Eavesdropping on the Elements We Know What Chemists Think of the Elements, but What Do the Elements Think of Each Other?

December 1994

MysteryMatters 4 Smuggling Bear Galls When chemists team up with wildlife agents, it’s good news for bears and bad news for poachers. Poison That Heals 7 The most toxic substance in the world has been turned into a cure. Life on Mars? 10 Twenty years after landing a robot on Mars, one scientist still believes the answer is yes. Fine Art Fraud 14 Was the masterpiece painted by a seventeenth-century artist or a twentieth-century criminal? The Puzzle Page 16 Eavesdropping on the Elements We know what chemists think of the elements, but what do the elements think of each other?

MYSTERY MATTERS In 1991 Sergeant Joseph Campbell of the Division of Fish and Wildlife SMUGGLING Protection in Anchorage, Bear gallbladders are used in traditional Asian medicine to treat gall- Alaska, received a tip from “one of my sources” stones, liver disease, and a variety of other ailments. Unlike rhinoc- that two Asian men would be arriving shortly at eros horn, and many of the other animal-derived substances used in the airport carrying a shipment of undeclared Asian medicine, bear gallbladders do contain a compound that has therapeutic value. This compound is ursodeoxycholic acid, one of the gallbladders from Canadian bears. Traffic in bear bile acids produced by the bear liver, concentrated and stored in the gallbladders is internationally regulated and is gall bladder and ultimately released into the intestine to help digest fats. A synthetic form of this bile acid, ursodiol (“ursa” means “bear” largely illegal. Campbell’s source turned out to in Latin), has been shown to be effective in treating both gallstones be right. “I met the men at the and a liver disease called primary biliary cirrhosis. Although a synthetic substitute for bear gallbladders is available, airport,” says Campbell. many Asians prefer the real thing. In China and Korea, people “They had 174 bear collect bile from bears kept in small cages. These bear gallbladders in farms operate like dairies—the bears have catheters implanted in their gallbladders so their bile can be a handbag.” “milked.” As cruel as this may sound, farming bears is the most humane way of getting their bile. The other option is to kill them. The black market demand for bear gallbladders has so depleted Asian bear populations that they are in danger of extinction. Because of the decline in Asian bears, people are killing more and more North American bears for their gallbladders. In Canada, black bear carcasses have been found with just their gallbladders cut out. Although many Asians prefer real bear bile, only the wealthy can afford it. Devotees of traditional Asian medicine will pay $250 per gram for dry bear gallbladders or “galls,” the most commonly sold form. Galls weigh between 30 and 50 grams, which means a single gall has a street value of $7,500 to $12,500, making the trade lucrative enough to tempt people to flout the laws. Importing bear gallbladders into the United States requires a permit, and most states prohibit selling bear gallbladders.

American black bear. Many are killed solely to harvest the gallbladder.

PHOTO COURTESY JOSEPH CAMPBELL, ALASKA FISH AND WILDLIFE PROTECTION

4 CHEM MATTERS, DECEMBER 1994 BEAR GALLS by Robin Meadows

The two men apprehended by Campbell in the Anchorage Airport This 20-centimeter (8-inch) bear gall was confiscated by the had bought their galls in Canada for an estimated $35,000 (which Alaskan Fish and Wildlife works out to the bargain price of $200 per gall), and had brought Protection Agency. In a recent them to first Chicago and then to Anchorage, which is a convenient article in the Journal of point of departure for Asia. The men had intended to go on to Korea, Forensic Sciences, Edgard Espinoza wrote, “A where the galls could have fetched the staggering sum of more than desiccated bear $1 million. If convicted, the men would face a penalty of up to gallbladder will sell for $250,000 in fines and five years in prison for not declaring the bear $15 in Idaho, $1500 in gallbladders when they entered the United States. Hawaii, and $55,000 in Korea. These To make a case against the men, Campbell had to prove two prices have things: first, that the gallbladders had been for sale and, second, that created an illegal they had indeed come from bears. The first part was easy. “I asked international them if the galls were for sale and they said yes,” recalls Campbell. black market for bear gallbladders The second part, proving the origin of the galls, was harder. When by individuals dry, bear gallbladders are the size of a large fig and are “dark, dark involved in criminal brown with some gold. They have a very distinctive smell and taste. wildlife commerciali- Once you’ve smelled them—or tasted them, if you’re brave—you zation. The trade in bear parts has placed the don’t forget it.” But no matter how certain he is, Campbell needs Asiatic bears in danger of more than his opinion to make a case. For proof that is admissible in extinction and the demand has PHOTO COURTESY JOSEPH CAMPBELL, court, he relies on Edgard Espinoza, chief of the Criminalistics Section now turned to North America.” ALASKA FISH AND WILDLIFE PROTECTION of the U.S. National Fish and Wildlife Forensic Laboratory in Ashland, Oregon. To determine whether the 174 confiscated gallbladders came from A bile sample is added to a stream of liquid solvent (in this case, a bears, Espinoza analyzed the gall’s bile using a method that he devel- mixture of water and methanol) that flows through a narrow metal oped with Lee Hagey, a graduate student who worked with gastroen- tube called the column. The solvent must be pumped under high terologist Alan Hofmann at the University of California, San Diego. pressure because the column is densely packed with C-18-coated sil- The method uses high-pressure liquid chromatography (HPLC) to ica (octadecyl silane). The bile compounds stick to the surface of the separate and identify the various compounds found in bile. silica beads because they are soluble in octadecyl silane, then redis- solve into the liquid stream. Because the various bile compounds differ in solubility in liquid solvent versus octadecyl silane, they spend CH3 different amounts of time attached to the beads and, therefore, HO emerge from the column at different times. “A good analogy is mixing CH3 SO3H honey and lemon juice, putting the mixture through a hose, and then O N turning on the water. Naturally the lemon juice comes out first CH3 because the honey sticks to the inside of the hose,” explains Espinoza. Espinoza and Hagey had previously used their method to establish that bears have a bile profile (the kinds and amounts of bile com- HO OH pounds) that is unique in the animal kingdom. Bears have three main bile acids: tauro-ursodeoxycholic acid (UDCA), tauro-chenodeoxycholic CHOLYL-TAURINE acid, and tauro-cholic acid. Analyses of 600 non-bear species showed that none of them have the bile acid profile characteristic of bears. Figure 1. Cholyl-taurine is the chief ingredient of the bile of These species include giant pandas and red pandas (thought by some carnivorous animals. Bear bile contains this compound and biologists to be closely related to bears) and pigs (gallbladders from two others of very similar chemical structure which, together, pigs look just like those from bears and are often substituted for them facilitate absorption of fat in the small intestine. on the black market).

