I started ‘CommonScienceSpace’ over 8 years ago, and this will probably be the last post. Of the 66 posts, a number are related to the broad theme of how new stuff is discovered, and the science behind it. In that vein, let me tell you a story.
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The smell of sweet clover
Sixty years ago, back in the last millennium, I was a student at the University of Alberta, finishing up a four-year degree in Chemistry. ‘Qualitative Organic Analysis’ was a final year course that tested everything you had learned. It was a lab course, in which we were given a series of somewhat complex chemicals to be identified. Once identified, you were to synthesize some derivatives of the mystery material to prove your identification. It was grown-up chemistry. And cheating wasn’t possible, because each student had different unknowns to solve.
My last unknown that final semester was a fluke. I sniffed it, carefully, as you were told to do, and had a Proustian moment: I recognized the smell of sweet clover hay from my summer months as a farmhand. The smell, I read, was due to a chemical called coumarin. So, instead of a lot of chemical tests to identify my unknown, which is hard work, I took my sample to the man who ran the analytical Nuclear Magnetic Resonance (NMR) facility, which was easy. (Remember, we were told to use everything we had learned; I had learned that as students in Honours Chemistry, we had access to the analytical NMR lab.) He ran the spectrum, and it confirmed my guess: it was coumarin. Slam dunk. I looked up the literature on coumarin, where I came upon research by a laboratory at the University of Wisconsin some twenty years earlier, and followed a couple of their recipes for making derivatives. Case closed. I needed the time to study for finals anyway.
A few months later, I was off to graduate school. Thanks to the interest of one of my professors, I had made the wise choice to join the Department of Biochemistry at the University of Wisconsin in Madison, which was considered to be a very good graduate department. The next chapter was about to begin.
The sunshine patent
One of the events that raised the profile of UW Biochemistry was its involvement in the story of Vitamin D, which is related to the disease rickets. Rickets was described hundreds of years ago as the failure, in babies and young children, to build strong bones. Untreated, it leads to lifelong problems of weak bones and malformation of the ribs, and often to death. Rickets became more prevalent during the industrial revolution, due to the mass movement of people from rural to (smoke-beclouded, sunless) cities in Europe: lack of sun exposure. In 1861 a French doctor named Trousseau thought that rickets was caused by a lack of sun and a faulty diet, and could be reversed by cod-liver oil. But it continued to afflict children. At the turn of the twentieth century, rickets was an epidemic in the United States — estimates of its prevalence in inner city children ranged to greater than 50%. It is caused by a deficiency in vitamin D, and as Dr. Trousseau thought, can be reversed by cod liver oil and sunlight.
Laboratory animals deprived of fat-soluble micronutrients show the same symptoms as children suffering from rickets, and both cod liver oil and exposure to sunlight can prevent or reverse their symptoms. Several groups of researchers were curious about whether ultraviolet irradiation of food (fake sunlight) would prevent rickets in animals. It did, and this led to a process invented by Professor Harry Steenbock at University of Wisconsin in 1924: the ultraviolet irradiation of milk. UV treatment of milk increases the immediate precursors of vitamin D, and established the connection between cod liver oil and sunlight, which both increase the level of vitamin D.
Subsequent work showed that Vitamin D increases the uptake of calcium from the diet and thereby the formation of strong bones. The beauty of irradiating milk was that it contains lots of calcium. For just pennies, children who had access to treated milk were protected from rickets, even if they lived in the slums of industrial cities. Between 1925 and 1945, irradiation of milk was used to provide children with vitamin D.
WARF
At the time that Harry Steenbock invented his method of adding vitamin D to milk, the usual procedure for university research that had commercial potential, as this process clearly did, was to sell the intellectual property to an interested commercial entity, which would patent it. Many companies were willing to shell out for the rights to the vitamin D process, including Quaker Oats, who offered a million 1925 dollars dollars to use the method on their cereal products.
But a group of nine UW alumni wanted to do something more profitable; in 1925 they created a university agency that would retain ownership of the irradiation patent. They named it the ‘Wisconsin Alumni Research Foundation’, WARF for short. WARF would out-license the milk-irradiation technology to commercial interests and charge them royalties. This was prescient, but also a great rarity at the time. It was proposed that future inventions at UW could also be taken up by WARF. Under the terms of such arrangements, the inventor would get 20% of the gross royalty revenue, and WARF would use the rest to pay the filing costs and the legal costs of protecting the intellectual property. Any additional earnings would go to benefit research on the campus. The cost to the nine supporters to establish WARF was $100 each.
