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Chemical studies on vitamin BT. Isolation and characterization as carnitine
Chemical Studies on Vitamin B,.‘J Isolation and Characterization as Carnitine Herbert
E. Carter, P. I(. Bhattacharyya, Katharine G. Fraenkel
From the Departments of Chemistry and Entomology, Urbana, Illinois
R. Weidman3 and
University
of Illinois,
Received February 4, 1952 INTRODUCTION
Fraenkel and co-workers (2) have demonstrated that the yellow mealworm, Tenebrio molitor, requires a new growth factor, vitamin BT. They found the vitamin to be present in milk, yeast, and many animal tissues (3) and developed a method of purification involving as a final step extraction of the active material with phenol from a neutral aqueous solution (4). The “second phenol extracts” thus obtained were brown gums active at 3-6 pg./g. of food,4 representing roughly a 300-fold concentration of B, from the original whey. The present paper deals with further studies which led to the isolation of vitamin B, in pure form and its characterization as carnitine. Second phenol extracts with vitamin BT activity of 3-6 pg./g. were prepared according to the method of Fraenkel (4) from Borden’s condensed whey solids (active at 250-500 pg./g.) and from a special vitamin Blz-free Wilson’s liver extract (active at 100 pg./g.). About 20 ‘% of the original activity was recovered in the second phenol extracts. Further purification of the second phenol extracts was attempted by * Part of the material presented in this paper was taken from the thesis submitted by Katharine R. Weidman to the Graduate College of the University of Illinois in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Chemistry. A preliminary report of this work has been published (1). * Aided by a grant from the Graduate College Research Fund of the University of Illinois. 3 Present address: Mrs. Katharine Weidman McLean, Rohm and Haas Co., Huntsville, Alabama. 4 Vitamin Br activity is expressed as micrograms of sample required per gram of purified diet to give good survival and growth of Tenebrio larvae (3). 405
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CARTER,
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adsorption and partition chromatography and by countercurrent distribution. Of the many techniques studied only two-chromatography over alumina and Craig countercurrent distribution-proved useful. Applied in the order indicated, essentially pure vitamin B, was obtained from second phenol extract in yields of 60-80%. In a study of the behavior of vitamin BT on various types of alumina it was found that the vitamin was strongly adsorbed by acid alumina and was effectively removed only by alkaline solvents (methanolic
VITAMIN -*-
ET (LIVER)
EXPERIMENTAL
.-....--THEORETICAL
FIG. 1. Weight distribution of a 53-transfer Craig countercurrent fractionation of crude vitamin BT from liver. Solvent system: phenol-dilute HCI (pH 1.5); maxim&n BT activity (0.37475 pg./g.) in tubes 15-25.
ammonia). Alkaline alumina had much less affinity for the vitamin, and little or no material was adsorbed from 60% aqueous methanol. However, good separation was obtained over alkaline alumina columns using 80% methanol as the developing solvent. Second phenol extracts of both liver and whey gave very similar behavior (Tables II and III). After variable amounts of unidentified material passed through the column, a series of four partially overlapping, fractions was slowly eluted. The first and fourth bands yielded crystalline products which could be purified from aqueous alcohol. Compound A (fourth fraction) and Compound B (first fraction) were identified as creatine and
VITAMIN
407
BT
creatinine, respectively, on the basis of analytical data, melting points, and color reactions. The second fraction was a gum, whose main constituent was established to be choline by preparation of the reineckate. The third fraction, also a gum, consisted mainly of vitamin BT together with contaminating choline and creatine. In one experiment with liver the vitamin was obtained in crystalline form directly from this fraction. However in all other cases further purification was required to obtain the pure vitamin. 120IOOr, 9
8~
‘\
j/i
VITAMIN
BT CWHEYI
-- EXPERIMENTAL -.- THEORETICAL
-
FIQ. 2. Weight distribution of a 53-transfer Craig countercurrent fractionation of crude vitamin BT from whey. Solvent system: phenol-dilute HCl (pH 1.5); maximum BT activity (0.37-0.75 pg./g.) in tubes 1624.
Vitamin B, preparations from the alumina columns (active at 0.75 pg./g.) were subjected to a 53-transfer Craig distribution in a phenol-dilute HCl (pH 1.5) system. Both liver and whey concentrates gave similar distribution curves (Figs. 1 and 2). Maximum B, activity (0.37-0.75 pg./g.) was located in tubes 15-25, with a weight peak for the vitamin occurring at tube 20 or 21. The major impurity, an inactive compound, was concentrated in tube 9 and consisted mainly of choline. The close agreement between the theoretical and experimental distribution curves for B, indicates that the active material consisted of a single component and that good resolution had been attained.
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The vitamin was obtained as the hydrochloride from the peak Craig fractions. This salt was recrystallized from acetone-ethanol giving a white crystalline product melting at 137-139°C (specific rotation, ]a]Dz2 = -20.4”). The free vitamin, formed from the hydrochloride with anion exchange resin IRA 400, was recrystallized:from anhydrous acetone-ethanol giving purified vitamin B, melting at 195-197” and active at 0.37 pg./g. The pure crystalline material was highly hygroscopic, was soluble in water and the lower alcohols, gave only end absorption in the ultraviolet, and had a specific rotation of [al22 = -23.5”. Analytical data and molecular weight studies indicated an empirical formula of CrH16N03. Vitamin BT is highly stable toward acids but is slowly inactivated by refluxing with strong bases. The latter reaction was investigated further in the hope of gaining some insight into the structure of the vitamin. Prolonged alkaline degradation of pure BT gave a volatile amine accounting for over 80% of the original nitrogen. The crude base gave a positive test for secondary amine (Simon test) and yielded a picrate melting at 198-200” and an oxalate melting at 156-158”. Comparison of these derivatives with authentic samples of the corresponding salts of trimethylamine established the identity of the two bases. The crude degradation product also contained a small amount of dimethylamine which was identified as the dinitrophenyl derivative. This base was responsible for the positive Simon test. The production of trimethylamine on alkaline degradation suggests the presence of a quaternary nitrogen in vitamin B,. Since the analytical data for BT were in agreement with those for carnitine, a careful comparison was made of the properties and distribution of the two substances. The results of this study (Table I) left little doubt that Vitamin B, is, indeed, carnitine. Furthermore, B, like carnitine, yields crotonobetaine [chloroaurate, m.p. 196-200” (8)] on dehydration with sulfuric acid. (CHs) aN+-CH&H(OH) carnitine
CH&O, -+ (CH,)3N+-CH&H=CH-C0~crotonobetaine
(-)-Carnitine, isolated from meat extracts by the method of Strack et al. (5) showed vitamin B, activity at levels of 0.37-0.75 pg./g. of diet, 5 Unless determined
otherwise indicated on a micro-block.