CHEM MATTERS, DECEMBER 1994 5 Although there are other species (including people) S 2 that have UDCA, most of them have only small 150 1 amounts of this bile acid. The 100

only non-bear species known mAU 3 to have significant amounts of 50 UDCA is the nutria, a beaver- 0 like South American rodent. 2 4 6 8 10 12 However, nutrias and bears Time (min) have different forms of UDCA that can be easily distin- Figure 2. High-performance liquid chromatography, HPLC, was used to separate and identify the guished by HPLC (the UDCA major chemicals in bile. Bear bile was mixed with solvent and a standard marker compound, then in nutrias is bound to the injected into the HPLC column. Five minutes later the standard emerged (peak S on the graph), followed by ursodeoxycholyl-taurine after six minutes, cholyl-taurine at eight minutes, and cheno- amino acid glycine whereas deoxycholyl-taurine at 12 minutes. When chemists analyzed bile from a domestic pig, they found the UDCA in bears is bound to different compounds, which formed peaks at different times, making it easy to distinguish bear the amino acid taurine). galls from pig galls. Interestingly, North American bears have far more UDCA than Asian bears. Although seized as criminal evidence in Hong Kong, Taiwan, and Malaysia, as UDCA accounts for only up to 8% of the total bile acids in Asian well as in the United States and Canada. Although only about 20% of bears, the compound accounts for an average of 39% of the bile the gallbladders seized in Asia and the United States actually came acids in American black bears. Brown bears and polar bears, which from bears (the rest were from pigs), 85% of those seized in Canada are both closely related to American black bears, also have relatively were from bears. high amounts of UDCA (19% and 17%, respectively). The North When Espinoza analyzed the 174 gallbladders that Campbell had American species evolved more recently than the Asian species, seized at the Anchorage Airport, he found that 169 of them were which may have some effect on the difference in UDCA levels indeed from bears; tests of the other five gallbladders were inconclu- between the two groups. sive. “If it hadn’t been for the forensics lab we wouldn’t have had a Espinoza has analyzed hundreds of purported bear gallbladders case,” says Campbell. “We depend on it more and more.” The two men caught with the handbag full of bear gallbladders were prosecuted by federal authorities in Chicago, their port of entry into the United States, and fined for importing undeclared goods. “They also lost their $35,000 [what they had spent on the bear gallbladders],” says Campbell. “The prosecutors figured that altogether that was enough punishment.”

Robin Meadows is a freelance science writer Photograph not Available living in Fairfield, CA. A previous article written by Ms. Meadows for Chem Matters magazine (“Saint’s Blood,” February 1993) won the top award from the Society for Technical Communication.

FOR FURTHER INFORMATION: Photograph not Available Beardsley, Tim. “Galling the bears.” Scientific American 1992, 267 (1), 24. Mills, J. “Milking the Bear Because Asian bears are hunted for their bile, which is used to Trade.” International prepare a medicine, their population in the wild has decreased Wildlife, May/June 1992, dramatically. A fully effective synthetic drug is available, but many p.38. Asians prefer the “natural” medicine. This Asian bear, photo- Poten, C.J. “A shameful har- graphed in a Chinese “bear farm” in 1993, was cruelly restrained in vest.” National Geographic a cage so small that it could not move, and a catheter was surgi- 1991, 180 (3),106. cally implanted in its bile duct. Periodically, a tube is attached to Pain, Stephanie. “Witness for the deceased.” New the catheter valve (right) to milk the valuable fluid. Bear farming is Scientist, August 27, 1994, a lucrative business, but when bile production slacks off, the once- p. 21. valuable animal becomes a liability and is killed.

