Advertisement

Comparison of four documents describing adrenaline purification, and the work of three important scientists, Keizo Uenaka, Nagai Nagayoshi and Jokichi Takamine

Open AccessPublished:April 28, 2020DOI:https://doi.org/10.1016/j.janh.2020.04.001

      Highlights

      • Keizo Uenaka was the scientist who practically isolated adrenaline as crystal.
      • Adrenalin purification methods released in 1901 was based on Uenaka's hand-written document, “On adrenalin, Memorandum, July to December, 1900”.
      • Nagayoshi Nagai, Jokichi Takamine and Keizo Uenaka had crucial roles in chemical isolation of ephedrine and adrenaline.

      Abstract

      The name of Keizo Uenaka has not been documented in textbooks. However, Uenaka was the scientist who worked on ephedrine and played a practical role in the purification and crystallization of adrenaline. His handwritten memorandum, “On Adrenaline, Memorandum, July to December, 1900” is now stored in a Buddhist temple, Kyougyou-ji in Nashio, Japan.
      In the present report, we compared Uenaka's original description and Jokichi Takamine's published scientific reports, and examined how each statement in four documents are related to each other in terms of successful adrenaline crystallization.
      Uenaka's memorandum contained precise procedures and experimental tips for successful purification. The experimental steps were considered to transcribed in the first published document in The American Journal of Pharmacy by Takamine in 1901, and summarized in another document in ``Journal of Physiology'' in 1901. A Japanese version was published in ``Yakugakuzasshi'' in 1903 by translating the English paper in the American Journal of Pharmacy published in 1901.
      Reading Uenaka's memorandum, we realized that he tirelessly and diligently undertook routine experiments that to some of us might seem boring and laborious. Although the name of Uenaka is not globally well known, he was the main scientist who did the actual work of purifying adrenaline.

      Keywords

      Introduction

      Ephedrine and adrenaline are indispensable drugs for hemodynamic management in anesthesiology practice. Physicians involved in acute medical practices, including anesthesiologists, have significant experience in using these drugs. In the history of pharmaceutical and clinical medicine, Nagayoshi Nagai is recognized as the scientist who purified ephedrine from an oriental herbal medicine, "má huáng", and Jokichi Takamine was credited with developing adrenaline (epinephrine in the United States) from bovine adrenal grands.
      • Ishida M.
      Hormone hunters: the discovery of adrenaline.
      However, the name of Keizo Uenaka has not been documented in textbooks. Uenaka was the scientist who worked on ephedrine and played a practical role in the purification and crystallization of adrenaline (Figure 1). More than 100 years ago, he performed repeated experiments under the supervision of Takamine and finally obtained crystalized adrenaline in 1900.
      • Nakayama S.
      Adrenalin and Keizo Uenaka.
      Uenaka's technique as a chemist was highly sophisticated, and he took special care to prevent the oxidization of adrenaline in the purification process.
      A handwritten memorandum by Uenaka, “On Adrenaline, Memorandum, July to December, 1900” is now stored in a Buddhist temple, Kyougyou-ji in Nashio, Japan.
      • Uenaka K.
      On adrenalin, Memorandum, July to December 1900.
      The Chemical Society of Japan considers this document as historically important evidence of adrenaline purification by Uenaka and Takamine. Also, the National Science Museum of Japan registered this document as part of the cultural heritage of Japan in 2010.
      • Nakayama S.
      The cultural heritage of Japan registered by the National Science Museum of Japan. No. 002: “On adrenalin, Memorandum, July to December 1900” by Keizo Uenaka; the reason why stored in Kyogyouji temple.
      In the present study, the authors traced back the life and works of Uenaka, through survey of historical documents and practical visits to his memorial site. We visited the Buddhist temple "Kyougyou-ji" and examined details of the original document, comparing the statements in the hand-written document
      • Nakayama S.
      The cultural heritage of Japan registered by the National Science Museum of Japan. No. 002: “On adrenalin, Memorandum, July to December 1900” by Keizo Uenaka; the reason why stored in Kyogyouji temple.
      with those in Jokichi Takamine's published papers describing adrenaline purification.
      • Takamine J.
      Adrenalin the active principle of the suprarenal glands and its mode of preparation.
      ,
      • Takamine J.
      The isolation of the active principle of the suprarenal gland.