all melting
points
reported
in this
paper
were
VITAMIN
409
BT
and nn-carnitine synthesized by a combination of the procedures of Bergmann et al. (9) and Tomita (10) was active at a level of 0.37-0.75 pg./g. These data conclusively establish the identity of vitamin BT and carnitine. (-)-Carnitine, long known as a constituent of muscle, is present in meat extract to the extent of 1.5-3.0%. The latter material is a good preparative source of carnitine which can be obtained in excellent yields and in crystalline form simply by chromatographing “second phenol extracts” over alumina. The purification of carnitine from this TABLE
I
Comparison of Vitamin BT and Carnitine Vitamin
Amount in meat extract (%) Physical properties: M.p. Specific rotation (hydrochloride) Derivatives: m.p. Hydrochloride Chloroaurate Chloroplatiuate Acetyl, hydrochloride Acetyl, chloroaurate (recryst. from water) Ethyl ester chloride Ethyl ester chloroaurate
BT
Carnitine
1.5-3.0
1.8-2.1
196-198” -21.5”
-20.9”
137-139” 153-155O 216-219’ 188-190° 128-130” 160-163” 144-146” 106”
(5,6)
142” 155” 214-218” 187” 129-130” 160-162” 146” 105”
(7) (5) (5) (5) (5) (5) (5) (6) (6)
source is simplified by the relatively small amounts of choline present in muscle extracts. The discovery that the mealworm requires carnitine affords an interesting opportunity for metabolic studies. Some preliminary data have already been obtained along these lines. Crotonobetaine has no vitamin BT activity, but nL+hydroxy--y-aminobutyric acid will replace Br at a level of 12-24 pg./g. of diet. This activity could be explained in various ways but suggests that Tenebrio can synthesize carnitine by methylation of /I-hydroxy-y-aminobutyric acid and that carnitine may participate in the transmethylation reactions of the animal body. The question still remains as to whether the (+)-isomer of carnitine is biologically active. The assay results suggested that or,-carnitine might
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be about one half as active as the (-)-isomer. It will, however, be necessary to prepare the pure “unnatural” isomer to establish this point conclusively. EXPERIMENTBL
Preparation
of Crude BT Concentrates
The crude starting material for isolation studies was prepared essentially according to the method of Fraenkel (4). In a typical experiment, 1.6 kg. of Borden’s condensed whey solids (200-500 bg./g.) yielded 2.2 g. of second phenol extract active at 3 pg./g. of diet (18% recovery of activity). Wilson’s Blx-free liver extract (100 pg./g.) when processed by the same method with the exclusion of the preliminary alcohol-extraction step gave a second phenol extract also active at 3 pg./g., but the yields were slightly higher (20-25s recovery of activity). The second phenol extracts were light-brown gums which often contained some crystalline material.
Isolation
of Compound A from Second Phenol Extracts
Attempts to crystallize second phenol extract of liver or whey from water gave a crystalline material devoid of BT activity. The following is a typical experiment. A 2.91-g. aliquot of second phenol extract from liver was dissolved in 8 ml. of boiling water. Repeated cooling and concentrating gave three crops of crystals totalling 1.85 g. A 737-mg. portion of the first two crops of crystals was recrystallized six times from hot water yielding 185 mg. of purified compound A melting at 256-258” dec. (transition point 246-247” to brilliant birefrigent needles). Anal. ChHBN302 (131.1). Calcd.: C 36.63, H 6.92, N 32.05; found: C 37.02, H 6.77, N 31.72. Compound A was obtained by the same procedure from whey extracts. It was devoid of BT activity and was characterized as creatine on the basis of the analytical data and conversion to creatinine. In preparing second phenol extracts for chromatography, the creatine was removed as completely as possible by fractionation from ethanol. In a typical experiment, 18.9 g. of second phenol extract from liver was extracted with 50 ml. of hot absolute ethanol. On cooling the solution, 1.3 g. of creatine separated. The filtrate was evaporated to dryness and the residue was chromatographed over alumina as described below.
Chromatography of Crude BT over Alumina In these experiments water-washed heat-activated Alcoa alumina was used. A 15-g. portion of the residue described above was dissolved in 15 ml. of 80% aqueous methanol and applied to a 3-in. column containing 1500 g. of alumina. The column was developed with 80% aqueous methanol, and arbitrary 50-ml. fractions were collected and evaporated to dryness. The results are summarized ip Table II. A similar experiment with second phenol extract from whey is summarized in Table III. The results recorded in Table II and III are typical of the several columns which
VITAMIN
TABLE
(15 g. active VdlJme
1 2-6 7-21 22-29 30-40 41-54 55-60 61-95 96-126 127-150 15 l-200
250 750 400 550 700 300 1750 1500 1250 2500
at 3-6 Mg./g. of diet)
Weight
PQ.lQ.
-
606 3900 1347 949 737 234 891 584 275 283
12-24 3.0 0.75-l. 5 0.37 0.37-0.75 0.75 0.75-1.5 1.5 TABLE
(4.8 g. active Volume
l--4 557 8-10 11-13 14-16 17-19 20-35 26-32 33-40 41-56 57-71
$0 75 75 75 75 75 150 175 200 650 1400
Crystalline Et?m, (233) 428; Compound B Ultraviolet absorption very low Gum Gum + crystals Do. Do. Do. Do. Do.
III
of Second Phenol Extract from Whey
Chromatography
Fraction
Remarks
Activity
mo.
125
II
of Second Phenol Extract from Liver
Chromatography
Fraction
411
BT
Weight
at 3-6 pg./g. of diet) Activity
Remarks
TW.
33 467 493 456 198 479 430 343 525 431
,‘Q./Q. of diet
-
6-12 3-6 0.75-1.5 0.75 1.5-3.0
E:E. (233) 200 (compound E:z, (233) 480 (compound E:z. (233) 187 (compound Gum + crystals Gum Gum Gum Gum
B) B) B)
were run on both liver and whey. The most active fractions from the columns were combined and purified further by the Craig procedure as described below. Pure compound B was obtained from fractions 7-21 (Table II). The mixture of gum and crystalline solid was washed with cold absolute ethanol to remove the gum. A 538mg. portion of the solid residue was recrystallized three times from hot water and once from 80% aqueous ethanol giving a colorless, crystalline nonhygroscopic product melting at 257-258” dec. [Et &. (200) 1136; (234) 524.1
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Anal. &H,N,O. C&d.: C 42.47, H 6.24, N 37.15; found: C 42.20, H 6.15, N 36.91. This material was characterized as creatinine on the basis of the analytical data, physical properties, color reactions, and properties of the picrate.
