The history of vitamin C research in India - Lecturer Note - Indian History - I B Chatterjee -, Study notes of Indian History

Download The history of vitamin C research in India - Lecturer Note - Indian History - I B Chatterjee - and more Study notes Indian History in PDF only on Docsity! The history of vitamin C research in India 1 J. Biosci. 34(2), June 2009 1. Prelude Vitamin C is perhaps the most controversial, highly publicized yet least understood of all vitamins. Eighty years have passed since its discovery, but till now its precise biological function has remained an enigma. It is perceived by the general public as a panacea, a miracle pill that is capable of curing myriad diseases. Humans, in contrast to many animals, are incapable of synthesizing this vitamin and are totally dependent on its dietary intake for their survival. The biosynthetic pathway of vitamin C in animals and its defective counterpart in humans have been completely elucidated, mainly by the pioneering work done in the USA and India. In this essay, besides the discovery of vitamin C by Szent-Györgyi (1928) as well as King and Waugh (1932), I shall elaborate on vitamin C research done exclusively in India. 2. Discovery of vitamin C The distinctive features of scurvy resulting from vitamin C defi ciency were described in the Ebers Papyrus around 1700 BC. They are also found in the texts of Susruta, the great Indian surgeon, around 400 BC; the writings of Hippocrates; and the works of Chang Chi of China ça 200 AD. The knowledge that humans cannot synthesize vitamin C was gained by experience. Between 1500 and 1800 AD, scurvy took a toll of at least two million sailors (McCord 1959). In that period, more seamen died of scurvy than of all other causes combined, including other diseases, shipwrecks, accidents and battle wounds. Scurvy also ravaged whole armies and inhabitants of besieged cities. It was perhaps the main disease resulting from an occupational hazard and, as a nutritional defi ciency disease, has caused the most suffering in recorded history (McCord 1959). It was the genius of James Lind (fi gure 1) that banished this merciless killer of seamen. In 1753, in his book A treatise of scurvy, Lind pointed out the importance of lemons, oranges and fresh green vegetables for the prevention and cure of scurvy. It took another 175 years to discover the antiscorbutic factor vitamin C. The discovery was accidental. In 1927, Albert Szent-Györgyi (fi gure 2a), a young Hungarian scientist, came to work for one year in the laboratory of F G Hopkins at Cambridge, England, with a fellowship from http://www.ias.ac.in/jbiosci J. Biosci. 34(2), June 2009, 000–000, © Indian Academy of Sciences 0 Perspectives The history of vitamin C research in India I B CHATTERJEE Dr B C Guha Centre for Genetic Engineering and Biotechnology, Calcutta University College of Science, Kolkata 700 019, India (Email, ibc123@rediffmail.com) Keywords. Biological function; biosynthesis; emphysema; genetic defects in human; vitamin C Figure 1. Scottish doctor James Lind with the book “A Treatise of the Scurvy” in his hand, published in 1753 discovered the importance of lemons, oranges and fresh green vegetables for the prevention and cure for scurvy. I B Chatterjee2 J. Biosci. 34(2), June 2009 the Rockefeller Foundation. Szent-Györgyi’s fi eld of interest was oxidation reactions of compounds present in both plant and animal tissues. In Hopkins’s laboratory, his objective was to isolate some redox substances present in ox adrenal glands. He measured the amount of reducing factor in samples by their power to decolourize iodine. While extracting and concentrating redox compounds from ox adrenal gland, he isolated some sugar-like crystals that he knew nothing about. This ignorance was apparent from the title of the paper he submitted to Biochemical Journal: “Observation on the function of peroxidase systems and the chemistry of the adrenal cortex: description of a new carbohydrate derivative” (Szent-Györgyi 1928). He was so ignorant of the nature of the carbohydrate derivative that he fi rst named it ignose (ign for ignorance and ose for sugar). However, the Editor of Biochemical Journal did not accept the name. Later, Szent- Györgyi named it godnose (God only knows). However, the Editor objected to this name as well. Finally, the structure of the carbohydrate was elucidated by the famous English chemist W M Haworth at the University of Birmingham and was named hexuronic acid (hex = six carbon uronic acid) (fi gure 3). During the same period, King and Waugh (1932) of Columbia University isolated the antiscorbutic factor from lemon juice and named it vitamin C. It had all the characteristics of Szent-Györgyi’s hexuronic