      Methods

      A stone monument in memory of Uenaka, which is inscribed with his achievements, is located at Kyougyou-ji temple, Nashio-Nishinomiya in Japan. The memorial was established in 1981 by Professor Sosogu Nakayama, who is a physiologist, historian and Buddhist priest at the temple
      • Nakayama S.
      Adrenalin and Keizo Uenaka.
      ,
      • Nakayama S.
      The cultural heritage of Japan registered by the National Science Museum of Japan. No. 002: “On adrenalin, Memorandum, July to December 1900” by Keizo Uenaka; the reason why stored in Kyogyouji temple.
      (Figure 2). Uenaka's handwritten memorandum, “On Adrenaline, Memorandum, July to December, 1900” is stored here.
      • Nakayama S.
      The cultural heritage of Japan registered by the National Science Museum of Japan. No. 002: “On adrenalin, Memorandum, July to December 1900” by Keizo Uenaka; the reason why stored in Kyogyouji temple.
      Fig 2
      Fig. 2Stone monument erected in 1981 in memory of Keizo Uenaka at Kyougyou-ji Temple, Nashio-Nishinomiya in Japan, established in 1981.
      We examined the contents of the memorandum in detail, courtesy of the administrators of Kyougyou-ji. The contents were compared with two English papers published by Jokichi Takamine in 1901,
      • Takamine J.
      Adrenalin the active principle of the suprarenal glands and its mode of preparation.
      ,
      • Takamine J.
      The isolation of the active principle of the suprarenal gland.
      and a Japanese paper published in 1903.
      Watanabe
      Gakuji-ihou: adrenalin purification by Jokichi Takamine method.
      We listed the experimental procedures step by step, linking each step in the four documents and examining the relationship between each statement. We also repeated the purification experiment following the steps written in the Japanese paper published in 1903 (3, Figure 3), using porcine adrenal glands.
      • Saito S.
      • Kamiyama J.
      Adrenaline: isolated 111 years ago by an energetic Japanese scientist.
      Fig 3
      Fig. 3Steps of purification of adrenaline.
      Watanabe
      Gakuji-ihou: adrenalin purification by Jokichi Takamine method.

      Results

      The memorandum, “On Adrenaline, Memorandum, July to December, 1900”, is 10 × 15 cm in size, and consists of 47 pages of hand-written notes (Figure 4). The hand-written letters were translated into modern characters by Yamashita in 1966.
      • Yamashita A
      Adrenaline experiment note.
      Fig 4
      Fig. 4(a) and (b) “On adrenaline, Memorandum, July to December, 1900.
      The earliest entry in the memorandum was on July 20, 1900. Uenaka was given a crude extract of the adrenal gland by Takamine. Although previous descriptions of adrenal extract stated that biologically active substances could not be obtained by alkalization, he suspected that scientists who reported previous data might not have examined the chemical characteristics accurately. On the same day, he added a small amount of sodium hydrate to the extract and obtained crude crystals by the next morning, positive by the Vulpian reaction, which tested bright green in color with FeCl3, suggesting activity of the target substance, as he noted on July 21.
      • Vulpian A.
      Note sur quelques réactions propres à la substance des capsules surrénales.
      He also found that the adrenal gland extract was easily oxidized and changed its color, and noted that prevention of oxidization during experimental steps was important for successful purification.
      Uenaka applied sophisticated chemical techniques mastered in Nagai's laboratory, in which mild acids and alkalis were gradually added for purification. Uenaka diligently repeated the time-consuming experiments day after day, and successfully purified adrenaline from adrenal grands for the first time on around August 5, 1900. Since ephedrine and adrenaline have high structural similarity (Figure 5), Uenaka's previous work at Tokyo university facilitated successful purification of adrenaline in a relatively short period.
      Fig 5
      Fig. 5(a) and (b) Chemical structures of ephedrine (a) and adrenaline (b), respectively, indicating structural similarities.
      Following the first successful purification using watch glasses and a small amount of adrenal gland tissue, Takamine ordered a scale-up of the experiment aiming at industrial business. Although numerical values are very limited in Uenaka's memorandum, they apparently placed repeated orders of 8–23 kg of bovine adrenal glands.
      Takamine did not publish details of the adrenaline purification methods, and instead focused on obtaining a patent and released a simple summary at some academic meetings. The summarized methods without numerical values were published both in English and Japanese (The American Journal of Pharmacy,
      • Takamine J.
      Adrenalin the active principle of the suprarenal glands and its mode of preparation.
      Journal of Physiology
      • Takamine J.
      The isolation of the active principle of the suprarenal gland.
      and Yakugakuzasshi
      Watanabe
      Gakuji-ihou: adrenalin purification by Jokichi Takamine method.
      (Figure 3). The descriptions in The American Journal of Pharmacy and Yakugakuzasshi are almost identical, except for the language. The experimental steps shown in the two English publications are listed in Table 1. All the experimental steps shown in the two documents had compatible statements to the original hand-written document written by Uenaka. Hence, it is considered that Uenaka or Takamine prepared the published documents, on the basis of “On Adrenaline, Memorandum, July to December, 1900”.
      Table 1Comparison of Uenaka’s original description and Jokichi Takamine’s published scientific reports.
      On adrenalin, Memorandum, July to December, 1900 (reference #3)The American Journal of Pharmacy. 73: 523–535, 1901. (reference #5) Yakugakuzassi 260; 1044–1045, 1903 (in Japanese, reference #7)Journal of Physiology (Proceedings of the Physiological Society) 1901; 27: XXIX – XXX. (reference #6)
      Purification steps(original date of description in the "memorandum" and summarized experimental note)purification stepadditional comments
      1July 20th: Extracting in water.