Purification
of Crude BT by Craig Countercurrent Distribution
The final purification of the vitamin was achieved by countercurrent distribution in a phenol-dilute HCl (pH 1.5) system in a 54-tube all-glass Craig apparatus (11). The tubes held approximately 80 ml. of each phase. Phenol and water were equilibrated and adjusted to pH 1.5 (aqueous layer) by the addition of concentrated HCl. Eighty ml. of the phenol phase was added to each of tubes l-53. The material to be distributed was dissolved in a mixture containing 65 ml. of each phase and the pH of the aqueous layer was adjusted to 1.5 with concentrated HCl. The volume of each phase was made up to 80 ml. from stock, and the two layers were poured into tube 0. A 53-transfer distribution was then carried out in the usual way (ll).O After the run was completed the entire contents of each tube were removed and extracted twice with 250-ml. quantities of ether to remove the phenol. The aqueous layers were taken to dryness under reduced pressure, and the residues were dried and weighed. The weight distribution curves as determined in this way were not highly accurate, but proved adequate for preparative purposes. Distribution of Crude BT from Whey. A 1.9-g. sample of Br (active at 0.75-1.5 pg./g.) from an alumina column was subjected to a 53-transfer distribution. The results are summarized in Fig. 2. The fraction with a weight peak at tube 20 (K = 0.606) contained most of the BT activity. Solids from tubes 16-23 were active at 0.37-0.75 pg./g. The peak 9 fraction showed very low Br activity (6-12 P&/&l. Distribution of BT from Liver. A similar run was made on a 1.98-g. sample (active at 0.75 pg./g.) of crude Br from liver. The Br weight peak came at tube 21 (K = 0.656), and the solids in tubes 15-24 showed an activity of 0.37-0.75 pg./g. The similarity in distribution pattern of the vitamin from liver and whey constituted strong evidence for the identity of the two substances. Vitamin BT Hydrochloride. Fractions 15-24 from the Craig distributions consisted mainly of vitamin BT hydrochloride, and on standing in vacua the dried residues partially crystallized. The material in tube 22 from a liver preparation (115 mg.) was washed with 2.5 ml. of a cold anhydrous mixture of acetone and alcohol (3:2) which removed the gum. The residue was recrystallized twice from 10 ml. of acetone-alcohol (10: 1) giving 11 mg. of colorless, extremely hygroscopic, crystalline material melting at 137-139” ([&*a = -20.4” (aqueous solution)). This product retained ionic chlorine even after repeated recrystallization and agreed in properties with carnitine hydrochloride (7). 6 In running Craig distributions with phenol-water systems we customarily add SO-ml. portions of aqueous phase to tubes 2 and 3. These aqueous layers move ahead of aqueous phase from tube 0 leaving one tube between free of aqueous phase. In this way decrease of volume of the first aqueous phase due to fluctuations of temperature is minimized.
VITAMIN
BT
413
Vitamin Br. A 750-mg. aliquot of combined fractions 15-24 from liver was dissolved in 5 ml. of water (pH of the solution 1.5) and passed over 10 ml. of Amberlite IRA-400 in the hydroxyl phase. The percolate and aqueous washings from the resin (55 ml., pH 7.0) were vacuum dried giving 470 mg. of crystalline residue melting at 187-193” (active at 0.37 pg./g.). This material was dissolved in 25 ml. of a boiling mixture of anhydrous acetone-ethanol (3 : 2). The extract was cooled, filtered, and treated with 70 ml. of ice-cold anhydrous acetone with vigorous scratching. The supernatant liquid was decanted, leaving 270 mg. of colorless, crystalline, hygroscopic solid (m.p. 194-197’). This material was recrystallized from 14.5 ml. of acetone-alcohol (3:2) by the addition of 41.5 ml. of acetone giving 187 mg. of pure vitamin Br [m.p. 196-198”; [(Y]E? = -23.5” (0.5% solution in water)]. An identical product was obtained from fractions 15-24 from whey. Pure Br showed only end absorption in the ultraviolet and gave negative results in all the common tests for nitrogen-containing groups. The biological activity was not destroyed by treatment with HNOZ, nor by prolonged refluxing with concentrated HCl. Refluxing with NaOH, however, destroyed the activity completely, with the liberation of a volatile amine. Anal. CrHnNOs(161.2). Calcd.: C 52.15, H 9.38, N. 8.69; found: C 52.32, H 9.03, N 8.40; molecular weight (ebullioscopic) 179.
Alkaline
Degradation of BT
Crystalline vitamin Br (503 mg.) was refluxed with 15 ml. of 3 N NaOH for 24 hr. and the volatile amine evolved was removed from the reaction mixture by a current of Cot-free air and collected in 15 ml. of 0.5 N HCl. The HCl solution was evaporated under reduced pressure leaving a very hygroscopic, crystalline compound (248 mg.; m.p. 177-213”) which gave a positive Simon test (12) for a secondary amine. Picrate. A 50-mg. aliquot of the hydrochloride was treated with a solution of 130 mg. of picric acid in 2.5 ml. of ethanol. On cooling, 119 mg. of a crystalline piorate was obtained (m.p. 198-200”). Three recrystallizations from ethanol failed to change the melting point. The amine regenerated from this picrate gave a negative Simon test. And. CaHr,N.CaHaNsOr. Calcd.: C 37.51, H 4.19, N 19.44; found: C 37.52, H 4.13, N 19.35. Ozalate. A 68mg. sample of the hydrochloride was decomposed with 2 ml. of 40% NaOH and distilled into 10 ml. of water containing 102 mg. of oxalic acid. On evaporating to 0.5 ml., the solution deposited some free oxalic acid (63 mg.). The filtrate was taken to dryness and a crystalline solid was obtained (94.6 mg.; m.p. 135-152”). Five recrystallisations from ethanol yielded the pure, crystalline acid oxalate (25.3 mg.; m.p. 156-158’). Anal. C3HgN.C2H20c Calcd.: C 40.26, H 7.43, N 9.39; found: C 40.53, H 7.69, N 9.40. I,Q-Dinitrophenyl Derivative. 2+Dinitrofluorobensene (174 mg.) was added to a solution of 95.5 mg. of the amine hydrochloride in 3.0 ml. of ethanol, and 200 mg. of NaHCOs was added. After 3 hr. at room temperature the mixture was filtered
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and the filtrate diluted to 12 ml. with water. An oil separated. This oil was taken up in 1 ml. of hot ethanol and the solution was allowed to cool slowly, giving 33 mg. of a yellow crystalline precipitate (m.p. 73-83”). After three recrystallizations from ethanol, 8.9 mg. of long, light yellow needles were obtained (m.p. 87”). Anal. (CH,),N.G,H,(NO,),. Calcd.: C 45.50, H 4.30; found: C 45.58, H 4.04. An authentic sample of dimethylamine gave t,he same dinitrophenyl derivative (m.p. 86-87”), whereas the dinitrophenyl derivative of ethylmethylamine melted at 55”.
