Rosetta Stones

On May 25, 1939, the "University Explorer," reaching deep to advertise the Laboratory, announced the arrival of radioactive fertilizer in Hawaii. A professor at the University there had imported P³² from Berkeley by Pan American clipper to test its power on pineapples. An explorer of the consequences of the innovations of the Laboratory had (and has!) his work cut out: "The influence of the cyclotron has been felt in so many different fields of science that no one can predict its ultimate value to mankind."^[137]

The influence began to spread at home, in 1937/38, among members of Berkeley's Chemistry Department. The first practitioners of radioactive tracing were Libby, Seaborg, and, above all, Ruben, through whose efforts University biologists took up the technique. Purely chemical applications, which were not irrelevant to the biologist, included the vast fields of exchange reactions and reaction mechanisms; the nature of the inquiry may be indicated by studies by Libby's students of exchanges between various valence forms of sulphur and of photochemical processes in solution, and by Libby himself of the reactions of recoil nuclei activated by neutrons. Another conspicuous line, detection of impurities by their radioactivity, which had been an unwelcome and misleading annoyance, was practiced for a time by Seaborg and Livingood, who found copper in nickel, iron in cobalt, and phosphorus and sulphur everywhere.^[138]

Ruben and P³² inspired a major direction of research in the Physiology Department on the metabolism of phospholipids or phosphatides, which occur in all living tissues in connection with fatty deposits. Ruben, Chaikoff, and their students and colleagues, who included the chemist I. Perlman and, occasionally, John Lawrence, ground up rats and birds fed radiophosphorus under various regimes—fasting, normal diets, fatty diets—to learn the loci of the creation and destruction of phospholipids. Following John

[137] "University Explorer," 25 May 1939 (40/15).

[138] Kamen, Science, 140 (1963), 587, re Ruben; Seaborg, Chem. rev., 27 (1940), 250–73, esp. 256, 263, 266; Voge and Libby, JACS, 59 (1937), 2474; Rollefson and Libby, Jl. chem. phys., 5 (1937), 569–71; Seaborg and Livingood, JACS, 60 (1938), 1784–6.

Lawrence's interests, they also examined cancerous mice. They extended the findings of Segré's associates in Palermo, who, with an Italian touch, had fed olive oil and Berkeley phosphorus to their rats; which, when anatomized, disclosed that the liver is the fastest metabolizer of phospholipids. The Berkeley group further showed that excised bits of liver, kidney, and intestine continued to work at phospholipid metabolism in vitro, and that the rate of its metabolism in tumors transplanted from one animal to another was characteristic of the tumor, not the animal.^[139]

Two other sustained programs in radioactive tracing that thrived on the Laboratory's output deserve mention. One grew in the Biochemistry Department around David Greenberg's ongoing investigations of mineral metabolism. Inspired, he said, by the "revolutionary nature and potential importance" of radiotracing, Greenberg and his associates began as others did, feeding radiophosphorus to rats and examining deposits in tissue and feces. They went on to the phosphorus metabolism of rachitic animals. They were the first to use radiocalcium (Ca⁴⁵ ), a commodity more costly than Fe⁵⁹ , as a tracer. In 1940 and 1941 they published information on the metabolism, deposition, and elimination of manganese, iron, and cobalt, the last, as they found, a possible cause of polycythemia.^[140] The second program, conducted by S.C. Brooks at the Medical School, studied the transport of ions into and within plant cells. Here radioactive indicators brought light where darkness had long prevailed. According to an authoritative reviewer, the results of Brooks and his associates released biologists from "the necessity of postulating mysterious properties for cellular membranes and protoplasm."^[141] Further

[139] Artom et al., Nature, 139 (15 May 1937), 836–7, and ibid. (26 June 1937), 1105–6; Perlman, Ruben, Chaikoff, and others, in various combinations, Jl. biol. chem., 122 (1937), 169–82, 123 (1938), 587–93, 124 (1938), 795–802, 126 (1938), 493–500, 127 (1938), 211–20, 128 (1939), 631–44, 735–42. This and similar work from other laboratories is reviewed in Greenberg, Ann. rev. biochem., 8 (1939), 276–7; Hevesy, ibid., 9 (1940), 649–54; and Hamilton, Jl. appl. phys., 12 (1941), 445, and Radiology, 39 (1942), 545–8.

[140] Greenberg reviewed his own work in Ann. rev. biochem., 8 (1939), 269–70, quote, and in Wisconsin, Univ., Symposium (1948), 263–9, 270–3, 279–81. Cf. Kohler, From medical chemistry , 329; Greenberg, "Recollections" (UCA); Hamilton, Radiology, 39 (1942), 566–7.

