the isolation of formaldehyde from … · the isolation of formaldehyde from dimethyl-...

THE ISOLATION OF FORMALDEHYDE FROM DIMETHYL- AMINOETHANOL, DIMETHYLGLYCINE,

SARCOSINE, AND METHANOL*

BY COSMO G. MACKENZIE, JOHN M. JOHNSTON, AND WILHELM R. FRISELL

(From the Department of Biochemistry, University of Colorado School of Medicine, Denver, Colorado)

(Received for publication, February 3, 1953)

Radioactive formaldehyde has recently been isolated as a product of the oxidation of methyl-labeled sarcosine by partially sedimented liver homogenates and by liver and kidney slices (1). A portion of the formaldehyde that accumulated in these preparations was shown to be present in the free or volatile form. Radioformate and carbon dioxide were also found to be products of radiosarcosine oxidation. In an extension of these results to the whole animal, radioformate appeared in the urine after ad- ministration of either radiobetaine or radiosarcosine. In concomitant experiments (2) it was shown that sarcosine is a metabolite in the animal body, its methyl carbon being derived from methyl groups ingested in the form of methionine or betaine. It was concluded, therefore, that “bio- logically labile” methyl groups as a class are sources of formaldehyde and formate in the animal organism.

Some 10 years earlier, Handler, Bernheim, and Klein (3) had observed an increased oxygen uptake and a positive color test for formaldehyde when sarcosine and dimethylglycine were incubated with a washed liver sediment. They reported that sarcosine was not oxidized by a similar preparation obtained from kidney. In the course of further investigations on l-carbon compounds, we have found that formaldehyde can be isolated as the dimedon derivative after incubation of dimethylaminoethanol, dimethylglycine, sarcosine, or methanol with an unfractionatid liver homogenate. In the case of sarcosine and dimethylglycine, the enzyme systems responsible for their conversion to formaldehyde have been located in the mitochondria. Furthermore, they have been distinguished by inhibition experiments. Formaldehyde was also isolated from sarcosine when a kidney homogenate was employed,

* A preliminary report on this work was presented before the American Society of Biological Chemists at New York, April 15, 1952 (Federation Proc., 11, 252 (1952)). This work has been supported by a grant from the United States Public Health Service.

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744 FORMALDEHYDE 1SOLATION

RESULTS AND DISCUSSION

Whole Homogenates-In our initial experiments in which liver was homogenized in Serensen’s 0.05 M sodium and potassium phosphate buffer, formaldehyde was isolated from dimethylglycine and sarcosine when the incubation was carried out at either pH 7.8 or 8.8. In the case of dimethylaminoethanol, formaldehyde was only isolated in appreciable amounts following incubation at pH 8.8. However, when a 0.075 M

potassium phosphate buffer, containing calcium and magnesium, but devoid of sodium was substituted for Sorensen’s buffer, formaldehyde was obtained from dimethylaminoethanol at both hydrogen ion concentrations. Moreover, with the potassium phosphate buffer, which more closely re- sembles the intracellular fluid of liver than does Serensen’s buffer, formaldehyde was isolated in higher yields from both dimethylglycine and sarcosine. The quantity of formaldemethone isolated from these substrates in a typical experiment with both buffers at both hydrogen ion concentrations is shown in Table I. The isolation experiments were carried out in duplicate or triplicate and were repeated from two to six times with each compound under the several conditions described above. In the case of all three substrates the isolated formaldemethone, following recrystallization from alcohol and water, melted at 188-189”, uncorrected. The melting point was not depressed on admixture with an authentic sample of formaldemethone.

When aminoethanol, monomethylaminoethanol, choline, betaine, gly- tine, monomethylamine, dimethylamine, serine, or methionine was incubated at pH 7.8 or 8.8 with liver homogenized in either the potassium phosphate or Serensen’s buffer, formaldehyde was not obtained. Sar- cosine, which was used as a control in each of these tests, invariably yielded formaldehyde. Consequently, the compounds listed above are eliminated as major intermediates in the conversion of dimethylaminoethanol, dimethylglycine, and sarcosine to formaldehyde in the system employed. It appears, therefore, that dimethylaminoethanol gives rise to formaldehyde either directly or following its oxidation to the aldehyde or acid, that is, to dimethylglycine.’