Comparison of Vitamin
BT with Carnitine
Derivatives of BT Chloroaurate. Eighty mg. of crystalline BT was treated with a solution of 158 mg. of AuC13 in 2.0 ml. of 1 y0 HCI. On cooling the solution, 178 mg. of yellow elongated plates separated (m.p. 153-155”). The melting point was not altered by repeated recrystallizations from dilute HCl. Anal. CTHI~NO~AUC~~. Calcd.: C 16.77, H 3.22, N 2.79, Au 39.34; found: C 17.44, H 3.55, N 2.99, Au 39.57. Chloroplatinate. One hundred mg. of BT was treated with 2 ml. of ethanolic solution containing 200 mg. of PtCL and 0.5 ml. of concentrated HCI. On cooling the solution, 117 mg. of microcrystalline yellow powder separated. This material was recrystallized once from 60% aqueous ethanol and once more from water (m.p. 216-219”). The chloroaurate and chloroplatinate agree in properties with the corresponding derivatives of carnitine (5,6). Other derivatives of BT, prepared in the usual way, are listed in Table II.
Conversion of BT to Crotonobetaine A 300-mg. sample of BT was heated on an oil bath at 120-130” for 1 hr. with 5.2 ml. of concentrated H2S04 (sp. gr. 1.84). The solution was poured into 20 g. of ice and the HzSOa was neutralized with an excess of BaCOa. The filtrate was evaporated to dryness and the residue was extracted with 3.0 ml. of water. The filtered solution was treated with a solution of 600 mg. of AuC13 in 0.3 ml. of concentrated HCl and 3.0 ml. of water. The precipitate (291 mg.) was recrystallized from hot 0.01 y0 HCl giving 100 mg. of the golden-yellow, crystalline chloroaurate of crotonobetaine (m.p. 196-200”). Anal. C~Hl~NO~AuC1~. Calcd.: C 17.40, H 2.92, N 2.90, Au 40.81; found: C 17.52, H 2.82, N 2.96, Au 40.64. This product appeared to be identical with the chloroaurate of crotonobetaine reported by Engeland (8). The free crotonobetaine, obtained by removing the gold with H&J, showed no vitamin BT activity.
Preparation
of Carnitine from Meat Extract
The majority of the experiments with crystalline vitamin BT were carried out on material isolated from whey or liver. When the work was almost completed it was discovered that meat extract is a much richer source of the vitamin. By applying to meat extract the techniques developed for whey and liver, vitamin BT (carnitine)
VITAMIN
BT
415
can be isolated in good yields and with much less work. The following procedure has given uniformly satisfactory results in our hands. One hundred grams of Difco Beef Extract (active at 12-24 pg./g.) was dissolved in 700 ml. of water, and the solution was shaken with 300 ml. of molten phenol. The emulsion was filtered from suspended matter and the phenol layer was separated. The aqueous layer was re-extracted with 200 ml. of phenol. The first and second phenol layers were successively washed with 100 ml. of water using the same aqueous layer for both the phenol layers. The combined phenol layers were extracted with 1500 ml. of ether. After separation from the thick dark aqueous layer, the ether layer was washed three times with 100 ml. of distilled water and discarded. The aqueous layer and the washings were combined (total solids, 22 g.; active at 3-6 rg./g.), acidified to pH 1 with HCl, and decolorized by shaking with 475 g. of activated carbon (Norit). The filtrate and washings from carbon (total solids, 11.9 g.; active at 1.5-3.0 pg./g.) were neutralized with NH,OH (pH 6.5), evaporated to 400 ml. under reduced pressure, and extracted three times with 200-ml., lOO-ml., and loo-ml. portions of phenol, respectively. The phenol layers were washed successively with the same loo-ml. quantity of distilled water, combined, and freed from phenol in the usual manner by two successive extractions with 3 vol. of ether. The colorless aqueous layers from the last operation (second phenol extract) gave a colorless gum on evaporation (3.44 g., active at 0.75 pg./g.; recovery, 50-100%). Two grams of second phenol extract was dissolved in 4 ml. of 80% aqueous methanol and chromatographed over a column of 300 g. of water-washed Alcoa alumina. The column was developed with 80% aqueous methanol, and IO-ml. eluate fractions were collected. Fractions 25-75 on evaporation gave almost pure carnitine (0.95 g., m.p. 192-196”). This product on one further recrystallization gave 0.8 g. of essentially pure carnitine (m.p. 196-198”, active at 0.37 pg./g.).
ACKNOWLEDGMENTS The authors wish to express their appreciation to Dr. Claire Graham of the Wilson Laboratories for his assistance in making available special liver extracts for this work. We also are indebted to the Borden Company for supplies of whey and special whey preparations and for support in the isolation of a quantity of crude vitamin BT. We are happy to acknowledge research grants from the U. S. Public Health Service and from Merck and Co., Inc., in support of this project. The vitamin Br assays reported in this paper were performed by Allison Whittingham, Harriet R. Stern, and Elizabeth Nilson.
SUMMARY Vitamin B, has been isolated as a pure, crystalline substance from liver and whey, Analytical data and production of trimethylamine on alkaline degradation suggested that vitamin BT might be identical with carnitine. A careful comparison of the properties of derivatives and of reactions of the two substances established conclusively that vitamin BT is carnitine.
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REFERENCES 1.
CARTER, H.E.,
BHATTACHARYYA,P.K.,
WEIDMAN, K.R., ANDFRAENKEL,G., 36, 241 (1952). G., BLEWETT, M., AND COLES, M.,Nature161,981 (1948). G., Arch. Biochem. Biophys. 32, 457 (1951). G., Arch. Biochem. Biophys. 34, 468 (1951). WORDEHOFF,P., AND~CHWANEBERG,H., Z.physiol.Chem.236,183
Arch. Biochem. Biophys. 2. FRAENKEL, 3. FRAENKEL,
4. FRAENKEL,
5. STRACK,E., (1936). 6. STRACK, E., AND FORSTERLING,K., Ber. 71B, 1143 (1838). 7. KRIMBERG, R., 2. physiol. Chem. 66, 466 (1908). 8. ENGELAND, R., Ber. 64B, 2208 (1921). 9. BERGMANN, M., BRAND, E., AND WEINMANN, F., Z. physiol.
Chem. 131,
1
(1923).
TOMITA, M., 2. physiol. Chem. 124, 253 (1922-3). CRAIG, L. C., Anal. Chem. 22, 1346 (1950). 12. SCHNEIDER, F., Qualitative Organic Microanalysis, 10. 11.