      July 30th: Steeping in 75–85° water for 8hours.
      Suprarenal capsules finely disintegrated by suitable means, are steeped in water or acidulated water for a period of about five hours at a temperature varying from 50–80° centigrade, with frequent agitation and with the addition of water as it evaporates.Tips #1: As the active principle of the glands is prone to absorb the oxygen from the air, to form inactive substance, it is necessary to avoid exposure of the liquid to the air as much as possible.Disintegrate the suprarenal glands of sheep and oxen, extracting them in water.
      2July 30th: Press and filter to separate lipid.

      September 10th: Keep at 60° water for 6hours, then heat to 95° to coagulate proteins.

      September 30th: boil in 80° for 12hours and heat up to 90–95° at the end to coagulate proteins.
      The temperature of the mass is now raised from 90 to 95° centigrade for the period of one hour so as to coagulate as much albuminoid as possible. (with tips #1–3)Tips #2: A layer of fat floating on the surface of the mass acts very conveniently for this purpose and at the same time it has an effect of retarding evaporation of water as well.Rendered weakly acid by the addition of a few drops of acetic or hydrochloric acid, at 95 °C
      3September 11th: Press to separate lipid.

      September 30th: Press and separate solution.
      The mass is now pressed and separated from the liquid portion which contains the active principle. (with tips #4)Tips #3: Other methods of preventing oxidation may be employed at this stage, such as conducting the steeping process in an atmosphere of carbonic acid gas.The solid residue is pressed and reextracted.
      4September 30th: Concentrate in vacuum.

      October 20th: The squeezed solution is evaporated in vacuum.
      The clear extract is now evaporated in a vacuum pan to a suitable strength.Tips #4: The mass is again steeped for hours in warm water slightly acidulated with acetic or hydrochloric acid, in order to extract the residual amount of active principle. The liquid separated from the mass is now added to the first extract and allowed to separate from the oil.The liquid is filtered and then concentrated by evaporation.
      5July 30th: Add triple volume of 95% alcohol.

      September 11th: Add 95% alcohol to make 63% alcohol solution.

      September 30th: Add alcohol to precipitate inorganic salts.

      October 10th: Removal of inorganic salts with alcohol should not be skipped for successful purification.
      To this concentrated solution about two to three times its own volume of strong ethyl alcohol is added or more economically wood spirit, which will precipitate both inert organic and inorganic substances. The inert substances thus separated are washed with alcohol so as to free them from the active principle.Alcohol is then added until no further precipitation occurs.
      6September 30th: The filtrate is evaporated in vacuum.The alcohol solution is now evaporated preferably in vacuum still, whereby the alcohol used is duly recovered.The filtrate is then evaporated in vacuo
      7July 20th: Alkalization with sodium hydrate.

      September 11th: Add sodium hydrate and ammonia to obtain crystal.

      September 30th: Precipitate crystal with sodium hydrate and ammonia.

      October 20th: Precipitation can be achieved solely with ammonia, without use of sodium hydrate and ammonium chloride.

      November 6th: Precipitation method with ammonia is more effective than the method with sodium hydrate and ammonium chloride.
      To the residual liquid, ammonia is now added until the solution gets distinctly alkaline and left over for several hours. (Tips #5)Tips #5: Instead of using ammonia, sodium hydrate may be used as a precipitant, but care must be taken not to use in excess, which redissolves adrenalin. In order to counteract caustic alkali, ammonium chloride or carbonic acid may be conveniently used.Treat with ammonia or ammonium chloride or sodium hydrate until the solution is distinctly alkaline.
      8August 11th: Adrenalin crystal is easily combined with phosphate salts.

