Luminaries Submitted 2.14.11 | Revision Received 3.8.11 | Accepted 3.10.11

Albert Szent-Györgyi, MD, PhD

Discoverer of Vitamin C and a Pioneer of Cellular Respiration, Muscle Physiology, and Cancer Development Mark J. Ranek, BS,1 Steven W. Cotten, PhD,2 Monte S. Willis, MD, PhD2 ( 1University of South Dakota, Vermillion, SD, 2University of North Carolina, Chapel Hill, NC) Albert Szent-Györgyi (1893-1986)

DOI: 10.1309/LMM23KS8NKQMHEHE

“Think boldly, don’t be afraid of making mistakes, don’t miss small details, keep your eyes open, and be modest in everything except your aims.” — Albert Szent-Györgyi Dr. Szent-Györgyi traveled by train to a number of laboratories throughout Europe. He first studied pharmacology with G. Mansfeld in Pozsony (now Brataslavia, Slovakia), then electrophysiology with Armin von Tschermak in Prague, then in Berlin with L. Michaelis, followed by training in physical chemistry for 2 years in Hamburg at the Institute for

Corresponding Author Monte S. Willis, MD, PhD [email protected]

Abbreviations ATP, adenosine triphosphate; G-actin, globular actin; F-actin, fibrous actin; ADP, adenosine diphosphate; MBL, Marine Biological Laboratory

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Tropical Medicine.1 His first position was as an assistant in Leiden at the University Institute; from 1922-1926 he studied with H.J. Hamburger in Groningen (Netherlands).1 It was in Groningen that he became interested primarily in cellular respiration—that myriad of reactions in cells allowing them to use nutrients to create adenosine triphosphate (ATP). While his focus was primarily on cellular energy production, he also began to investigate the browning that occurs with plants when they are in the process of dying. He discovered that this process occurred when plants could not supply enough hydrogen necessary to prevent oxidation.2 With this discovery, he went on to investigate plants that did not brown when they died to discover the mechanism behind this oxidation reaction. While investigating citrus plants, he found that peroxidase, known at the time to be involved in oxidation, could enhance the browning in plants, while the addition of citrus juice could prevent this browning.2 His research on biological oxidation and intracellular respiration was noticed by Sir Frederick Gowland Hopkins, who subsequently offered Dr. Szent-Györgyi a Rockefeller fellowship at Cambridge University in 1927. In the year Dr. Szent-Györgyi was at Cambridge University, he successfully isolated this reducing substance from orange juice, cabbage, and the adrenal glands of animals.2-3 He measured the amount of the reducing substance in samples by their ability to decolorize iodine.3 He identified this reducing agent as C6H8O6 and named it hexuronic acid, a 6 carbon uronic acid (Figure 1), a chemical with properties of both a sugar and an acid (Figure 1A).3 The identification and isolation of hexuronic acid as the reducing agent in adrenal glands was performed by a series of elegant experiments. First, he minced the adrenal cortex he previously determined to have a high activity of the reducing agent, then he mixed it with alcohol. He then stirred it with carbon dioxide, then acetate, filtered it, and repeated the alcohol and acetate additions followed by further filtrations. This procedure yielded a small amount of precipitate in the form of crystals.2 Further investigations revealed this substance was a highly reactive carbohydrate derivative, isomeric with glycuronic acid, as evidenced by a positive Molisch reaction and orcinol test, among others.2 Subsequently, he isolated hexuronic acid from the adrenal cortex, indicating that hexuronic acid was the reducing factor responsible for the reducing reactions in the adrenal cortex.2 labmedicine.com

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Albert Imre Szent-Györgyi was born in Budapest, Hungary, on September 16, 1893, to Miklós and Jozefin Szent-Györgyi. Miklós was a businessman who ran a large estate outside of Budapest, while Jozefin was a talented musician whose family consisted of notable scientists József Lenhossék (father) and Mihály Lenhossék (brother), both professors of anatomy at the University of Budapest. In 1911, SzentGyörgyi entered the Budapest Medical School. Shortly thereafter, he became more interested in his uncle’s laboratory than his medical classes.1 Szent-Györgyi’s medical education was temporarily suspended by the outbreak of World War I, where he served as an army medic for 2 years. He earned the Silver Medal of Valor for bravery before being discharged in 1917 after being wounded in the arm. He was sent back to Budapest, and while his arm healed he completed his MD in 1917. After graduation, Dr. Szent-Györgyi was sent to his next military post where he remained until the war ended.1

