Journey of a Late Blooming Biochemical Neuroscientist

I graduated from tuition-free College of the City of New York (CCNY) in 1933. At that time the country was in the depths of the Great Depression, and there were few positions available for City College graduates. Its students were mainly children of immigrants who were bright and highly motivated. CCNY graduates included eight future Nobel Prize awardees and several distinguished biochemists. After graduating from City College, I obtained a position at the Harriman Research Laboratories at New York University. Though it paid $25 a month, I was delighted to work in a laboratory. I assisted Dr. K. G. Falk in his research on enzymes in malignant tumors. Dr. Falk was trained as a biochemist in the laboratories in Europe. In 1924, Falk wrote a monograph on "The Chemistry of Enzyme Action." Falk was greatly influenced by Richard Willstäter, an eminent German biochemist. Willstäter believed that enzymes were catalysts of low molecular weight adsorbed on colloidal carriers such as proteins. In 1935 I was lucky to get a position as a chemist in the Laboratory of Industrial Hygiene for $40 a week. This laboratory was a nonprofit organization and was set up by the New York City Department of Health to test the amount of vitamin supplements added to foods. In the 1930s vitamins were a hot subject in biochemistry. My duties were to modify published methods for vitamins so that they could be assayed in various food products. It required some ingenuity to modify methods for the measurement of vitamins from the literature. This experience proved useful in my later research. In 1946, there was an unexpected change in my career. At that time, analgesic drugs such as acetanilide and phenacetin were widely used. Some people who used excessive amounts of these drugs became habituated and developed methemoglobinemia. An independent organization, the Institute for the Study of Analgesic and Sedative Drugs, approached Dr. George B. Wallace, then president of the Laboratory of Industrial Hygiene and retired Chairman of the Department of Pharmacology at New York University, for advice. Dr. Wallace asked me if I would like to work on this problem and he suggested that I consult Dr. Bernard B. Brodie. Bernard Brodie (Fig. 1) was a professor in the Pharmacology Department at New York University doing research at Goldwater Memorial Hospital in New York City. Goldwater Memorial Hospital was set up during World War II to clinically test newly synthesized antimalaria drugs. Brodie was responsible for developing methods for measuring blood levels of these drugs to establish the most effective dosage regimen. Soon after the end of the war, Brodie and his assistant Sidney Udenfriend published a series of influential papers in the Journal of Biological Chemistry on "The Estimation of Basic Organic Compounds in Biological Materials." I met with Brodie in February 1946 to discuss the cause of toxicity of acetanilide. It was a fateful meeting for me. Brodie invited me to spend time in his laboratory to work on this problem. One of the possible transformation products of ingested acetanilide causing the toxic effects could be aniline (Fig. 2). One important lesson I learned from my discussions with Brodie was to ask the right questions at the right time and devise the means to answer these questions. From my previous experience, I learned how to develop methods. Within a few weeks I developed a colorimetric method to measure aniline in blood and urine by diazotizing the amino group and coupling with a dye. After taking acetanilide orally, I identified aniline in my urine. This was one of the most exhilarating experiences in my life, making an important new discovery. Experiments in dogs showed that there was a direct relationship between the concentration of plasma aniline and methemoglobinemia after the administration of acetanilide. After the oral administration of acetanilide to humans acetanilide was almost completely metabolized. Aniline represented only about 4% of the ingested acetanilide. A common transformation of compounds containing a benzene ring is hydroxylation on the para position. Thus a possible metabolic product after the ingestion of acetanilide could beN-acetyl-p-aminophenol. Within a few weeks we identified the major metabolic product in humans after the oral administration of acetanilide asN-acetyl-p-aminophenol and its conjugates, sulfate and glucuronide. The route of metabolism of acetanilide in humans was found to proceed as shown in Fig. 2.N-Acetyl-p-aminophenol was found to be as potent as acetanilide in analgesic activity. By taking serial plasma samples, acetanilide was rapidly transformed toN-acetyl-p-aminophenol. After the administration of N-acetyl-p-aminophenol, negligible amounts of methemoglobin were formed. In