He could see the replicating forks (described as looking like the Greek letter theta – θ). He also showed that replication had a single origin and was bidirectional [23] . Tritium later went on to play a central role in elucidating the workings of both DNA and RNA polymerases [24] . Radioisotopic tracers have been equally important in elucidating the metabolism of small molecules as well as macromolecules. For small molecules (under ca 1000 Da) 14C can usually be incorporated by chemical synthesis (e.g., using the aforementioned Grignard reagents) whilst tritium has been favored for macromolecules because of the relative ease of tritium exchange labeling. Tritium labeling of amanitin, a peptide toxin found in mushrooms and a potent inhibitor of RNA polymerase II and III for example, was used in the elucidation of the function of the aforementioned polymerases [25] . Although 3H was, and still is, used to label macromolecules, other isotopes with halflives much shorter than tritium (the half-life of 3H is 12.32 ± 0.02 years) have also been used. Because of the high molecular weight of macromolecules, high molar specific activity is required in order to obtain the necessary assay sensitivity when measuring the mass of a compound. To achieve this, isotopes with short halflives are required, but with half-lives still long enough so that there is sufficient time to conduct the necessary experiments. Aforementioned 11C, for example, with a half-life of 20 min is too unstable for most laboratorybased tracer applications. It has to be generated in a cyclotron and then immediately used in an experiment. For the isotopic labeling of macromolecules, half-lives

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A historical perspective on radioisotopic tracers in metabolism & biochemistry

of a few weeks offer the ideal compromise between assay sensitivity and experimental convenience. In 1973, a protein radioisotopic labeling method was proposed using iodine-125 (125I, half-life 59.4 days) which subsequently became the widely used Bolton–Hunter reagent [26] . Interestingly, the very first radioisotopic tracer study involving the labeling of proteins was conducted using radioactive iodine as early as 1943 [27] just 4 years after Kamen’s seminal publication using 11CO2 to investigate photosynthesis [9] . It was a study performed in dogs involving radioactively tagged plasma proteins in the study of hemorrhagic shock. The precise isotope is not mentioned in the paper, other than it was an isotope of iodine. It must have been 131I, however, as this was the only radioactive isotope of iodine known at that time. (131I was discovered by Glenn Seaborg in the 1930s, whilst 125I was not discovered until 1946.) On a more practical level, the potential for molecules to bind to DNA has been of great interest to the chemical and pharmaceutical industries for many years as unintentional DNA binding is generally bad news for business. The study of DNA binding is complicated principally by three factors: the molecular structure of the adduct, assuming it is present, might not be known and therefore it is difficult to locate and quantify; the magnitude of binding is likely be relatively low and assays have to be highly sensitive; and although the formation of DNA-adducts is analytically plausible when it occurs, it is virtually impossible to prove that no binding has occurred (i.e., to prove a negative). In 1979 a paper was published by Werner Lutz, which detailed methods for the quantitative assessment of DNA adduct formation using radioisotopically labeled test compounds. The labeled compound was administered to animals, the DNA extracted and purified, then the radioactivity assayed (see scintillation counting, below). Lutz proposed a covalent binding index (CBI), which is defined in Equation 1. CBI =

nmol chemical bound per mol DNA nucleotide mmol chemical dosed per kg bodyweight Equation 1

The method exploited the innate quantitative nature of radioactivity measurement, which is unaffected by the environment (pH, temperature, etc.) and does not require an analytical standard, rendering knowledge of the chemical structure of the adduct unnecessary. CBI was at one time widely used but was unpopular with the industry because it was virtually impossible to prove that no DNA binding occurred and therefore regulatory submissions were invariably ambiguous. The author became involved in the investigation of the potential DNA binding of an agricultural fungicide called captan, whereby initial studies appeared to show

