formation of formaldehyde and formate … · formation of formaldehyde and formate in the...

FORMATION OF FORMALDEHYDE AND FORMATE IN THE BIOOXIDATION OF THE METHYL GROUP*

BY COSMO G. MACKENZIE

(From the Department of Biochemistry, Cornell University Medical College, New York City)

(Received for publication, April 15, 1950)

When the oxidation of the “biologically labile” methyl group to carbon dioxide was first reported several years ago, it was pointed out that the identity of the intermediates produced in this reaction and their roles in metabolic processes were matters of considerable interest (1). It seemed probable that these intermediates included single carbon compounds whose normal occurrence and function in .higher animals had received comparatively little attention. As an initial approach to this problem, the assumption was made that formaldehyde and formic acid are among the compounds produced in the oxidation of methyl groups found in the body attached to S and N, and experiments were conducted to test the validity of this hypothesis.

In exploratory investigations, it was found that the methyl groups of Ci4-labeled methionine, betaine, choline, and .sarcosine were oxidized to Cl402 by rat liver slices. However, only the methyl group of sarcosine was oxidized to carbon dioxide when liver homogenates were employed. Since homogenates offer advantages over tissue slices with respect to the isolation of metabolic products, sarcosine was used as the source of methyl groups in the subsequent isolation experiments.’

Evidence of the demethylation of sarcosine to glycine in vivo was reported by Abbot and Lewis (3) in 1939. Shortly thereafter, Bloch and Schoenheimer (4) fed N16-labeled sarcosine to rats and showed by isolation experiments that it was rapidly and directly demethylated to form glycine without deamination. In 1941, Handler, Bernheim, and Klein

*A research grant from the Lederle Laboratories Division of the American Cyanamid Company has made the present investigation possible.

1 Although ssrcosine was prepared in 1847 by Liebig (2) as a degradation product of creatine, no reference could be found in the literature to its occurrence in higher animals. Experiments were therefore undertaken to obtain evidence for the production of sarcosine in the animal body. The experiments have shown that sarcosine is formed in the rat and that the methyl groups of methionine and betaine contribute to its formation (Horner, W. H., and Mackenzie, C. G., J. Biol. Chem., in press). These results indicate that sarcosine may be used to study intermediates formed in the oxidation of methyl groups provided by “biologically labile” methyl compounds.

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352 FORMATION OF FORMALDEHYDE AND FORMATE

(5) observed that sarcosine was oxidatively demethylated by liver homogenates. On the basis of the increase in oxygen consumption, the increase in glycine, and a positive color test for formaldehyde, they concluded that 1 mole of sarcosine reacts with 1 atom of oxygen wit(h the production of 1 mole of glycine and 1 mole of formaldehyde.

The present paper is a report of the isolation of formaldehyde and the determination of formate as intermediates in the oxidation of the methyl group of sarcosine by liver homogenates and liver slices, and the extension of these results to the metabolism of the intact animal.

A preliminary report of the isolation of formaldehyde has already appeared (6). At the time that this result was presented at the Detroit meeting of the American Society of Biological Chemists, it was also reported that formate is an oxidation product of the methyl group of sarcosine. Recently Ling and Tung (7) have observed a positive color test for formaldehyde when N-methyl-L-amino acids are oxidized by a flavo- protein prepared from liver or kidney. Sarcosine is not oxidized by this enzyme.

Preparations and Methods

Synthesis of Radiosarcosine-Barium carbonate2 containing Cl4 was converted to radiomethyl iodide by the procedure of Melville, Rachele, and Keller (8). The methyl iodide was condensed with the p-toluenesulfonyl- glycine at 70” to yield p-toluenesulfonylsarcosine (9). The latter compound was hydrolyzed in concentrated HCI, and the radiosarcosine was isolated as the p-toluenesulfonic acid salt. This salt melted at 186-187”, corrected. There was no depression in the melting point following admixture with an authentic sample.

CioHi60sNS. Calculated, N 5.36, S 12.26; found, N 5.66, S 12.30

The p-toluenesulfonic acid salt of radiosarcosine was converted to sarcosine hydrochloride.3 The melting point was 170-171°, corrected. The radiosarcosine hydrochloride was neutralized with buffer prior to its use in the biological experiments. Hereafter all mention of radiosarcosine will refer to the free base. 1 mg. of radiosarcosine, when oxidized and converted to BaC1403, gave 1.45 X lo6 c.p.m., corrected for the background and self-absorption.

Preparation of Honzogenates-Non-fasted adult rats of the Rockland

2 The radioactive barium carbonate used in this investigation was supplied by the Monsanto Chemical Company, on allocation from the Isotopes Division, United States Atomic Energy Commission.

JThe author wishes to express his appreciation to Dr. Donald B. Melville for his collaboration in the synthesis of radiosarcosine.