Luminaries A         B

Donorox

Donorred

OH

CH2OH

H

O

O O Dehydroascorbic acid

O2

OH

Flavonoidred L-ascorbic acid oxidase E 1.10.3.3

H OH

HO OH H Hexuronic acid (Ascorbic acid)

OH

Ascorbic acid

H

H

O

O

HO H

HO O

H O

HO

O

H

HO

HO

OH

O

HO

OH

Flavonoidox

2H2O OH

Glucose

O

HO

O

L-ascorbic acid peroxidase E 1.11.1.11

OH O

HO

O

H

H O

O

Dehydroascorbic acid

HO 2H2O

H2O2

OH

Ascorbic acid

Dr. Szent-Györgyi speculated that hexuronic acid was a catalyst involved in cellular respiration.4 The cabbage leaf, which also contains a fair amount of hexuronic acid, was his model to test this hypothesis. When cabbage leaves were placed in a respirometer in the presence of potassium hydroxide, there was a very strong utilization of oxygen. However, if the cells were injured, very little oxygen was used. He followed the reduction of hexuronic acid with the use of color reactions and found that hexuronic acid can be both oxidized and reduced.4 He concluded that hexuronic acid is a hydrogen carrier, involved in the oxidation-reduction reactions associated with the oxidation of foodstuffs (Figure 1B).4 These observations fueled Dr. Szent-Györgyi’s desire to understand the biologic role of hexuronic acid. Understanding the full significance of his discovery was yet to come. For his work isolating hexuronic acid at Cambridge, Dr. Szent-Györgyi was awarded a PhD in 1927.1 In 1928 he went to the Mayo Clinic in Rochester, MN, where he had an ample supply of adrenal glands from local slaughterhouses, which he used to isolate 25 g of hexuronic acid. In 1930, he became professor of medicinal chemistry at the University of Szeged (Hungary). Hexuronic Acid Happens to Cure Scurvy Today scurvy is a rare disease, resulting from the inability of collagen to be formed in the absence of vitamin C leading to spongy gums, bleeding from mucous membranes, and a host of other symptoms. In 1912, the Polish American biochemist, Casimir Funk, introduced the concept of vitamins, referring to them as “vital” “amines” because of their necessary function in health, and his hypothesis that they contained amine groups.5 After dropping the “e” with growing evidence that amine groups were not involved, the word “vitamin” was labmedicine.com

born. One of the vitamins discovered by Holst and Frølich in 1928 was called “water-soluble C” and had anti-scorbutic properties, although its structure had not been determined.6,7 Subsequently, Dr. Szent-Györgyi speculated that hexuronic acid was the component of citrus fruits that prevented scurvy, at the time just recently identified as vitamin C. He was not able to test this hypothesis, however, because he lacked sufficient hexuronic acid and a suitable animal model. So, in 1930, he recruited a post-doctoral fellow, Joseph Svirbely, to his lab from Dr. Charles Glen King’s lab, a vitamin expert from the University of Pittsburgh, to determine if hexuronic acid could cure scurvy.8 Svirbely used a guinea pig model of scurvy to test if hexuronic acid could cure the disease. Like humans, guinea pigs do not produce their own vitamin C and develop scurvy if adequate vitamin C is not ingested in their diet. Svirbely’s experiment placed guinea pigs into 3 groups: 1) those receiving 1 mg of isolated hexuronic acid daily; 2) the positive controls receiving 1 cc of lemon juice daily; and 3) the negative controls receiving a basal diet only.9 The negative controls experienced weight loss and died within 20-34 days and an autopsy showed severe scurvy, while the animals receiving hexuronic acid, similar to those receiving lemon juice, showed normal growth and no pathological signs of scurvy.9 While only small amounts of hexuronic acid could be isolated from citrus fruits and adrenal glands, Dr. SzentGyörgyi would later find that paprika (Capsicum annuum), aka peppers, was a rich source of hexuronic acid that could be isolated on a large scale, as paprika is very prevalent in Hungary. Paprika juice was also noted for its ability to prevent scurvy.9 These results indicated hexuronic acid was identical with vitamin C, which is now also known as ascorbic acid.9 Dr. Szent-Györgyi asked for the assistance of Dr. Walter Haworth, an expert in carbohydrate chemistry from November 2011 ■ Volume 42 Number 11 ■ LABMEDICINE