[141] Loofbourow, RMP, 12 (1940), 275, with references to the literature.

attacks on these mysteries occurred at the School of Agriculture, where Perry Stout and his co-workers made the uptake of alkali and halide salts by growing trees and shrubs their subject of study. They settled the vexed question whether salts rise through the bark as well as through the xylem (the answer is no) and demonstrated by persuasive autoradiography the deposit of P³² in the leaves and fruit of growing plants (plate 8.5).^[142]

It did not take much, apart from the material, to set up with radioactive tracers and to get quick, publishable results. There were so many animals and plants, so many elements, so many permutations of experimental circumstances. Hence the Laboratory received many requests for its products, which, if they did not save lives, might improve careers. Lawrence honored requests from outside the University on the basis of merit, other factors being equal. His most generous support went to workers in Rochester and in Copenhagen, the two places where, in the opinion of Merle Tuve, no fan of the Laboratory, work with tracers second only to Berkeley's was being done.^[143] In Copenhagen, Hevesy's group continued work with P³² , following it throughout the body and its products, into blood, eggs, milk, across cell walls, to and from the liver, and into the brain. Like Chaikoff's group, they paid much attention to phospholipid metabolism; and they worked out other biochemical pathways, notably the role of phosphorus compounds in the enzymatic breakdown of carbohydrates (glycolisis). Lawrence encouraged Hevesy to ask for "as much [P³² ] as he can use. . . . We are more than eager to help his important work."^[144]

Hevesy received the Nobel prize in chemistry in 1944 for his contributions to the tracer method. The leader of the Rochester

[142] Stout and Hoagland, Am. jl. botany, 26 (1939), 320–4; Arnon, Stout, and Sipos, ibid., 27 (1940), 791–8; J.H. Lawrence, Nature, 145 (1940), 125–6, and "Summary of biological and medical investigations," Jan 1940 (22/11); Hamilton, Radiology, 39 (1942), 550–2.

[143] Tuve, "Status of the use of radioactive tracers," 10 May 1939 (MAT, 25/"Hamilton Club"), 2–3.

[144] Hevesy, Selected papers , 111–74, 440–1; Hevesy et al., Acta biol. exp., 12 (1938), 34–44; Lawrence to Bohr, 17 Feb, quote, and 14 Nov 1938, and Bohr to Lawrence, 25 Jul 1939 (3/3); correspondence with Hevesy, 1937–40 (9/7); Hevesy in Frisch et al., Trends , 115.

group, George Whipple, already had a Nobel prize, that of 1934 in physiology and medicine, for work on anemia. Radioiron sent from Berkeley allowed him to wallow more deeply in his favorite subject. His group, in which P.F. Hahn took the lead, showed that anemic dogs accepted iron in any form, collected it rapidly in the bone marrow, and disbursed it rapidly in blood cells. A normal dog would absorb almost no iron at all. It appeared that ingested iron entered the blood stream only if the body's iron store had been depleted; otherwise it passed directly through the intestines. Whipple expected to devise a satisfactory treatment of anemic patients using ordinary iron on the basis of the knowledge labelled iron gave him.^[145] The information did not come easily. Most of the time Whipple's group were as deprived of iron as their dogs. The cyclotron could not keep up the supply: the 37-inch made less than 1 mµCi of Fe⁵⁹ per µAh, the 60-inch only 0.03 µCi. A radioiron with suitably high specific activity, Fe⁵⁵ (t = 4 years), can be made by (d,n) on manganese, but it was not available before the war.^[146]

The Rochester iron men hoped that the radioisotopes furnished by physicists would turn out to be the "'Rosetta Stone' for the undertaking and study of body metabolism." That was to be very optimistic. The Stone then had nothing to say about the largest part of the bodies of plants and animals: no useful radioactive tracer for hydrogen, carbon, oxygen, or nitrogen had yet been found. No one felt this difficulty more than Kamen and Ruben, who had boldly set forth in 1938 to find the way through photosynthesis armed with C¹¹ , which has a half-life of about 21 minutes. An experiment consisted of making the isotope, burning it to carbon dioxide, feeding it to plants, chopping the leaves into a beaker, adding as carrier any substance they guessed might have been labelled with C¹¹ by the plant, and examining the various

[145] Hahn et al., Jl. exp. med., 69 (1939), 739–53, 70 (1939), 443–51, and 71 (1940), 731–6; Whipple's ambition, recorded by Tuve, "Status of the use of radioactive tracers," 10 May 1939 (MAT, 25/"Hamilton Club"), 7; Hamilton, Radiology, 39 (1942), 564–6, and Jl. appl. phys., 12 (1941), 455–6.