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C. G. MACKENZIE 353

Farms strain, that had been maintained on a commercial dog chow (“big red dog food,” Co-op Grange League), were stunned by a blow on the head and killed by exsanguination. The liver was removed and homogenized in the Potter-Elvehjem apparatus (10) with sodium phosphate buffer, 0.05 M, pH 7.8, equal in volume to the weight of liver employed. This is the buffer employed by Handler, Bernheim, and Klein (5) in their investigation of sarcosine oxidase. Homogenization and subsequent operations were carried out in a cold room at 7”.

The homogenate was centrifuged for 5 minutes at approximately 2500 X g. All, or a portion, of the upper phase, depending on the indi- vidual experiment, was then removed and replaced with an equal volume of buffer. The homogenate was mixed and the above process was repeated. The preparation was used immediately.

Warburg Bcperiments-1 ml. of homogenate and 1 ml. of buffer were added to each vessel. The desired quantity of radiosarcosine dissolved in 0.2 ml. of buffer was placed in a side arm. The incubation was carried out at 37” in an atmosphere of air. In experiments with liver slices 200 mg. of slices in 2 ml. of Krebs-Ringer phosphate buffer, pH 7.4, were placed in each vessel. The incubation was carried out at 37” in an atmosphere of oxygen.

In both the enzyme and liver slice experiments the center well of the Warburg vessel contained 0.2 ml. of 2.5 N sodium hydroxide. Hydro- chloric acid was generally used to stop the reaction and to liberate carbon dioxide. Shaking was continued for 3 hour after the addition of acid.

Large Scale Experiments-The incubation was carried out in a 250 ml. filter flask immersed in a water bath at 37”. The ratio between the total volume of buffer and homogenate (or liver slices) was the same as that used in the Warburg experiments. Substrate and reagents were added to the flask by way of a separatory funnel. Carbon dioxide-free air (or oxygen) was drawn through the reaction vessel and thence through a scrubber containing an aqueous solution of dimedon adjusted to pH 4.5 with acetic acid.

Measurement of C1402---The sodium hydroxide contained in the center well of each Warburg vessel was transferred to an Erlenmeyer flask and the well was washed out with 5 ml. of water. 21.5 mg. of non-isotopic sodium carbonate were added to the combined solutions, the volume was made up to approximately 25 ml., and the carbon dioxide was precipitated as barium carbonate. The latter compound was deposited on a filter paper disk and counted iti a thin mica window Geiger-Miiller counter against a barium carbonate standard prepared from the combustion of radiosarcosine. The Cl4 present in the experimental sample was calculated in terms of radiosarcosine, and the percentage of the incubated


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-CY4H3 oxidized to U402 was derived from this value. A detailed account of the preparation of samples for counting will be found in a previous publication (1).

Isolation of C14HzO-The radioformaldehyde present in the incubation mixture at the end of an experiment was isolated as the dimedon (di- methyldihydroresorcinol) derivative (1 l), also referred to as formaldemethone. In the homogenate experiments the incubation mixture from each Warburg vessel was transferred, with the aid of 3 ml. of water, to a 15 ml. centrifuge tube. When slices were employed, the contents of each vessel were homogenized in the Potter-Elvehjem apparatus at the end of the incubation period and then transferred to a centrifuge tube. 5 ml. of 20 per cent trichloroacetic acid (TCA) were added and the protein was spun down in the centrifuge. The supernatant was decanted, and the precipitate was washed twice with 5 ml. portions of 10 per cent TCA. The combined TCA solutions were adjusted to pH 4.5 with sodium hydroxide and filtered.

3.7 mg. of non-isotopic formaldehyde were added to the filtrate, followed in a few minutes by 20 ml. of 0.4 per cent aqueous dimedon. After 24 hours the formaldemethone was removed by filtration. Examination of the filtrate showed that this procedure removed 99 per cent of the metabolic C14Hz0 present in the TCA-soluble fraction. The isolated formaldehyde was counted directly against the barium carbonate standard prepared from radiosarcosine. The counts were corrected for self-absorption but not for any difference that may exist in the counting rate of formaldemethone and barium carbonate. The dimedon derivative was recrystallized to constant radioactivity. The results were expressed as the per cent of incubated -CL4H3 recovered as CY4Hz0.

‘Determination of HC1400H-10 mg. of carrier formic acid and 16 ml. of 0.1 M phosphoric acid were added to the formaldehyde-free TCA- soluble fraction and the solution was distilled into dilute alkali. When the volume in the distilling flask reached 20 ml., 50 ml. of water were added and the distillation was continued. This process was repeated a second time. The recovery of formic acid from solution by this procedure was 95 per cent.

The distillate was adjusted to pH 6 and treated with 20 ml. of the mercuric chloride reagent of Benedict and Harrop (12), which oxidizes formate to carbon dioxide. The reaction mixture was acidified with phosphoric acid and the evolved carbon dioxide was collected in sodium hydroxide solution. The CL4 content of the carbon dioxide was determined following its conversion to barium carbonate. The results were expressed as the per cent of incubated --C14H3 recovered as HCY400H. The above procedure was used in the routine determination of HU400H.