our paper published in 1948 (1Brodie B.B. Axelrod J. J. Pharmacol. Exp. Ther. 1948; 94: 29-38PubMed Google Scholar), Brodie and I stated "the results are compatible with the assumption that acetanilide exerts its actions throughN-acetyl-p-aminophenol (now known as acetaminophen); it is possible, therefore, that it might have distinct advantages over acetanilide as an analgesic." This was my first taste of research and I loved it. "The Fate of Acetanilide in Man" (1Brodie B.B. Axelrod J. J. Pharmacol. Exp. Ther. 1948; 94: 29-38PubMed Google Scholar) was my first paper and I was determined to continue doing research. Several pharmaceutical companies subsequently began to sell products containingN-acetyl-p-aminophenol. However, aspirin still dominated the analgesic market. In the early 1970s Johnson & Johnson marketedN-acetyl-p-aminophenol as Tylenol. Because aspirin might produce gastrointestinal ulcers, Tylenol became one of the best selling analgesics. Brodie invited me to stay on at Goldwater Memorial Hospital to study the fate of other analgesic drugs. We received a small grant from the Institute for the Study of Analgesic and Sedative Drugs. Another analgesic drug we studied was antipyrine. We found that this drug distributes like body water. The first paper I published as coauthor in the Journal of Biological Chemistry in 1949 was "The Use of the Antipyrine in the Measurement of Total Body Water in Man" (2Soberman R. Brodie B.B. Levy B.B. Axelrod J. Steele J.M. J. Biol. Chem. 1949; 179: 31-42Abstract Full Text PDF PubMed Google Scholar). Because I did not have a doctoral degree, advancement was unlikely in a hospital associated with an academic institution. In 1949, James Shannon was appointed director for intramural research at the newly formed National Heart Institute in Bethesda, Maryland. I applied for a position at the National Heart Institute and Shannon accepted me. In 1949 the government expanded the original National Institute of Health to a number of medical institutes to form the National Institutes of Health. At that time, many scientists believed that medical research in government laboratories was mediocre. When Sid Udenfriend, a postdoctoral fellow at Washington University, was offered a position in the National Heart Institute, he asked Carl Cori, his laboratory chief, for advice. Cori told Udenfriend that working in a government laboratory would be the end of his research career. Shannon persuaded Brodie to come to Bethesda as the Chief of the Laboratory of Chemical Pharmacology at the National Heart Institute. I was assigned to the Laboratory of Chemical Pharmacology in Building 3. This three-story building on the Bethesda campus of the NIH became one of the most fertile research settings in the world. Among the scientists working in Building 3 in the early 1950s, more than half became members of the National Academy of Science, five became Nobel laureates, and three were appointed directors of the NIH. The ambience in Building 3 was highly stimulating. Everyone knew each other and their research. As the immunologist and essayist Lewis Thomas so eloquently stated, "The National Institutes of Health is not only the largest institution for biomedical science on earth, it is one of the nation's great treasures. As social interventions for human betterment go, this is one standing proof that, at least once in a while, government possesses the capacity to do something unique, imaginative, useful, and altogether right." The first problem I chose was the physiological disposition of the widely used compound caffeine in man. I developed a sensitive and specific method for measuring caffeine in biological material. The plasma half-life of caffeine in man and the distribution in dog tissues were determined (3Axelrod J. Reichenthal J. J. Pharmacol. Exp. Ther. 1953; 107: 519-523PubMed Google Scholar). I soon became intrigued with the sympathomimetic amines. In 1910, Barger and Dale (4Barger G. Dale H.H. J. Physiol. ( Lond. ). 1910; 41: 19-59Crossref PubMed Scopus (248) Google Scholar) found that β-phenylethanolamine derivatives simulated the effects of sympathetic nerve stimulation with varying degrees of intensity and precision, and they coined the term sympathomimetic amines. Some sympathomimetic amines produced unusual behavioral effects. Amphetamine and methamphetamine in large doses produced symptoms of paranoia. Mescaline, the active principle of peyote, caused hallucinations. In 1952 little was known about the metabolism of these amines. Because of my experience in drug metabolism, I decided to study the metabolism of ephedrine and amphetamine in a number of animal species. The first amine I examined was ephedrine. Ephedrine, the active principle of Ma Huang, an herb used by ancient Chinese physicians, was introduced to modern medicine by Chen and Schmidt (5Chen K.K. Schmidt F. Medicine ( Baltimore ). 