Fractionation of Liver Homogenate-The distribution of the enzyme systems responsible for the production of formaldehyde from dimethylaminoethanol, dimethylglycine, sarcosine, and methanol by the whole homogenate was next investigated. Liver was homogenized in the potassium phosphate buffer and separated into supernatant and sediment frac-

1 This does not imply that in other preparations, or in the whole animal, dimethylaminoethanol is not converted to formaldehyde via choline, betaine, or other compounds. Rather, it calls attention to a possible metabolic shunt in dimethylaminoethanol metabolism.


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MACKENZIE, JOHNSTON, AND FRISELL 745

tions by centrifugation. The sediment, or particulate fraction, was further freed of soluble components by resuspending in fresh buffer and recentrifuging several times. When either fraction was incubated with dimethylaminoethanol alone, formaldehyde did not accumulate in quantities that could be isolated as the dimedon derivative. However, as shown in Table II, the combination of the supernatant and particulate fractions resulted in the isolation of formaldehyde.2

In contrast to these results, formaldehyde was isolated from both dimethylglycine and sarcosine incubated with the washed sediment alone. Indeed, the yields were appreciably higher than those obtained with the whole homogenate. No formaldehyde was isolated from either substrate

TABLE I

Production of Formaldehyde by Unfractionated Liver Homogenate

0.225 mM of substrate was incubated for 1 hour at 37” in’ air with the equivalent of 2.5 gm. of liver. The total volume of the mixture was 22 ml. The Serensen’s buffer was 0.05 M and the potassium phosphate buffer was 0.075 M. The contents of the flasks were adjusted to pH 7.8 or 3.8 prior to incubation. The yields of formaldehyde are expressed as mg. of the dimedon derivative isolated. Triplicate determinations agreed within 10 per cent.

Substrate S~rensen’s buffer Potassium phosphate

pH 7.8 i pH 8.8 pH 7.8 1 pH 8.8

Dimethylaminoethanol ........ Dimethylglycine. ..............

Sarcosine ......................

0.1 0.9 0.5 1.0 1.2 1.6 2.3 2.3 3.5 4.2 6.1 / 6.4

plus the supernatant fraction. Furthermore, the addition of this fraction to the sediment lowered the accumulation of formaldehyde to the level that prevailed with the whole homogenate (Table II).

On the other hand, when methanol was incubated with the supernatant fraction, formaldehyde accumulated and was isolated. Furthermore, no formaldehyde was obtained from this compound plus the washed sediment. Thus all possible distributions of enzyme systems producing formaldehyde from the four methyl compounds were realized. These distribution studies indicate that, unlike dimethylglycine and sarcosine, dimethylaminoethanol requires one or more factors present in the supernatant fraction in addition to the washed particles for its conversion to formaldehyde, a finding in harmony with the hypothesis that oxidation of

2 When the unwashed sediment was employed alone, some formaldehyde was obtained and the recombination of this preparation and the supernatant fraction resulted in yields comparable to those obtained with the starting homogenate.


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746 FORMALDEHYDE ISOLATION

the alcohol group precedes demethylation. These experiments also elimi- nate methanol as an intermediate in the formation of formaldehyde from dimethylglycine and sarcosine.

Formaldehyde was also isolated after the incubation of sarcosine with the sediment fraction of rat and chick kidney homogenates. The concentrations of substrate and enzyme preparations were those employed for liver. The yields of formaldemethone obtained with rat and chick kidney were 1.3 and 2.8 mg., respectively. These direct isolation experiments are in accord with previous results obtained with radiosarcosine and rat kidney slices (1).

TABLE II Fractionation of Liver Enzyme Systems Yielding Formaldehyde

Liver homogenate was fractionated by centrifuging at 1670 X g. 0.075 M potassium phosphate buffer of pH 7.8 was employed. The incubation mixture was adjusted to pH 8.8 in the case of dimethylaminoethanol. The homogenate fraction in each flask was equivalent to 2.5 gm. of liver, and each flask contained 0.225 mM of substrate. The conditions of incubation are given in Table I. The results are expressed as mg. of formaldemethone isolated.

Substrate Homogenate Sediment supematant Reconstituted*

Dimethylaminoethanol 1.2 0 0 0.5 Dimethylglycine. 2.9 5.8 0 2.8 Sarcosine...................... 6.7 12.6 0 6.2 Methanol. . . . . 2.5 0 2.2 1.9

* The reconstituted homogenate consisted of the first supernatant fraction and the twice washed sediment.