6 CHEM MATTERS, DECEMBER 1994 PoisonThatPoisonThat HealsHeals by Harvey Black n a June night in 1971, George Cochran Jr. sat down to eat botulism con- a bowl of canned vichyssoise. The cold potato soup, he may tamination in have thought, was just the right thing for a warm summer recent history. evening.O But it tasted spoiled, so after a few spoonfuls, he and his The strict wife threw it out. government The next day, Cochran, a banker who lived in the New York City requirements to suburb of Westchester County, was dead, his wife hospitalized. The prevent contami- cause: botulism poisoning that resulted from eating the soup. nation were not Less than 20 years later, Howard Thiel, a retired insurance carried out at Bon agency owner from Elm Grove, Wisconsin, Vivant, Inc., a says he owes his life to the Newark, New very same poison that Jersey, soup manu- killed Cochran. Thiel facturer. Cochran’s uses it to treat a neuro- death triggered a muscular disease. Called massive nationwide the deadliest toxin in the effort to get all the world, the botulism toxin soups made by the is secreted by Clostridium firm off store shelves. botulinum bacteria. These Tests by government bacteria, which are found scientists revealed that throughout the environment, a number of cans con- even in our food, are indeed tained botulinum toxin. a real hazard, and the govern- That, plus Cochran’s ment has strict regulations to death and the resulting PHOTO: NEAL CLODFELTER make sure the poison they publicity, were too much for secrete doesn’t contaminate the the firm, which had been in business for more than 100 processed food we eat. years. It went out of business later that summer. An Botulism poisoning is very investigation by the Food and Drug Administration rare. Before Cochran’s death (his revealed that the company had not been adequately wife survived), Westchester heat treating its canned soups, thus allowing bacteria County’s last case of botulism poi- to thrive. soning occurred in 1934. (Botulism Unlike most living things, C. botulinum shun continues to be a rare disease. See oxygen. It is poison to these bacteria, which are “Protecting your food” page 9.) known as anaerobes, meaning they live without Scientists have harnessed that same oxygen. Eric Johnson, a University of Wisconsin– poison, and now physicians are using Madison bacteriologist and food scientist, it to treat people suffering from a wide explains that they survive by fermentation. They range of neuromuscular diseases. In secrete enzymes that break down organic matter fact, the effort to learn how to do that to provide energy. had begun before Cochran was poisoned But it’s the chemical they secrete that in one of the most publicized cases of makes these bacteria both saviors and devils.

CHEM MATTERS, DECEMBER 1994 7 This compound paralyzes muscles. It blocks a chemical called acetylcholine from carrying messages from nerves to muscles. Acetylcholine is a neurotransmitter, a chemical that carries the impulse from the nerve, across a tiny gap called a synapse, to the muscle, making it contract or extend. Block that transmission and the muscle is useless. Victims of botulism poisoning die because the poison paralyzes the muscles responsible for breathing. The victims suffocate. But the paralysis of muscles can sometimes be beneficial if, for instance, they are too active and cause spasms. And trying to control such misbe- having muscles is what led to this deadly poison being turned into a medical treatment. Alan Scott, a San Francisco surgeon, had been trying to develop a The most nonsurgical technique to cure strabismus (crossed eyes). In this con- poisonous dition certain muscles in the eye are more active than others, and they pull one eye About 0.060 g of toxin can be refined from this substance in out of alignment with the other. 11-liter (3-gallon) batch Consequently victims of strabismus can’t of Clostridium botulinum the world is see normally. What if there were some way, bacteria. This may not being produced without going into the operating room, to sound like much but it is enough to kill 60,000 weaken the overactive muscle? That would people. A lethal dose for in this bottle. avoid delicate surgery. humans is about one ten- In the late 1960s Scott got in touch with millionth of a gram. Edward Schantz, a biochemist who had studied the toxin during World War II to be able to develop antidotes to it should it be Toxin development used as a biological warfare agent. “I sug- gested he try botulinum toxin, because we All the botulism toxin currently available for treat- knew it weakened muscles,” Schantz said. ing disease today came out of the laboratory of Scott tried it on monkeys in which he had Edward Schantz at the University of Wisconsin– experimentally created strabismus. “It was Madison’s Food Research Institute. Two hundred just the ticket,” said Schantz, now an emer- milligrams of it was brewed up in 1979. About itus professor at the University of half went to Dr. Scott for his research and half Wisconsin in Madison. remained in Dr. Schantz’s lab. Schantz was one of the first scientists Schantz says the bacteria were grown in a cul- to purify the toxin. After a decade of suc- ture of hydrolyzed casein, yeast extract, and glucose. The poison was purified and crys- PHOTOS COURTESY ERIC JOHNSON cessful research on monkeys, with Scott tallized in several steps, using acid and grain alcohol to precipitate crystals of the toxin. using botulinum toxin supplied by Schantz, The toxin is a combination of toxic and nontoxic proteins that are held together electro- the Food and Drug Administration let the statically. The toxin proteins impart stability to the toxic portion. San Francisco surgeon try the toxin on In 1989, the Food and Drug Administration licensed the toxin for treatment. human volunteers. Since the 1980s the It is now available to doctors from Allergan, Inc., in Irvine, California. toxin has been injected into more than The white crystals of the toxin come in a small bottle and are put 20,000 people to help treat a wide range of in a saline solution for injection into patients. relatively rare muscular diseases. Dr. Schantz (left photo) and fellow biochemist Thiel is one person who has been Eric Johnson (right photo) continue to work on helped enormously by the toxin. He suffers the toxin at the University of Wisconsin–Madison. from a disorder called spasmodic torticollis, a muscular spasm that twists his neck muscles, forcing his head down onto his chest. The painful disorder Wisconsin, who suffers from Clostridium botulinum bacteria defied diagnosis for nearly two decades, and left Thiel at the brink of the same disease as Thiel, credits the x50,000 suicide, because he could find no way to treat it and relieve the unre- toxin with allowing her to continue lenting pain. The only way he could correct the spasm was to clasp her livelihood. Without it, she says, “I his hands on his head and straighten it out. Finally in 1986 a Mayo might have existed, but I wouldn’t be living.” Clinic doctor diagnosed the problem, and Thiel was able to get treated Mitchell Brin, a neurologist at Columbia–Presbyterian Medical with the botulinum toxin, then still in the experimental stages. Center in New York City, and his colleagues have been treating people Treatments with the toxin, he says, have made his life worthwhile. with the toxin since 1984. One disease they have been able to combat Similarly, Susan Laux, an interior designer from Mequon, is called blepharospasm. This disease forces the eyelids shut.