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Figure 1_Hexuronic acid and its role in cellular respiration. (A) The properties of hexuronic acid (left) were initially found to be closely related to glucose (right), which is not surprising given their chemical similarity. Most animals and plants are able to synthesize vitamin C enzymatically by converting glucose to vitamin C. Among the animals that have lost this ability to convert glucose to vitamin C are the primate suborders anthropoidea or haplorrhini, which are composed of simians, tarsiers, and humans. Simians (monkeys) generally take in through their diet 10-20 times the dose of vitamin C required by humans. (B) The role of hexuronic (ascorbic) acid in cellular respiration as initially identified by Dr. Szent-Györgyi. Adapted from Dr. Szent-Györgyi’s Nobel lecture.9

Luminaries

O

subsequently to fumaric acid and finally, succinic acid, which ultimately transferred its hydrogen atoms to cytochromes.14 Dr. Szent-Györgyi added much clarity to the process of cellular respiration by correctly identifying many of the steps in this process determined later by Hans Krebs15,16 to constitute the series of cyclic reactions bearing his name (Figure 2).14 For Dr. Szent-Györgyi’s discovery of vitamin C and the importance of the catalysis of fumaric acid in cellular respiration, he was awarded the 1937 Nobel Prize in Physiology or Medicine. The Discovery of Actin In 1938, Dr. Szent-Györgyi investigated the biophysics of muscle contraction. His first breakthrough in this field came in 1942 with his trainee, Dr. Ilona Banga.17 They observed that the exposure of muscle tissue to a high salt concentration for 20 minutes could be used to extract a low-viscosity protein, myosin A, while overnight exposure solubilized a highviscosity protein, myosin B.18 They also observed the addition of ATP reduced myosin B, whereas the viscosity of myosin A remained unchanged. Dr. Szent-Györgyi, using H.H. Weber’s method to prepare myosin threads, noticed the addition of boiled muscle juice (a source of ATP) to myosin B elicited contraction of the myosin threads (Figure 3).19 Using a hypodermic needle, he was able to extrude myosin from rabbit muscle (prepared in a low-ionic strength solution) and then press it out into fine threads.18,19 Dr. Szent-Györgyi said in his autobiography, “to see myosin threads contract and see one of the oldest and most mysterious signs of life, motion, reproduced for the first time in vitro, was perhaps the most exciting experience of my research career.”20 This observation suggested that myosin B contained another protein in addition to myosin. While working in Dr. Szent-Györgyi’s lab, Brúnó F. Straub isolated an additional protein from muscle tissue that, in combination with myosin, was responsible for its high viscosity and contractility.17 Because myosin could not contract or become activated without this additional protein, they named this new protein

O

HO OH O oxaloacetate Proposed Cellular Respiration malate of Muscle Tissue (1939) dehydrogenase (as per Dr. Szent-Györgyi) OH O HO OH O malate O

Oxaloacetate

Citrate

Malate

Isocitrate

The Citric Acid/ Acid Krebs Cycle

2

Figure 2_The steps of cellular respiration that Dr. Szent-Györgyi correctly identified, which were used subsequently by Sir Hans Krebs to understand the series of cyclic reactions producing ATP from the catabolism of glucose known as the Krebs or tricarboxylic acid cycle. Adapted from Dr. Szent-Györgyi’s Nobel lecture.9

fumarase

Warburg’s Respiratory Enzyme (now known as iron oxygenase)