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CBIs in the range of 38–91 (to put this into context, these values are approximately 10% of the carcinogen benzo-[a]-pyrene). Following extensive purification of the DNA and the use of 35S-captan, it was shown that the apparent CBI was likely to be an artifact arising from residual protein bound to the DNA that was impossible to remove without complete molecular hydrolysis [28] . In 1981, a method of detecting DNA adducts using 32 P was proposed whereby enzymatic hydrolysis of non-radioactive adducted DNA was carried out to produce the 3′-phosphonucleosides [29] . Polynucleotide kinase mediated phosphorylation at the free 3′OH moiety with 32P-ATP generated radiolabeled adducts that could then be chromatographically separated. The technique had the advantage that the test compounds did not have to be specially synthesized to contain 14C (the 32P reagents were generic) and the method was potentially sensitive enough to detect one adduct in 107 bases. The method has been widely utilized but a number of potential false-negatives have been reported due to the loss of the adduct during hydrolysis, particularly with methyl and ethyl substitutions [30] . DNA binding studies remain controversial and difficult to perform to this day. The final (an admittedly selected) example concerns the incorporation of 14C-amino acids into protein. Given the examples above, this may not initially appear to be of particular note, but it was the first time radioisotopic tracers were used to determine metabolic turnover. By using a 14C-tracer, labeled amino acids could be distinguished from the naturally occurring pools and the kinetics of amino acid incorporation into protein could be measured. The original work was conducted by Schoenheimer in 1942 using stable isotopes [31] and was then extended by Eagle et al. in the late 1950s with 14C [32] . Eagle et al. labeled proteins by adding amino acids to in vitro cultures, then monitored the loss of the 14C from the protein over time. By measuring both the uptake and loss of 14C, the turnover rates of protein were measured. The concept of metabolic turnover is taken for granted today but at that time little was known about the constant breakdown and synthesis of metabolic components such as proteins, and by inference tissues. Metabolic turnover has more recently been extended to specific cell-types in the body but this is discussed in the next section on human studies. Radioisotopic studies in humans In addition to animal, plant and bacterial studies, radioisotopic tracers were also applied to studies in humans, which raised obvious questions regarding risks from radioactive exposure. In the early days after the

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Key terms Accelerator MS : Originally developed for radiocarbon dating, it measures the isotopic ratio between 12C/13C/14C and does not rely on the detection of energy emitted from relatively infrequent atom decay. Consequently it is about a million times more sensitive than scintillation counting. Coincidence circuit:An Electronic circuit on a scintillation counter that distinguishes between signals derived from two photomultipliers. If the signal is generated in both tubes within a given time-window (typically a few μs) then the count is deemed real. If only one tube generates a signal, or the time between the signals exceeds some threshold, the signal is deemed to have originated from a background event. The coincidence circuit was the first major advancement in noise scintillation reduction.

discovery of radioactivity, its dangers were not appreciated. There are well-known stories such as Pierre Curie carrying a vial of radium in his top pocket for several days and then showing the resulting reddened skin to curious colleagues. Radium became the rage of its age and was put into everything from toothpaste to suppositories, from cosmetics to condoms! The first indications of the dangers of radiation came in the early 1900s when women employed to apply radium paint to luminous watch dials sued the US Radium factory based in Orange New Jersey. The women, who became known as the radium girls, used their mouths to form a fine point on the radium-loaded paintbrushes, which caused a condition known at the time as ‘radium jaw’ – a euphemism for oral cancer. There were also ‘quack cures’ of the time based on radium, the best known being Radithor, made by William Bailey (a Harvard dropout who claimed to be a doctor) which sold for around $0.63 an ounce. Radithor was made from a total of 37 MBq of radium dissolved in distilled water and was marketed as a health drink. One consumer of Radithor was Eben Byers, a popular, rich and powerful Pittsburgh sportsman and chairman of the A. M. Byers Company, makers of pipes with connections to coal and banking. He died of oral cancer in 1932, with his jaw virtually dissolved away. With such high profile cases, the dangers of exposure to ionizing radiation became evident. Marie Curie herself died in 1934 from leukemia, thought to be the result of a lifetime exposure to raised levels of radioactivity. She was nevertheless, 67 when she died, not a bad age for the time. A supreme irony was that the Curie’s daughter, Irene, also died of leukemia at the age of 58, also very likely brought on from radioactive exposure, some of it received as a child in the very laboratory where her mother had worked. During the late 1920s through the 1930s, the biological effects of radioactive exposure began to be investigated, which gave birth to a new scientific discipline of radiobiology, generally accepted to be founded by the

