Anatoly Bezkorovainy
Rush-PresbyterionSt. Luke's Medical Center Chicago, Winois 60612
Both camosine and camitine are widely distributed in various animal tissues; however, the function of carnitine has been elucidated only recently and that of camosine remains yet to be determined. Carnitine (I) was discovered in 1906, but i t was not until the late 1940's and early 1950's that its identity with vitamin BT,an insect growth factor was established by Fraenkel and coworkers (I). The physiological function of carnitine is now well understood: I t acts as a means to transport fatty acids from the cytoplasm into the mitochondria. where the fatty acids are oxidized to yield chemical energy. Each fatty acid appears to require a-specific enzyme for the conversion into its carnitine derivative, and the camitine palmitoyl transferases have so far been the best characterized of such enzymes (2.3). B
(CHAN-CHrCH-as-COOH
I
OH (0 camitine
The biological precursor of carnitine has been shown to be lysine in both the Neurospora microorganism (4) and the rat (5). Lysine is first methylated a t the c-nitrogen atom, the first two carbon atoms of this compound are then lost to yield y-butyrobetaine, and the latter is finally hydroxylated in the 3-position to yield carnitine. A metabolic disease characterized by a deficiency of muscle carnitine palmitoyl transferase has recently been described (6). The patient also had myoglobinuria, which was mnsidered to be secondary to the muscle palmitoyl transferase defect. Carnosine, a dipeptide consisting of bistidine and p-alanine (11) was discovered before camitine, though there is yet no idea as to its precise function in the vertebrate organism. It is primarily a constituent of the muscle, and suggestions have been made that i t functions in a buffering capacity, that i t may have an effect on muscle contraction, or that it participates in transphosphorylation reactions (7). I t is well established, however, that camosine is metabolized by a specific enzyme called carnosinase (8), and several cases of a hereditary disease characterized by carnosinase deficiency have been reported (9). Camosinase also cleaves a related dipeptide called anserine (III), and patients stricken with the carnosinase deficiency excrete increased amounts of both carnosine and anserine in their urines. 0
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Carnosine, Carnitine, and Vladimir Gulevich I t is thus clear that both camitine and camosine are compounds that are extremely important in biochemistry and medicine, even though the precise function of one of them (carnosine) is not yet known. The man who, with his students, is responsible for the discovery of these two compounds, as well as for the elucidation of a number of other problems in the biochemistry of nitrogenous compounds, was Vladimir Gulevich, a professor of biochemistry a t Moscow University. He was born in the city of Riazan (USSR) in 1867. He graduated from the medical school of Moscow University in 1890 and obtained his Doctor of Medicine degree in 1896. After a pilgrimage to Western European laboratories, as was then the custom of young Russian scientists, Gulevich was appointed to the faculty of Kharkov University in 1899, and in 1901 moved to Moscow where he remained to his death in 1933. Gulevich also organized the biochemistry department of the Moscow Women's Medical College, and taught organic chemistry a t the Moscow Commercial Institute. He was, prior to World War I, an editor of HoppeSevler's Zeitschrift . .fiir Phvsioloeische Chemie. a leadine in&mational journal of biochemical research. Gulevich's soiourn in forelen laboratories resulted in a number of pubiications, the most important of which, by today's standards, was probably the report on his attempt to define the specificity of proteolytic enzymes using small molecular weight substrates (10). He was not generally successful in this endeavor simply because the basic structure of proteins was not yet firmly established and because of the unavailability of the appropriate small mw lecular weight compounds. He tested the following mmDounds for their abilitv :(I be o l i t bv. twosin: .. ~henvlethvl ether, biuret, acetanifide, sulianilic acid, hippuric acid, salol, diphenylurea, diphenylthiourea, acetylsalicylic acid, salicylic acyl anilide, acetyl-p-0-ethyl aminophenol, Nethylaniline, acetylphenylhydrazine, and N,O-diacetylaminophenol. Only the latter was split by trypsin, though it is not clear whether the ester, the peptide bond, or both, was broken. The occurrence of arginine in animals was also extensively investigated by Gulevich. He isolated arginine from the herring testicle, and characterized both the free amino acid and a number of its derivatives. Its molecular weight. determined cryoscopically, was 166-185; hence it f