[146] Whipple to Lawrence, 23 Nov, and reply, 30 Nov 1937, and Lawrence to DuBridge, same date (15/26); letters to Lawrence from Evans, 6 Mar 1939 (7/8), and from Van Voorhis, 5 Feb 1939 (17/14).

carriers. All in an hour or two. The pace forced out their first collaborator, Zev Hassid, who suffered from high blood pressure. They made three of these hectic runs a week for three years.^[147]

They made some progress. A plant fed in the dark attached labelled CO₂ to a big molecule RH, R is an unknown radical, making a compound RCOOH. The reaction appeared to be reversible. In the light, however, and the presence of chlorophyll, RCOOH gained water and lost oxygen, irreversibly, to become RCH₂ OH. This structure may be considered a molecule R'H, which can fix another CO₂ molecule in the same way to become RCH₂ OCH₂ OH, and so on. R might then break off, leaving a sugar. The scheme was novel and, in keeping with the requirements of philosophers, had an easily testable consequence that distinguished it from older theories. The consequence: that RH be a very big molecule. For evidence, Kamen and Ruben turned to the ultracentrifuge. The best local setup was at Stanford. Ruben stationed himself there, awaiting with counters ready the delivery of the labelled samples Kamen rushed down from Berkeley. Their lives eased with the discovery that Shell Development Company in Emeryville, adjacent to Berkeley, had a similar centrifuge. The machine showed RH to have a molecular weight between 500 and 1,000.^[148] To go farther, to identify the heavy molecule and the intermediates in photosynthesis, Kamen and Ruben needed a longer-lived isotope of carbon. A bout with N¹³ , which has a half-life of 10.5 minutes and which did not quite enable them to decide whether nonleguminous plants can fix nitrogen, further indicated the limits on biochemical research placed by lack of tracers for organic reactions.^[149]

Another path lay open. For many years biochemists had been following reactions by tagging compounds with naturally occurring isotopes. They would introduce a substance artificially enriched with deuterium or with C¹³ , which makes up a little over 1 percent of ordinary carbon. They had then only to take the final

[147] Hahn et al., Jl. exp. med., 69 (1939), 739; Kamen, Radiant science , 84–7.

[148] Ruben, Hassid, and Kamen, JACS, 61 (1939), 661–3, 62 (1940), 3443–55; Kamen and Ruben, Jl. appl. phys., 12 (1941), 326; Kamen, Radiant science , 105–9; Hamilton, Radiology, 39 (1942), 567–9, and Jl. appl. phys., 12 (1941), 457–8.

[149] Ruben, Hassid, and Kamen, Science, 91 (14 June 1940), 578–9.

products of interest and assay them for the relative abundance of the rare isotope in a mass spectrograph. Urey's colleague at Columbia, Rudolf Schoenheimer, the great master of the technique, routinely detected changes of 1 percent in isotopic abundance, which, in the case of carbon, meant one part in ten thousand. With some encouragement from the Research Corporation, Urey had perfected a method to enrich the concentration of C¹³ , which he turned over to Columbia University to patent at the end of 1939.^[150] At about the same time, the Eastman Corporation sought advice about the likely market for the stable isotope N¹⁵ as a tracer for nitrogen. Urey formed and did not hide the opinion that any foundation truly wishing to advance biochemistry and biology should assist in making rare natural isotopes available and not throw its money into cyclotrons.

Lawrence was then at the beginning of his negotiations with the Rockefeller Foundation for what became a request for a million dollars. Around October 1, 1939, Lawrence summoned Kamen and ordered him to find a radioactive carbon, nitrogen, and/or oxygen to silence Urey. "He said I could have both the 37-inch and the 60-inch cyclotrons and all the time I needed, as well as help from whomever I requested—Segrè, Seaborg, anyone!" Naturally Kamen chose to center his all-out search on carbon, and all the more after he had demonstrated with Segrè's help that no useful oxygen or nitrogen could be made by irradiating oxygen with alpha particles or deuterons. He pinned his hopes on an internal target of graphite, which he baked with deuterons for 5,700 µAh during the first six weeks of 1940. On February 13, Kamen terminated his exposure and left the hot target for Ruben to analyze.^[151]

Kamen recalled that he had turned to deuterons on graphite in "desperation and resignation." The only likely candidate for a useful radiocarbon was C¹⁴ made by (d,p) on the rare isotope C¹³ .