Formaldehyde Production by Cell Components-Having observed that such a reactive, and reputedly mutagenic, compound (4, 5) as formaldehyde was produced from the metabolites dimethylaminoethanol (6), dimethylglycine (7), and sarcosine (2) by sediment preparations, which contain nuclei, cell fragments, and intact cells in addition to mitochondria, we undertook to determine which of these structural units contained the enzymes responsible for this reaction. Accordingly, the mitochondrial, nuclear, and supernatant fractions of liver were prepared in sucrose by the method of Schneider and Hogeboom (8). With dimethylaminoethanol as a substrate, none of these fractions produced formaldehyde in quantities that could be detected. However, the incubation of dimethylglycine or sarcosine with the mitochondria resulted in the isolation of formaldehyde (Table III). Some formaldehyde was obtained also with the nuclear fraction, but none was isolated from the supernatant fraction, which contained the microsomes. When the three fractions were recombined, the


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yield of formaldehyde was considerably less than was obtained with the original unfractionated homogenate. However, the whole and reconstituted homogenates differed considerably with respect to the relative pro- portions of sucrose and buffer that they contained, inasmuch as the whole homogenate, while prepared in sucrose, was brought to the desired volume with buffer, whereas the reconstituted homogenate contained a much higher per cent of sucrose contributed by the supernatant fraction.

After determining that formaldehyde could be recovered quantitatively from either buffer or sucrose, the incubation of dimethylglycine and sarcosine was repeated with the whole homogenate, the reconstituted homog-

TABLE III

Cellular Distribution of Enzymes Producing Formaldehyde from Dimethylglycine and Sarcosine

Each incubation mixture consisted of 0.225 mM of substrate and the material prepared from 2.5 gm. of liver. The results are expressed as mg. of formaldehyde isolated as formaldemethone.

Substrate

Dimethylglycine “

Sarcosine “

Medium

Buffer* Sucroset Buffer* Sucroset

2.7 0.2 3.2 0 1.8 2.2 0.4 1.8

/ 0 2.2

6.4 2.5 6.8 0 2.8 3.0 2.1 5.6 0 3.0

* The per cent of the potassium phosphate buffer in the medium for the several fractions was as follows: homogenate, 88; nuclei and mitochondria, 100; supernatant and reconstituted fractions, 50. The remaining volume in each fraction consisted of the sucrose solution.

f The medium for all fractions consisted of 83 per cent sucrose solution and 17 per cent buffer.

enate, and the several fractions suspended in a medium comprised of 83 per cent sucrose solution and 17 per cent buffer. Under these conditions, the quantity of formaldehyde isolated from the reconstituted homogenate agreed with the quantity obtained from the original homogenate (Table III), thus demonstrating that there had been no loss of activity during the fractionation. These experiments show that the enzymes that oxidize dimethylglycine and sarcosine to formaldehyde reside primarily in the mitochondria or large granules of the liver cells. Microscopically, the mitochondria were free from nuclei and cells and consisted of granules and aggregates of granules. This material was stained by Janus green as shown by high power examination. When viewed under oil immersion, many of the granules were seen to be brightly stained, while others, par- ticularly those in the aggregates, were more faintly and diffusely colored.


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As mentioned above, formaldehyde was isolated also when sarcosine and dimethylglycine were incubated with the so called nuclear fraction. Microscopic examination of this fraction revealed that it contained, in addition to nuclei, some red blood cells, fragments of liver cells (containing mitochondria, as judged by staining with Janus green), and small clumps of intact liver cells. Resuspension and recentrifugation of the nuclear fraction in sucrose for as many as seven times failed to reduce the accumulation of formaldehyde by more than 25 per cent. Red blood cells were eliminated as the source of the formaldehyde-producing enzymes by in- cubating rat erythrocytes with sarcosine with negative results. Accord- ingly, nuclei were prepared by the more recent procedure of Hogeboom, Schneider, and Striebich (9). In confirmation of their description, this nuclear preparation was found to be uncontaminated with liver cells and discernible cell fragments, to contain no material staining with Janus green, and to contain only a small percentage of red blood cells. When sarcosine or dimethylglycine was incubated with this nuclear preparation, no formaldehyde was isolated.