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British physicist, Louis Harold Gray (1905–1965). Gray was an undergraduate at the Cavendish Laboratory in Cambridge and was taught by the Nobel Laureates, Rutherford, Thomson and Chadwick. Gray studied the effects of radioactivity on various materials and worked on the measurement of cosmic radiation, a subject where very little was known at the time. He worked with William Bragg (1862–1942) to develop the Bragg–Gray equation in 1936, which described how the energy of radioactivity was absorbed by matter. Gray went on to work at Mount Vernon Hospital in Norwood, London, to study the effects of radiation on living tissue. He devised a new unit to describe the absorbed dose of radioactivity, which was named after him, the Gray (Gy). One Gray was defined as one Joule of energy absorbed by 1 kg of matter. Another radiobiologist, Rolf Sievert (1896–1966) working at the Karolinska Institute in Sweden, recognized that different types of radioactivity (e.g., α, β and γ) had different effects on living tissue and assigned what was known as a quality factor to account for the potential of biological damage. The modern unit of radioactive dose, which accounts for the quality factor, is the Sievert (Sv). Nowadays, regulatory authorities throughout the world limit human exposure to ionizing radioactivity for research to approximately 1 mSv, although a case can be made to increase this, typically to a maximum of 5 mSv [33,34] . For the majority of ADME studies with humans, however, researchers try not to exceed 0.5 mSv. For a drug with a half-life of around 6 h that does not exhibit any tissue binding, then 0.5 mSv is the equivalent of about 20 kBq/kg (0.54 μCi/kg) bodyweight. Not that long ago, however, alarmingly large amounts of radioactivity were used in human studies. For example, in a study looking at the metabolic turnover of sitosterol performed in the late 1960s, 855 kBq (21.3 μCi) of tritiated compound per day per subject were administered for 83 days [35] . These levels are unlikely to be allowed today. In an attempt to reduce the radioactive exposure associated with the administration of radioisotopically labeled compounds to human subjects, particularly those in drug development, the application of a technique known as Accelerator MS (AMS) was first reported in 1997 [36] . AMS was originally developed in the mid-1970s for use in archeological radiocarbon dating. It is an extremely sensitive method of measuring isotope ratios and is capable of reliably measuring as little as the equivalent of 0.001 dpm per g tissue [37] . The first study reported where a 14C-labeled compound was administered to human volunteers and AMS was used in the sample analysis, was a DNA binding study looking at the binding of environmental carcinogen, 2-amino-3,8-dimethylimidazo[4,5-f] quinoxaline (MeIQx). The study was a collaboration

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A historical perspective on radioisotopic tracers in metabolism & biochemistry

between the Lawrence Livermore National Laboratory (LLNL) in California, USA, and the University of York in the UK. Collaborators from both LLNL and the University of York subsequently established the first, and competing, commercial companies offering AMS services. In DNA binding studies with rodents, animals were administered 10 mg MeIQx/kg. These doses, far in excess of those encountered from environmental exposure, were necessary in order to achieve the required sensitivity when assaying DNA adducts using scintillation counting (see below). Studies were then conducted in the mouse using AMS for 14C analysis [38] followed by a study in humans [36] . In the human study, 228 μg 370 kBq (10 μCi, equivalent to 0.3 μSv) of MeIQx was administered which was much closer to those encountered environmentally. The sensitivity of AMS then allowed binding to be measured not just in isolated DNA but as the individual adducts following hydrolysis of the macromolecule and separation by HPLC. It was shown that humans have approximately 10-fold more MeIQx–related DNA adducts present than rodents, although the biological significance remains unclear. AMS has since been used in a wide range of studies involving radioisotopic tracers (mostly 14C) in humans [39] . A recent study with AMS re-examined the metabolic turnover of sitosterol (mentioned above) but was conducted using a total of just 4 kBq (100 nCi) of compound per subject as a tracer in sitosterol in the diet [40] . The study of protein turnover (discussed above) was first conducted in vitro but this has now been extended to looking at cellular turnover in humans. In the late 1950s and early 1960s pulses of 14C were emitted into the global atmosphere by atomic bomb tests. The peak of the 14C-bomb pulse was around 1967 and people born at that time incorporated the raised levels of radiocarbon into their tissues. In fact the global 14C levels increased by around 50% at that time, which is an extraordinary amount considering this was on a global scale. As the cells in the body died and were replaced, so new cells contained levels of 14C congruent with the atmosphere at that time. Thus, as the levels of 14C declined, so newly generated cells reflected the lower concentrations. The Human Regenerative Map Project run at the Karolinska Institute in Sweden analyses samples of extracted DNA from donated corpses, where the exact date of birth is known. The presence of the 14C-bomb pulse is measured using very precise AMS methods developed at the University of Uppsala [41] which can then be correlated to the rate of cellular flux. The project has, for example, shown that contrary to common belief, brain neurons exhibit significant lifetime regeneration [42] and the rate of triglyceride storage is higher, but the turnover is slower, in obese compared with non-obese subjects [43] .