      September 30th: Wash and dry with alcohol in vacuum.
      A yellow brownish precipitate will be formed which is the crude adrenalin in a basic form. (Tips #6)Tips #6: The impure adrenalin usually precipitates in a light yellow brownish tomato-shaped form, but not infrequently in needles. The former is an agglomeration of needle crystals and is more or less contaminated with coloring matters and some inorganic substance, chiefly phosphates.Adrenalin crystallizes out in the course of a few hours.
      9July 21th: Adrenalin crystal can be dissolved in weak acetic acid, not in water, alcohol or ether.

      August 4th: Ether is added to remove lipid, and add sodium hydrate carefully for alkalization to facilitate crystallization.

      August 4th: Adrenaline crystal is easily dissolved in weak sulfuric acid, not in water and alcohol.

      August 5th: Adrenaline crystal is easily dissolved in weak acid and carbonated water.

      August 13th: Crude crystal can be dissolved in acetic acid and precipitated with lead acetate, or crystalized by alkalizing with ammonia.

      September 19th: For further purification, crude crystal is dissolved in acetic acid, and then 5–6 times volume 95% alcohol is added. Following precipitation of inorganic salts, the filtrated solution is alkalized with ammonia.

      November 12: For further purification, crude precipitate is re-dissolved in weak acetic acid and precipitated with ample volume of alcohol, then half volume of ether is added. Adrenaline is precipitated by adding ammonia to the filtrate. The precipitate is again re-dissolved in acetic acid and re-precipitated with ammonia. The obtained crystal is washed and dried.
      The precipitate is now filtered, washed with water and dried.Further purification: The crude adrenalin is dissolved in acid and alcohol and ether is added to a sufficient quantity. A brown colored precipitate is produced which chiefly consists of coloring matter and inorganic impurities. The precipitate is separated both by decantation and filtration. The filtrate is now treated by one of the above mentioned processes, when white crystalline precipitate of adrenalin will be obtained. It is quickly filtered, washed with water and then with alcohol and dried. The process of purification may be repeated, if desired, two or three times.Adrenalin may be purified by dissolving in acid and re-precipitating.
      For confirmation, we re-examined the methods using porcine adrenal glands, and reported the details in a Japanese journal.
      • Saito S.
      • Kamiyama J.
      Adrenaline: isolated 111 years ago by an energetic Japanese scientist.
      We repeated the methods roughly listed in the Japanese Pharmaceutical Journal,
      Watanabe
      Gakuji-ihou: adrenalin purification by Jokichi Takamine method.
      by using 20 kg fat covering the kidneys which contained 179 g porcine adrenal gland tissue (bovine adrenal grands are relatively difficult to obtain, because of Bovine Spongiform Encephalopathy-related strict regulations), and obtained 35.6 ng of a mixture of adrenaline and noradrenaline crystals. According to Uenaka's memorandum, his extraction ratio from wet adrenal glands on September 19, 1900 was 3.08 × 10−2%, while our extraction ratio was 1.99 × 10−8%. Through our experiments, we recognized Uenaka's outstanding ability as a practicing chemist. We were also able to reconfirm several comments mentioned in Uenaka's memorandum, such as "the tissues surrounding the adrenal gland are very rich in fat", "re-squeezing of tissues and repeated precipitation of crude solvents are effective in increasing the harvest ratio", and "prevention of oxidization using a surface lipid layer or carbon dioxide gas insufflation is important to obtain active precipitate". These cautious approaches seem to be based on Uenaka's knowledge as a chemist and his experience in ephedrine purification in Nagai's laboratory. Apparently, his craftsmanship contributed largely to successful adrenaline purification.