1930; 9: 1-117Crossref Scopus (65) Google Scholar) in 1930 to elevate blood pressure. I soon found that ephedrine was metabolized in animals (dogs, guinea pigs, rats) by two pathways, demethylation and hydroxylation on the benzene ring, to yield metabolites that had pressor activity (6Axelrod J. J. Pharmacol. Exp. Ther. 1953; 109: 62-73PubMed Google Scholar). I then examined the metabolism of amphetamine and methamphetamine (7Axelrod J. J. Pharmacol. Exp. Ther. 1954; 110: 315-326PubMed Google Scholar). These compounds were transformed by a variety of pathways including hydroxylation, demethylation, deamination, and conjugation. Marked species variations in the transformation of these drugs were also found. Over the past 150 years biochemists and pharmacologists have observed that almost all chemical compounds ingested are metabolized by a variety of biochemical changes. Depending on the chemical structure, the body can inactivate or activate drugs and foreign compounds by chemical transformation. In some cases toxic as well as pharmacologically active metabolites can be formed. In 1953 little was known about the enzymes involved in metabolizing drugs and foreign compounds. The ability of animals to metabolize amphetamines and ephedrine by a variety of metabolic pathways stimulated my interest in finding the enzymes involved in these transformations. I was hesitant to do enzymology; I believed it required special training and aptitude. Gordon Tomkins, then a postdoctoral fellow who shared my laboratory, gave me good advice. He told me all I needed to start was a method for measuring amphetamine and ephedrine, an animal liver, and a razor blade. In January, 1953 I did my first in vitro experiment. To my great pleasure, amphetamine almost completely disappeared when I incubated this drug with rabbit liver slices in a Krebs-Ringer solution. In the following experiment I homogenized the rabbit liver and found that the metabolism of amphetamine was increased when a cofactor, TPN (NADP), was added. I decided to examine which subcellular fraction was transforming amphetamine. Schneider (8Schneider W.C. J. Biol. Chem. 1948; 176: 259-265Abstract Full Text PDF PubMed Google Scholar) had developed a method for separating the various subcellular fractions by homogenizing tissues in isotonic sucrose and subjecting the homogenate to differential centrifugation. After separation of the nuclei, mitochondria, microsomes (homogenized endoplasmic reticulum), and the cytosol, none of these fractions were able to metabolize amphetamine even in the presence of added TPN. However, when microsomes and the cytosolic fractions were combined, amphetamine rapidly disappeared upon the addition of TPN. Before going any further I decided to identify the metabolic products of amphetamine. When the combined microsomal and cytosolic fractions were incubated with amphetamine and TPN, ammonia and phenylacetone were identified (9Axelrod J. J. Biol. Chem. 1955; 214: 753-763Abstract Full Text PDF PubMed Google Scholar). These results indicated that amphetamine was deaminated by an oxidative enzyme in rabbit liver requiring TPN. This experiment also suggested that the enzyme was present either in the microsomes or the cytosol, and one subcellular fraction supplied factors to the other fraction containing the enzyme. Because of the structure of amphetamine and the requirement of TPN, it was apparent that this enzyme was different from monoamine oxidase. An approach that I thought was likely to give me a clue to the intracellular location of the amphetamine-deaminating enzyme was to subject each subcellular fraction to elevated temperatures. When I heated the cytosolic fraction to 55 °C for 10 min and then added unheated microsomes, amphetamine, and TPN, amphetamine was metabolized. However, after heating the microsomes to 55 °C and then adding unheated cytosolic fraction and TPN, amphetamine was no longer metabolized. This experiment told me that a heat-labile enzyme was located in the microsomes, and the cytosol provided factors necessary for the deamination of amphetamine. I suspected that the cytosolic fraction was involved in the action of TPN. Bernard Horecker (then at the NIH) had prepared several substrates for his classic experiments on the pentose phosphate pathway. These enzymes required TPN. He generously supplied me with several substrates that I could test. The addition of glucose 6-phosphate, isocitric acid, or phosphogluconate together with TPN and dialyzed cytosol fraction to microsomes resulted in the metabolism of amphetamine. These substrates had one thing in common: they all generated TPNH (NADPH) even in the presence of oxygen. It became obvious that the cytosolic fraction was supplying a dehydrogenase and substrates to reduce