Sons, Inc., New York,
1946.
p. 189. John
Wiley
anP
E. Carter, P. I(. Bhattacharyya, Katharine G. Fraenkel
From the Departments of Chemistry and Entomology, Urbana, Illinois
R. Weidman3 and
University
of Illinois,
Received February 4, 1952 INTRODUCTION
Fraenkel and co-workers (2) have demonstrated that the yellow mealworm, Tenebrio molitor, requires a new growth factor, vitamin BT. They found the vitamin to be present in milk, yeast, and many animal tissues (3) and developed a method of purification involving as a final step extraction of the active material with phenol from a neutral aqueous solution (4). The “second phenol extracts” thus obtained were brown gums active at 3-6 pg./g. of food,4 representing roughly a 300-fold concentration of B, from the original whey. The present paper deals with further studies which led to the isolation of vitamin B, in pure form and its characterization as carnitine. Second phenol extracts with vitamin BT activity of 3-6 pg./g. were prepared according to the method of Fraenkel (4) from Borden’s condensed whey solids (active at 250-500 pg./g.) and from a special vitamin Blz-free Wilson’s liver extract (active at 100 pg./g.). About 20 ‘% of the original activity was recovered in the second phenol extracts. Further purification of the second phenol extracts was attempted by * Part of the material presented in this paper was taken from the thesis submitted by Katharine R. Weidman to the Graduate College of the University of Illinois in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Chemistry. A preliminary report of this work has been published (1). * Aided by a grant from the Graduate College Research Fund of the University of Illinois. 3 Present address: Mrs. Katharine Weidman McLean, Rohm and Haas Co., Huntsville, Alabama. 4 Vitamin Br activity is expressed as micrograms of sample required per gram of purified diet to give good survival and growth of Tenebrio larvae (3). 405
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adsorption and partition chromatography and by countercurrent distribution. Of the many techniques studied only two-chromatography over alumina and Craig countercurrent distribution-proved useful. Applied in the order indicated, essentially pure vitamin B, was obtained from second phenol extract in yields of 60-80%. In a study of the behavior of vitamin BT on various types of alumina it was found that the vitamin was strongly adsorbed by acid alumina and was effectively removed only by alkaline solvents (methanolic
VITAMIN -*-
ET (LIVER)
EXPERIMENTAL
.-....--THEORETICAL
FIG. 1. Weight distribution of a 53-transfer Craig countercurrent fractionation of crude vitamin BT from liver. Solvent system: phenol-dilute HCI (pH 1.5); maxim&n BT activity (0.37475 pg./g.) in tubes 15-25.
ammonia). Alkaline alumina had much less affinity for the vitamin, and little or no material was adsorbed from 60% aqueous methanol. However, good separation was obtained over alkaline alumina columns using 80% methanol as the developing solvent. Second phenol extracts of both liver and whey gave very similar behavior (Tables II and III). After variable amounts of unidentified material passed through the column, a series of four partially overlapping, fractions was slowly eluted. The first and fourth bands yielded crystalline products which could be purified from aqueous alcohol. Compound A (fourth fraction) and Compound B (first fraction) were identified as creatine and
VITAMIN
407
BT
creatinine, respectively, on the basis of analytical data, melting points, and color reactions. The second fraction was a gum, whose main constituent was established to be choline by preparation of the reineckate. The third fraction, also a gum, consisted mainly of vitamin BT together with contaminating choline and creatine. In one experiment with liver the vitamin was obtained in crystalline form directly from this fraction. However in all other cases further purification was required to obtain the pure vitamin. 120IOOr, 9
8~
‘\
j/i
VITAMIN
BT CWHEYI
-- EXPERIMENTAL -.- THEORETICAL
-
FIQ. 2. Weight distribution of a 53-transfer Craig countercurrent fractionation of crude vitamin BT from whey. Solvent system: phenol-dilute HCl (pH 1.5); maximum BT activity (0.37-0.75 pg./g.) in tubes 1624.
Vitamin B, preparations from the alumina columns (active at 0.75 pg./g.) were subjected to a 53-transfer Craig distribution in a phenol-dilute HCl (pH 1.5) system. Both liver and whey concentrates gave similar distribution curves (Figs. 1 and 2). Maximum B, activity (0.37-0.75 pg./g.) was located in tubes 15-25, with a weight peak for the vitamin occurring at tube 20 or 21. The major impurity, an inactive compound, was concentrated in tube 9 and consisted mainly of choline. The close agreement between the theoretical and experimental distribution curves for B, indicates that the active material consisted of a single component and that good resolution had been attained.
408
CARTER,
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AND
FRAENKEL
The vitamin was obtained as the hydrochloride from the peak Craig fractions. This salt was recrystallized from acetone-ethanol giving a white crystalline product melting at 137-139°C (specific rotation, ]a]Dz2 = -20.4”). The free vitamin, formed from the hydrochloride with anion exchange resin IRA 400, was recrystallized:from anhydrous acetone-ethanol giving purified vitamin B, melting at 195-197” and active at 0.37 pg./g. The pure crystalline material was highly hygroscopic, was soluble in water and the lower alcohols, gave only end absorption in the ultraviolet, and had a specific rotation of [al22 = -23.5”. Analytical data and molecular weight studies indicated an empirical formula of CrH16N03. Vitamin BT is highly stable toward acids but is slowly inactivated by refluxing with strong bases. The latter reaction was investigated further in the hope of gaining some insight into the structure of the vitamin. Prolonged alkaline degradation of pure BT gave a volatile amine accounting for over 80% of the original nitrogen. The crude base gave a positive test for secondary amine (Simon test) and yielded a picrate melting at 198-200” and an oxalate melting at 156-158”. Comparison of these derivatives with authentic samples of the corresponding salts of trimethylamine established the identity of the two bases. The crude degradation product also contained a small amount of dimethylamine which was identified as the dinitrophenyl derivative. This base was responsible for the positive Simon test. The production of trimethylamine on alkaline degradation suggests the presence of a quaternary nitrogen in vitamin B,. Since the analytical data for BT were in agreement with those for carnitine, a careful comparison was made of the properties and distribution of the two substances. The results of this study (Table I) left little doubt that Vitamin B, is, indeed, carnitine. Furthermore, B, like carnitine, yields crotonobetaine [chloroaurate, m.p. 196-200” (8)] on dehydration with sulfuric acid. (CHs) aN+-CH&H(OH) carnitine
CH&O, -+ (CH,)3N+-CH&H=CH-C0~crotonobetaine
(-)-Carnitine, isolated from meat extracts by the method of Strack et al. (5) showed vitamin B, activity at levels of 0.37-0.75 pg./g. of diet, 5 Unless determined
otherwise indicated on a micro-block.