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Development of scintillation counting No account of the history of radioisotopic tracers would be complete without looking at scintillation counting, which is the most widely used and routine method of measuring radioactivity. The origin of the first scintillation counter might be a surprise to many readers. It was sold in 1903 as a children’s toy! William Crookes (1832–1919) designed a system that he called the Spinthariscope (from the Greek ‘spintharis’ – a spark), which consisted of a phosphorescent screen of zinc sulfide placed over a minute trace of a radium salt, which had been supplied to Crookes by Marie Curie. The α-particles emitted by the radium salt caused visible flashes of light (scintillations). The Spinthariscope was used like a kaleidoscope but one that today might not get past British Standards Kitemark! Rutherford and his coworkers in the 1900–1920s used the same basic principle of a scintillating zinc sulphide crystal when they were investigating the inner workings of the atom. Researchers sat in a dark room, prior to running experiments, to acclimatize their eyes before peering down a tube and counting the flashes of light for hours on end. The process was very error prone as it relied on the ability of individuals to count the flashes. Indeed, it was found that the experimental details and expected results had to be hidden from those doing the counting, which could otherwise subconsciously influence the data [44] . It was the advent of the photomultiplier tube that represented the big breakthrough in scintillation counting. There is some debate as to who invented the photomultiplier tube but it is generally attributed to the Russian physicist Kubestsky in the 1930s. The Photomultiplier Tube replaced the human eye for counting the light flashes emanating from scintillation events, thereby removing one of the major sources of error. In 1951, the idea of liquid scintillants was proposed by Raben and Bloembergen [45] . They proposed that liquid scintillants would be more efficient than solid scintillants such as zinc sulphide for α and β emissions as the analyte was in solution and hence in very close proximity to the source of scintillation. Work in the 1950s at Los Alamos developed liquid scintillants, which were based largely on 2-phenyl-5-,4-biphenylyl oxazole (PBO) developed by Hayes and his coworkers [46] and this remained the principal component of many scintillation cocktails for many years afterward. At the time when the first radioisotopic tracer studies were being conducted (e.g., by Calvin, see above), there were no commercial scintillation counters and researchers had to make their own. Calvin’s homemade counter was shrouded in concrete to reduce background (prior to coincidence circuits – see below). Although these early counters (and the scintillants) resulted mainly from the work conducted at Los Alamos, the

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Review  Lappin first commercial counters came from the University of Chicago. It was here that Arthur Compton (Nobel Prize in 1927) had established a laboratory and Enrico Fermi (Nobel Prize in 1938) had built the first nuclear reactor. As an aside, Fermi’s first nuclear reactor was built in the heart of Chicago and virtually none of the photographs taken of the reactor have survived as they were fogged by the leaking radioactivity. Lyle Packard gained his degree at Illinois Institute of Technology in Chicago and after training as an engineer in the US Navy, he joined the Institute of Radiobiology and Biophysics at the University of Chicago in 1946. Lyle Packard was, of course, the founder of the Packard Company where the first commercial liquid scintillation counter was built in 1954. The TriCarb, as the first counter was called, had dual photomultiplier tubes, a coincidence circuit and a light-tight shutter where samples were introduced. Packard had had some practice, before he built the first commercial instrument by constructing a custom-made counter for the University of Chicago as a prototype in 1953. Packard also developed the first automated instrument in 1957 where vials were loaded into a carousel holding 100 samples. Other manufacturers, such as Wallac and Beckman, followed soon after but Packard remained the world leader in liquid scintillation counting for decades afterward, until they were finally bought out by Perkin Elmer in 2001. Concluding comments & future perspective The use of radioisotopic tracers in biochemistry is today a routine technique based upon an unbridled pedigree and a string of Nobel Prizes. Although the regulatory guidelines covering ADME studies required for the regulatory submission of new drugs do not specify the use of radioisotopic tracers, the technique is nevertheless the gold standard and used almost universally. AMS is an extremely sensitive method for isotopic measurement (primarily 14C) and was first used in biomedicine in the late 1990s but has not been adopted as a widespread technique. For full disclosure the author is involved in applications of AMS to drug development and so fully accepts this may be seen as a bias opinion but nevertheless believes it is a highly underused technology. There are many possible reasons why AMS has not been widely adopted within the industry. AMS as an application was quickly commercialized at its outset and there are still no academic AMS research facilities dedicated to human biochemistry or pharmacology. Instruments are instead typically held in physics departments. This lack of research pedigree may have inhibited its adoption by a somewhat conservative industry. Undoubtedly, some of the very early AMS studies performed for the indus-