[150] Poillon to Lawrence, 21 Sep, and reply, 28 Sep 1937 (15/17A); Urey to Douglas C. Gibbs, secretary of University Patents, Inc., 22 Nov 1939, 1 Mar 1940 (Urey P, 8). The patent was filed 3 Aug 1940. The agreement, as usual with University Patents, gave 7 percent of gross receipts to the inventor; letters to Urey from Gibbs, 8 Aug, and from Howard S. Neiman, 13 Aug 1940 (Urey P, 8).

[151] Kamen, Radiant science , 127–30, and Science, 140 (1963), 588–9.

But for reasons similar to those that inhibited the discovery of the activity of H³ , C¹⁴ did not appear to possess the characteristics desired by radiobiologists. For C¹⁴ had already been discovered at the Laboratory. In 1934, in one of his first observations with his cloud chamber, Kurie had seen half a dozen tracks that indicated a new sort of radiation stimulated by neutrons. In addition to the then known (n,a ) process, he saw what he interpreted as (n,p) reactions on air, either N¹⁴ (n,p)C¹⁴ or O¹⁶ (n,p)N¹⁶ . His attribution, challenged by the Cavendish, was established by Bonner and Brubaker in 1936.^[152] Kurie and Kamen then studied the profusion of (n,p) events made possible by the cyclotron's enlarged neutron flux; they found the recoil tracks of the C¹⁴ ions and the liberated protons to be plentiful and conspicuous enough to serve as a measure of the stopping power of the air in the cloud chamber.^[153]

From general considerations set forth by Bethe and Bacher, not more than one of a set of isobars can be stable. But N¹⁴ is stable. The beta decay of C¹⁴ to N¹⁴ would depend upon their difference in mass, which Bethe and Bacher estimated at 100 or 200 MeV. "Assuming the b -transitions to be allowed, the lifetimes would be between 1/2 and 20 years." In the same bit of beryllium irradiated by deuterons in which he identified Be¹⁰ (in fact H³ ), McMillan had also noted a weak activity of about three months, which he ascribed to C¹⁴ made via (d,p) on a carbon impurity in the target. He then tried to make C¹⁴ in quantity by (n,p) by exposing ammonium nitrate to neutrons from the 37-inch cyclotron; the experiment ended with the accidental breakage of the salt's container and was not renewed.^[154] Livingston and Bethe accepted McMillan's estimate of a half-life of several months and Oppenheimer's student Phillip Morrison refined and confirmed their calculations. But careful search, notably by Ernest Pollard of Yale, who tried (d,p) on C¹³ and (a ,p) on B¹¹ , found the product

[152] Kurie, PR, 45 (1934), 904, letter of 15 June, and PR, 46 (1934), 330; Chadwick and Goldhaber, Camb. Phil. Soc., Proc., 31 (1935), 612; Bonner and Brubaker, PR, 49 (1936), 778; Livingston and Bethe, RMP, 9 (1937), 344.

[153] Kamen, Radiant science , 123–4, and Science, 140 (1963), 585.

[154] Bethe and Bacher, RMP, 8 (1936), 101–3, 201; McMillan, PR, 49 (1936), 875–6; Kamen, Science, 140 (1963), 586.

protons, and calculated that C¹⁴ should yield soft beta rays, disclosed no activity ascribable to any carbon isotope.^[155] Hence Kamen's doubt that anything interesting would result from bombarding charcoal with deuterons.

The first tests confirmed his pessimism. Carbon removed from a target of calcium carbonate did not excite a thin-walled Geiger counter. When placed inside a screen-walled counter devised by two of Ruben's colleagues, however, it gave some weak signs, about half the usual background count. That equivocal message marked the discovery of the most important radioactive tracer found at the Laboratory. On leap-year day 1940 the good news was sent for publication.^[156] The reason that the decay of C¹⁴ had been so difficult to detect is that its period is very long. Ruben and Kamen first estimated "years;" a larger irradiation (some 13,000 µAh) of a better probe target enriched in C¹³ allowed a much higher and better estimate, thousands of years. That was a puzzle for the theorists, who, however, in the persons of Oppenheimer and L.I. Schiff, helpfully remarked that the nuclei of C¹⁴ and N¹⁴ must differ enough in angular momentum to retard the decay by the amount observed.^[157] In any case, the long-sought long-lived carbon was in hand, and it remained only to discover how to make it in sufficient amounts to satisfy the expected large demand. Kamen and Ruben returned to Kurie's process, N¹⁴ (n,p)C¹⁴ , irradiating large carboys of concentrated ammonium nitrate with neutrons from the 60-inch cyclotron. Although the yield was greater and the recovery easier than with deuteron-irradiated graphite, Lawrence ordered the process discontinued. He had heard that ammonium nitrate presented a serious hazard of explosion and no weight of chemical opinion could persuade him of its safety in solution.^[158]