HO

Cytochrome Electron Transport

HO

O

OH

fumarate

succinate dehydrogenase

696

H+

α-Ketoglutarate

Fumarate

O

Succinate

Succinyl-CoA

O O

OH succinate

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the University of Birmingham in England, to determine the chemical structure of ascorbic acid. Dr. Haworth was able to determine its structure, and for his work he shared the 1937 Nobel Prize in chemistry with Dr. Paul Karrer.3,9 A controversy persisted between Dr. Szent-Györgyi and Dr. King over the rights to the discovery of vitamin C. The chronological order of events is as follows. Dr. Szent-Györgyi first isolated hexuronic acid as a reducing substance in 1928.2 McKinnis and Dr. King first published the suggestion of a possible correlation between hexuronic acid and vitamin C in 1930.10 Dr. Szent-Györgyi gave a lecture before the Hungarian Medical Association on March 18, 1932, stating that hexuronic acid is vitamin C.11 In March 1932, Dr. Szent-Györgyi’s post-doctoral fellow Svirbely wrote to his former mentor Dr. King telling him everything that he and Dr. Szent-Györgyi had found and that they were going to submit their report to Nature.1 Then, on April 1, 1932, Dr. King and his researchers published in Science that vitamin C is identical to Dr. Szent-Györgyi’s hexuronic acid.12 Finally, on April 16, 1932, and May 7, 1932, Dr. Szent-Györgyi and Svirbely published 2 papers in Nature reporting that hexuronic acid is vitamin C.13 Ultimately, Dr. Szent-Györgyi was credited with the discovery of vitamin C. In addition to his work with vitamin C, Dr. SzentGyörgyi continued his earlier investigation of cellular respiration in skeletal muscle. At the time, much was still left to be understood about cellular respiration. His work focused on the role of the dicarboxylic acids: malate, succinate, and fumarate. Dr. Szent-Györgyi made an important discovery when he observed that these acids were not consumed by cells as fuels when he added small amounts of them to minced pigeon muscle. He also noticed that much more oxygen was consumed than would be needed to oxidize them. These 2 observations allowed him to come to the hypothesis that these acids served as catalysts during respiration.14 He proposed that the oxidation of foodstuffs is a cyclical process involving the transfer of hydrogen atoms between donors and carriers starting with hydrogen from a cellular carbohydrate reducing oxaloacetic acid yielding malonic acid, which was reduced

Luminaries actin. Straub would later show that this protein existed in 2 forms, globular actin (G-actin) and fibrous actin (F-actin).21 Myosin A and actin were later purified.22,23 Myosin A would hold onto the myosin name, while myosin B would be renamed actomyosin due to interaction with actin.24 “Discovery consists of seeing what everyone else has seen and thinking what no one else has thought.” — Dr. Szent-Györgyi

The Move to the United States In addition to his research, Dr. Szent-Györgyi was actively involved in anti-fascist activities. He was a member of Hungary’s anti-Nazi underground during World War II and helped his Jewish colleagues, including Sir Hans Krebs, escape certain death in a Nazi concentration camp.1,16 Dr. Szent-Györgyi was asked by the Hungarian prime minister in 1943 to initiate secret negotiations with the Allies. At the time, Hungary was loosely associated with the Axis powers, Germany, Italy, and Japan. When the Germans heard of this plan in 1944, Dr. Szent-Györgyi was placed under house arrest. Subsequently, he escaped and hid in Hungary from the Gestapo for the remainder of the war. At the end of World War II, Soviet troops occupied Hungary, which gradually became a communist satellite nation of the Soviet Union.28 All the while, after many frustrations with his travel requests to the United States being delayed and denied, he decided to leave Hungary and its Soviet rule for good.28 In August 1947, Dr. Szent-Györgyi arrived in the United States and set up his lab at the Marine Biological Laboratory (MBL) in Woods Hole, MA. With the help of a wealthy Hungarian friend, the Szent-Györgyi Foundation was established to support him and his research team at MBL. Funding from the Armour Meat Company and a grant from the American Heart Association gave Dr. Szent-Györgyi the necessary financial resources to continue his research. Dr. SzentGyörgyi changed the name of the Szent-Györgyi Foundation to The Institute for Muscle Research, of which, he was of course, named the Director of Research.28 Using electron microscopy, he and his research team made many important discoveries and published many books during their tenure at the Institute. In 1949, Dr. SzentGyörgyi and his team of researchers discovered that muscle tissue could be stored in a cold 50% glycerin solution and still maintain its contractility, thereby decreasing the demand to always have supplies of fresh muscle tissue on hand.29 For his contributions to the understanding of cardiovascular diseases through basic muscle research, Dr. SzentGyörgyi received the 1954 Lasker Award, and in 1956 he was elected to the National Academy of Sciences. labmedicine.com