Inhibition of Sarcosine Oxidation-It will be noted from Table III that the ratio of formaldehyde isolated from the impure nuclear fraction to formaldehyde isolated from the mitochondria was higher for sarcosine than for dimethylglycine. This finding suggested that the systems oxi- dizing sarcosine and dimethylglycine were not identical in their distribution. Consequently, a compound was sought that would inhibit the conversion of sarcosine to formaldehyde without interfering with the oxidation of dimethylglycine. Because of its structural resemblance to sarcosine, it seemed that methoxyacetate might be such a compound, and this proved to be the case. As is shown in Table IV, methoxyacetate markedly reduced the yield of formaldehyde from sarcosine, but did not reduce the accumulation of formaldehyde when dimethylglycine was the substrate. It has been found in other experiments (10) that methoxyacetate lowers the oxygen consumption and the production of glycine and serine from sarcosine. Acetate, propionate, and butyrate have a similar action in decreasing order of effectiveness.

When dimethylglycine is the substrate, methoxyacetate also inhibits the formation of glycine and serine, even though formaldehyde accumulation is not affected. Moreover, when methoxyacetate is incubated with dimethylglycine, there occurs an accumulation of sarcosine which otherwise is not demonstrable by paper chromatography. These results indicate that dimethylglycine is oxidized in the particulate fraction of liver to formaldehyde and sarcosine by an enzyme that is either not identical with sarcosine oxidase or, alternatively, that sarcosine oxidase possesses a second and different active center for the oxidation of dimethylglycine. Further-


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more, the results indicate that both of the methyl groups of dimethylgly- tine are sources of formaldehyde.

While the experiments reported in this paper were designed to demon- strate sources of formaldehyde by direct isolation (or of l-carbon compounds exhibiting its oxidation level), and to determine the cellular distribution of enzyme systems responsible for its production, they also provide information concerning pat,hways for the metabolism of methyl groups. It has been shown by du Vigneaud and his coworkers (11) that the methyl groups of choline, betaine, and methionine are biochemically interchangeable and that monomethyl- and dimethylaminoethanol are converted to choline in the body (6). Dubnoff (12) has found that choline is oxidized to betaine prior to the methylation of homocysteine, and Muntz

TABLE IV

E$ect of Methoxyacetate and Acetate on Formaldehyde Production from Sarcosine and Dimethylglycine

0.225 rn~ of substrate and 1.125 mM of inhibitor were incubated with the washed sediment from 2.5 gm. of liver in the potassium phosphate buffer at pH 7.8. The final volume was 22 ml.

Inhibitor

None ................................ Methoxyacetate ..................... Acetate .............................

Yield of formaldemethone’

Sarcosine I Dimethylglycine

mg.

6.2 1.1 3.0

* Averages of triplicate determinations; yields agreed within 10 per cent.

(7) has presented evidence that dimethylglycine is a product of the trans- methylation reaction. Evidence is presented in the present paper that, in the rat, dimethylglycine is oxidized by the mitochondria to formaldehyde and sarcosine. Sarcosine in turn is converted to formaldehyde by the mitochondria. Thus, a pathway has been established for the oxidation of two of the three methyl groups of choline and betaine. This finding is in harmony with our earlier observation that the methyl carbons of betaine and of methionine are sources of the methyl group of sarcosine in the living animal (2).

EXPERIMENTAL

Xubstrates-The sarcosine was the free base supplied by Hoffmann-La Roche. The methanol, dimethylaminoethanol, and monomethylaminoethanol were distilled at atmospheric pressure and the fractions distilling at 60-64”, 125-126’, and 14%150”, respectively, were collected. The


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electrotitration curves of the aminoethanols corresponded to the theo- retical curves. Dimethylglycine was synthesized by the method of Mi- chaelis and Schubert (13) from monochloroacetic acid and dimethylamine and, after five recrystallizations from alcohol, gave the following analysis.

C4Hs02NNa. Calculated, C 38.40, H 6.45, N 11.20; found, C 38.46, H 6.48, N 11.21

None of these compounds contained formaldehyde as indicated by an absence of a precipitate when treated with dimedon.