i t t h e formula CeHlrN40z. It melted with decomposition a t 2(n°C, and had a specific rotation of +9-10' a t the D-line of sodium. At fist he felt that his preparation was a stereoisomer of arginine isolated from plants by Schultze and Steiger, but upon recalculating his optical rotation data he found that the two compounds were identical. He later discovered arginine in the spleen of the ox, thus being the first to show the presence of arginine in higher animals (11). He also implied that arginine was important in the formation of urea by the mammalian organism (12). Gulevich's most productive period came after he had returned to Russia, first to Kharkov University (18991901), then to Moscow University. In 1900 he and his student Amiradzibi described a new compound which they had isolated from meat extracts and which they called carnosine (13). As a nitrate, camosine had a specific rotation of +22.3", a melting point of 211-212"C, and gave an ~
~
~
~~~
. - -
empirical formula of CSHMN~O~.HNOS. The free hase was prepared from the nitrate, and was found to he stmngly basic with a decomposition point of 239'C. Other derivatives of camosine prepared by Gulevich were its silver and copper complexes. The silver complex had an empirical formula of CgHllN403.Ag20 and a decomposition point of 195"C, whereas the copper complex had a formula of either CsHlrNr03.CuO or CsH16N403.CuO with a decomposition point of 220°C. The structure of camosine was also established by Gulevich (14). By alkaline hydrolysis of the latter, he was ahle to isolate histidine as a silver salt and as a free amino acid. Its identity was deduced from the melting point of 253-254°C and itsempirical formula of C~H9N.102.BY difference, the other component of camosine d s deduced to he alanine, and Gulivich proposed that camosine was either histidyl alanine or alanyl It is todav known that camosine is beta-alanvl histidine. -~~~~~ histidine (11). Another hioloeical base isolated hv Gulevich and his students was carkitine, which was prepared as the chloroplatinate salt from the by-products of carnosine isolation (15). The salt had a melting point of 214-218°C and an empirical formula of C I ~ H ~ Z N Z O ~ C IThe S P ~free . hase was assumed to have a formula of CTHINO~. . ... . Further work on the composition of muscle extracts was carried out hv Gulevich's associatt. at Moscow Lniversitv, R. ~ r i m h e r i ( l 6 ) . In order to dispose of the possibility that both carnosine and camitine were artefacts producted by putrefication or proteolysis, Krimherg prepared his extracts within a half hour of the animal's death. He found hoth camosine and carnitine in such extracts and, in addition, also found methyl-guanine that had been discovered simultaneously hy Gulevich and Kutscher in 1906. Krimherg was ahle to obtain 5.8 g of pure carnosine fmm 4.5 kg of meat (i.e., 1.3% of the total muscle tissue), whereas creatine made up 1.5-2.0% of the total weight of muscle tissue. The correct structure of camitine was also determined by Krimberg and one can, as Fraenkel did (I), only marvel a t this man's ability to come up with the structure from the meager data available to him. Krimherg had gathered the following information (17, 18): Carnitine, for which various metal complexes were available (C,HlsNO3.2HgClz; C14H3~N206C16Pt;C T H I J N O ~ HC1.6HgC12; and C7Hl6NO3.AuC14) was known to he a 7-carbon compound. Upon heating in a sealed tube with water a t high temperatures or in the presence of harium hydroxide, the free hase yielded trimethyl amine that was identified by its odor and its auric chloride complex (C3HloN.AuCl4). In addition, a barium salt of an organic acid was also isolated. This indicated that camitine had a quaternary nitrogen as well as an acidic group. The acidic group accounted for two of the three oxygen atoms present on the camitine molecule. The thud oxygen atom had to he in the form of a hydroxyl group; and, moreover, since camitine rotated plane polarized light, the hydroxyl group was present on an asymmetric carbon atom. He concluded that the structure of camitine was similar to that of hetaine, and placed (correctly) the hydroxyl group on C-3 of camitine, though he expressed some uncertainty regarding this decision. Krimherg's structure is given in (N), which represents an internal salt of carnitine. ~
/O (CH,,),iN 'cH,-CH.OH-CH,
I
03
(N)Krirnberg'a caroitlnc
In a later publication (19), Krimberg identified the above barium salt as that of crotonic acid. He was also ahle to reduce camitine to y-trimethylhutyrohetaine without proving, however, the position of the hydroxyl group. The discovery of camitine is said to have been accom-