8 CHEM MATTERS, DECEMBER 1994 “Many of these people were functionally blind because they couldn’t keep their eyelids open,” he says. The effects of the toxin are “quite Protecting your food dramatic. After treatment with botulinum toxin most of them were able to go back and drive and work and do the sorts of things that you The Food and Drug Administration requires that canned and I do,” he says. Eighty percent of the patients he and his col- foods, which are low in acid (that is, with a pH greater than leagues treat are satisfied with the results, says Brin. 4.6) be heated to 121 ˚C (250 ˚F) for at least three minutes to Blepharospasm and torticollis are examples of a group of diseases kill botulinum spores. Without that intense heat, the spores called dystonias—disorders marked by muscular spasms. Nearly 400 would germinate inside the airless can. The bacteria then out of a million people suffer from dysto- would release their deadly toxin, as nias, according to one study. Botulinum happened in the Bon Vivant vichys- toxin is the favored treatment for them. soise eaten by George Cochran. Other Early in 1994, doctors reported using the precautions include adding salt to limit same toxin to treat a disorder that interferes the water that allows the bacteria to with swallowing. In this disorder, known as grow, adding vinegar to lower the pH, achalasia, a muscle in the esophagus can’t and adding nitrites to foods such as relax to let food go into the stomach. And hot dogs to prevent C. botulinum from recently, physicians have started to use the growing. Refrigeration also prevents toxin to remove facial wrinkles. Children the bacteria from growing. have also been treated successfully for In spite of such precautions, botu- muscle deformities in cerebral palsy. lism poisoning cases do occur. Although scientists have known the gen- According to the U.S. Centers for eral action of botulinum toxin for some Disease Control and Prevention there time, they have been working to understand are about 120 cases a year of botu- the mechanism in more detail. Johnson, lism, about 20 caused by poisoned who has continued to work with Schantz to food and about 100 from botulism in study the toxin, says recent research shows PHOTO: NEAL CLODFELTER infants up to a year old. Infant botu- that it cleaves, or breaks up, the proteins that actually transport lism is caused by the botulinum spores infecting the infant’s acetylcholine between the nerve endings and the muscles. In effect undeveloped digestive tract, germinating, and producing the toxin torpedoes the tugboat towing the ocean liner. toxin. The toxin is absorbed into the lymph and migrates to Although the toxin can produce dramatic results, it’s not a cure. nerve endings. The symptoms are identical to food-borne Brin says that patients must return to their doctor for injections of the botulism. toxin every few months. And there are side effects. The toxin can par- A person treated for botulism poisoning is typically put on alyze some muscles that are working perfectly. The poison injected to a respirator, because the toxin paralyzes the breathing mus- treat muscles in the neck can diffuse and paralyze some muscles cles, and is checked very carefully by physicians. An anti- important for swallowing. Such effects are transient, however, lasting toxin, a substance to prevent paralysis, can be injected but it only several days. A more serious concern is that patients usually isn’t. That’s because botulism poisoning is so rare become immune to the toxin’s effect, meaning it simply that most physicians don’t recognize the symptoms quickly doesn’t work anymore. That’s happened to about a enough for the antitoxin to be effective. dozen patients. “We try to keep the dose as low According to Dr. Denise Koo, botulism poisoning victims as possible, to try to minimize the possibility can recover after four to six weeks of hospitalization. of antibodies developing,” says Brin. Schantz “Eventually they will regenerate new nerve endings and they says the doses are extremely small, in the will be able to recover,” she says. In 1991 and 1992 two peo- range of a tiny fraction of a billionth of a gram. ple died from botulism poisoning. And even though the toxin has been available for about a decade, researchers are still concerned about what long-term effects, if any, it might have. Bernard, wrote, “Poisons can be employed as a means for the Nevertheless, says Brin, the toxin has advantages over alter- destruction of life or as agents for the treatment of the sick.” And the natives. Drugs, he says, often are not very effective, working for medieval physician Paracelsus three centuries before him offered this only 25 to 35 percent of patients. And drugs bring with them their bit of scientific wisdom: “The dose makes the poison.” own array of side effects, including drowsiness and forgetfulness. Surgery on the muscles can also be a problem, he says. “It’s hard for Harvey Black is a freelance science writer living in Madison, WI. His the surgeon to judge how much the muscle has to be weakened when other publications in Chem Matters magazine include “The Exploding the patient is asleep.” Cabin” (October 1994) and “Carnivorous Plants” (December 1993). The tale of the toxin used as a treatment is not as strange as it FOR FURTHER INFORMATION: sounds. In fact, the notion of using potentially harmful organisms and Waters, Tom. “The fine art of making poison.” Discover 1992, 13 (8), 28. substances as treatments is not new. Vaccines are one such example. Jankovic, Joseph, and Brin, Mitchell F. “Therapeutic uses of botulism Furthermore, the renowned nineteenth-century physiologist, Claude toxin.” The New England Journal of Medicine 1991, 324 (17), 1186.