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try encountered unforeseen problems, likely due to extending the technique into areas where there was little previous experience. Industry may have also been inhibited by the initial cost of instrumentation, which was in excess of two million US dollars. The instruments first used for biomedical AMS studies were of a high voltage tandem design, taking up in excess of 300 m2 of floor space. It is also the case that until very recently, significant sample preparation was required, whereby the carbon from a sample was purified by chemical oxidation to CO2 then reduction to graphite. This process, which takes several days, has been compared with mass spectrometry where data can be acquired in hours. This makes AMS analysis comparatively slow and, because the process is labor-intensive, relatively expensive. Instrument design and methods of sample analysis are rapidly advancing, however, primarily due to the efforts at the Eidgenössische Technische Hochschule at the University of Zürich in Switzerland. There are now AMS instruments specifically designed for biomedical work that cost little more than a quadrupole LC–MS and occupy about the same footprint. Automated sample preparation methods and the application of CO2 sources enable analysis times approaching regular mass spectroscopy. Considerable advances are also being made in coupling AMS to HPLC through CO2 sources, although challenges remain mostly because of the difficulties in removing the last vestiges of atmospheric CO2 from the system [47] . Nevertheless, at the time of writing, there is still only one pharmaceutical company that has an AMS instrument (GlaxoSmithKline in the UK) [48] . Whilst there are a number of possible reasons why the industry might remain skeptical of the value of AMS in the conduct of studies for regulatory submission in drug development, there is (in the view of the author) great potential in fundamental research. In this environment, some of the aforementioned issues are not seen as such a hurdle. For example, the Universities of Kentucky and Purdue very recently adopted AMS to track complex engineered nanoparticles through biological tissues [49] . Because of its extreme sensitivity there is potential to administer radiotracers to humans to study fundamental biochemistry and perhaps mechanisms of disease that were only previously feasible in animal models due to the limitations of radioactive exposure. Information from the aforementioned Human Regenerative Map project, for example, may prove valuable in understanding fatty acid turnover in the treatment of obesity and in stem cell therapy from knowledge of the turnover of specific tissues. Despite biomedical AMS being over 15‐years old, there is still a huge unexploited potential for the technique. An account on the history of radioisotopic tracers written

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A historical perspective on radioisotopic tracers in metabolism & biochemistry

in 20 years will surely include major advances in our understanding of human biochemistry based upon applications of AMS. Financial & competing interests disclosure G Lappin is a consultant to the pharmaceutical industry on isotopic tracer studies and is a consulting scientist for Xceleron Inc.

Review

He has no stock ownership or options in any of these companies. The author has no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. No writing assistance was utilized in the production of this manuscript. 

Executive summary • Radioisotopic tracers are used routinely in the modern biochemistry laboratory to track organic molecules through complex biological systems. • Modern methods, largely taken for granted today, have a history that started about 75 years ago with the discovery of radioisotopes of carbon and hydrogen. • The history of radioisotopic tracers is one of groundbreaking science and a string of Nobel Prizes. The article selects some pivotal studies and explores how applications of radioisotopes have contributed to our modern understanding of biochemistry and metabolism. • Along with its meager beginnings, the article explores current state-of-the-art applications of radioisotopic tracers, how the modern scintillation counter developed and the contribution of Accelerator MS.

References

12

Tsukamoto H, Oguri K, Watabe T, Yoshimura H. Metabolism of drugs. XlI. The metabolic fate of strychnine in rabbit liver. J. Biochem. 55, 394–400 (1964).

Papers of special note have been highlighted as: • of interest 1

Creager ANH.  Life Atomic: A History of Radioisotopes in Science and Medicine. University of Chicago Press, Chicago, IL, USA (2013).

13

Wilson AT, Calvin M. The photosynthetic cycle. CO2 dependent transients. J Am. Chem. Soc. 77(22), 5948–5957 (1955).

2

Soddy F. The Interpretation of Radium. John Murray, London  (1909). 

•

The paper that led to the Nobel Prize for Melvin Calvin for the elucidation of the photosynthetic pathway.

•

A fascinating book capturing a key moment in the history of radioactivity written at the same time Rutherford was awarded the Nobel Prize for the elucidation of atomic structure.