In two experiments, radioformate was also obtained from incubation


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C. G. MACKENZIE 355

mixtures of radiosarcosine and homogenized liver by vacuum distillation. The enzymatic reaction was stopped by the addition of 0.6 ml. of 2.5 M

phosphoric acid to the main compartment of the Warburg vessel. The contents of the vessel were transferred to a distilling flask, and 115 ml. of water, 3.6 mg. of formaldehyde, and 12.3 mg. of formic acid were added. The mixture was distilled at 22-24” and at a pressure of Fpproximately 15 mm. of Hg. The vapors were collected and frozen in a flask immersed in a cellosolve-dry ice bath.

When 100 to 110 ml. of distillate had been collected, 20 ml. of 0.4 per cent dimedon solution were added to the melted distillate, and the formaldemethone was removed by filtration 24 hours later.

The filtrate, pH 4.5, was aerated with carbon dioxide-free air for 1 hour. At the end of this time 13 mg. of sodium carbonate were added, and the aeration was continued for another hour. As was shown by analysis, this procedure removed any residual Cl402 present in the solution. The presence of HC400H was demonstrated by the production of CY402 when the solution was treated with mercuric ions. The quantity of HCY400H found by this procedure was approximately 15 per cent less than the quantity found in the routine procedure described above.

EXPERIMENTAL

In the first experiment, in which 460 mg. quantities of radiosarcosine were incubated with a washed liver homogenate, two procedures were employed in isolating CY4Hz0, as the dimedon derivative, from the contents of the Warburg vessels. In one of these, carrier formaldehyde was added to the TCA-soluble fraction prepared from an incubation mixture, and the formaldehyde was then precipitated as the dimedon derivative. In the second procedure, the contents of the main well of the Warburg vessel were transferred to a flask containing 200 ml. of 0.1 M phosphoric acid, and the mixture was distilled into aqueous dimedon. Carrier formaldehyde was added to the distillate and the formaldemethone that crystallized was removed by filtration.

The formaldemethone prepared by both of these procedures gave over 1000 c.p.m., uncorrected. The material obtained from the TCA-soluble fraction, and twice recrystallized from ethanol-water, possessed an activity of 148 c.p.m. per mg., corrected for background and self-absorption. There was no change in specific activity following a third recrystallization from this solvent pair. The melting point was 18%189”, corrected. The formaldemethone was finally recrystallized from hot isopropyl alcohol. The crystals so obtained gave 148 c.p.m. per mg. When formaldemethone that had been thrice recrystallized from ethanol-water was sublimed at 100” in vucuo (1 mm. of Hg), there was no decrease in the specific activity.

In view of the foregoing results, the’ formaldemethone prepared from


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356 FORMATION OF FORMALDERPDE AND FORMATE

the TCA-soluble fraction and recrystallized three times from ethanol- water was used in the routine determination of CP4H20. The results of triplicate determinations on the same homogenate preparation agreed to within 10 per cent.

Liver Homogenates---The quantity of C4Hz0 that was isolated when radiosarcosine- was incubated for 1 hour with different liver homogenates is shown in Table I. The experiments are listed in the order of i&-easing yield of CY4Hz0. An increase in the accumulation of CY4Hz0 was ac- companied by a decrease in the accumulation of HC1400H and a decrease

Oxidation of Methyl Group

Experiment No.

41 4.77 20 75 3.36 30 71A 5.07 20 11 5.18 60 71B 5.07 50t

Controls~ 4.77-5.7 20

- I I

VOlUllX removed

Radiosarcosine ‘g$E$ homogenate*

- I

TABLE I of Radiosarcosine by Liver Homogenates

OW&- consumption

Oxidation product2Ct;xr cent incubated 8

Homog- enate alone

PM

7.2 6.8 6.5 2.7 2.5 0

--

PM

3.0 2.4 2.1 3.8 2.7 8.1 2.8 12.9 2.4 17.4 0 0

EW’OOH

15.1 14.0 12.7

10.3 0

8.6 26.1 4.6 22.4 2.6 23.4 1.4 0.5 28.2 0 0

* The figures in this column indicate the per cent of the total volume removed from the top of the homogenate and replaced with buffer after the second of two centrifugations. Equal or smaller volumes had been removed after the first centrifugation.

‘t Thii homogenate was centrifuged three times; otherwise the general procedure was unchanged.

$ Homogenates heated for 5 minutes in a boiling water bath.

in the production of CY402. However, the sum of these three oxidation products agreed reasonably well in different experiments.

These results suggested that approximately the same amount of C14Hz0 was produced by different preparations, but that the quantity that ac- cumulated depended on the extent to which formaldehyde oxidase (and ,formic acid dehydrogenase) had been removed from the sedimented fraction of the homogenate. Evidence for this supposition was found in Experiments 71A and 71B (Table I), in which the homogenates were prepared from the same rat liver, and hence differed only in the extent to which the particulate fraction was freed from the supernatant phase. With the more thoroughly washed homogenate (Experiment 71B), the accumulation of C14Hz0 was double that obtained with the less thoroughly


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C. Q. MACKENZIE 357

washed. homogenate (Experiment 71A). It was observed that the dis- carded upper phase of the centrifuged homogenates contained formaldehyde oxidase, as indicated by Warburg experiments.

