On The Trail Of Vitamin A With A Distinguished Biochemist

(Ed. note: After a distinguished career devoted to plant biochemistry and the study of vitamin synthesis, Trevor Goodwin retired in 1983 as Johnston Professor of Biochemistry at the University of Liverpool. He was highly influential in shaping the course of British science, serving in such key science groups as the University Grants Committee and the Council of the Royal Society. He also authored widely used textbooks and recently completed a history of the U.K Biochemical Society. Here, Goodwin, now 72, casts his eye backward on some of his most memorable early research.] When I look back over more than 40 years of research and more than 400 papers, the investigation that always comes to mind first is one I carried out with Rod Gregory in 1946, very early in our careers. Working together in a shabby basement laboratory in Liverpool, we demonstrated that in mammals vitamin A is formed from beta-carotene as it crosses the intestinal wall. Today, this may seem an elementary observation. Yet it gave us a tremendous thrill, and was important because it overturned the dogma of the day—that the transformation occurred in the liver. The event was significant for me too because its original inspiration was a lecture I had attended as an undergraduate eight years earlier. Although interested in biochemistry even when at school, I had opted to study “straight” chemistry at Liverpool University because a condition of my bursary was that I should take up teaching as a profession. Then, during my final year, I heard R. A. Morton lecturing on vitamin A, and this fired my imagination about doing research myself. I soon persuaded the Department of Education to defer my teacher training for a year so that I could study for an M.Sc. with Morton. Thanks to some national savings accumulated at school, the princely sum of 10 pounds per term for demonstrating to medical students, and remission of fees (Morton’s influence, no doubt), I just about managed to get through financially. Vitamin A disappeared from view, however, because in those days students were allowed little or no choice in the selection of research topics—even under the most benevolent supervisor. Instead, I found myself investigating the use of spectroscopy to determine the amounts of certain amino acids in proteins. In fact, our photographic methods proved insufficiently accurate—but at least our technique correctly demonstrated the absence of tryptophan from insulin. Incidentally, I took up this problem again in 1945 when a Beckman DU photoelectric spectrophotometer became available to us under lease-lend arrangements with the United States. The resulting paper became a “Citation Classic” [because the method of analyzing for trypotphan and tyrosine proved to be simple and widely used] in 1981—sadly, two years after Morton died. When war broke out in September 1938,1 was exempted from national service for one year to complete my studies for a Diploma in Education. Because of the threat of air raids over Liverpool, the Department of Education and certain other parts of the university were evacuated to Coleg Harlech, an education college nestled beneath Harlech Castle in Wales. I thus spent the so-called “phony war” learning to be a teacher and getting to know my future wife, who had been evacuated too as a member of the Arts Department. Then, out of the blue in June 1939, just as I was contemplating an imminent call to join the armed forces, I received a letter from Morton. He wanted me to join him as an assistant for a Ministry of Food project to control the vitamin status of Britain’s civilian population. I accepted with alacrity—and returned to Liverpool on the very day when the first air raid of the war took place in the city. The Food Ministry had decided that margarine should be supplemented with vitamins A and D, to prevent any deficiency in the wartime diet. I was particularly concerned with measuring vitamin A and assessing its stability in oils and fats from different manufacturers. At last I was involved in vitamin A biochemistry. The analysis of relatively low levels of the vitamin added to margarine posed considerable problems, but the first paper I ever read to a scientific society—the Society of Public Analysis—described a reasonably satisfactory method that we had devised. Apart from fire watching and teaching air cadets the principles of flight (although I had never been inside an airplane), my other main activity was taking part in “The Sheffield Experiment.” A group of conscientious objectors to military service had volunteered as subjects for nutritional research, and had been collected in Sheffield. The first investigation was to determine the human requirement for vitamin A. The human guinea pigs were under the daily control of Hans Krebs, and a scientific committee met each month to monitor progress. I attended these meetings regularly and brought back samples for analysis in Liverpool. The containers full of ground, dried human feces did not endear me to my fellow passengers in the crowded wartime trains. Nevertheless, the final publication from this investigation is still cited as an authoritative contribution to the field. In 1944, Morton was appointed to the Johnston Chair of Biochemistry at Liverpool, and I became Lecturer in Biochemistry. During the Sheffield Experiment, I had begun to question the conventional view that the transformation of beta-carotene into vitamin A in mammals occurs in the liver. The only “evidence” for this was that the vitamin accumulated in that organ in large amounts. Yet apart from humans and cattle, most mammals had insignificant quantities of beta-carotene circulating in the blood even after receiving massive doses. How could it possibly travel to the liver for conversion? Perhaps the transformation really took place as beta-carotene crossed the intestinal wall after being acquired from food? In this case, vitamin A, being fat-soluble, would be transported not in the blood but in the lymph. Once the lymph was mixed with blood as it re-entered the circulation through the thoracic duct, however, the resulting dilution would be so great that only a slight rise above the resting vitamin A blood levels could be expected. Here was an idea that I should be able to test experimentally. Unfortunately, the accuracy and sensitivity of existing methods for measuring vitamin A in blood were such that quantitatively significant results could not be expected. However, it should have been possible to find out if the conversion had occurred in the way I imagined by examining the lymph produced after an animal had received a dose of beta-carotene. I called upon my friend Rod Gregory (who later became Professor of Physiology at Liverpool, and a Fellow of the Royal Society). Using great surgical expertise, he inserted cannulae into goats’ thoracic ducts, so that lymph could be collected. He also used penicillin—which was then just about to come onto the market—to prevent the massive infections that would otherwise have oc- curred around the cannulae. I shall never forget our first experiment in 1946. I had been married just over two years, we had recently acquired a baby and a flat, and I was decorating the kitchen one Sunday evening in June when, at about 10:00 p.m., the phone rang. Rod had prepared the goat, which was providing an excellent supply of lymph. I immediately put down my brush, jumped on a bicycle, and cycled the two miles to the lab. Rod had already begun collecting lymph (in condoms, so as to put as little strain as possible on the thoracic cannula), which could be analyzed later to tell us the baseline level of vitamin A. I took a beta-carotene preparation from the refrigerator and, to speed up absorption and avoid the unlikely possibility of its being transformed by bacteria, we transferred the preparation slowly through a cannula directly into the goat’s duodenum. As Rod had a bed in the lab, he then insisted that I go home while he kept watch and stored samples of lymph in the fridge over the next eight hours. When I returned in the morning, there, waiting for me, were a dozen samples ready to be tested for vitamin A. I saw at once that all were colorless and milky. This seemed to augur well—at least no yellow beta-carotene had appeared. So I began the tests, using a procedure in which vitamin A would be indicated by the production of a blue color and a measurement of its intensity at a wavelength of 617 nanometers. The moment of truth had arrived. The first sample produced the most glorious blue, far too intense to be measured in the spectrophotometer. All of the other samples reacted in the same way. But samples taken from the same animal before beta-carotene administration produced only the faintest traces of blue. We conducted many more experiments, of course, before our paper appeared in the Biochemical Journal in 1948. Yet those years’ first tests had clearly established the conversion of beta-carotene into vitamin A as it crossed the intestinal wall. We had experimentally ruled out a well-entrenched belief that the liver played a vital part in the conversion. I still recall those experiments in our tatty basement laboratory with the greatest pleasure.

On The Trail Of Vitamin A With A Distinguished Biochemist

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