      Discussion

      Personal history of Keizo Uenaka and adrenaline purification

      Keizo Uenaka was born June 29, 1876, in Nashio-Nishinomiya, Japan. His father, Jihei Uenaka, managed a paper company. Keizo moved to Osaka in 1886 and learned chemistry and English for 3 years at Taiseigakkan school. Following his education, he started to work at a local medicine wholesale shop, Ishizu-Shouten, located at Doshou street in Osaka.
      • Nakayama S.
      Adrenalin and Keizo Uenaka.
      ,
      • Saito S.
      Keizo Uenaka: the main researcher in adrenaline purification.
      In 1891, he entered Osaka pharmaceutical school (currently Faculty of Pharmaceutical Science, Osaka University), and obtained an authorized pharmacist license. Further, he enrolled in a Pharmaceutical Specialty Course at Tokyo University Faculty of Medicine, and studied under the supervision of Nagayoshi Nagai. He completed the two-year course and worked another 8 months with Professor Nagai. After his work with Nagai, he was recruited by Tokyo Hygiene Laboratory, and worked on tetrodotoxin and environmental pollution at Ashio copper mine.
      In December 1899, Uenaka moved to the United States from the international port at Yokohama, with a reference prepared by Dr. Tamemasa Horiwa at Tokyo University Faculty of Science. He visited Jokichi Takamine on 109th street, Central Park West boulevard in New York on February 6th 1900, and started laboratory experiments using bovine adrenal grand extract on July 20, 1900.
      • Iinuma K.
      • Sugano T.
      The Life of Jokichi Takamine, the truth of adrenalin discovery: Asahi Sensho.
      Following the successful purification of adrenaline, Uenaka was employed by Park Davis Inc. and the then Takamine Farmament Inc. in the United States as an executive member, and worked on a Taka-diastase related project. In 1916, he moved back to Japan and was employed by Sankyo Pharmaceutical Inc., which was established by Matasaku Shiobara in 1916 and presided by Takamine, as the first chief executive officer. At Sankyo, Uenaka managed Taka-diastase, adrenaline and bakelite-related industrial projects. Further, he was given the role of auditor of the company, and retired from the company in 1933. He died on January 11, 1960, at the age of 83 years.
      • Saito S.
      • Kamiyama J.
      Adrenaline: isolated 111 years ago by an energetic Japanese scientist.
      ,
      • Saito S.
      Keizo Uenaka: the main researcher in adrenaline purification.

      Uenaka's supervisor, Nagayoshi Nagai, and ephedrine

      Nagayoshi Nagai was born in Tokushima Japan in 1845, and was the son of a traditional medical doctor. He had the opportunity to learn about western medicine at the German Medical School of Nagasaki in 1864. Although Japanese society was under drastic cultural changes from a medieval samurai era to a modern imperial nation, Nagai continued his scientific studies at Tokyo Imperial University. He became the first doctor of pharmaceutical science in Japan and was sent to the University of Berlin under government sponsorship. In Berlin, he completed a doctorate in 1873 with a study on eugenol, while working as an assistant in August Wilhelm von Hofmann's laboratory. Nagai returned to Japan in 1883 and first isolated ephedrine from Ephedra vulgaris in 1885 (Figure 5(a)). He recognized ephedrine as being the active constituent of the traditional Chinese herbal medicine, má huáng (Ephedra sinica).
      • Kaneo S.
      Nagai Nagayoshi den.
      ,
      • Nohara H.
      Konohito Nagai Nagayoshi.
      Although Nagai himself did not study the biological action of this substance in detail, his followers completed the synthesis and structural elucidation of ephedrine in 1929.
      • Kaneo S.
      Nagai Nagayoshi den.
      Following their work, ephedrine was established as a sympathomimetic amine commonly used to treat the hypotension associated with nerve blocks and anesthesia.
      • Ockerblad N.F.
      • Dillon T.G.
      The use of ephedrine in spinal anesthesia: preliminary report.
      ,
      • Dripps R.D.
      • Deming M.V.
      An evaluation of certain drugs used to maintain blood pressure during spinal anesthesia: comparison of ephedrine, paredrine, pitressin-ephedrine and methedrine in 2500 cases.
      Doctors in the field of internal medicine have also variably categorized ephedrine as a bronchodilator, decongestant and appetite suppressant.
      • Chen K.K.
      • Schmidt C.F.
      The action of ephedrine, the active principle of the Chinese drug, Ma Huang.
      Nagai obtained a position at Tokyo Imperial University, where he went on to become a Professor of Chemistry and Pharmacy in 1893. After the isolation of ephedrine, his research focused on the chemical extraction and analysis of various Japanese and Chinese traditional herbal medicines.
      • Nohara H.
      Konohito Nagai Nagayoshi.
      Keizo Uenaka was one of the scientists working on such chemical purification. Nagai established the Pharmaceutical Society of Japan (PSJ) and became the first president of the authorized society. In honor of his achievements and contributions to Japanese Chemistry and Pharmaceutical Science, he is still known as the “father” of this field. Statues of Nagai can be seen in the entrance hall of the PSJ office building and at Tokushima University. His name still appears on the ampoules of ephedrine sold in Japan, to commemorate his name.