The increase in oxygen consumption that was produced by the addition of sarcosine to a liver homogenate (Table I) was in each instance more than double the calculated quantity required for the production of the observed oxidation products. In Experiment 41, an attempt was made to locate the incubated radiocarbon that was unaccounted for as formaldehyde, formate, and carbon dioxide.

The sodium hydroxide solution obtained from the center well of the Warburg flasks was examined for the presence of CY4Hz0 and HCY400H. The combined quantity of these two compounds that had been absorbed in the alkali was equivalent to approximately 0.1 per cent of the incubated radiomethyl groups.

The completeness with which C14H~0 had been removed from the TCA- soluble fraction was checked by adding a second quantity of carrier formaldehyde to the filtrate obtained from the first formaldehyde precipitation. This second sample of carrier formaldehyde was reisolated as the dimedon derivative and found to contain less than 0.05 per cent of the Cl4 originally added as radiosarcosine.

The protein fraction (TCA precipitate) was extracted twice with 10 per cent TCA, three times with acetone, and twice with ether. The average Cl4 content of the protein obtained from the experimental vessels was equal to 1.8 per cent of the Cl4 added as radiosarcosine. In contrast, the protein prepared from the boiled homogenate incubated with radiosarcosine contained 0.1 per cent of the added CY4.

The radiosarcosine still present in the incubation mixture was determined by the carrier procedure. 50 mg. of carrier sarcosine were added to aliquots of the TCA-soluble fraction obtained from each vessel. The sarcosine was reisolated as the hydrochloride and recrystallized from ethanol-ether to constant specific activity. From the radioactivity determinations, it was calculated that the average experimental vessel contained no more than 1.1 per cent of the original radiosarcosine.

A portion of the acidified TCA-soluble fraction, from which HCY400H had been removed by distillation, was neutralized and evaporated to dryness in vacua over phosphorus pentoxide. The residue was oxidized by the method of Van Slyke and Folch (13), and the evolved carbon dioxide was absorbed in alkali, converted to barium carbonate, and counted. When corrected for the size of the aliquot employed, the total Cl4 present in the TCA-soluble fraction was found to represent 60 per cent of the incubated radiomethyl carbon, of which 1.1 per cent at the most could be accounted for as radiosarcosine, The remainder was present


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as a compound (or compounds) that had been retained in acid solution during distillation.

The total recovery of Cl* from the experimental vessels, in the form of formaldehyde, formate, carbon dioxide, protein, sarcosine, and one or more unidentified compounds, was 85 per cent. With the boiled homogenate, the total recovery of Cl4 was 92 per cent. 78 per cent was present as radiosarcosine, and the remainder was present as an unidentified compound (or compounds) retained in acid solution during distillation.

The determination of the total Cl* and the radiosarcosine present in the TCA-soluble material, obtained from both the control and the experimental vessels, was made after the TCA-soluble material had been stored in a refrigerator for approximately 1 month.

Liver Slices-C1*H20 was isolated, as the dimedon derivative, when liver slices were incubated with radiosarcosine. The formaldemethone

TABLE II Oxidation of Methyl Group of Radiosarcosine by Liver Slices

_. Oxidation products, as per cent incubated --CVh

Radiosarcosine C“HLl HCY’OOH C”O1

-_ -~__ Y

300 0.55 4.9 300 0.88 1.9 5.0 300 0.54 1.8 4.8 300* 0 0 0

- * Incubated with liver slices heated for 5 minutes in a boiling water bath.

obtained in different experiments gave approximately 75 c.p.m. above the background. No CY*HzO was formed when liver slices were heated in a boiling water bath before the addition of radiosarcosine.

HCY*OOH and CY402 were produced in these experiments, as is shown in Table II. The oxygen uptake of liver slices was not increased signifi- cantly by either radiosarcosine or non-isotopic sarcosine.

The conversion of the methyl group of sarcosine to formaldehyde by kidney cortex slices was also demonstrated.

Detection of Free (Volatile) C’*HzO-In the foregoing experiments with liver homogenates and slices, the possibility existed that C’*HzO was not elaborated as such but was formed from a labile precursor during the precipitation of proteins with trichloroacetic acid. The following experiment was carried out to obviate this possibility.

1.03 mg. of radiosarcosine were incubated with a washed liver homogenate in the apparatus described in the section on methods. A slow


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C. G. MACKENZIE 359

stream of carbon dioxide-free air was drawn through the reaction vessel and thence through an aqueous solution of dimedon. After 30 minutes, 3.74 mg. of non-isotopic formaldehyde were added to the reaction mixture. 5 minutes later the flow of air was discontinued, and a rapid stream of nitrogen (approximately 0.5 liter per minute) was drawn through the apparatus for 4 hours. During this time crystals of formaldemethone appeared in the dimedon solution. The scrubber containing dimedon was disconnected and TCA was run into the reaction vessel. CY4Hz0 was isolated from both the dimedon solution and from the TCA-soluble fraction of the incubation mixture, and the two samples of formaldemethone were recrystallized to constant radioactivity. The results are shown in Table III.