CHEM MATTERS, DECEMBER 1994 9 by Dan Scott n 1976, landers from the Viking 1 and Viking 2 space- I craft touched down on the surface of the Red Planet. Their mission: to run experiments to answer an age-old question — Is there life on Mars? Their answer? It depends on whom you ask. Many scientists are confident the on Viking landers found no evidence Life for life, but Dr. Gilbert Levin, chief designer of one of the three Viking life-detection experiments, is one of a small group of scientists who strongly disagrees. Today, Levin still believes that his labeled release experiment, performed on several samples of Martian soil, has most probably arsAFTER ALMOST TWENTY YEARS, ONE detected microorganisms on Mars. “No objective scientist VIKING SCIENTIST STILL ?MAINTAINS looking at the test results can deny thatM the LR [labeled release THE ANSWER MAY WELL BE “YES”! experiment] produced strong evidence for life on Mars,” Levin has stated publicly. and pyrolytic release (PR) experiments]. All this hardware was Why, after all these years, does Levin’s minority continue to argue crammed into a volume of only one cubic foot — no larger than a for the prospect of life on Mars? This article, based in part on an student’s stack of books. Each Viking lander also carried a combined exclusive Chem Matters interview with Levin, explores the reasons gas chromatograph and mass spectrometer for detecting and identi- behind his controversial stand. fying the organic compounds expected to be found. Viking 1, launched on August 20, 1975, entered Mars orbit on Destination: A desolate environment June 19, 1976. On July 20, the Viking 1 landing craft separated from The three life-detection experiments, like the rest of the Viking cargo, its orbiter and touched down on the slope of a dry basin known as began their long journey to Mars from planet Earth. Within the span Chryse Planitia in the northern hemisphere of Mars at a latitude of 22 of several weeks in the summer of 1975, two Viking spacecraft were degrees. Viking 2, consisting of an identical orbiter/lander pair, set launched from Cape Canaveral, each carrying identical sets of equip- off for Mars on September 9, 1975. Its lander arrived at a more ment for Levin’s LR experiment and two others [gas exchange (GEx) northerly site (48 degrees latitude), a rocky plain in a region called Utopia Planitia, on September 3, 1976. (Viking 1 and Viking 2 are the first — and only — spacecraft to have successfully landed and performed experiments on the surface of Mars.) After landing, the sam- pling arm on each Viking lander scooped up a small amount of

The landing site for Viking I (indicated in red) was an ellipse 300 by 80 kilometers (180 x 48 miles) in the northern hemisphere. Near the mouth of a long rift canyon, the terrain promised interesting geology and a relatively smooth landing surface. PHOTOS COURTESY NASA.

10 CHEM MATTERS, DECEMBER 1994 Martian soil and delivered the sample to the lander’s sample proces- used radioisotopes to look for signs of metabolism — chemical reac- sor, where it was mixed, sieved, and delivered to the test chambers of tions that take place in living cells to produce work and synthesize all four instruments. During the course of its experiments, the Viking food. The GEx experiment sought metabolism also; however, it did 1 lander scooped up two samples of soil; Viking 2 scooped up three. not use radioisotopes but looked for microbial respiration by measur- On the way down to the Mars surface, the Viking landers analyzed ing changes in gases in the test chamber. The PR experiment tested the Martian atmosphere. They determined that Martian air near the for photosynthesis. “The four national scientific committees that ground consisted of about 95 percent carbon dioxide, 2.5 percent selected these experiments agreed that any one of the experiments nitrogen, and 1.5 percent argon, with traces of oxygen, carbon showing a positive response and a negative control would be evi- monoxide, neon, krypton, xenon, and water vapor. They measured a dence for microbial life on Mars,” recalls Levin. surface atmospheric pressure about one percent of that of Earth’s at sea level, and temperatures averaging about 222 K (–50 ˚C or –60 ˚F), The labeled release experiment with daytime temperatures of the surface material probably briefly The word “labeled” in Levin’s labeled release experiment referred to above freezing. Though all the necessary elements for life were there, using radioactive carbon atoms to mark reaction products. Normally, the environment was very harsh. carbon atoms consist of 6 protons and 6 neutrons, having an atomic Unprotected, humans would certainly die on Mars, but microbes, weight of 12, and are therefore called “carbon-12.” Another form, or living organisms such as bacteria, algae, and protozoa, too “isotope,” of carbon is carbon-14, consisting of 6 protons and 8 small to be seen with the naked eye, may be able to neutrons. Carbon-14 is a radioactive, unstable form of carbon; survive in such an environment. Microbes were the it becomes carbon-12 through a process called radioactive first living things on Earth, and today they are decay, which can be measured in both the reactants and found where nothing else can live — in the dry products of a reaction. valleys of frigid Antarctica; along the floors of “A small piece of the Mars soil was placed in a container, the Earth’s deepest oceans, where volcanic and a tiny bit of the radioactive [carbon-14-labeled] nutrient activity creates temperatures exceeding the solution was squirted onto it,” recalls Levin. boiling point of water; in aquifers thousands of Nutrient compounds in the solution contained feet below the surface of the Earth; and even VIKING carbon-14 atoms in place of carbon-12 atoms. inside rocks and salt crystals. Levin’s labeled LANDER Levin theorized that if something in the Martian release experiment, like the other two experiments, soil could sought to detect the presence of microbial metabolize life. Meteorology the nutrients, sensors On Earth, microbial life the metabo- either metabolizes (eats) or lism reaction photosynthesizes would break (uses sunlight to down the nutrients grow). Both types and the carbon-14 respire (breathe). atoms would show up Logically, microbial in the evolved carbon- life on Mars based gas would also per- form these functions, too. Temperature Levin’s LR sensor experiment

Biology Gas chromatographÐ processor Funnel for X-ray mass spectrometer fluorescence experiment