      Jokichi Takamine and isolation of adrenaline with Keizo Uenaka

      Jokichi Takamine was born in the small town of Takaoka in Japan in 1854.
      • Iinuma K.
      • Sugano T.
      The Life of Jokichi Takamine, the truth of adrenalin discovery: Asahi Sensho.
      ,
      • Yamashima T.
      Jokichi Takamine (1854–1922), the samurai chemist, and his work on adrenalin.
      ,
      • Mille A.D.
      Where the wings grow.
      At the age of 12 years, he began to study European science in Nagasaki, and at the age of 24, he was sent by the government to study technology at the University of Glasgow in the United Kingdom. After completing his studies there, he worked on chemical industrial business with the Japanese government and moved to the United States in 1890, with a contract with Whiskey Trust Inc. Takamine first worked in Chicago and then in Peoria, Illinois, applying the Japanese sake-making process to the beer and whiskey industry by substituting the fungal starch-digestive enzyme with malt. Distressed by a mysterious fire in his laboratory and a conflict with local distillery manufacturers, however, he was forced to give up his fermentation business.
      • Kawakami K.K.
      • Finley J.H.
      Jokichi Takamine: a record of his American achievements.
      Takamine isolated the yeast enzyme diastase, and in 1894 he was granted a patent titled “Process of Making Diastatic Enzyme” (US Patent 525,823), the first patent on a microbial enzyme in the United States He licensed diastase to Parke-Davis of Detroit under the brand name Taka-diastase. Probably because there was a large population suffering from abdominal discomfort following a carbohydrate-rich diet, this medicine, which contains a highly effective digestive enzyme, sold very well, and Takamine became a big success in the pharmaceutical industry.
      In 1895, George Oliver and Edward Schäfer discovered that when adrenal gland secretions were injected into experimental animals, the animals’ blood pressure increased.
      • Bennett M.R
      Historical review: one hundred years of adrenaline: the discovery of autoreceptors.
      Parke-Davis encouraged Takamine to work on the isolation and purification of this vasoactive substance from the adrenal gland. Purification of the substance became a matter of great interest. In 1897, John Jacob Abel in the United States, Furth in Germany and Schafer and Moore in Britain all aimed to be the first to crystallize the substance.
      • Davenport H.W.
      Historical Articles: epinephrin(e).
      Abel thought that he had succeeded when he obtained a crystalline active, and named it epinephrine. (It was later shown that Abel had isolated a benzoyl derivative rather than the pure adrenaline).
      • Abel J.J.
      Chemistry in relation to biology and medicine with especial reference to insulin and other hormones.
      Meanwhile, using the profits from Taka-diastase, Takamine hired a young chemist from Japan, Keizo Uenaka, to assist him. Uenaka moved to the U.S. after completing his education in chemistry under Nagayoshi Nagai. With the help of Uenaka's logical perspective and careful experiments, Takamine and Uenaka successfully isolated and purified the active substance from the adrenal gland in July-August 1900
      • Uenaka K.
      On adrenalin, Memorandum, July to December 1900.
      (Figure 5(b)).
      Although Uenaka was the main scientist and technician in adrenaline purification, his name is not mentioned as an author in scientific meetings nor documented explicitly in publications. His name was only shown in the November 1901 issue of the American Journal of Pharmacy as Mr. Wooyenaka, as Takamine's associate who energetically assisted Takamine in his accomplishment.
      • Takamine J.
      Adrenalin the active principle of the suprarenal glands and its mode of preparation.
      This might be explained by the fact that Takamine was more focused on industrial applications than pure science. Although Takamine did not openly announce that Uenaka was the main chemist who actually successfully performed adrenaline purification, he treated Uenaka very cordially in his and his related companies throughout his life. Also, Uenaka did not complain about the fact that his name was not formally noted in scientific descriptions as a key person in the purification of adrenaline.
      • Saito S.
      Keizo Uenaka: the main researcher in adrenaline purification.
      ,
      • Iinuma K.
      • Sugano T.
      The Life of Jokichi Takamine, the truth of adrenalin discovery: Asahi Sensho.
      Takamine used the royalties from adrenaline and Taka-diastase to enlarge his pharmaceutical business. Together with Matasaku Shiobara, he expanded the pharmaceutical company Sankyo Inc. in Tokyo in 1913.
      • Iinuma K.
      • Sugano T.
      The Life of Jokichi Takamine, the truth of adrenalin discovery: Asahi Sensho.
      In his later life, Takamine made efforts to improve the position of Japanese immigrants in the U.S. He was a key person in the newly founded “Nippon Club”, a Japanese Society in the U.S. He also tried to foster better relations between Americans and Japanese people. In 1909, Takamine funded a gift of 2000 cherry trees to the city of Washington, D.C., to decorate the Tidal Basin area around the Potomac River. This symbol of Japanese–American friendship has become a tourist attraction.
      • Iinuma K.
      • Sugano T.
      The Life of Jokichi Takamine, the truth of adrenalin discovery: Asahi Sensho.
      ,
      • Mille A.D.
      Where the wings grow.