TABLE III Detection of Free (Vohtile) C14Hp0 as Oxidation Product of Radiosarcosine

Sedimented fraction from 1 gm. liver...........................

0.6 liver slices.. . . gm. . .

w. hrs.

1.03 0.5 3.9 16.6 1.51 2.5 1 0.2 1.2

* At the end of the incubation period a stream of nitrogen was drawn through the homogenate preparation for 4 hours, and a stream of air was drawn through the liver slice preparation for 1 hour. For further details see the text.

A similar experiment was carried out with 600 mg. of liver slices and 1.5 mg. of radiosarcosine incubated in an atmosphere of oxygen. 5.6 mg. of carrier formaldehyde were added to the reaction mixture during a period of 2+ hours. The mixture was then adjusted to pH 2 with hydrochloric acid to stop the reaction and to increase the rate of diffusion of formaldehyde (14), and carbon dioxide-free air was drawn through the apparatus at 1 liter per minute and at a pressure of -70 mm. of Hg. for 1 hour. The quantities of CY4Hz0 isolated from the dimedon scrubber and from the reaction vessel are given in Table III. The Cl402 produced in the course of this experiment was also determined and found to be equivalent to 5.9 per cent of the incubated -CY4H3.

Direct Isolation of Formaldehyde-Because of the importance that is attached to the identification of formaldehyde as a normally occurring compound in the metabolism of animals, it was desirable to exclude com-


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360 FORMATION OF FORMALDEHYDE AND FORMATD

pletely the possibility that its isolation with carrier formaldehyde was an artifact attributable to the presence of a radioactive contaminant. Ac- cordingly, an attempt was made to isolate formaldehyde as an oxidation product of the methyl group without the use of a carrier.

The livers from four rats were homogenized in the Potter-Elvehjem apparatus. The homogenate was centrifuged twice, the entire upper phase being removed and replaced with buffer following each centrifugation. The particulate fraction was incubated for 1 hour with 49 mg. of non- isotopic sarcosine plus 1.26 mg. of radiosarcosine. 8 mg. of formaldemethone were isolated from the TCA-soluble fraction of the incubation mixture. The material was recrystallized three times from ethanol-water. Admixture with an authentic sample of formaldemethone did not depress the melting point. The specific activity of the formaldemethone was 730 c.p.m. per mg.

In view of the relatively low yield of formaldehyde (Table IV), and the . . - . ^ difficulties encountered in homogenizing large volumes of liver m the Potter-Elvehjem apparatus, the experiment was repeated on a homogenate prepared in the Waring blendor. The particulate fraction from 29 gm. of homogenized liver was incubated with 25 mg. of non-isotopic sarcosine for 3 hour. At the end of this time a second 25 mg. portion of sarcosine was added. The reaction was stopped after 1 hour by the addition of TCA. Dimedon was added to the TCA filtrate, whereupon crystals of formaldemethone appeared. After one recrystallization from ethanol- water, the isolated formaldehyde derivative weighed 34.8 mg. The derivative was recrystallized two more times from ethanol-water, and a portion was mixed with an authentic sample of formaldemethone. There was no depression in the melting point.

CITHSO~. Calculated, C 69.86, H 8.22; found, C 69.58, H 8.33

Formaldehyde was not isolated when the sedimented fraction of homogenized liver was incubated alone or in the presence of any of the following compounds: glycine, betaine, monomethylaminoethanol, or choline. However, formaldehyde was formed when 16 mg. of methanol were employed as the substrate. The recrystallized formaldemethone, obtained from the oxidation of methanol by the sedimented fraction of a liver homogenate, melted at 188’, corrected.

ChHr04. Calculated, C 69.86, H 8.22; found, C 69.60, H 8.59

This experiment was repeated several times and formaldehyde was isolated on each occasion. In control experiments, in which the liver preparation was omitted, methanol was not converted to formaldehyde. The experiments described in this section are summarized in Table IV.

Rate of Oxidation of Methyl Group in Intact Animal-An adult rat,


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C. 0. MACKENZIE 361

maintained on a diet containing 1.2 per cent methionine and 0.2 per cent choline chloride (15), was given a single 14.6 mg. dose of radiosarcosine intraperitoneally. At the same time 2 gm. of the basal diet were administered by stomach tube. The animal was then placed in a metabolism

TABLE IV Direct Isolation of Formaldehyde without Use of Carrier Formaldehyde

Weight of live1 before bomo- geniz.ation

P. 30* 29 53 31 33 31 27 31

Substrate

50 mg. radiosarcosine 50 I‘ sarcosine None added 172 mg. glycihe 92 mg. choline chloride 174 mg. betaine hydrochloride 18 mg. methanol 36 ‘I I‘

fler cent 5

34.8 21 0 0 0 0 9.8 6

10.1 3

* Homogenized in the Potter-Elvehjem apparatus. In subsequent experiments the homogenate was prepared in the Waring blendor.