So WARF kept the patent on the Vitamin D process, and licensed it to Quaker Oats, and other companies. By the time that patent expired, rickets had been essentially eliminated in the USA, and WARF had earned 8 million dollars from it (equivalent to over a hundred million dollars today). (Since the 1940s, Vitamin D is added directly to milk.) Other discoveries from University of Wisconsin labs were added to the WARF portfolio over the years; it became, and remains, one of the most effective university technology transfer agencies. Today, it is the focus of biotechnology based on recombinant DNA technology.
Ed Carlson’s problems
There are strong connections between coumarin, the University of Wisconsin, and WARF. That part of the story began on a cold blustery Saturday in February, 1933. On that day, a farmer named Ed Carlson drove his truck from his farm near Deer Park, Wisconsin, to Madison, some 190 miles away. On the back of his truck were a dead cow, a milk can full of cow’s blood that would not clot, and 100 pounds of spoiled sweet clover hay.
Farmer Carlson had two problems. One was that his cattle, the basis of his farm, were bleeding to death, either internally or through normally insignificant external wounds. Their blood would not clot, and he had a milk can full of un-clotted blood to prove it. The local veterinarian recognized the symptoms of ‘Spoiled Clover Disease’, which resulted from cattle eating spoiled sweet clover hay, and losing their blood’s ability to clot. Farmer Carlson had a hundred pounds of spoiled sweet clover on his truck as well. There was no medicine to treat Spoiled Clover Disease, but there was a simple solution: replace the spoiled sweet clover with healthy feed. Carlson’s second problem, the lack of money, prevented him from taking this course of action. He, like so many others, had been beaten down by the Great Depression and had hardly a dollar to his name. He didn’t want to believe the diagnosis of his local vet, and hoping against hope, decided to get a second opinion from the state agricultural agent, who worked on the UW campus in Madison.
When Carlson arrived, the Agricultural Experimental Station was closed, and the agent was at home. It was Saturday. He wandered around the campus and came upon a building marked ‘Agricultural Chemistry’. That must have sounded promising. He went in, and by chance ran into Professor Karl Paul Link.
Running into K. P. Link was fortuitous; he was probably the only person in Madison who knew something about the science of sweet clover. A few months earlier, he had been offered a faculty position at the University of Minnesota, which would include the study of sweet clover disease. Link didn’t take the Minnesota position, but he soon began a collaborative project to develop a variant of sweet clover with a reduced coumarin content. Coumarin, as I earlier described, gives sweet clover its characteristic smell. It also imparts a bitter flavour, which cattle don’t like. KP, as he was colloquially known, was collaborating with a geneticist to develop sweet clover with lower coumarin content.
Farmer Carlson’s story had an impact on Professor Link. As a scientist, he was of course intrigued. But he was also profoundly affected by its human aspects. He came from a poor rural background like that of Carlson’s, and he could empathize with him. However, there was nothing he could tell the farmer that would help him with his problem, and Carlson returned to Deer Lake, without the 100 pounds of sweet clover and the can of unclottable blood.
An unusual man
It would be unusual to find an academic scientist responding directly to such an encounter, then as now, but K. P. Link was not a conventional scientist or person.
Karl Paul Gerhard Link was born in LaPorte, Indiana in 1901, the eighth of ten surviving children of a Lutheran minister and his wife. Although the family was poor, the children benefitted from their father’s fine library, and a piano that had originally been brought from Germany by Karl’s maternal grandparents. His father, who had to leave the ministry when Karl was two years old because of a throat condition, became a local court clerk, but died when Karl was 12. Life became more difficult, but the children were imbued with an appreciation of literature and classical music by what must have been an indefatigable single mother. Of the ten children, one son received a PhD in Botany from the University of Chicago and became a Professor there; one graduated from the law school at the same university and became a circuit court judge; one became a politician in LaPorte; one of the children taught voice and piano; one designed and sold millinery; one received a PhD in petroleum geology, also from the U. of C.; one was a registered nurse; one became chief geologist for Standard Oil of New Jersey, and one became manager of the Tucson Red Cross.