Bu$ers-In our initial experiments 0.05 M Sldrensen’s phosphate buffer (containing Na2HP04 and KH2POI) of pH 7.8 was employed. As the investigation progressed, it appeared desirable to construct a buffer that resembled the constitution of the intracellular fluid of rat liver more closely both with respect to ionic species and ionic strength. It was calculated from the data of Fenn (14) that rat liver contains 212 m.eq. of K and 147 m.eq. of acid-soluble phosphorus per liter of cell water. According to Gamble (15) the anionic groupings on cell protein are equivalent to 33 per cent of the total cations of cell water. On this basis 70 m.eq. of cell K would be matched against protein and 142 m.eq. against other anions, a figure in agreement with the 147 m.eq. of P found by Fenn. Accordingly, our buffer was made up of 0.075 M potassium phosphate which contains approximately 150 m.eq. each of K and phosphate ions at pH 7.8. In addition, MgS04 and CaS04 were added in quantities that were found to be soluble at pH 8.8; i.e., 0.001 M and 0.0001 M, respectively. Thus, the buffer contained the major inorganic ionic constituents of intracellular fluid and was devoid of Na and Cl ions.

Liver Fractions-Livers from decapitated male and female rats, weigh- ing from 150 to 200 gm., were used as the source of the enzyme preparations. The whole homogenate was prepared by homogenizing a given weight of liver with a corresponding volume of buffer at pH 7.8 in the Waring blendor for 1 minute and straining the mixture through a double layer of cheese-cloth. It was assumed that 1 ml. of this mixture contained 0.5 gm. of liver. 5 ml. portions of this preparation were pipetted into 125 ml. Erlenmeyer flasks and 15 ml. of buffer were added. The pH of the mixture was checked and, when desired, it was raised to 8.8 by the addition of 0.1 N KOH. The contents of each flask were warmed to approximately 37” in a hot water bath and the flasks were then immediately clamped to a shaker in a 37” constant temperature room. After shaking for 10 minutes, 0.225 mM of substrate, dissolved in 2 ml. of buffer at 37”, was added to the flasks.

The sediment or particulate fraction of liver was prepared by centrifuging the whole homogenate, diluted so that 4 ml. contained 1 gm. of liver, in an International PR-1 refrigerated centrifuge with the angle head No. 823 at 1670 X g (calculated for the end of the centrifuge tube) for 10 min-


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utes at 0”. At the end of this time four layers were discernible in the centrifuge tube; a bright red button at the bottom, a larger dark red layer, a viscous buff-colored layer, and a relatively clear top layer. The upper- most layer and the buffy layer were removed by decanting. This material constituted the supernatant fraction. The sediment remaining in the tube was resuspended in a volume of buffer equal to the volume of the supernatant fraction and centrifuged for 3 minutes and the supernatant fluid was poured off. The sediment was washed two more times in this fashion. Both the washed sediment and the supernatant fractions were added to incubation flasks in quantities equivalent to 2.5 gm. of liver and the volume was adjusted to 20 ml.

The mitochondria, nuclei, and microsomes of liver were separated by centrifugation in 0.25 M sucrose according to the method of Schneider and Hogeboom (8) after homogenization in sucrose in the Potter and Elvehjem (16) apparatus. Because of the relatively large volume of the supernatant (microsomal) fraction obtained in this procedure, the volume of the incubation mixture for all fractions and for the reconstituted preparation was brought to 42.5 ml. In each instance this volume contained the material obtained from 2.5 gm. of liver.

Formaldehyde Isolation-All of the preparations from either the buffer or sucrose homogenates were incubated with substrate for 1 hour, after which time the reaction was stopped by precipitating the protein with an equal volume of 20 per cent trichloroacetic acid. The protein was separated by centrifugation and washed once with 10 ml. of 10 per cent trichloroacetic acid. The supernatant fluid from the washing was added to the original supernatant fraction and the combined solutions were neutralized with 10 per cent NaOH. The pH was adjusted to 4.5 with acetic acid with methyl red as the indicator. The solution was filtered through Whatman No. 52 paper and 20 ml. of a 0.4 per cent solution of dimedon were added to each flask. After standing at room temperature for 18 to 24 hours, the precipitate was collected on a filter paper disk by filtering through the apparatus of Henriques et al. (17). The precipitate was washed twice with 25 ml. portions of water and was then dried for 5 minutes at the pump under an infra-red lamp. The disk containing the precipitate was then removed and dried for 1 hour under the lamp.