Hours FIG. 1. The rate of oxidation to CO% of the methyl group of sarcosine. Aaingle

14.6 mg. dose of sarcosine, labeled with Cl4 in the methyl group, was injected intra- peritonedly into a 160 gm. male rat, and the Cr4Ot eliminated in the expired air during the next 30 hours was determined.

apparatus (1) and the quantity of CY40a that appeared in the expired air during the next 30 hours was measured.

The maximum rate of oxidation of the methyl group to carbon dioxide .occurred during the 1st hour (Fig. 1). At the end of 6 hours, 40 per cent, and at the end of 24 hours, 50 per cent of the injected radiocarbon had


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appeared in the expired air as C1*02. 4.8 per cent of the administered methyl carbon was excreted in the urine, and 0.35 per cent was eliminated in the feces, in 24 hours. Thus, 55 per cent of the administered methyl carbon was eliminated in 24 hours, and the remainder was still present in the body in the form of methyl groups and their oxidation products.

It should be noted that the oxidation curve for radiosarcosine reflects not only the rate of formation of C140z but also the rate of formation of the intermediates produced in the oxidation of the methyl carbon.

Conversion of Methyl Groups to Formate in Intact AnimaP-Rats weighing approximately 200 gm. were placed on a purified diet (Table V) and transferred to metabolism cages. The urine was collected daily in

TABLE V Excretion of Formic Acid by Rats Following Intraperitoneal Injection of Sarcosine,

Glycine, and Methanol*

Weight of rat Compound injected

w.

237 223 248 218 234 190 159

Sarcosine I‘ “

Methanol “

Glycine “

-

- Formic acid excreted in urine

Quantity in- during injection period jetted daily

I 1st day 2nd day

gm. w.

1 17 1 6 1 9 0.8 2 0.8 I 5 1 0.4 1 0.1

.-

-

fw. 24

13 7

24 0 1.3

* The percentage composition of the experimental diet was as follows: sucrose 55.4, casein 20.0, Covo 19.0, corn oil 1.0, salts 4.0 (15), L-cystine 0.4, choline chloride 0.2, and vitamins (15).

15 ml. of 0.1 N sodium hydroxide. The average daily excretion of formate, as determined by the method of Benedict and Harrop (12), was 1 (0.2 to 2.0) mg.

The intraperitoneal injection, twice daily, of 0.5 gm. of sarcosine, dissolved in 1 ml. of warm water, was followed by a prompt and definite increase in formate excretion (Table V). The administration of methyl groups in the form of methanol also increased the urinary excretion of formate. The injection of glycine on the other hand produced no dis- cernible increase in urinary formate.

Direct evidence that the methyl group of sarcosine is converted to formate was obtained by adding 3.58 mg. of radiosarcosine to the non-

4 The author wishes to express his appreciation to Miss Rose Lubschez for her collaboration in the experiments reported in thii section.


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C. 0. MACKENZIE 363

isotopic sarcosine administered on the 3rd day of an injection experiment. The urine collected in the ensuing 24 hours was acidified and distilled into dilute alkali. The alkaline solution was then distilled into aqueous dimedon. When three-quarters of the original volume had been distilled, the solution that remained in the distilling flask was adjusted to pH 4 and aerated for 2 hours with carbon dioxide-free air. At the end of this time the solution was shown by analysis to be devoid of CY40z. The solution was t,hen adjusted to pH 6 and oxidized with mercuric chloride reagent. The mixture was acidified and the carbon dioxide, that had been formed in the oxidation, was trapped in dilute alkali and converted to barium carbonate. The activity of the barium carbonate was 253 c.p.m., corrected. It was calculated from this figure that 0.3 per cent of the injected radiomethyl groups had been excreted as HCY400H.

The distillate that had been collected in aqueous dimedon (see above) was treated with carrier formaldehyde and the formaldemethone was removed by filtration. The formaldemethone was counted until 1700 counts had been obtained on four different occasions. The average count above the background was 6.5 (6.0 to 7.3) c.p.m., uncorrected. This level of activity was equivalent to 0.6 y of the injected radiosarcosine. The formaldemethone was sublimed in YUCUO (1 mm. of Hg) at 100” without a loss in specific activity. In a control experiment in which 0.75 mg. of radiosarcosine was added to rat urine and allowed to stand for 24 hours, the formaldemethone obtained from the urine by the procedure outlined above contained no detectable C14. HCY400H was also absent (<0.002 per cent).

The excretion of radioformate following the administration of 3.3 mg. of radiobetaine (16) was also demonstrated. The betaine was added to the non-isotopic sarcosine injected on the 3rd day of a metabolism experiment. 0.8 per cent of the administered radiomethyl carbon appeared in the urine as HCY400H in 24 hours. Prior to the determination of formate any C14H30 present in the urine was removed as the dimedon derivative. Cursory examination of the formaldemethone indicated that it possessed several counts per minute above background. When 3.5 mg. of radiobetaine were added to acidified urine in a control experiment, and the mixture was distilled, a small amount of CY4Hz0 was formed. The C14Hz0 present in. the distillate was equivalent to 0.02 per cent of the added radiobetaine. The radioformate present in the distillate was equivalent to 0.002 per cent of the added betaine.