Extendible arm

Soil collection scoop Labeled Release Experiment byproduct — carbon dioxide or carbon Nutrient with Radiation It is not easy to detect the life labeled C-14 counter processes of microscopic organ- monoxide. Because the byproduct gases isms from 100,000 kilometers away. would contain carbon-14 atoms — the The Labeled Release experiment same carbon-14 atoms that were in the was based on the hypothesis that original nutrients — the radioactivity read- Martian microbes might be able to eat and oxidize simple organic com- ing would indicate how much gas was pounds. A sample of Martial soil was being evolved. A low reading would indi- 1. Nutrient deposited in the central reaction cate little or no metabolism, a high reading labeled with chamber, 1, and was sprayed with a could indicate that metabolism — or per- C-14 sprayed solution containing nutrients labeled onto soil with radioactive carbon-14. If haps some process that was mimicking microbes were present, and their metabolism — was taking place. In short, metabolism were similar to those on this experiment could measure the gases Earth, they might oxidize the nutri- exhaled by any organisms that ate the ents, 2, and break them down into CO or CO2 gas, which would contain nutrients, assuming that the Martian some of the radioactive C-14. Later, organisms could digest the nutrients used 3, the gas was sent to a radiation in the test and would exhale carbon-con- 2. Nutrient counter that would signal the pres- taining gases. metabolized ence of any radioactive carbon. to CO To improve the chances of a positive 2 metabolic response from whatever was in Since Levin invented the labeled the Martian soil, Levin supplied radioactive release approach in the mid-1950s, he versions of simple compounds that possi- has tested it in all kinds of conditions. bly comprised the first “meal” on Earth. Tests have been performed on sand “All the compounds we selected had been dunes from Death Valley, California; on made in the Miller–Urey experiments in soil from the Gobi Desert; on mountain- the laboratory, which means that those 3. Any C-14 tops 12,000 feet above sea level; in in CO2 is things were probably formed on primitive counted Antarctica; on soil from the moon; even Earth with sunlight, maybe lightning, car- on samples in a “Mars Box” at the NASA bon monoxide, methane, water vapor, and Ames Research Center that simulated carbon dioxide,” says Levin. (In 1953 conditions on Mars. “Throughout our Stanley Miller and Harold Urey formed these compounds by simulat- extensive test program over the years, the labeled release approach ing conditions that they theorized existed on Earth right before life never produced any false positive or false negative results,” says began.) “My theory was, that if Mars had gone through an evolution Levin. In addition, the labeled release approach proved to be very like Earth’s, it probably would have formed the same compounds and sensitive, “detecting as few as ten colony-forming microbial units in a the same kind of biological evolution would have occurred.” sample.” Levin selected the following five radioactively labeled (indicated by* in formulas) carbohydrates and amino acids: 14C-glycine Labeled release results on Mars 14 (NH2•*CH2•*COOH), C-DL-alanine (*CH3•*CH[NH2]•*COOH), When nutrient was added to the sample Viking 1 scooped up from 14 14 C–sodium formate (H*COONa), C–DL–sodium lactate the soil of Chryse Planitia, the response was positive. The carbon-14 14 (*CH3•*CHOH•*COONa), and C–calcium glycolate detector read a radiation count of 10,000 counts per minute, much

([*CH2•*OH•*COO]2Ca). The “DL” designation in the amino acid ala- higher than the background level of 400. “For the first 10 hours, the nine and the carbohydrate sodium lactate means that both “right- gas evolution in the samples closely followed that obtained from handed” (D) and “left-handed” (L) orientations of these molecules Earth samples using an LR model that resembled the test instrument were provided. (D and L configurations of a molecule are mirror on Viking 1,” recalls Levin. “While the counts per minute level was images of each other. This property, called “chirality,” can be com- one-tenth that of Earth soils with moderately high microbial popula- pared with your right and left hands; they seem identical, yet you can- tions, the magnitude of the Mars test was similar to that obtained not fit your right hand into a left-handed glove.) “Earth life uses and from low-population Antarctic soils.” makes essentially only right-handed carbohydrates and left-handed What if this response was caused by some nonbiological reac- amino acids,” says Levin. “No reason is known for that; it could have tion? One way to see if some kind of strange nonbiological chemistry been happenstance. So in order to guard against sending, for exam- were taking place would be to see what effect microbe-killing heat ple, a right-handed carbohydrate and having nothing on Mars that would have on the results. Exposing the soil to high temperatures could use that configuration, we sent both right-handed and left- should kill the Martian microbes, and supplying nutrients to this handed forms of alanine and sodium lactate.” [For more information “sterilized” soil should result in no evolved gas. “To test this hypoth- on chiral molecules, see “Mirror Molecules,” Chem Matters, April esis, a separate portion of the soil that had been held in the hopper 1989.] was put in an identical test cell and heated to 160 ˚C (320 ˚F) for three