Karl attended the University of Wisconsin, and earned a PhD in plant biochemistry. After receiving his degree, he carried out postdoctoral studies in Scotland, Austria and Switzerland before returning to Madison in 1927 as an Assistant Professor in the Department of Agricultural Chemistry. (A few years later, it was re-named The Department of Biochemistry. But the marble lintel over the side door still had ‘Agricultural Chemistry’ carved into it when I attended UW.) The young professor K. P. Link soon became known as an effective lecturer and a dedicated researcher.
KP (his photo appears at the top of this post) was something of a maverick throughout his academic life. He frequently engaged in anti-establishment laments and was a pro-student advocate on almost any issue. One student later wrote “He dressed to attract attention, and spoke with a booming voice . . . he kept people’s attention while he transferred a remarkable amount of information. He sometimes injected outrageous statements: slurs at the university administration, pithy quotations, or his own aphorisms, to keep minds from wandering. He affected a spontaneity in his lectures, but in truth, he worked diligently on them to gain the optimal effect. Link was accepted as a showman, but he accomplished the goal of teaching…”.
Rabbits and rats
Although KP was unable to help Mr. Carlson, he soon did start a research project to identify the anticoagulant factor in spoiled sweet clover because of meeting him. A test for it was developed using rabbits, which had to be specially bred for the purpose, and which were ‘recycled’ as test subjects. (One champion rabbit was used for over 200 individual assays in a period of 5 years.) The material to be tested was injected into a rabbit, and the clotting time of plasma drawn from the animal was determined. Using this cumbersome bioassay, the team recovered a few milligrams in crystalline form of the anticoagulant from spoiled sweet clover in 1939; it had taken 6 years. Within a year, KP and his team had identified it as a derivative of coumarin, the sweet-smelling, bitter-tasting component of sweet clover that was my last unknown in Qual Organic Analysis. Spoilage of the sweet clover under moist conditions converted coumarin to a closely-related chemical called dicoumarol (a fungus that grew on the wet clover was involved). Coumarin had no detectable effect on blood; dicoumarol was a potent anticoagulant, and was soon shown to be responsible for the deaths of cattle suffering from ‘Sweet Clover Disease’.
The anti-clotting property of dicoumarol suggested that it might be useful in treating thrombosis in heart-attack victims. Starting in 1941, clinicians began to look into this. Progress was slow, and dicoumarol could only be used in a carefully-monitored clinical environment. Back in the biochemistry department, KP and his team were producing variants of dicoumarol, over 100 eventually, and testing them for something else entirely: the ability to kill rats (it was this chemistry that I had read about, and which helped me finesse my last unknown in Qualitative Organic Analysis). WARF supported this work, in hopes of a practical outcome. And there was one. Compound 42 was the most toxic rodenticide yet, and in honour of the support of the foundation, it was named ‘Warfarin’ (WARF plus the end of ‘coumarin’). A patent was applied for, and granted, and Warfarin became, and remains, the best rodenticide available.
A President needs help
There matters might have ended: the clinicians were unable to solve the technical problems of using dicoumarol as a human anti-clotting agent on a broader scale. Then, in 1951, fate stepped in. A US army inductee decided to kill himself. He read up on Warfarin, and using a dose that would kill a rat his size, started eating it mixed into starch. He knew that Warfarin was cumulative, so he took his dose over several days. But during that time, he had second thoughts, rushed to the Emergency Department, and was saved by blood transfusion and massive doses of Vitamin K (a known antagonist of Warfarin). But the lesson learned was, that the most toxic derivative of dicoumarol to rats, compound 42, Warfarin, was also much less toxic to humans than the original. And that was the birth of Warfarin as the safe blood clot prevention drug.
There was some resistance to using a known rat poison in human patients, but this was overcome by an event in 1954 involving the 34th President of the United States of America, Dwight David Eisenhower. Eisenhower was the five-star General of the United States Army by the end of World War II, the commander of Allied forces during the invasion and liberation of Europe. After the War, he became the supreme commander of NATO. His public profile and effectiveness, together with his personability, made him an attractive political candidate, and the Republican Party recruited him as their nominee for the Presidency in the 1952 election. His nickname, ‘Ike’, was on campaign buttons ‘We Like Ike’. Ike won in a landslide, and was re-elected in 1956, again by a wide majority.