DISCUSSION

In the experiments described in this paper radioformaldehyde was re- peatedly isolated as the dimedon derivative from liver homogenates and


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surviving liver slices incubated with radiosarcosine. Radioformate was determined in these preparations by the classical oxidation-reduction reaction between formate and mercuric ions. Methyl alcohol was found to resist oxidation by the mercuric chloride reagent, and other investigators have shown that oxalate, acetate, lactate, pyruvate, etc., are not oxidized by mercuric ions (17, 18). Accordingly, the oxidation of the methyl group of sarcosine may be schematicdly represented as follows:

R-NHCHa --+ [Xl -+ CHzO --) HCOOH + CO*

where X represents the initial product (or products) formed in the reaction. It is quite possible that both formaldehyde and formate are hydrated prior to dehydrogenation and that bicarbonate ion is thus the end-product of the oxidation. With respect to the compound (or compounds) produced in the conversion of the methyl group of sarcosine to formaldehyde and formate, two observations are of interest. First, it was shown, in experiments of a purely qualitative nature, that the sedimented fraction of liver homogenates oxidizes both sarcosine and methanol to formaldehyde. Secondly, the incubation of radiosarcosine with a liver homogenate for 1 hour gave rise to a product that was soluble in trichloroacetic acid and accounted for a major fraction of the added C4. This compound (or compounds) was not removed from acid solution by distillation. Both of these findings are the subjects of further investigations. Ratner, Nocito, and Green (19) have shown that sarcosine is converted to meth- ylamine by glycine oxidase. With respect to methyl alcohol, it is of interest that recent investigations by du Vigneaud and Verly (20) have shown that the administration of CY4Hz0H to the rat results in the appearance of Cl4 in the methyl groups of choline and creatine.

The significance of the series of reactions depicted above is not confined to sarcosine. Collateral experiments have shown that the methyl groups of methionine and betaine contribute to sarcosine formation in the body. It follows from this observation that at least part of the methyl groups provided by these compounds are oxidized by way of the same series of reactions. The same is true for methyl compounds (such as choline and dimethylthetin) that contribute methyl groups, directly or indirectly, to methionine and betaine. Thus all “biologically labile” methyl groups are sources of formaldehyde and formate in the body. When radiobetaine was administered to a rat with formaturia, HCY400H was excreted in the urine.

The results presented in this paper appear to provide the first direct evidence that formaldehyde is a normally occurring compound in the animal body. While nothing is known as yet concerning the fate of formaldehyde in the body, other than its oxidation to formate, it is highly


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CL 0. MACKENZIE 365

probable that such a reactive molecule will. be found to play an important role in synthetic reactions, particularly in reactions involving proteins and amino acids.

Formicacid, on the other hand, has long been known to occur in small amounts in the urine of man and other animals, although information concerning the identity of its precursors, or the extent of its formation in the body, has been scanty. Dakin, Janney, and Wakeman (17) studied the effect of diet on formate excretion, and reached the conclusion that it was produced in the breakdown of carbohydrates and proteins. When Shemin (21) found that serine was converted directly to glycine in the body, he suggested that formate was a product of this reaction. Recently, Siekevitz, Winnick, and Greenberg (22) and Siekevitz and Greenberg (23) reported the presence of HCY400H in liver slices incubated with glycine labeled in the a-carbon. HC1400H was identXed by oxidation to carbon dioxide with mercuric ions.

The identification of both methyl groups and glycine as sources of formate in the animal body is of considerable interest in view of the papers that have appeared in the past few years on the participation of exogenous formate in purine and serine syntheses. The first of these papers was the report by Buchanan and Sonne (24) and Sonne, Buchanan, and Delluva (25) that the carbon of exogenous formate was incorporated into positions 2 and 8 of uric acid by the pigeon. Thii was confirmed by Karlsson and Barker (26), who showed that the a-carbon of glycine also entered these positions. Greenberg (27) found that the addition of radioformate to pigeon liver homogenates resulted in the synthesis of radio- hypoxanthine, and Marsh (28) observed that the administration of radioformate to pigeons resulted in the appearance of Cl4 in the adenine and. guanine of the nucleic acids. However, in similar experiments. with rats, Marsh (28) found that all of the Cl4 that entered into purines could be accounted for by C1402 fixation.6

Evidence that glycine is converted to serine by rat liver homogenates was obtained by Winnick, Moring-Claesson, and Greenberg (29), although

6 In an experiment conducted in this laboratory in collaboration with Dr. Wil- liam H. Horner a rat was injected with 24 mg. of n-methionine, labeled with CY in the methyl group, and the animal was sacrificed 30 hours later. Adenine was isolated from the visceral nucleic acids. The specific activity of the adenine carbon was double the specific activity of the respiratory carbon. Consequently, the specific activity of the newly synthesized adenine must have been consid- erably greater than that of the expired carbon dioxide. It may be concluded that the methyl carbon of methionine contributed to adenine synthesis, probably by way of formaldehyde or formate. In a counter-current distribution of the isolated adenine, performed through the kindness of Dr. George B. Brown, the distribution of Cl* paralleled the distribution of the adenine, thus indicating that the 04 was a part of the adenine molecule and not present as a contaminant.