12 CHEM MATTERS, DECEMBER 1994 hours. Then we let it cool, let fresh Mars atmosphere in, sealed it up, hydrogen peroxide, either on the surface of Mars or through the and ran the experiment again,” recalls Levin. “This is the ‘control’ entire atmospheric column of Mars,” says Levin. “Neither have more run, an essential part of all three Viking life detection experiments. sensitive observations made recently from Earth.” The peroxide the- We got no response from the control.” ory was based on the presence of a substance that could not be The labeled release experiments were repeated several times at detected. Chryse Planitia and at the Viking 2 landing site of Utopia Planitia. All One of the most interesting Viking tests was on a sample that was the labeled release results were consistent with the presence of stored away from the Mars environment for a number of weeks. microorganisms. Although the GEx and PR release experiments were Before storage, the sample generated a high radioactivity count when inconclusive, it would seem that these labeled release experiments given the nutrient; after storage, it did not. “Just holding the sample would be considered convincing evidence for life on Mars. in a box for two to three months at a temperature of 7 ˚C to 10 ˚C, shielded from any sunlight and removed from the Mars soil, seemed Explaining the results to stop the metabolic reaction,” observes Levin. “No scientist has However, results from the gas chromatograph/mass spectrometer proposed a nonbiological mechanism that explains why storage away that each Viking lander carried were disquieting. They were unable to from the Martian environment stops gas production.” detect any organic carbon in the soil samples. How could life exist if Many other scientists have come up with explanations for the no organic matter could be detected? The answer, according to results of the labeled release experiments, but none have satisfied Levin, is simple: GCMS was not sensitive enough. “Even the experi- Levin: “None of them have duplicated the positive and control menters in charge of the GCMS measurements acknowl- responses of the soil samples tested by the Viking labeled edged that they would require at least a million release experiments on Mars.” microorganisms in their test cell to get a In spite of Levin’s arguments, many sci- response, meaning the sample would entists still believe the Viking experi- have to have a concentration of 10 mil- ments detected no life on Mars. lion to 100 million microorganisms Perhaps it’s the nature of scientific per gram,” says Levin. Even when method to not believe something The lack of sensitivity of viewed through the until it is proved beyond a GCMS was corroborated by shadow of a doubt. Perhaps tests on Earth. Both the most powerful lens— the “no-life” advocates won’t GCMS and the labeled the Hubble Space believe there’s life on Mars release experiment tested unless they can see the the same sample of soil from Telescope—the Red microorganisms for them- Antarctica. The labeled Planet guards its secrets. selves, squirming under a release experiment clearly microscope. detected living organisms in The question of life may So, is there life on the soil, but GCMS was not be answered Mars? Conventional wis- unable to detect any organic dom says “No.” Levin and matter at all. However, a stan- conclusively until a his colleagues say “Yes, prob- dard organic assay performed manned laboratory ably.” When will we know for by GCMS did detect organic matter sure? When the next craft armed in the Antarctic sample. “By compar- is positioned with life detection equipment lands ing the results on soil sample Antarctic on Mars. on the Red Planet? Maybe yes, #726, we were able to determine a differ- maybe no. ence of about a million-fold in sensitivity between the two instruments,” says Levin. Dan Scott is a science writer who lives in New Some scientists think that ultraviolet light from the sun arriving on Mexico and writes technical documents for Sandia National the surface of Mars created changes in the Martian soil that activated Laboratories. His contributions to Chem Matters include “Lost in the labeled release reactions. (Mars has no protective ozone layer to Space: Apollo 13’s Fight for Survival” (February 1994) and “Designer block out ultraviolet light.) However, one of the Viking test samples Catalysts” (April 1994). was from soil underneath a rock that had not been exposed to sun- FOR FURTHER INFORMATION: light for probably millions of years. Its behavior was the same as that Schwartz, Barth David. “Muddy evidence: it keeps alive the case for life of the other samples. on Mars.” Scientific American 1989, 260 (6), 28.. Other scientists have offered the theory that hydrogen peroxide “Salty life on Mars.” Discover 1991, 12 (6), 12. Sagan, Carl. “The Search for Extraterrestrial Life.” Scientific American present in the Martian soil broke down the nutrient solution, releasing 1994, 271 (4), 92. carbon dioxide and carbon monoxide. “Measurements taken by Keiffer, H.H., Jakosky, B.M., Snyder, C., and Mathews, M.S., editors. Mariner 9’s infrared interferometer spectrometer did not detect Mars. University of Arizona Press: Tuscon, 1992.

CHEM MATTERS, DECEMBER 1994 13 FineArt

ixty years ago Hans van Meegeren,s in the secrecy of his studio, examined a 300-year-old canvas raud that gleamed with new paint. The F by Mike McClure restoration revealed a stunning, lifelike figure of Christ surrounded by his disciples. Little did he real- believing that a contemporary painting was a 300-year-old master- ize then the riches and misery the painting would bring. piece? Artists, historians, and scientists were called in to reevaluate Van Meegeren was an artist living and working in Holland. the authenticity of the paintings. During van Meegeren’s trial much Although a talented painter, he had achieved little public recognition physical and historical evidence was presented to substantiate his and even less money for his art. To improve his situation he began claim of forgery. Van Meegeren even took brushes and canvas into sidelining as an art dealer. In 1937 he went to the office of a lawyer the courtroom to demonstrate his technique for copying Vermeer’s friend with a painting he claimed to be old and valuable. The painting magnificent style. However, no definitive chemical tests were con- was called “Christ and His Disciples at Emmaus.” The painting, he ducted. explained, had been collecting dust in an old castle for centuries. The Decades later, in 1968, Bernard Keisch, a nuclear chemist at lawyer agreed to help van Meegeren find a buyer for the painting. But Brookhaven National Laboratories, realized that radioactive dating first it had to be studied and authenticated to judge its worth. After a could be used to establish the age of van Meegeren’s painting. “The period of intense scrutiny, art experts excitedly announced that the idea of using radioactivity to date works of art was thought of in the painting was a genuine Vermeer. A painting by the gifted seven- 1930s,” says Keisch. “But back then they didn’t even have Geiger teenth-century artist Johannes Vermeer was considered a rare find counters, and you couldn’t measure radioactivity accurately.” Keisch and extremely valuable. Eventually van Meegeren, with the help of knew that white paint was manufactured from lead compounds. his friend, sold the painting to a museum in Rotterdam for $280,000. Furthermore, naturally occurring lead ore contains some uranium- Unknown to art historians of the time, van Meegeren continued to 238, a radioactive isotope of uranium. Uranium-238 has a nucleus uncover and sell Vermeers for a number of years. His uncanny ability containing 92 protons and 138 neutrons. The neutron to proton ratio to discover lost artworks proved financially makes it unstable, and the uranium-238 rewarding. But when he sold to the Nazis nucleus disintegrates by emitting an alpha his luck ran out. When World War II ended, particle. The products are also radioactive, Dutch authorities discovered that van giving rise to the uranium-238 decay series. Meegeren had sold one of the Vermeers to The final product in the series is a stable Hermann Goering, the architect of Hitler’s isotope of lead (lead-206). air force. Van Meegeren was taken immedi- Keisch’s work focused on two key iso- ately into custody and charged with trea- topes in the uranium decay series. son. But after spending six weeks in Radium-226 and lead-210 were present in detention van Meegeren made an astound- the white lead pigments used by Vermeer ing claim. He had not really sold a Dutch and other seventeenth-century artists. The national art treasure to the Nazis because Ra-226 slowly decays with a half-life of the Vermeers were fakes! Van Meegeren 1600 years. Its descendant, Pb-210, confessed that he had secretly painted decays much faster with a half-life of only “Christ and His Disciples at Emmaus” and “Christ and His Disciples at Emmaus” was 22 years. “In nature the two isotopes are in other works, copying Vermeer’s artistic originally attributed to artist Johannes equilibrium and the disintegration rates are style. Vermeer (1632Ð1675), but Hans van Meegeren equal,” says Keisch. But when the lead ore Art experts were stunned—and skeptical. confessed that he had forged the painting in is mined from the earth and manufactured the 1930s. Decades later, radioactive dating Many believed that van Meegeren was lying techniques proved conclusively that the into white pigment most of the Ra-226 is to avoid prosecution for treason. How painting could not have been painted by removed. The Pb-210 remains, but could anyone fool the specialists into Vermeer in the seventeenth century. because of its relatively short half-life, the