The crystals of formaldemethone dissolved readily in alcohol and were recrystallized by the addition of water. Material recrystallized three times in this fashion was used for melting point determinations. The yields of formaldehyde reported are for non-recrystallized material cor- rected for blanks obtained from the same liver fraction incubated in the absence of a substrate. The correction was approximately 0.1 mg. and was due to an amorphous material that was insoluble in alcohol. In suspension this material was easily distinguished from formaldemethone,


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whose crystals are readily discernible in quantities of less than 0.1 mg. in a 100 ml. volume. In experiments in which 6 mg. of formaldehyde were added to whole homogenates and the mixture immediately deproteinized, 90 per cent of the formaldehyde was recovered as the dimedon derivative.

The authors wish to acknowledge the technical assistance of Mrs. E. Gregory Ashe.

SUMMARY

Formaldehyde has been isolated, as the dimedon derivative, from dimethylaminoethanol, dimethylglycine, sarcosine, and methanol incubated with liver homogenates prepared in buffer.

Fractionation of the homogenate by centrifugation revealed that both the supernatant fraction and washed sediment were required for the conversion of dimethylaminoethanol to formaldehyde. On the other hand, formaldehyde was isolated when methanol was incubated with the supernatant fraction alone or when dimethylglycine and sarcosine were incubated with the washed sediment. With the latter substrates, the addition of the supernatant fraction reduced the accumulation of formaldehyde.

When the components of liver cells were prepared by differential centrifugation in sucrose, the enzyme systems responsible for formaldehyde production from dimethylglycine and sarcosine were located in the mitochondria. Formaldehyde was not isolated when either of these substrates was incubated with a purified nuclear fraction or with the supernatant fraction containing the microsomes.

The enzyme reactions that catalyze the oxidation of dimethylglycine and sarcosine to formaldehyde have been distinguished through inhibition experiments. It has been found that methoxyacetate and acetate inhibit the oxidation of sarcosine to formaldehyde. However, when dimethylglycine is incubated in the presence of these ions, its oxidation is not in- hibited and formaldehyde and sarcosine accumulate. Thus both of the methyl groups of dimethylglycine appear to be sources of formaldehyde in mitochondrial preparations, one by direct oxidation and the other via sarcosine formation.

When monomethylaminoethanol, choline, betaine, and methionine were incubated with liver homogenates, formaldehyde was not isolated. The experimental results are discussed with respect to pathways of oxidation of labile and non-labile methyl groups.

BIBLIOGRAPHY

1. Mackenzie, C. G., J. Biol. Chem., 186, 351 (1950). 2. Horner, W. H., and Mackenzie, C. G., J. Biol. Chem., 187, 15 (1950).


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3. Handler, P., Bernheim, M. L. C., and Klein, J. R., J. Biol. Chem., 138,211 (1941). 4. Rapoport, I. A., J. Gen. Biol., U. S. S. R., 8, 359 (1947); Chem. Abstr., 42, 1977

(1948). 5. Kaplan, W. D., Science, 108, 43 (1948). 6. du Vigneaud, V., Chandler, J. P., Simmonds, S., Moyer, A. W., and Cohn, M.,

J. BioZ. Chem., 184, 603 (1946). ‘7. Muntz, J. A., J. Biol. Chem., 182, 489 (1950). 8. Schneider, W. C., and Hogeboom, G. H., J. Biol. Chem., 183, 123 (1950). 9. Hogeboom, G. H., Schneider, W. C., and Striebich, M. J., J. BioZ. Chem., 196,

111 (1952). 10. Frisell, W. R., and Mackenzie, C. G., Federation Proc., 12, 206 (1953). 11. du Vigneaud, V., A trail of research, Ithaca (1952). 12. Dubnoff, J. W., Arch. Biochem., 24, 251 (1949). 13. Michaelis, L., and Schubert, M. P., J. BioZ. Chem., 115, 221 (1936). 14. Fenn, W. O., J. BioZ. Chem., 128, 297 (1939). 15. Gamble, J. L., Chemical anatomy, physiology and pathology of extracellular

fluid, Cambridge (1951). 16. Potter, V. R., and Elvehjem, C. A., J. BioZ. Chem., 114, 495 (1936). 17. Henriques, F. C., Jr., Kistiakowsky, G. B., Margnetti, C., and Schneider, W. G.,

Ind. and Eng. Chem., Anal. Ed., 18, 349 (1946).


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Wilhelm R. FrisellCosmo G. Mackenzie, John M. Johnston and

AND METHANOLDIMETHYLGLYCINE, SARCOSINE,

FROM DIMETHYLAMINOETHANOL, THE ISOLATION OF FORMALDEHYDE

1953, 203:743-753.J. Biol. Chem.

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