      Further researches on adrenaline after its purification

      Takamine named the substance “adrenaline” and filed a patent application for his discovery on November 5, 1900. Thomas Aldrich published the correct chemical structure as C9H13NO3 in 1904,
      • Aldrich T.B.
      Is adrenalin the active principle of the suprarenal gland?.
      and Parke-Davis began marketing the product under the trade name Adrenalin. The use of adrenaline rapidly became widespread in surgery, where it was used to control hemorrhage. Also, the discovery of adrenaline enjoyed medical and popular attention; a well-known champion boxer always kept adrenaline on hand when he went into the ring to treat minor bleeding and swelling after being punched.
      • Kawakami K.K.
      • Finley J.H.
      Jokichi Takamine: a record of his American achievements.
      Following its isolation, the physiological and pharmacological actions of adrenaline (also known as epinephrine) were established as a hormone and neurotransmitter that increases heart rate, constricts blood vessels and dilates air passages.
      • Davenport H.W.
      Early history of the concept of chemical transmission of the nerve impulse.
      Subsequently, adrenaline was applied in cardiology, obstetrics and the treatment of asthma and other allergies. In the 1920s, Cannon WB et al. proposed that adrenaline is the main activator of the sympathetic nervous system.
      • Cannon W.B.
      • Uridil J.E.
      Studies on the conditions of activity in endocrine glands. VIII Some effects on the denervated heart of stimulating the nerves of the liver.
      Further biochemical studies revealed the chemical structure and biosynthetic pathway of adrenaline, as a catecholamine, a monoamine produced only by the adrenal glands from the amino acids phenylalanine and tyrosine.
      • Davenport H.W.
      Historical Articles: epinephrin(e).
      The differentiation of adrenaline and noradrenaline was established in the 1940s, and it was reported that the original adrenaline purified by Uenaka and Takamine probably contained approximately 36% noradrenaline.
      • Euler U.S.V.
      A specific sympathomimetic Ergone in Adrenaergic Nerve Fivebers (Sympathin) and its relations to Adrenaline and Noradrenaline.
      After Takamine's death (1922), controversy emerged over who was actually the first to purify adrenaline. John Jacob Abel persisted in the belief that he was the first and that Takamine's product was not pure.
      • Aronson J.K.
      Where name and image meet” - the argument for “adrenaline”.
      Almost 100 years after Takamine and Uenaka isolated adrenaline, the debate was settled and Takamine and Uenaka's claim was confirmed.
      • Davenport H.W.
      Early history of the concept of chemical transmission of the nerve impulse.
      ,
      • Parascandola J.
      The Allergy Archives. Pioneers and Milestones: abel, Takamine, and the isolation of epinephrine.
      Adrenaline was first synthesized in the laboratory by Friedrich Stolz and Henry Drysdale Dakin, each working independently, in 1904, which further expanded its market.

      Conclusion

      Uenaka was the scientist who worked on ephedrine and played a practical role in the purification and crystallization of adrenaline.
      Many Japanese anesthesiologists working in the field of medical research are proud of Takamine and Uenaka, who accelerated progress in medical science and whose endeavors have helped countless people world-wide, and are especially proud of Uenaka for not aiming for heroism. Reading his memorandum, we realize that day after day, he tirelessly and diligently undertook routine experiments that to some of us might seem boring and laborious. Adrenaline purification stories truly remind us that "There is no royal road to learning".

      Financial disclosure

      This work is financially supported by Gunma University to Shigeru Saito. This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

      Declaration of Competing Interest

      All authors do not have any actual or potential conflict of interest including any financial, personal or other relationships with other people or organizations within three years of beginning the submitted work that could inappropriately influence, or be perceived to influence, their work.

      Acknowledgement

      The work described has not been published previously, is not under consideration for publication elsewhere. And its publication is approved by all authors and tacitly or explicitly by the responsible authorities where the work was carried out. If accepted, it will not be published elsewhere in the same form, in English or in any other language, including electronically without the written consent of the copyright-holder.