14 CHEM MATTERS, DECEMBER 1994 After van Meegeren was arrested for trying to sell the “Vermeer” masterpiece to the Nazis, he tried to prove that the painting was a fake to avoid the serious charge of treason. In prison, van Meegeren was given canvas and brush, and asked to prove to the court that he had the skill to create such a convincing 238 forgery. 92U α concentration of Pb-210 declines over a few Meegeren’s painting were not in equilibrium. decades. Because the tiny amounts of Ra-226 234 234 234 Calculations indicated the painting was less than 90Th Pa U that remained with the lead pigment continue β 91 β 92 50 years old. Keisch’s work proved conclusively to decay into Pb-210, the two iso- α that Johannes Vermeer did not paint “Christ and His topes come into balance again in 230 Disciples at Emmaus.” Of course, radioactive dating alone URANIUM Th a couple of centuries. The time RADIOACTIVE 90 could not prove that van Meegeren was the forger. necessary for the two isotopes α In 1947 van Meegeren testified in court how he had faked the to return closely to this state of DECAY SERIES Vermeers. He had meticulously removed most of the paint from an 226 equilibrium is about 150 years. If Johannes Ra unimportant seventeenth-century canvas. Using pigments similar to Vermeer had painted “Christ and His Disciples 88 those Vermeer would have used, and skillfully duplicating Vermeer’s at Emmaus,” more than 300 years would α distinctive style, van Meegeren painted “Christ and His Disciples at have elapsed—enough time, according Emmaus.” Finally, to make new paint look centuries old, he baked the 222 to Keisch, for the concentration of the 86Rn painting in an oven to chemically harden the pigments. Eventually Pb-210 isotope to be in balance the charge of treason against van Meegeren was dropped and with its Ra-226 ancestor. α replaced with a charge of forgery. Van Meegeren was found guilty

Keisch obtained small 218 and condemned to prison, but he died samples of white pig- 84Po A nucleus decreases by 2 protons shortly before beginning his sentence. and 4 mass units in alpha decay. ment from unimpor- α Although van Meegeren was a talented tant areas of van A nucleus increases by 1 proton artist in his own right, he is better 214 214 Meegeren’s Pb Bi remembered for being the most suc- 82 β 83 with no loss of mass in beta decay. painting. α cessful art forger of all time. Samples from 210 210 210 210 Ti Pb Bi Po Mike McClure teaches chemistry at other paintings of 82 βββ82 83 84 known age were also αHopkinsville Community College in Hopkinsville, KY. His interest in animal taken to act as controls. 206 Each sample was dissolved in nitric acid and the isotopes precipitated 82Pb forensic science is evident in another Chem out on small metal plates called planchettes. The planchettes were Matters article, “Deer Kill” (October 1992). analyzed using solid-state detectors sensitive to alpha particles. FOR FURTHER INFORMATION: When alpha particles strike the solid atoms of the detector, Rogers, F. E. “Chemistry in Art: Radiochemistry and Forgery”. electrons are kicked out of valence orbitals. This ioniza- Journal of Chemical Education 1972. 49 (6), 418. tion process produces free electrons and positively “Art of Forgery? The strange case of Hans van Meegeren: the videodisc investigation of a real life art charged holes in the solid crystal lattice. The number forgery.” Order # 4SCH-631-A; from of electron–hole pairs can then be detected or counted by Intellimation, Library for the Macintosh the instrument and are proportional to the number of alpha parti- (phone:805-968-2291). Requires a laserdisc player. cles. Because the number of alpha particles is related to the number of atoms undergoing decay, the concentration of the isotopes can be found. All of the control samples agreed with this theory — their isotope ratios matched their age. The lead and radium concentrations were in equilibrium. But the isotopes in the paint taken from van Eavesdropping John Ihde, chemistry teacher in Eavesdropping Wausau, WI, overheard some rare on the Elements conversations between atoms.

+ - K Na Na CI H CI Sn Hg I'VE TRIED IT BOTH WAYS AND BELIEVE ME, IONIC IS MUCH MORE SATISFYING FOR SOME REASON, Ni NICK, I CAN NEVER REMEMBER THEIR Al NAMES Cl Br

Ne Ar 14 6C HOW OLD DO YOU THINK HE IS, KAY?

THOSE TWO ARE ALWAYS HANGING AROUND DOWNTOWN AT NIGHT 20 19 40Ar 39K

Cm Fe Tc Fm I DON'T KNOW WHAT IT IS ABOUT HIM, KAY, BUT HE'S GOT SOME KIND OF Mo I DON'T KNOW, MOLLY, ANIMAL MAGNETISM THERE'S SOMETHING VERY ARTIFICIAL ABOUT THAT NEW FAMILY Mo K K

16 CHEM MATTERS, DECEMBER 1994