14

Calvin M. The Website of the Nobel Prize (1961). www.nobelprize.org

15

Heidelberger C, Jones HB. The distribution of radioactivity in the mouse following administration of dibenzanthracene labeled in the 9 and 10 positions with carbon 14. Cancer 1(2), 252–260 (1948).

•

For anyone involved in animal or human absorption, distribution, metabolism and excretion (mass balance) studies, this paper is of great historical interest. It is the first study of its type but the methods and results are entirely analogous with a modern study. The authors reported a 98.8% mean recovery in 1948, an achievement which remains a challenge to this day.

16

Dauben WG, Reid JC, Yankwich PE. Techniques in the use of Carbon 14. J. Am. Chem. Soc. 19(11), 828–832 (1947).

17

Elliott HW, Chang FNH, Abdou IA, Anderson HH. The distribution of radioactivity in rats after administration of C-14 labeled methadone. J. Pharmacol. Exp. Ther. 95, 494–501 (1949).

18

Morris HP, Weisburger JH, Weisburger EK. The distribution of radioactivity following the feeding of carbon 14 labeled 2-acetylaminofluorene to rats. Cancer Res. 10, 620–624 (1950).

3

Chadwick HC. Letters to the editor: possible existence of a neutron. Nature 130, 572 (1932).

4

Rötgen WC.  Auf eine neue Art von Strahlen (On a New Kind of Rays) . Wilhelm Conrad, University of Würzburg, Germany, (1895).

5

Thompson JJ. Paper read before the Cambridge Philosophical Society: a Friday Evening Discourse at the Royal Institution, London. Philosophical Magazine 44, 293 (1897).

6

Becquerel MH. Sur les radiations invisibles émises par les corps phosphorescents (The invisible radiation from phosphorescent bodies). Reports of Séanes de l’Academie de Sciences T-122, 501 (1896).

7

Oliphant MLE, Harteck P, Rutherford E. Transmutation effects observed with heavy hydrogen. Proc. R. Soc. 144, 692 (1934).

8

Ruben S, Kamen MD. Radioactive carbon of long half-live. Phys. Rev. 57, 549 (1940).

9

Ruben S, Hassid WZ, Kamen MD. Radioactive carbon in the study of photosynthesis. J. Am. Chem. Soc. 61, 661–663 (1939).

19

Hatcher RA, Eggleston C. The fate of strychnine in the body. J. Pharmacol. Exp. Ther. 10, 281–319 (1917).

Tokumitsu Y, Michio UI. Separation and determination of 14C-labelled intermediates of the citric acid cycle and related compounds. Anal. Biochem. 59(1), 110–121 (1974).

20

Watson JD, Crick FH. Molecular structure of nucleic acids; a structure for deoxyribose nucleic acid. Nature 171(4356), 737–738 (1953).

10 11

Adamson RH, Fouts JR. Enzymatic metabolism of strychnine. J. Pharmacol. Exp. Ther. 127, 87–91 (1959).

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Review  Lappin 21

Hershey AD, Chase M. Independent functions of viral protein and nucleic acid in growth of bacteriophage. J. Gen. Physiol. 36(1), 39–56 (1952).

•

The first use of accelerator mass spectrometry in biomedicine – an interesting paper for anyone involved in this technique.

22

Sheline RH, Holtzman RB, Fan CT. The Nuclide 33P and the 32P spectrum. Phys. Rev. 85, 919–925 (1951).

37

23

Cairns J. The bacterial chromosome and its manner of replication as seen by autoradiography. J. Mol. Biol. 6, 208–213 (1963).

Lappin G, Garner RC. Ultra-sensitive detection of radiolabelled drugs and their metabolites using accelerator mass spectrometry. In: Handbook of Analytical Separations. Wilson I (Ed.). Elsevier, Amsterdam, The Netherlands, 331–349 (2003).

24

Weinmann R, Raskas HJ, Roeder RG. Role of DNAdependent RNA polymerases II and III in transcription of the adenovirus genome late in productive infection. Proc. Natl Acad. Sci. USA 71(9), 3426–3439 (1974).

38

25

Garrity GM, Brown A. Competition binding assay using o-methyl-[3H]-demethyl-gamma-amanitin for study of RNA polymerase B. Proc. Soc. Exp. Biol. Med. 159(1), 98–104 (1978).