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366 E'ORMATION OF.FORMALDEHYDE. AND FORMATE

previous experiments by Greenberg and Wnmick (30) had failed to demonstrate this reaction in the whole animal. Sskami (31) showed that glycine, labeled in the carboxyl group, was converted to serine in the intact animal, and that the carbon of exogenous formate appeared in the p-carbon of serine. Subsequently, Goldsworthy, Winnick, and Greenberg (32) also were able to demonstrate the systhesis of serine from glycine in the in&t animal.

The condensation of the a-carbon of 1 molecule of glycine with a 2nd molecule of glycine to yield serine was reported independently by Sieke- vita, Winnick, and Greenberg (22, 23), on the basis of experiments with liver slices, and by Sakami (33), on the basis of experiments in the whole animal. Sakami (34) has recently shown that the methyl carbon of choline is also found in the @-carbon of serine, a finding that is in accord with the observation that methyl groups are converted to formaldehyde and formate. Wittenberg and Shemin (35) have recently reported that the (Y- carbon of glycine enters the methine bridge of heme, apparently as a single carbon molecule.6

The experiments cited above suggest that formate, or a derivative of formate, participates directly in the synthesis of purines, porphyrins, and serine. On the other hand, it is possible that the single carbon compound involved in these reactions is formaldehyde, or a radical or compound derived from formaldehyde. In either case, it seems probable that our present knowledge of single carbon compounds provides us with but a glimmering of the Ale they eventually will be found to play in synthetic reactions in the animal body.

Plaut, Betheil, and Lardy (37) have reported that folic acid functions in the metabolism of formate, and evidence is rapidly accumulating that vitamin B1z is concerned with the metabolism of methyl groups (3841).

The author wishes to express his appreciation to Dr. Vincent du Vig- neaud for his advice and counsel during the course of this investigation.

6 In an experiment performed in this laboratory several years ago, hemin crystals were isolated from the blood of a rat that had received 52 hours earlier a single 200 mg. dose of L-methionine, labeled with 04 in the methyl group. The activity of the administered methionine was 1.12 X lo6 c.p.m. per mg. The carbon dioxide expired throughout the period of the experiment showed an average activity of 244 c.p.m. per mg. of carbon. The carbon of the hemin possessed 161 c.p.m. per mg. Presumably about 2 per cent of the total heme in the blood was synthesized in the course of the experiment (36). On the basis of this figure, the activity of the newly formed heme was approximately 8000 c.p.m. per mg. of carbon. If it is assumed, in the light of the results of Wittenberg and Shemin (35), that most of this activity was in the methine carbons, it may be calculated that approximately 4.6 per cent of these carbon atoms were furnished by the administered methyl carbon.


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C. Q. iUAC!KENZIE 367

Appreciation is expressed to Mrs. Josephine T. Marshall for the micro- analyses, and to Mr. Harry L. Isrow for technical assistance in all phases of the work.

SUMMARY

The experiments reported in this paper indicate that formaldehyde is a naturally occurring compound in the animal organism. CY4Hz0 was isolated, as the dimedon derivative, from incubation mixtures composed’ of radiosarcosine and either liver homogenat,es or surviving liver slices. Free formaldehyde was formed in these preparations, as was shown by blowing CY4Hz0 directly out of incubation mixtures with a stream of gas. Cr4Hz0 was isolated from the sedimented fraction of liver homogenates both with and without the use of non-isotopic carrier formaldehyde. 1

HC1400H and Cl402 were also produced in the oxidation of, radiosarcosine by liver homogenates and liver slices. The results obtained with different homogenate preparations were consonant with the view that most or all of the formate and carbon dioxide formed in the oxidation of the methyl group arose by way of formaldehyde.

In the whole animal, the administration of radiosarcosine was followed by the elimination of HCY400H in the urine. A trace of CY4Hz0 was isolated from the urine as the dimedon derivative. The methyl groups of sarcosine were oxidized at a rapid rate in the body, as was shown by following the CY402 eliminated in the expired air.

Since collateral experiments have shown that the methyl groups of methionine and betaine contribute to sarcosine formation in the rat, it follows that “biologically labile” methyl groups as a class are sources of formaldehyde and formate in the body. When radiobetaine was administered to a rat with formaturia, HCY400H appeared in the urine.

The significance of these results with respect to the r61e of single carbon compounds in biosynthetic reactions is discussed, and a brief account is given of experiments which indicate that the carbon of methyl groups contributes to the synthesis of the adenine of nucleic acids and the por- phyrin of hemoglobin.

BIBLIOGRAPHY

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368 FORMATION OF F&tMALDEHYDE AND FORMATE

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Cosmo G. MackenzieGROUP

BIOOXIDATION OF THE METHYLAND FORMATE IN THE

FORMATION OF FORMALDEHYDE

1950, 186:351-368.J. Biol. Chem.

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