TPN to TPNH. When microsomes were incubated in air with glucose-6-phosphate dehydrogenase, glucose 6-phosphate, TPN, and amphetamine, ammonia and phenylacetone were generated. To confirm that the deaminating enzyme was in the microsomes and that TPNH was a necessary cofactor, I incubated TPNH, microsomes, and amphetamine. Ammonia and phenylacetone were formed in stoichiometric amounts. DPNH could not be substituted for TPNH. In the absence of air there was no metabolism of amphetamine. By 1954 I felt quite confident that I had found an enzyme localized in rabbit liver microsomes that deaminated amphetamine in the presence of TPNH and O2. At about the same time I also found that ephedrine was demethylated to norephedrine and formaldehyde by an enzyme present in rabbit microsomes that required TPNH and oxygen. I reported these finding in 1954 at the fall meeting of the American Society of Pharmacology and Experimental Therapeutics. Complete papers appeared in the Journal of Biological Chemistry (amphetamine) and the Journal of Pharmacology and Experimental Therapeutics(ephedrine) in 1955 (9Axelrod J. J. Biol. Chem. 1955; 214: 753-763Abstract Full Text PDF PubMed Google Scholar, 10Axelrod J. J. Pharmacol. Exp. Ther. 1955; 114: 430-438PubMed Google Scholar). Soon after my report on the TPNH-dependent microsomal enzymes was published many drugs that are metabolized by a similar enzyme system were described. In 1957, it was found that enzymes in the microsomes that required TPNH and oxygen could also catalyze the oxidative metabolism of normally occurring compounds such as androgens to estrogen (11Ryan K.G. Engel L. J. Biol. Chem. 1957; 225: 103-114Abstract Full Text PDF PubMed Google Scholar). In studies on the N-demethylation of narcotic drugs in a variety of species, it became apparent that there were several microsomal enzymes involved in the metabolism of foreign and normally occurring compounds (12Axelrod J. J. Pharmacol. Exp. Ther. 1956; 117: 322-330PubMed Google Scholar, 13Lu A.Y.H. Pharmacol. Rev. 1979; 31: 277-295PubMed Google Scholar). In 1965 Omura et al. (14Omura T. Sato R. Cooper D.Y. Rosenthal O. Estabrook R.W. Fed. Proc. Am. Soc. Exp. Biol. 1965; 24: 1181-1189Google Scholar) reported that cytochrome P450 was present in liver rat microsomes, and about the same time Estabrook and co-workers demonstrated that this hemoprotein was responsible for the oxidative metabolism of drugs and steroids (14Omura T. Sato R. Cooper D.Y. Rosenthal O. Estabrook R.W. Fed. Proc. Am. Soc. Exp. Biol. 1965; 24: 1181-1189Google Scholar). After many years of intensive work in several laboratories, many cytochrome P450 enzymes were purified (15Nebert W. Gonzalez F. Annu. Rev. Biochem. 1987; 56: 945-993Crossref PubMed Scopus (1416) Google Scholar). By 1954 I had published about 25 papers, most of them independently and many of them as a solo author. I applied for a promotion at the National Heart Institute and was turned down because I did not have a doctorate. I decided to get a Ph.D. in pharmacology at George Washington University and in 1955 at the age of 42, I received a Ph.D. I decided to leave the National Heart Institute and soon was appointed to a position at the National Institute of Mental Health. Just before I left the National Heart Institute, I was intrigued by a paper that found that uridine diphosphate glucuronic acid (UDPGA) was a necessary cofactor for the formation of phenolic glucuronides in a cell-free preparation of liver (16Dutton G.J. Storey I.D.E. Biochem. J. 1954; 57: 275-283Crossref PubMed Scopus (37) Google Scholar). In a chance meeting with Jack Strominger, then a biochemist at the NIH, we discussed the possible mechanisms for the enzymatic synthesis of UDPGA. We suspected it would be formed by the oxidation of uridine diphosphate glucose (UDPG) by either TPN or DPN. In a preliminary experiment we measured the formation of morphine glucuronide after incubating microsomes and the cytosolic fraction of rat liver with UDPG and either TPN or DPN. Morphine glucuronide was formed in the presence of DPN but not TPN. An enzyme, uridine diphosphate glucose dehydrogenase (17Strominger J.L. Maxwell E.S. Axelrod J. Kalckar H.M. J. Am. Chem. Sec. 1954; 76: 6411-6412Crossref Scopus (60) Google Scholar), was purified 180-fold which carried out the following reaction: UDPG + 2DPN+ → UDPGA + 2DPNH + H+. The work on glucuronide conjugation led to studies on the role of bilirubin glucuronide formation in jaundice. Rudi Schmidt, then at the NIH, and I observed that bilirubin was detoxified by enzymatic transformation to bilirubin glucuronide in the liver, a reaction requiring UDPGA. This led to an interesting clinical observation relating glucuronide formation and jaundice. In patients with congenital jaundice there is a marked increase in free bilirubin in the blood. This