Addition of boiled muscle juice (ATP source)

ATP medicated contraction

Figure 3_The contraction of muscle myosin threads upon the addition of boiled muscle juice containing ATP. Myosin threads before and after the addition of ATP. Based on studies performed by Dr. Szent-Györgyi and his colleagues, 1942.21

A New Era of Cancer Research In the late 1950s, Dr. Szent-Györgyi’s research focus turned toward cancer. He was one of the first scientists to explore the connection between free radicals and the development of cancer. He thought free radicals could push cells into an uncontrolled proliferative state.30 Dr. Szent-Györgyi was primarily funded by the National Foundation for Cancer Research, which was initially set up just to support him but evolved to support many scientists. Dr. Szent-Györgyi made many important discoveries contributing to our understanding of the pathophysiology of cancer, including the damaging effects of free radicals on the cellular milieu and the protection antioxidant vitamins like vitamin C and vitamin E could impart.31-34 Dr. Szent-Györgyi was married 3 times, first to Cornelia (Nelly) Demeny in 1917. They had a daughter in 1918 (Cornelia Szent-Györgyi, aka “Little Nellie”). They were divorced in 1941. He married Marta Borbiro Miskolczy SzentGyörgyi in 1941, who died in 1963 of cancer. He married Marcia Houston in 1975. He died on October 22, 1986, at the age of 93 due to leukemia, involving kidney and heart complications. He never retired and continued working in his lab until just before his death. Throughout his career Dr. Szent-Györgyi published more than 300 articles and 11 books. The Medical University of Szeged was renamed the Albert Szent-Györgyi Medical University. Dr. Szent-Györgyi is most known for the discovery of vitamin C, but his discovery of actin, parts of the Krebs cycle, and his vision about the importance of free radicals during the development of cancer, all contribute to his stature as a luminary of science and medicine. LM Suggested Readings 1. Free Radical: Albert Szent-Györgyi and the Battle Over Vitamin C. By Ralph W. Moss, Paragon House, 1988. 2. Contraction in Body and Heart Muscle. By Albert Szent-Györgyi, 1953. 3. The Crazy Ape. By Albert Szent-Györgyi, 1970. 4. What Next? By Albert Szent-Györgyi, 1971 (Memoir)

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Dr. Szent-Györgyi would further elucidate the importance of ATP in muscle contraction. He found that adenosine diphosphate (ADP) cannot elicit muscle contraction, and it is the ATP-actomyosin interaction triggering the contractile event.25,26 Moreover, the addition of Mg2+ ATP to extracted muscle was shown to yield muscle tissue with comparable tension to that of living muscle.17 He also completed initial investigations into the mechanisms controlling the contraction of cardiac muscle.27 It was these initial investigations by Dr. Szent-Györgyi that laid the foundation for generations of scientists who studied muscle tissue.

Extrusion of myosin threads prepared from rabbit muscle

Luminaries 5. The Living State With Observations on Cancer. By Albert Szent-Györgyi, 1972. 6. Electronic Biology and Cancer: A New Theory of Cancer. By Albert SzentGyörgyi, 1976.

1. The Albert Szent-Györgyi Papers. National Library of Medicine, 2011. Available at: http://profiles.nlm.nih.gov/ps/retrieve/narrative/WG/p-nid/149. Accessed January 2011. 2. Szent-Györgyi A. Observations on the function of peroxidase systems and the chemistry of the adrenal cortex: Description of a new carbohydrate derivative. Biochem J. 1928;22:1387-1409. 3. Chatterjee IB. The history of vitamin C research in India. J Biosci. 2009;34:185-194. 4. Szent-Györgyi A. On the function of hexuronic acid in the respiration of the cabbage leaf. J Biol Chem. 1930;90:385-393. 5. Piro A, Tagarelli G, Lagonia P, et al. Casimir Funk: His discovery of the vitamins and their deficiency disorders. Ann Nutr Metab. 2010;57:85-88. 6. Norum KR, Grav HJ. (Axel Holst and Theodor Frolich—Pioneers in the combat of scurvy). Tidsskr Nor Laegeforen. 2002;122:1686-1687.