Turteltaub KW, Vogel JS, Frantz C, Felton JS, McManus M. Assessment of the DNA adduction and pharmacokinetics of PhIP and MeIOx in rodents at doses approximating human exposure using the technique of accelerator mass spectrometry (AMS) and 32P-postlabeling. Princess Takamatsu Symp. 23, 93–102 (1995).

39

Bolton AM, Hunter RM. The labelling of proteins to high specific radioactivities by conjugation to a 1245I-containing alkylating agent. Biochem. J. 133, 529–539 (1973).

Lappin G, Stevens L. Biomedical accelerator mass spectrometry: recent applications in metabolism and pharmacokinetics. Expert Opin. Drug Metab. Toxicol. 4(8), 1021–1033 (2008).

40

27

Fine J, Seligman AM. Traumatic Shock VII. A study of the problem of the lost plasma in hemorrhagic tourniquet and burn shock by the use of radioactive iodo-plasma protein. J. Clin. Invest. 23(5), 720–730 (1943).

Duchateau G, Cochrane B, Windebank S et al. Absolute oral bioavailability and metabolic turnover of beta-sitosterol in healthy subjects. Drug Metab. Dispos. 40(10), 2026–2030 (2012).

41

28

Provan WM, Eyton-Jones H, Lappin G, Pritchard D, Moore RB, Green T. The incorporation of radiolabelled sulphur from captan into protein and its impact on a DNA binding study. Chem. Biol. Interact. 96(2), 173–184 (1995).

Salehpour M, Forsgard N, Possnert G. Biological AMS at Uppsala University: Status report. Nucl. Instrum. Methods Phys. Res. doi:10.1016/j.nimb.2009.10.055 (2009) (Epub ahead of print).

42

Randerath K, Reddy MV, Gupta RC. 32P-labeling test for DNA damage. Proc. Natl Acad. Sci. USA 78(10), 6126–6129 (1981).

Spalding KL, Bergmann O, Alkass K et al. Dynamics of hippocampal neurogenesis in adult humans. Cell 153(6), 1219–1227 (2013).

43

Arner P, Bernard S, Salehpour M et al. Dynamics of human adipose lipid turnover in health and metabolic disease. Nature 478(7367), 110–113 (2011).

44

Cathcart B. The Fly in the Cathedral. Farra Straus Giroux, NY, USA (2004).

26

29

30

540

Phillips DH, Farmer PB, Beland FA et al. Methods of DNA adduct determination and their application to testing compounds for genotoxicity. Environ. Mol. Mutagen. 35(3), 222–233 (2000).

31

Schoenheimer R. The Dynamic State of Body Constituents. Harvard University Press, Cambridge, MA, USA, (1942).

•

A popular science book that tells the story of how Rutherford and his team looked into the heart of the atom. Brilliant bedtime reading!

32

Eagle H, Piez KA, Fleischman R, Oyama VI. Protein turnover in mammalian cell culture. J. Biol. Chem. 234(3), 592–597 (1958).

45

Raben MS, Bloembergen N. Determination of radioactivity by solution in a liquid scintillator science. Science, 114(269), 363–364 (1951).

33

ICRP, International Commission on Radiological Protection. Radiological protection in biomedical research, ICRP Publication 62. Ann. ICRP 33, 3 (1991).

46

Peng C-T, Horrocks DL, Alpen EL. Liquid Scintillation Counting. Academic Press, MA, USA (1980).

47

34

World Health Organization, Use of ionising radiation and radionuclides on human beings for medical purposes, Technical report series, 611, Geneva, (1977). http://whqlibdoc.who.int

ETH Zurich - D-PHYS - Ion Beam Physics  www.ams.ethz.ch  

48

Young G, Ellis WJ. AMS in drug development at GSK. Nucl. Instrum. Methods Phys. Res. B 259, 752–757 (2007).

49

Wang B, Jackson GS, Yokel RA, Grulke EA. Applying accelerator mass spectrometry for low-level detection of complex engineered nanoparticles in biological media. J. Pharm. Biomed. Anal. 97, 81–87 (2014).

35

Salen G, Ahrens EH, Jr., Grundy SM. Metabolism of B-sitosterol in Man. J. Clin. Invest. 49, 952–967 (1970).

36

Turteltaub KW, Mauthe RJ, Dingley KH et al. MeIQx-DNA adduct formation in rodent and human tissues at low doses. Mutat. Res. 376(1–2), 243–252 (1997).

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