acid. In January 1931, Szent-Györgyi left Cambridge and returned to Hungary to chair the medical chemistry department at the University of Szeged. In the fall of 1931, an American post-doctoral fellow, Joseph Svirbely, joined Szent- Györgyi’s research team. Svirbely had been working with C G King (fi gure 2b) at the University of Pittsburgh, trying to isolate vitamin C. Szent-Györgyi gave him the remains of the hexuronic acid he had isolated at the Mayo Clinic and asked him to test it on guinea pigs with induced scurvy. Repeated trials proved that hexuronic acid was, in fact, vitamin C. Szent-Györgyi had suspected this, but had put the project aside rather than take up the messy, expensive and labour-intensive animal studies required. King, meanwhile, had also been close to reaching a similar conclusion. Svirbely wrote to his former mentor in March 1932, telling King what he had found at the Szeged laboratory, adding that he and Szent-Györgyi were submitting a report to Nature. On 1 April 1932, Science published King’s paper entitled “The chemical nature of vitamin C” claiming that he had discovered vitamin C, which was identical to hexuronic acid. King cited Szent-Györgyi’s earlier work on hexuronic acid but gave him no credit for the discovery of vitamin C. The story of the discovery was quickly picked up by the American press. Two weeks later, astonished and dismayed, Szent-Györgyi and Svirbely sent off their own report to Nature entitled “Hexuronic acid as the antiscorbutic factor” (Svirbely and Szent-Györgyi 1932), challenging King’s claim to the discovery. A bitter controversy ensued. King, as was well-known, had been working on the problem for over fi ve years; he had many supporters who were ready to vilify Szent-Györgyi as a plagiarist. Yet European and British scientists also knew of Szent-Györgyi’s long research on this antioxidant substance and accepted his claim. Ultimately, Szent-Györgyi’s claim prevailed. In October 1937, he was awarded the Nobel Prize for Physiology or Medicine “for his discoveries in connection with the biological combustion processes, with especial reference to vitamin C and the catalysis of fumaric acid”. In fact, Szent-Györgyi was surprised when informed of the Nobel Prize. Here was a scientist who worked only for a year on a problem with quite a different objective and without doing a single animal experiment but got full credit Figure 2. (a) Albert Szent-Györgyi: Born: September 16, 1893, Died: October 22, 1986. (b) Charles Glen King: Born: October 22, 1896; discovered hexuronic acid from adrenal gland in 1928. Died: January 23, 1988 discovered vitamin C from lemon juice in 1932 and showed that it had all the characteristics of hexuronic acid. Figure 3. Structure of hexuronic acid (vitamin C). The history of vitamin C research in India 5 J. Biosci. 34(2), June 2009 in 1960, I was further encouraged when I saw that our observations on vitamin C became the basis of an editorial written by F G Young, indicating a correlation between the defect of a single gene (L-gulonolactone oxidase) and the development of molecular pathology (Young 1960). I was also gratifi ed by the comment made on our work (Chatterjee et al. 1961) by C G King, the discoverer of vitamin C: “This discovery adds a new and very interesting landmark to the history of vitamin C” (King 1961). After being awarded the degree of Doctor of Science of Calcutta University in 1961. I proceeded for postdoctoral studies in the Department of Physiological Chemistry, University of California at Los Angeles (UCLA). In September 1961, I joined the laboratory of Ralph W McKee, a co-worker of Henrik Dam, who won the Nobel Prize for the discovery of vitamin K. On 20 March 1962, the heartbreaking message reached me of the sudden and untimely death of B C Guha at the age of 57 years. To me it was a bolt from the blue. As a mentor, Guha had induced in me the essence of research imbibed at London and Cambridge under Sir Jack Drummond and Sir Frederick Gowland Hopkins. In UCLA I had another learning experience about confi dence in research. McKee had given me a problem on the immunological aspect of Ehrlich ascites tumour. At that time, cancer research was in its early infancy and Ehrilich ascites cells occupied a major part of cancer research. McKee’s observation was that if irradiated ascites cells were injected into normal C7 black mice, the mice became immune. Injection of viable ascites cells into the immune mice did not produce any tumour. The hypothesis was that injection of irradiated cells produced tumour- specifi c antibodies. After working for six months I came to the conclusion that injection of irradiated cells produced only increased levels of γ-globulin and not tumour-specifi c antibody. When I reported these fi ndings to McKee, I found that he was reluctant to accept my inference, particularly because I had no experience in immunology. However, I was fi nally able to convince him of the veracity of my fi ndings. McKee was a large-hearted man and thereafter gave me the green signal to continue my research on vitamin C, my primary interest. From then on, vitamin C has been with me all along. While at UCLA, I published three papers on vitamin C (e.g. Chatterjee et al. 1965). 