plished simultaneously by Gulevich and Krimherg, on the one hand, and by Kutscher of Marburg University on the other (I). Kutscher named his compound novain and gave i t a formula depicted in (V). Immediately thereafter, Krimherg suggested that camitiie and novain were identical, hut Kutscher refused to accept this. Eventually, Krimherg was ahle to show conclusively that camitine and novain were identical (20): A number of metal complexes of camitine and novain had identical melting points and crystal structures, and the specific rotations of hoth in the hydrochloride form were also identical (near -21"). Kutscher and Gulevich had another point of controversy, where Kutscher, some years after Gulevich and Amiradzihi's report on the isolation of camosine, reported on the isolation of a "new" hase from the muscle, which he called ignotin. Gulevich immediately recognized it as being identical to carnosine (21). and thus elicited a vigoroui response from Kutscher in the form of several ar$cles entitled, "Zur Abwehr." Kutscher believed that carnosine and imotin were. a t the most. isomers of each other. I t is t&ay well settled that camitine and novain, on the one hand. and camosine and ienotin, on the other. are identical. C&-C&CH,-CH
I
/OH
O 'H
(CH,),N-OH
(v)Kutaehcr's novaln (carnitine) Some 20 years after the discovery of camitine, when Krimherg was a t the Riga University Physiological Institute in the then independent Latvia, one of his coworkers, S. A. Komarov, discovered that muscle extracts, when injected into an animal, would bring about hoth the gastric and intestinal secretions (22). Creatine and creatinine had no such effect, whereas carnosine, carnitine, and methylguanine did. Krimherg thereupon formulated a comprehensive theory to the effect that these bases were digestive hormones similar to secretin and gastrin which, instead of, or in addition to the vagal system, were responsible for the elaboration of appetite juice and the continuing activity of the secretory glands in the digestive tract (23). Komarov eventually moved to Canada to work with Babkin, and Krimherg did not further elaborate on his theory. The effect of the three bases from muscle extracts on the secretion of digestive hormones remains yet to he elucidated. The Soviet encvclonedia. "Bol'shaia Souetskaiia Entsiklopediia," (1957 edition) claims that anserine was also discovered in Gulevich's laboratory hy N. F. Tolkachevskaia in 1929. The credit for the dis&very of anserine is generally given to Ackermann and coworkers (24)and neither they, nor any of the papers (25, 26) immediately following the Ackermann et al. work, make any reference to T o k a chevskaia. Neither could any record to this effect he established by searching through the 1929-1930 issues of Index Medicus, The question regarding the contribution of Gulevich's laboratory to the discovery of anserine must thus remain open. As a concluding remark, i t may he stated that Gulevich was a distinguished biochemist, who was responsible for the discoverv of several hioloeicallv" imnortant bases and . who inspired some extremely capable students and associates to continue their investiaations of the role of nitmaenous compounds in the living organism.
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Literature Cited (1) Racokei, G., in "Recent Research on Carnitine," (Editor: Waif, G I , The MIT Press, Cambridge, 1965.p. 1. (2) Bmrnan.J.T..Kopec, B.. andFtit%,I.B.,J Biol Chem., 248,4075 119731. (3) K o p c , B., and Fritz, 1.B., J B i d Chem.. 248,4069 (1973). ( I ) Horne.D. W..endBmquist. H.P.,J. Biol Chrm.. 218.2170(1973). (51 Tauphabitr, V., and Bmquiat, H.P., J B i d Chem., 248,2176 (1973). (6) DiMsum, S.,sndDiMaum, P.M.M.,Scienca, l82,929(1973). (7) Woioe, A,. and Minakowski. W.. Rlepyfliochem.. 10.307 119731. (3) Murphey, W. H., Patchen, L., and Lindmark, D. G., Clin Chim. Arlo, 42. 303 (1972).
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(9) Mumbey, W. H.,Lindmsrk. D. G.. Patehen, L. I.. Housler, M.E.. Hanod, E. K., and Momrich, L., Md. Re*, 7, €01 (1913). (LO) Gulavieh. W.. Hoppe-Swl~r'sZ.Phy&l. C k m . , 27.54011899). Ill) Gulerivieh. W.. Hopps-Swier'sZ. Physlol. C k m . , 27, 178and 368 (1899). 112) Gulevieh. W . ,sndJockdaohn, A,. Hoppa-Swl~r'sZ.Phy3rol. C k m . , 30,533 (19m). (13) Gulcvich. W.. and Amiradzihi, S., Hoppe-Seykr'az. Phyriol. Chem., a0.566 119m). (14) Gulevich, W . ,Home-Seylor'aZ. Physzol. Chem., SO. 53511907). (16)Gulevieh. W.. and Krimbrg. R.. Hoppe-Swler'sZ. Physlol. Chem., 45,326 11805). (16) Krlmborg. R.. Hop~odeyier'sZ.Physiol. Chem.. 411.412 (19%). (I71 Krimbrg. R.,Hoppp-Seyler'lZ. Physml. Chem., 49,89119061.
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(24) Ackermnnn, D., T i m p . 0.. and Poller, K., H o p ~ S w l r r ' a Z Physiol, . Chem.. 183, ,11429, .,.. , (25) Linnawh, W., Keil. A. W.. and Hopp-Beylet. F. A., Hoppe-Seyler'a Z Phvaiol. Chrm.. 183.11 (1929). (26) Kcil. W.. HoppSeylsr'sZ. Phyaiol. Chem.. 208, 67 (1932).