18. Banga I, Szent-Györgyi A. Preparation and properties of myosin A and B. Stud Inst Med Chem Univ Szeged. 1942;1:5-15. 19. Szent-Györgyi AG. The contraction of myosin threads. Stud Inst Med Chem Univ Szeged. 1942;1:17-26. 20. Szent-Györgyi A. Lost in the Twentieth Century. Annu Rev Biochem. 1963;32:1-14. 21. Straub FB. Actin, II. Stud Inst Med Chem Univ Szeged. 1943;3:23-37. 22. Straub FB. Actin. Stud Inst Med Chem Univ Szeged. 1942;2:3-15. 23. Szent-Györgyi A. The crystallization of myosin and some of its properties and reactions. Stud Inst Med Chem Univ Szeged. 1943;III:76-85. 24. Szent-Györgyi A. Discussion. Stud Inst Med Chem Univ Szeged. 1942;I:67-71. 25. Szent-Györgyi A. Observations on actomyosin. Stud Inst Med Chem Univ Szeged. 1943;3:86-92. 26. Szent-Györgyi A. Free-energy relations and contraction of actomyosin. Biol Bull. 1949;96:140-161. 27. Szent-Györgyi A. Contraction in the heart muscle fibre. Bull N Y Acad Med. 1952;28:3-10. 28. Medicine NLo. The Albert Szent-Gyorgyi Papers. Profiles in Science 2010. 29. Mueller H, Szent-Györgyi A. Wet-freeze of muscle. Science. 1957;126:970971.

8. Szent-Györgyi A. Oxidation, energy transfer, and vitamins. Nobel Lecture 1937.

30. Szent-Györgyi A. Electronic biology and its relation to cancer. Life Sci. 1974;15:863-875.

9. Svirbely JL, Szent-Györgyi A. The chemical nature of vitamin C. Biochem J. 1932;26:865-870.

31. Gascoyne PR, Pethig R, Szent-Györgyi A. Electron spin resonance studies of the interaction of oxidoreductases with 2,6-dimethoxy-p-quinone and semiquinone. Biochim Biophys Acta. 1987;923:257-262.

10. McKinnis RB, King CG. THe nature of vitamin C: A study of its electrical transference. J Biol Chem. 1930;87:615-623. 11. Szent-Györgyi A. The identification of vitamin C. Science. 1938;87:214-215. 12. King CG, Waugh WA. The chemical nature of vitamin C. Science. 1932;75:357-358. 13. Svirbely JL, Szent-Györgyi A. A Hexuronic acide as the antiscorbutic factor. Nature. 1932;129:576. 14. Szent-Györgyi A. Biological Oxidation and Vitamins: Harvey Lecture, May 18, 1939. Bull N Y Acad Med. 1939;15:456-468. 15. Krebs HA. The citric acid cycle and the Szent-Györgyi cycle in pigeon breast muscle. Biochem J. 1940;34:775-779.

32. Pethig R, Gascoyne PR, McLaughlin JA, et al. Enzyme-controlled scavenging of ascorbyl and 2,6-dimethoxy-semiquinone free radicals in Ehrlich ascites tumor cells. Proc Natl Acad Sci U S A. 1985;82:1439-1442. 33. Pethig R, Gascoyne PR, McLaughlin JA, et al. Interaction of the 2,6-dimethoxysemiquinone and ascorbyl free radicals with Ehrlich ascites cells: A probe of cell-surface charge. Proc Natl Acad Sci U S A. 1984;81:2088-2091. 34. Otto P, Ladik J, Szent-Györgyi A. Quantum chemical calculations of model systems for ascorbic acid adducts with Schiff bases of lysine side chains: Possibility of internal charge transfer in proteins. Proc Natl Acad Sci U S A. 1979;76:3849-3851.

16. Wilson BA, Schisler JC, Willis MS. Sir Hans Adolf Krebs: Architect of metabolic cycles. LabMedicine. 2010;41:377-380. 17. Szent-Györgyi A. The early history of the biochemistry of muscle contraction. J Gen Physiol. 2004;123:631-641.

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7. Rosenfeld L. Vitamine-vitamin. The early years of discovery. Clin Chem. 1997;43:680-685.