6. Evolution and the biosynthesis of vitamin C After returning from the USA in 1964, I joined the Department of Biochemistry of Calcutta University as a lecturer and continued my research on vitamin C, addressing the question of the evolutionary loss of vitamin C-synthesizing capacity in humans. The ability to synthesize ascorbic acid is absent in insects, invertebrates and fi sh. Biosynthetic capacity evolved in amphibians and was tissue specifi c. Biosynthesis occurred fi rst in the kidney of amphibians, resided in the kidney of reptiles, was transferred to the liver of mammals, and fi nally disappeared from guinea pig, bats, monkeys, apes and humans (Chatterjee 1973). Similar observations were made in birds (Chaudhury and Chatterjee 1969). Primitive birds synthesize vitamin C in the kidney. The more evolved Passeriformes produce it in the liver, while a number of more evolved passerines are incapable of synthesizing the vitamin (fi gure 6). The inability of guinea pig, bats, monkeys, apes and humans as well as the highly evolved passerine birds to synthesize the vitamin is due to a common defect – the absence of the terminal enzyme L-gulonolactone oxidase (Chatterjee et al. 1960a,b, 1961, 1975; Chatterjee 1978). Amphibians were the fi rst to develop the capacity to synthesize vitamin C about 330–340 million years ago and the gene mutation leading to loss of the capacity took place in the common ancestor of humans and other primates about 25 million years ago. Loss of the gene in guinea pig and humans was probably due to a deletion mutation ((Nishikimi et al. 1994). The mutants did not become extinct because the environment furnished vitamin C and the species continued to survive (Nandi et al. 1997). On the basis of our observations, the famous nutritionist Nevin S Scrimshaw Figure 6. Vitamin C synthesizing ability of different species of animals in a schematic phylogenetic tree (Chatterjee 1973). Full reference in list of references at end. I B Chatterjee6 J. Biosci. 34(2), June 2009 wrote an article in Scientifi c American (Scrimshaw and Young 1976). He commented that the inability of humans to synthesize vitamin C is not only of nutritional signifi cance and part of their biological evolution, but also has shaped social evolution. It has been suggested that the migration of human groups to the northern regions of the earth was slowed by the limited amounts of ascorbic acid in the foods available in those areas during the long winter months (Scrimshaw and Young 1976). 7. Biological function of vitamin C After answering two questions: (i) how is vitamin C synthesized by most animals and (ii) why are humans incapable of producing vitamin C, I started on work to answer the third question: what is the precise biological function of vitamin C? Eight decades have passed since the discovery of vitamin C, but its biological function has remained a mystery. The biological function of vitamin C is probably related to the evolutionary signifi cance of the biosynthesis of vitamin C in terrestrial vertebrates. The evolution of amphibians from fi sh took place in the Devonian, when the oxygen concentration in water was about 0.5% but the atmospheric oxygen concentration was 15–18% (Graham et al. 1995). Thus, during evolution from an aquatic medium to the terrestrial regimen, newly evolved vertebrates were exposed to an environmental oxygen concentration 30–36 times that of their aquatic ancestors. Such a high concentration of extremely toxic oxygen acted as an acute constraint for respiratory adaptation and survival on land. Apparently expression of the L-gulonolactone oxidase (LGO) gene took place in early tetrapods under selection pressure to provide the newly evolved terrestrial vertebrates with adequate amounts of vitamin C to protect their tissues against oxygen toxicity (Nandi et al. 1997). This hypothesis supports the numerous reports about the well-established antioxidant function of vitamin C. However, some reports indicate that LGO activity is present in partly terrestrial lung fi sh (Chatterjee 2008). This would suggest that the ancestral fi sh LGO gene might have been passed on to amphibians about 400 million years ago. The concept