all melting
points
reported
in this
paper
were
VITAMIN
409
BT
and nn-carnitine synthesized by a combination of the procedures of Bergmann et al. (9) and Tomita (10) was active at a level of 0.37-0.75 pg./g. These data conclusively establish the identity of vitamin BT and carnitine. (-)-Carnitine, long known as a constituent of muscle, is present in meat extract to the extent of 1.5-3.0%. The latter material is a good preparative source of carnitine which can be obtained in excellent yields and in crystalline form simply by chromatographing “second phenol extracts” over alumina. The purification of carnitine from this TABLE
I
Comparison of Vitamin BT and Carnitine Vitamin
Amount in meat extract (%) Physical properties: M.p. Specific rotation (hydrochloride) Derivatives: m.p. Hydrochloride Chloroaurate Chloroplatiuate Acetyl, hydrochloride Acetyl, chloroaurate (recryst. from water) Ethyl ester chloride Ethyl ester chloroaurate
BT
Carnitine
1.5-3.0
1.8-2.1
196-198” -21.5”
-20.9”
137-139” 153-155O 216-219’ 188-190° 128-130” 160-163” 144-146” 106”
(5,6)
142” 155” 214-218” 187” 129-130” 160-162” 146” 105”
(7) (5) (5) (5) (5) (5) (5) (6) (6)
source is simplified by the relatively small amounts of choline present in muscle extracts. The discovery that the mealworm requires carnitine affords an interesting opportunity for metabolic studies. Some preliminary data have already been obtained along these lines. Crotonobetaine has no vitamin BT activity, but nL+hydroxy--y-aminobutyric acid will replace Br at a level of 12-24 pg./g. of diet. This activity could be explained in various ways but suggests that Tenebrio can synthesize carnitine by methylation of /I-hydroxy-y-aminobutyric acid and that carnitine may participate in the transmethylation reactions of the animal body. The question still remains as to whether the (+)-isomer of carnitine is biologically active. The assay results suggested that or,-carnitine might
410
CARTER,
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AND
FRAENKEL
be about one half as active as the (-)-isomer. It will, however, be necessary to prepare the pure “unnatural” isomer to establish this point conclusively. EXPERIMENTBL
Preparation
of Crude BT Concentrates
The crude starting material for isolation studies was prepared essentially according to the method of Fraenkel (4). In a typical experiment, 1.6 kg. of Borden’s condensed whey solids (200-500 bg./g.) yielded 2.2 g. of second phenol extract active at 3 pg./g. of diet (18% recovery of activity). Wilson’s Blx-free liver extract (100 pg./g.) when processed by the same method with the exclusion of the preliminary alcohol-extraction step gave a second phenol extract also active at 3 pg./g., but the yields were slightly higher (20-25s recovery of activity). The second phenol extracts were light-brown gums which often contained some crystalline material.
Isolation
of Compound A from Second Phenol Extracts
Attempts to crystallize second phenol extract of liver or whey from water gave a crystalline material devoid of BT activity. The following is a typical experiment. A 2.91-g. aliquot of second phenol extract from liver was dissolved in 8 ml. of boiling water. Repeated cooling and concentrating gave three crops of crystals totalling 1.85 g. A 737-mg. portion of the first two crops of crystals was recrystallized six times from hot water yielding 185 mg. of purified compound A melting at 256-258” dec. (transition point 246-247” to brilliant birefrigent needles). Anal. ChHBN302 (131.1). Calcd.: C 36.63, H 6.92, N 32.05; found: C 37.02, H 6.77, N 31.72. Compound A was obtained by the same procedure from whey extracts. It was devoid of BT activity and was characterized as creatine on the basis of the analytical data and conversion to creatinine. In preparing second phenol extracts for chromatography, the creatine was removed as completely as possible by fractionation from ethanol. In a typical experiment, 18.9 g. of second phenol extract from liver was extracted with 50 ml. of hot absolute ethanol. On cooling the solution, 1.3 g. of creatine separated. The filtrate was evaporated to dryness and the residue was chromatographed over alumina as described below.
Chromatography of Crude BT over Alumina In these experiments water-washed heat-activated Alcoa alumina was used. A 15-g. portion of the residue described above was dissolved in 15 ml. of 80% aqueous methanol and applied to a 3-in. column containing 1500 g. of alumina. The column was developed with 80% aqueous methanol, and arbitrary 50-ml. fractions were collected and evaporated to dryness. The results are summarized ip Table II. A similar experiment with second phenol extract from whey is summarized in Table III. The results recorded in Table II and III are typical of the several columns which
VITAMIN
TABLE
(15 g. active VdlJme
1 2-6 7-21 22-29 30-40 41-54 55-60 61-95 96-126 127-150 15 l-200
250 750 400 550 700 300 1750 1500 1250 2500
at 3-6 Mg./g. of diet)
Weight
PQ.lQ.
-
606 3900 1347 949 737 234 891 584 275 283
12-24 3.0 0.75-l. 5 0.37 0.37-0.75 0.75 0.75-1.5 1.5 TABLE
(4.8 g. active Volume
l--4 557 8-10 11-13 14-16 17-19 20-35 26-32 33-40 41-56 57-71
$0 75 75 75 75 75 150 175 200 650 1400
Crystalline Et?m, (233) 428; Compound B Ultraviolet absorption very low Gum Gum + crystals Do. Do. Do. Do. Do.
III
of Second Phenol Extract from Whey
Chromatography
Fraction
Remarks
Activity
mo.
125
II
of Second Phenol Extract from Liver
Chromatography
Fraction
411
BT
Weight
at 3-6 pg./g. of diet) Activity
Remarks
TW.
33 467 493 456 198 479 430 343 525 431
,‘Q./Q. of diet
-
6-12 3-6 0.75-1.5 0.75 1.5-3.0
E:E. (233) 200 (compound E:z, (233) 480 (compound E:z. (233) 187 (compound Gum + crystals Gum Gum Gum Gum
B) B) B)
were run on both liver and whey. The most active fractions from the columns were combined and purified further by the Craig procedure as described below. Pure compound B was obtained from fractions 7-21 (Table II). The mixture of gum and crystalline solid was washed with cold absolute ethanol to remove the gum. A 538mg. portion of the solid residue was recrystallized three times from hot water and once from 80% aqueous ethanol giving a colorless, crystalline nonhygroscopic product melting at 257-258” dec. [Et &. (200) 1136; (234) 524.1
412
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Anal. &H,N,O. C&d.: C 42.47, H 6.24, N 37.15; found: C 42.20, H 6.15, N 36.91. This material was characterized as creatinine on the basis of the analytical data, physical properties, color reactions, and properties of the picrate.