[155] Livingston and Bethe, RMP, 9 (1937), 344; Pollard, PR, 56 (1939), 1168, letter of 14 Nov; Kamen, Radiant science , 126. Pollard had irradiated his carbon for twelve hours with 0.5 µA, which, he supposed, would not give a detectable activity if the period of C were several years or more.

[156] Ruben and Kamen (1940), 549, letter of 29 Feb; Libby and Lee, PR, 55 (1939), 245.

[157] Kamen and Ruben, PR, 58 (1940), 194, late June 1940; Ruben and Kamen, PR, 59 (1941), 350–1, 354.

[158] Ibid., 351–2; Kamen to Urey, May 1940 (10/10); Kamen, Radiant science , 139–40, and Science, 140 (1963), 589–90.

Lawrence lost no time informing Urey of the miraculous appearance of C¹⁴ and in asking him for a sample of purified C¹³ to serve as a target for Kamen. That, he intimated, would be the true value of Urey's separation process; C¹⁴ would not depress the market for C¹³ ; "quite the contrary." The rules of science are strict. "Needless to say [Urey replied] we will surely send him the material he wants." But then the obvious question arose: why go to the trouble and expense of transmuting C¹³ into C¹⁴ for tracing if C¹³ will serve au naturel? Calculations by Urey, Kamen, Ruben, and Tuve concurred. As Kamen put it, C¹⁴ was "an ace in the hole," something for very special applications, such as the study of photosynthesis, where the big molecules involved might dilute the natural isotope past detection. To make a quantity of C¹⁴ useful for the purpose would require a long time on the machine. "It is quite beyond all probability to make more than one or two such strong samples in your or my lifetime as conditions are at present."^[159] Urey gave Kodak his apparatus and encouraged them to proceed. They agreed to make N¹⁵ and, if that succeeded, to try C¹³ . Kodak did not like Urey's method of separation, which used deadly hydrogen cyanide gas, and they worried that the more prolific route via N¹⁴ would make C¹³ unnecessary.^[160]

Although Lawrence, Kamen, and Ruben joined Urey and others in urging the commercial production of C¹³ ,^[161] Kodak did not move to serve the mass market of biotracers before the war. The frustration of having too little heavy carbon to unlock the secrets of life was eased by war. In 1945 Urey again tried to enlist Kodak. Again the question: if C¹⁴ can be made plentifully without separated C¹³ , why invest in a heavy-carbon plant? But if not,

[159] Lawrence to Urey, 24 Feb 1940; Urey to Lawrence, 29 Feb and 1 Mar 1940; Kamen to Urey, 13 May 1940, quote, all in 17/40; Tuve to Urey, 9 May and 3 June 1940 (MAT, 25/"biophys. 1940").

[160] Urey to C.E.K. Mees (Kodak), 27 Apr, 2 May, 12 Aug 1940; Mees to Urey, 1 and 29 May 1940, all in Urey P, 3/M; Urey to Hevesy, 25 Oct 1940 (Urey P, 2), and to Lawrence, 27 Oct 1940 (17/40).

[161] Lawrence to Urey, 3 Oct 1940 (17/40); Urey to Mees, 6 Nov 1940 (Urey P, 3/M).

Kodak should come to the rescue, lest the country "lose years in applying the tracer technique to chemical and biological problems."^[162] Kodak wisely remained out of the picture. The tremendous neutron fluxes in the piles built during the war created C¹⁴ in plenty. Thus the sciences of life gained "the most important tracer available among the artificial radioactive elements."^[163]

[162] Urey to Otto Beeck, Shell Development Co., 21 Mar, and to Paul H. Emmett, Mellon Institute of Industrial Research, 12 Apr and 9 Jul, and to A. Keith Brewer, National Bureau of Standards, 16 Jul 1945; letters to Urey from W.O. Kenyan, Kodak, 12 Sep, Mees, 11 Jul, and A.O.C. Nier, 20 Aug 1945, quote, all in Urey P, 1/"misc. corresp."

[163] Kamen in Wisconsin, Univ., Symposium , 150.