that megadoses of vitamin C could have a benefi cial effect was the brainchild of Linus Pauling, winner of two Nobel prizes (Chemistry Prize in 1954, Peace Prize in 1962) and a genius of the twentieth century. Based on some calculations such as (i) the capacity for biosynthesis of vitamin C by rats and (ii) vitamin C intake through various plant materials ingested by apes, Pauling suggested that the intake of vitamin C should be about 2.3 g a day for the maintenance of general health, and increased intake up to 10 g/day for combating infectious diseases, including the common cold as well as prevention and cure of cardiovascular diseases and cancer (Pauling 1970). Before Pauling stepped into the fi eld, research on vitamin C was based on the concept that the sole function of vitamin C was to prevent scurvy. The knowledge that vitamin C was needed for the prevention and cure of scurvy was amply satisfying for many scientists, particularly those in the medical profession. The prevalent notion was that the disease and its cure had been explained and therefore no longer needed investigation. But that was not accepted by either Pauling or Szent-Györgyi. In reply to a letter from Pauling, Szent-Györgyi wrote “As to ascorbic acid, right from the beginning I felt that the medical profession misled the public. If you do not take ascorbic acid with your food you get scurvy, so the medical profession said that if you do not get scurvy you are all right. I think that this is a very grave error. For full health you need much more. I am taking, myself, about 1 g a day. What I can tell you is that one can take any amount of ascorbic acid without the least danger” (Szent-Györgyi 1970). In fact, Pauling’s concept of the health benefi ts of vitamin C, particularly the benefi t of megadoses of vitamin C, has been the subject of debate and controversy. He was severely criticized by popular writings in the Medical Letter (25 December 1970), New York Times (3 January 1971), Consumer Report (February 1971) and Reader’s Digest (1971). The impact of these criticisms was so great that Pauling was debarred from communicating research papers of other scientists to the Proceedings of the National Academy of Sciences (PNAS). In 1992 Pauling regretfully wrote to me, “I must tell you that the Board of National Academy of Sciences has made a ruling that I am not permitted to submit papers for publications in the Proceedings, except those of which I myself am one of the authors.” The amount of vitamin C needed to prevent scurvy in humans is only about 10 mg/day. However, despite epidemiological and some experimental studies, it has not been possible to show conclusively that higher than an antiscorbutic intake of vitamin C has clinical benefi t (Padayatty et al. 2003). The pertinent point is how would one determine criteria to demonstrate the health benefi t of vitamin C? In fact, I also did not observe any extra benefi cial effect of megadoses of vitamin C in the maintenance of normal health in guinea pigs (Chatterjee 1973). On the other hand, megadoses of vitamin C appear to have benefi cial effects on the outcome of other medical conditions. Recently, using a guinea pig model, we have demonstrated that moderately large doses of vitamin C prevent cigarette smoke-induced emphysema. It should be mentioned that cigarette smoking is by far the commonest cause of emphysema, accounting for about 95% of cases (Barnes et al. 2003). 8. Research on cigarette smoke and vitamin C I consider that the prime biological function of vitamin C is associated with its redox property, which explains The history of vitamin C research in India 7 J. Biosci. 34(2), June 2009 its antioxidant effect. In the mid-nineties we performed some experiments to study the antioxidant effects of vitamin C. We developed an in vitro system where addition of nicotinamide adenine dinucleotide phosphate (NADPH) to microsomal suspensions of tissue (lung, liver, heart and kidney) led to oxidative damage accompanied by proteolysis of the microsomal proteins. This damage was exclusively prevented by vitamin C (Mukhopadhyay and Chatterjee 1994a, b). One evening, while I was walking down the corridor of the laboratory, I saw somebody smoking in the distance and observed that the smoke was curling up in the air. I knew that cigarette smoke contains several oxidants. At once, I thought of an important question: would the oxidative damage of proteins induced by cigarette smoke be prevented by vitamin C? The very next morning we performed an experiment to answer this question. We added a few microlitres of aqueous extract of cigarette smoke to guinea pig lung microsomal suspension and, to our surprise, we observed that cigarette smoke