      References

        • Ishida M.
        Hormone hunters: the discovery of adrenaline.
        Kyoto University Press, Kyoto2018: 1-203 (chapter 5)
        • Nakayama S.
        Adrenalin and Keizo Uenaka.
        Igakunoayum. 1987; 140 ([in Japanese]): 22
        • Uenaka K.
        On adrenalin, Memorandum, July to December 1900.
        Kyougyou-ji in Nashio. 1900 (Japan. [Japanese, hand-written document])
        • Nakayama S.
        The cultural heritage of Japan registered by the National Science Museum of Japan. No. 002: “On adrenalin, Memorandum, July to December 1900” by Keizo Uenaka; the reason why stored in Kyogyouji temple.
        Kagaku-to-Kogyo. 2010; 63 ([in Japanese]): 558-559
        • Takamine J.
        Adrenalin the active principle of the suprarenal glands and its mode of preparation.
        Am J Pharm. 1901; 73: 523-535
        • Takamine J.
        The isolation of the active principle of the suprarenal gland.
        J Physiol (Proc Physiol Soc. 1901; 27: 29-30
        • Watanabe
        Gakuji-ihou: adrenalin purification by Jokichi Takamine method.
        Yakugakuzassi. 1903; 260 ([in Japanese]): 1044-1045
        • Saito S.
        • Kamiyama J.
        Adrenaline: isolated 111 years ago by an energetic Japanese scientist.
        Jokichi Takamine. Masui. 2011; 60 ([in Japanese]): 1331-1341
        • Yamashita A
        Adrenaline experiment note.
        Kagakushi-kennkyuu. 1966; 79 ([in Japanese]): 143-148
        • Vulpian A.
        Note sur quelques réactions propres à la substance des capsules surrénales.
        Comp Rend l'Acad Sci. 1856; 43 ([in French]): 663-665
        • Saito S.
        Keizo Uenaka: the main researcher in adrenaline purification.
        J Jpn Soc Clin Anesthes. 2012; 32 ([in Japanese]): 573-581
        • Iinuma K.
        • Sugano T.
        The Life of Jokichi Takamine, the truth of adrenalin discovery: Asahi Sensho.
        Asahi-shinbun publication Inc., Tokyo2000 ([in Japanese])
        • Kaneo S.
        Nagai Nagayoshi den.
        The Pharmaceutical Society of Japan, Tokyo1960 ([in Japanese])
        • Nohara H.
        Konohito Nagai Nagayoshi.
        Human-creative Inc., Tokyo2008 ([in Japanese])
        • Ockerblad N.F.
        • Dillon T.G.
        The use of ephedrine in spinal anesthesia: preliminary report.
        J Am Med Assoc. 1927; 88: 1135-1136
        • Dripps R.D.
        • Deming M.V.
        An evaluation of certain drugs used to maintain blood pressure during spinal anesthesia: comparison of ephedrine, paredrine, pitressin-ephedrine and methedrine in 2500 cases.
        Surg Gynecol Obstet. 1946; 83: 312-322
        • Chen K.K.
        • Schmidt C.F.
        The action of ephedrine, the active principle of the Chinese drug, Ma Huang.
        J Pharmacol Exp Ther. 1924; 24: 339-357
        • Yamashima T.
        Jokichi Takamine (1854–1922), the samurai chemist, and his work on adrenalin.
        J Med Biogr. 2003; 11: 95-102
        • Mille A.D.
        Where the wings grow.
        Doubleday & Co., Westminster, MD1978 ([translated in Japanese by Yamashita A. Takamine Jokichi den -shofuden no kaisou-. Tokyo: Yushodo printing Inc.; 1991.])
        • Kawakami K.K.
        • Finley J.H.
        Jokichi Takamine: a record of his American achievements.
        William Edwin Rudge, New York, NY1928 (La Vergne: Kessinger Publishing's rare reprints; 2010)
        • Bennett M.R
        Historical review: one hundred years of adrenaline: the discovery of autoreceptors.
        Clin Autonom Res. 1999; 9: 145-159
        • Davenport H.W.
        Historical Articles: epinephrin(e).
        Physiologist. 1982; 25: 76-82
        • Abel J.J.
        Chemistry in relation to biology and medicine with especial reference to insulin and other hormones.
        Science. 1927; 66 (337-346): 307-319
        • Aldrich T.B.
        Is adrenalin the active principle of the suprarenal gland?.
        Am J Physiol. 1902; 7: 359-368
        • Davenport H.W.
        Early history of the concept of chemical transmission of the nerve impulse.
        Physiologist. 1991; 34 (178-190): 129
        • Cannon W.B.
        • Uridil J.E.
        Studies on the conditions of activity in endocrine glands. VIII Some effects on the denervated heart of stimulating the nerves of the liver.
        Am J Physiol. 1921; 58: 353-364
        • Euler U.S.V.
        A specific sympathomimetic Ergone in Adrenaergic Nerve Fivebers (Sympathin) and its relations to Adrenaline and Noradrenaline.
        Acta Physiolo Scand. 1946; 12: 73-97
        • Aronson J.K.
        Where name and image meet” - the argument for “adrenaline”.
        BMJ. 2000; 320: 506-509
        • Parascandola J.
        The Allergy Archives. Pioneers and Milestones: abel, Takamine, and the isolation of epinephrine.
        J Allergy Clin Immunol. 2010; (Feb:): 514-517