Purification
of Crude BT by Craig Countercurrent Distribution
The final purification of the vitamin was achieved by countercurrent distribution in a phenol-dilute HCl (pH 1.5) system in a 54-tube all-glass Craig apparatus (11). The tubes held approximately 80 ml. of each phase. Phenol and water were equilibrated and adjusted to pH 1.5 (aqueous layer) by the addition of concentrated HCl. Eighty ml. of the phenol phase was added to each of tubes l-53. The material to be distributed was dissolved in a mixture containing 65 ml. of each phase and the pH of the aqueous layer was adjusted to 1.5 with concentrated HCl. The volume of each phase was made up to 80 ml. from stock, and the two layers were poured into tube 0. A 53-transfer distribution was then carried out in the usual way (ll).O After the run was completed the entire contents of each tube were removed and extracted twice with 250-ml. quantities of ether to remove the phenol. The aqueous layers were taken to dryness under reduced pressure, and the residues were dried and weighed. The weight distribution curves as determined in this way were not highly accurate, but proved adequate for preparative purposes. Distribution of Crude BT from Whey. A 1.9-g. sample of Br (active at 0.75-1.5 pg./g.) from an alumina column was subjected to a 53-transfer distribution. The results are summarized in Fig. 2. The fraction with a weight peak at tube 20 (K = 0.606) contained most of the BT activity. Solids from tubes 16-23 were active at 0.37-0.75 pg./g. The peak 9 fraction showed very low Br activity (6-12 P&/&l. Distribution of BT from Liver. A similar run was made on a 1.98-g. sample (active at 0.75 pg./g.) of crude Br from liver. The Br weight peak came at tube 21 (K = 0.656), and the solids in tubes 15-24 showed an activity of 0.37-0.75 pg./g. The similarity in distribution pattern of the vitamin from liver and whey constituted strong evidence for the identity of the two substances. Vitamin BT Hydrochloride. Fractions 15-24 from the Craig distributions consisted mainly of vitamin BT hydrochloride, and on standing in vacua the dried residues partially crystallized. The material in tube 22 from a liver preparation (115 mg.) was washed with 2.5 ml. of a cold anhydrous mixture of acetone and alcohol (3:2) which removed the gum. The residue was recrystallized twice from 10 ml. of acetone-alcohol (10: 1) giving 11 mg. of colorless, extremely hygroscopic, crystalline material melting at 137-139” ([&*a = -20.4” (aqueous solution)). This product retained ionic chlorine even after repeated recrystallization and agreed in properties with carnitine hydrochloride (7). 6 In running Craig distributions with phenol-water systems we customarily add SO-ml. portions of aqueous phase to tubes 2 and 3. These aqueous layers move ahead of aqueous phase from tube 0 leaving one tube between free of aqueous phase. In this way decrease of volume of the first aqueous phase due to fluctuations of temperature is minimized.
VITAMIN
BT
413
Vitamin Br. A 750-mg. aliquot of combined fractions 15-24 from liver was dissolved in 5 ml. of water (pH of the solution 1.5) and passed over 10 ml. of Amberlite IRA-400 in the hydroxyl phase. The percolate and aqueous washings from the resin (55 ml., pH 7.0) were vacuum dried giving 470 mg. of crystalline residue melting at 187-193” (active at 0.37 pg./g.). This material was dissolved in 25 ml. of a boiling mixture of anhydrous acetone-ethanol (3 : 2). The extract was cooled, filtered, and treated with 70 ml. of ice-cold anhydrous acetone with vigorous scratching. The supernatant liquid was decanted, leaving 270 mg. of colorless, crystalline, hygroscopic solid (m.p. 194-197’). This material was recrystallized from 14.5 ml. of acetone-alcohol (3:2) by the addition of 41.5 ml. of acetone giving 187 mg. of pure vitamin Br [m.p. 196-198”; [(Y]E? = -23.5” (0.5% solution in water)]. An identical product was obtained from fractions 15-24 from whey. Pure Br showed only end absorption in the ultraviolet and gave negative results in all the common tests for nitrogen-containing groups. The biological activity was not destroyed by treatment with HNOZ, nor by prolonged refluxing with concentrated HCl. Refluxing with NaOH, however, destroyed the activity completely, with the liberation of a volatile amine. Anal. CrHnNOs(161.2). Calcd.: C 52.15, H 9.38, N. 8.69; found: C 52.32, H 9.03, N 8.40; molecular weight (ebullioscopic) 179.
Alkaline
Degradation of BT
Crystalline vitamin Br (503 mg.) was refluxed with 15 ml. of 3 N NaOH for 24 hr. and the volatile amine evolved was removed from the reaction mixture by a current of Cot-free air and collected in 15 ml. of 0.5 N HCl. The HCl solution was evaporated under reduced pressure leaving a very hygroscopic, crystalline compound (248 mg.; m.p. 177-213”) which gave a positive Simon test (12) for a secondary amine. Picrate. A 50-mg. aliquot of the hydrochloride was treated with a solution of 130 mg. of picric acid in 2.5 ml. of ethanol. On cooling, 119 mg. of a crystalline piorate was obtained (m.p. 198-200”). Three recrystallizations from ethanol failed to change the melting point. The amine regenerated from this picrate gave a negative Simon test. And. CaHr,N.CaHaNsOr. Calcd.: C 37.51, H 4.19, N 19.44; found: C 37.52, H 4.13, N 19.35. Ozalate. A 68mg. sample of the hydrochloride was decomposed with 2 ml. of 40% NaOH and distilled into 10 ml. of water containing 102 mg. of oxalic acid. On evaporating to 0.5 ml., the solution deposited some free oxalic acid (63 mg.). The filtrate was taken to dryness and a crystalline solid was obtained (94.6 mg.; m.p. 135-152”). Five recrystallisations from ethanol yielded the pure, crystalline acid oxalate (25.3 mg.; m.p. 156-158’). Anal. C3HgN.C2H20c Calcd.: C 40.26, H 7.43, N 9.39; found: C 40.53, H 7.69, N 9.40. I,Q-Dinitrophenyl Derivative. 2+Dinitrofluorobensene (174 mg.) was added to a solution of 95.5 mg. of the amine hydrochloride in 3.0 ml. of ethanol, and 200 mg. of NaHCOs was added. After 3 hr. at room temperature the mixture was filtered
414
CARTER,
BHATTACHARYYA,
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AND
FRAENKEL
and the filtrate diluted to 12 ml. with water. An oil separated. This oil was taken up in 1 ml. of hot ethanol and the solution was allowed to cool slowly, giving 33 mg. of a yellow crystalline precipitate (m.p. 73-83”). After three recrystallizations from ethanol, 8.9 mg. of long, light yellow needles were obtained (m.p. 87”). Anal. (CH,),N.G,H,(NO,),. Calcd.: C 45.50, H 4.30; found: C 45.58, H 4.04. An authentic sample of dimethylamine gave t,he same dinitrophenyl derivative (m.p. 86-87”), whereas the dinitrophenyl derivative of ethylmethylamine melted at 55”.