solution did cause oxidative damage and proteolytic degradation of the microsomal proteins, which was almost completely prevented by vitamin C (Ghosh et al. 1996; Panda et al. 1999, 2000). Since cigarette smoke- induced lung damage is apparently caused by destruction of the alveolar and septal cells, we addressed two more questions: (i) would cigarette smoke-induced oxidative damage of lung proteins lead to pulmonary emphysema that could be prevented by vitamin C, and (ii) what component of cigarette smoke was responsible for protein damage in the lung? Eventually, we solved both problems. This was possible only because I was completely ignorant about the nature and composition of cigarette smoke. If I knew then that cigarette smoke contains about 4000 compounds, I would never have dared to launch research on it. In 1995, when I formally retired from the post of Professor in the Department of Biochemistry at Calcutta University, R N Basu, the then Vice-Chancellor of Calcutta University, requested me to continue my research in the newly established Dr B C Guha Centre for Genetic Engineering and Biotechnology of the University of Calcutta (where I had been the founding coordinator). After fi ve years, we fi nally isolated from cigarette smoke a long-lived semiquinone radical and characterized it as p-benzosemiquinone (p-BSQ) (Banerjee et al. 2008). Earlier studies on the electron spin resonance of cigarette smoke condensate had indicated that semiquinones are present in cigarette smoke, but these had not been isolated and characterized separately (Pryor et al. 1998). An orthodox classical chemist would be skeptical as to how a semiquinone radical could be isolated. That is why it took a long time to get the paper published. The paper was accepted after several rejections. In spite of all the required experimental data, one reviewer believed that p-BSQ could not be isolated and purifi ed. We could do this because p-BSQ is a long-lived radical, the half-life of which in the solid state is about 48 h. We examined about 15 brands of cigarettes, both Indian and international. All the brands contain p-BSQ. At this point, I should express my gratitude to Dr R A Mashelkar, the then Director General of the Centre for Scientifi c and Industrial Research (CSIR). In 2001, during a leisurely conversation at the Bose Institute, Kolkata, I told Dr Mashelkar that we were probably on the verge of isolating a hazardous compound from cigarette smoke, which would explain the cause of lung damage due to smoking. Dr Mashelkar was very excited and told me that we should not publish the result; CSIR would patent this fi nding and bear all costs. Later, we observed that in a guinea pig model, p-BSQ very nearly mimics the protein damage, apoptosis and pulmonary emphysema caused by cigarette smoke (Banerjee et al. 2008). The process of isolation of p-BSQ from cigarette smoke has been patented (US Patent 2005) and is being patented in different countries of the world. At the request of CSIR, I had to reluctantly do another piece of research. We developed an activated charcoal fi lter which traps p-BSQ from the main smoke stream. The smoke emitted from this newly developed charcoal fi lter does not cause protein damage or emphysema in a guinea pig model. The cigarette fi lter has also been patented (US Patent 2006) and is being patented in different countries of the world. Dr Mashelkar and all of us had great hopes that the cigarette fi lter would be readily commercialized and we would be handsomely rewarded, as our charcoal fi lter cigarette would be a safer cigarette that would probably not produce lung cancer or emphysema, the two fatal diseases known to be caused mostly by cigarette smoking. We were further excited when we heard that a very big multinational cigarette company was seriously considering licensing our patent. Unfortunately, we did not realize that the company was actually trying to fi nd some commercial loopholes to circumvent our patent. 9. Vitamin C prevents cigarette smoke-induced emphysema Emphysema is a major and increasing global cause of mortality and morbidity. It is projected to become the third most common cause of death and the fi fth most common cause of disability in the world by the year 2020 (Lopez and Murray 1998; Pawels and Rabe 2004). There is no novel or even currently effective treatment aimed at preventing this irreversible, fatal disease. Although cigarette smoking is the commonest cause of emphysema, the cellular and molecular mechanisms of the disease are not yet clear. Recently, we have delineated the mechanism of cigarette smoke-induced emphysema and its prevention. This was possible only because we could produce emphysematous lung damage in