Some saw Dwight Eisenhower as a ‘do-nothing’ President. He was not. He maintained the social legislation introduced by Roosevelt’s New Deal and expanded Social Security, one of America’s most successful social programs. He championed the interstate highway system, and started a wave of civil rights legislation and actions that was furthered by Presidents John F. Kennedy and Lyndon Johnson. And in his last, prescient, speech, he warned of a growing ‘Military Industrial Complex’, which he feared would trap the country into soaring expenditures for military hardware even in peacetime.
Ike was also a man of the people, with a smiling demeanor and a love of golf. A tree on the 17th fairway at Augusta National golf course is named ‘The Eisenhower Tree’ because of the trouble he had avoiding it. On September 23, 1955, he was playing at the Cherry Hills golf course in Denver, one of his favourites. During the game, he complained of indigestion-like symptoms. That night he felt worse. He was taken to hospital and diagnosed with a mild heart attack.
Ike recovered and lived another 13 years. One reason he survived was the availability of effective anti-clotting therapy. Initially, he was administered heparin for this, but he was soon shifted to a drug that had been approved only two years earlier: Warfarin. With the Presidential seal of approval, Warfarin quickly became a heavily used anticoagulant, and remains so today.
Another vitamin
By the time I arrived in Madison as a new graduate student, KP was in his ‘60s, and significantly debilitated from several bouts of ‘wet pleurisy’ (tuberculosis) over his lifetime. But I remember him as a colorful character striding about the campus in his checked flannel shirts.
The mechanism of Warfarin’s effect on blood clotting was identified in the 1970s. It involves another vitamin, vitamin K, which plays an important role in several steps of the blood clotting cascade. Vitamin K isn’t an enzyme, but it is a critical cofactor for some of the enzymatic amplification steps of blood clotting. Without it, clotting doesn’t happen. When it takes part in the clotting cascade, vitamin K is converted to an inactive form, and then another enzyme re-activates it. Warfarin interferes with the reactivation of vitamin K, and that’s how it blocks blood clotting. Not surprisingly, vitamin K antagonizes Warfarin’s anti-clotting effect, and vitamin K is used to correct over-treatment with Warfarin.
One of the issues of using Warfarin as an anti-thrombosis agent is that people vary in their sensitivity to the drug. Diet is one source of variation – a diet high in vitamin K leads to higher dose requirement of Warfarin. A fundamental source of variation is that the enzyme that reactivates vitamin K comes in different genetically-inherited forms, which vary in their susceptibility to inhibition by Warfarin. A system that inactivates Warfarin is also genetically diverse. So patients differ in their response to the drug. Recently, tests have been developed that distinguish between genetic variant forms of those enzymes, and these can help tailor the dose to the genotype. This is individual-specific medication, in which genetic variation is analyzed, and drug administration is tailored to the results. This is 21st Century medicine.
A graduate student needs help
One outcome of the successful commercial development of Warfarin and other parts of the WARF portfolio was that money was available for on-campus research, which included financial support of graduate students. When I arrived in Madison, it was a little tricky for a foreign graduate student like me to gain financial support from federal American research funds. A WARF studentship came to my rescue. The $200 a month stipend was enough to live on in 1963 if you shared lodgings and had simple tastes. I owe a debt of gratitude to K. P. Link and the other contributors, beginning with Harry Steenbock, to the accumulated wealth of the Wisconsin Alumni Research Foundation. The value of that fund today stands at almost 2 billion dollars, and it currently provides $45 million a year for research on the Madison campus.
From the research laboratory of K. P. Link, to rat poison, to twenty-first century medicine, it’s been an interesting journey for the hemorrhagic factor in farmer Carlson’s spoiled sweet clover. Nobody predicted the course of events; it just followed from the curiosity and vision of the scientists involved.
References
‘The Discovery of Dicumarol and Its Sequels’. K. P. Link. Given February 25, 1958 at the New York Academy of Medicine, under the auspices of the Section of Medicine and the New York Heart Association, on the programme, ‘The Historical and Physiological Aspects of Anticoagulants’. Published in Circulation, Volume 19, January 1959, page 97.
Karl Paul Link, 1901-1978. A Biographical Memoir. R. H. Burris. National Academy of Sciences, Washington D. C.