Comparison of Vitamin
BT with Carnitine
Derivatives of BT Chloroaurate. Eighty mg. of crystalline BT was treated with a solution of 158 mg. of AuC13 in 2.0 ml. of 1 y0 HCI. On cooling the solution, 178 mg. of yellow elongated plates separated (m.p. 153-155”). The melting point was not altered by repeated recrystallizations from dilute HCl. Anal. CTHI~NO~AUC~~. Calcd.: C 16.77, H 3.22, N 2.79, Au 39.34; found: C 17.44, H 3.55, N 2.99, Au 39.57. Chloroplatinate. One hundred mg. of BT was treated with 2 ml. of ethanolic solution containing 200 mg. of PtCL and 0.5 ml. of concentrated HCI. On cooling the solution, 117 mg. of microcrystalline yellow powder separated. This material was recrystallized once from 60% aqueous ethanol and once more from water (m.p. 216-219”). The chloroaurate and chloroplatinate agree in properties with the corresponding derivatives of carnitine (5,6). Other derivatives of BT, prepared in the usual way, are listed in Table II.
Conversion of BT to Crotonobetaine A 300-mg. sample of BT was heated on an oil bath at 120-130” for 1 hr. with 5.2 ml. of concentrated H2S04 (sp. gr. 1.84). The solution was poured into 20 g. of ice and the HzSOa was neutralized with an excess of BaCOa. The filtrate was evaporated to dryness and the residue was extracted with 3.0 ml. of water. The filtered solution was treated with a solution of 600 mg. of AuC13 in 0.3 ml. of concentrated HCl and 3.0 ml. of water. The precipitate (291 mg.) was recrystallized from hot 0.01 y0 HCl giving 100 mg. of the golden-yellow, crystalline chloroaurate of crotonobetaine (m.p. 196-200”). Anal. C~Hl~NO~AuC1~. Calcd.: C 17.40, H 2.92, N 2.90, Au 40.81; found: C 17.52, H 2.82, N 2.96, Au 40.64. This product appeared to be identical with the chloroaurate of crotonobetaine reported by Engeland (8). The free crotonobetaine, obtained by removing the gold with H&J, showed no vitamin BT activity.
Preparation
of Carnitine from Meat Extract
The majority of the experiments with crystalline vitamin BT were carried out on material isolated from whey or liver. When the work was almost completed it was discovered that meat extract is a much richer source of the vitamin. By applying to meat extract the techniques developed for whey and liver, vitamin BT (carnitine)
VITAMIN
BT
415
can be isolated in good yields and with much less work. The following procedure has given uniformly satisfactory results in our hands. One hundred grams of Difco Beef Extract (active at 12-24 pg./g.) was dissolved in 700 ml. of water, and the solution was shaken with 300 ml. of molten phenol. The emulsion was filtered from suspended matter and the phenol layer was separated. The aqueous layer was re-extracted with 200 ml. of phenol. The first and second phenol layers were successively washed with 100 ml. of water using the same aqueous layer for both the phenol layers. The combined phenol layers were extracted with 1500 ml. of ether. After separation from the thick dark aqueous layer, the ether layer was washed three times with 100 ml. of distilled water and discarded. The aqueous layer and the washings were combined (total solids, 22 g.; active at 3-6 rg./g.), acidified to pH 1 with HCl, and decolorized by shaking with 475 g. of activated carbon (Norit). The filtrate and washings from carbon (total solids, 11.9 g.; active at 1.5-3.0 pg./g.) were neutralized with NH,OH (pH 6.5), evaporated to 400 ml. under reduced pressure, and extracted three times with 200-ml., lOO-ml., and loo-ml. portions of phenol, respectively. The phenol layers were washed successively with the same loo-ml. quantity of distilled water, combined, and freed from phenol in the usual manner by two successive extractions with 3 vol. of ether. The colorless aqueous layers from the last operation (second phenol extract) gave a colorless gum on evaporation (3.44 g., active at 0.75 pg./g.; recovery, 50-100%). Two grams of second phenol extract was dissolved in 4 ml. of 80% aqueous methanol and chromatographed over a column of 300 g. of water-washed Alcoa alumina. The column was developed with 80% aqueous methanol, and IO-ml. eluate fractions were collected. Fractions 25-75 on evaporation gave almost pure carnitine (0.95 g., m.p. 192-196”). This product on one further recrystallization gave 0.8 g. of essentially pure carnitine (m.p. 196-198”, active at 0.37 pg./g.).
ACKNOWLEDGMENTS The authors wish to express their appreciation to Dr. Claire Graham of the Wilson Laboratories for his assistance in making available special liver extracts for this work. We also are indebted to the Borden Company for supplies of whey and special whey preparations and for support in the isolation of a quantity of crude vitamin BT. We are happy to acknowledge research grants from the U. S. Public Health Service and from Merck and Co., Inc., in support of this project. The vitamin Br assays reported in this paper were performed by Allison Whittingham, Harriet R. Stern, and Elizabeth Nilson.
SUMMARY Vitamin B, has been isolated as a pure, crystalline substance from liver and whey, Analytical data and production of trimethylamine on alkaline degradation suggested that vitamin BT might be identical with carnitine. A careful comparison of the properties of derivatives and of reactions of the two substances established conclusively that vitamin BT is carnitine.
416
CARTER,
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WEIDMAN
AND
FRAENKEL
REFERENCES 1.
CARTER, H.E.,
BHATTACHARYYA,P.K.,
WEIDMAN, K.R., ANDFRAENKEL,G., 36, 241 (1952). G., BLEWETT, M., AND COLES, M.,Nature161,981 (1948). G., Arch. Biochem. Biophys. 32, 457 (1951). G., Arch. Biochem. Biophys. 34, 468 (1951). WORDEHOFF,P., AND~CHWANEBERG,H., Z.physiol.Chem.236,183
Arch. Biochem. Biophys. 2. FRAENKEL, 3. FRAENKEL,
4. FRAENKEL,
5. STRACK,E., (1936). 6. STRACK, E., AND FORSTERLING,K., Ber. 71B, 1143 (1838). 7. KRIMBERG, R., 2. physiol. Chem. 66, 466 (1908). 8. ENGELAND, R., Ber. 64B, 2208 (1921). 9. BERGMANN, M., BRAND, E., AND WEINMANN, F., Z. physiol.
Chem. 131,
1
(1923).
TOMITA, M., 2. physiol. Chem. 124, 253 (1922-3). CRAIG, L. C., Anal. Chem. 22, 1346 (1950). 12. SCHNEIDER, F., Qualitative Organic Microanalysis, 10. 11.
Sons, Inc., New York,
1946.
p. 189. John
Wiley
anP