suggested to us that there must be a defect in a enzyme in this The of a of rats) that were it possible to examine these animals had a enzyme R. L. 1957; PubMed Scopus Google Scholar). We showed that the had a low enzyme activity as with Brodie and I showed the was mainly metabolized by glucuronide formation in humans (1Brodie B.B. Axelrod J. J. Pharmacol. Exp. Ther. 1948; 94: 29-38PubMed Google Scholar). levels and its glucuronide were then examined in patients with jaundice. levels of were elevated in patients as with R. L. 1957; PubMed Scopus Google Scholar). In 1955 I the of my research to the of the system and the of drugs. The and of (Fig. influenced my to a was the first director of the intramural of the in He a in Bethesda that was and had an important on the of and biological In 1948 at the University of developed the method for the measurement of blood This had a on our of how the oxygen and glucose in a variety of and with his developed methods for the measurement of blood on of between and These led to the of by In the and co-workers a major study on the of the and used as a means of separating and factors in the of From these studies they that about of was of and the of appeared to be In addition to his was an When down as he became of the Laboratory of at the in This laboratory became a fertile organization and clinical research to and than were the of the Laboratory of When I the Laboratory of in there was no such as had an and separation scientists working in and the the these began to from molecular and The of new it possible to ask more and questions the system and the In a Society of was At the first meeting in there were about in there were When I the I was a small laboratory with a my laboratory chief, me to work on any problem that was and I thought that a study on the metabolism and distribution of would be an problem for my new laboratory. was then used as an drug by to produce at the NIH was in the of building a He was to me his which it possible to develop a sensitive for This later became the well known The of this it possible for many laboratories to devise sensitive methods for the measurement of and in the and other These newly developed methods for an important role in the in research. In a gave an of the of two found that when was to air it was to When was it produced hallucinations. Because of these behavioral they that might be caused by an metabolism of to In the I was to that little was known about the metabolism of at that time Because of the about the metabolism of in and my previous experience in research on compounds in structure to such as amphetamine, I decided to work on the metabolism of and was then believed to be metabolized and by monoamine oxidase. However, it was shown that after the administration of a monoamine the blood by to was still rapidly This indicated that enzymes other than monoamine metabolize and inactivate An in the of gave me a and Fed. Proc. 1957; Scholar) reported that patients with large amounts of product acid This suggested that could be formed by and deamination of or A could be When I and homogenized rat liver, was metabolized. to the structure of the most likely of would be in the group of to form The formed by incubating with was identified as J. R. J. Biol. Chem. Full Text PDF PubMed Google Scholar). The enzyme was purified and was found to and but not Because of its we the enzyme The enzyme was widely in tissues including the was found to in vitro and in J. PubMed Scopus Google Scholar). It was observed that mainly that had from animal resulted in the of the then a postdoctoral and I soon identified normally occurring metabolites of such as and in liver and As a of the of the metabolic pathways of metabolism were were metabolized by deamination, and conjugation to glucuronides and The work on gave me a interest in The metabolites of have used as a for many studies in biological of are used in the of with the of I became involved in several To more to then a postdoctoral fellow in the laboratory of a and I synthesized from rabbit Thus the of the group would the product of a The first enzyme by this was Axelrod J. R. J. Biol. Chem. Full Text PDF PubMed Google Scholar). were enzyme that to and This enzyme was found to a compound found in the to a The enzymes together with of specific activity were used to develop sensitive methods to measure amines in We were able to and measure and in the and other tissues J.M. Axelrod J. J. PubMed Scopus Google Scholar). Because of the of the enzymatic my and I were able to the of several in identified of J.M. Axelrod J. H.H. Proc. PubMed Scopus Google Scholar). demonstrated the of more than one in in T. H.M. PubMed Scopus Google Scholar). The experiments on stimulated my research on and chemical

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