STUDIES ON NATURALLY OCCURRING a-GLYCEROL ETHERS *

BY MANFRED L. KARNOVSKY AND BNNE F. BRUMM

(From the Department of Biological Chemistry and the Biophysical Laboratory, Harvard Medical School, Boston, Massachusetts)

(Received for publication, January 12, 1955)

The occurrence of the true ether linkage in nature is, in contrast to that of the acetal, amide, and ester links, very limited. The most prominent types of biological compound that are true ethers are (a) members of the group containing guiacol, vanillin, eugenol, and other methyl phenyl ethers; (b) t.hyroxine and allied compounds which are diphenyl ethers; (c) ethers of fatty alcohols and glycerol such as batyl and selachyl alco- hols, which are cu-monoethers of glycerol and have the general formula R-0-CHz-CH(OH)CHzOH.

Considerable attention has been devoted t.o the fatty ethers of glycerol with respect to their distribution in nature (1, 2-6) and their chemical const,itution (1, 7-9). Some work has been carried out on their pharma- cological nature; for example, their central depressant action (lo), erythro- poietic effect (ll), and tuberculostatic activity (12). Little, however, is known of their metabolic relations. This is somewhat surprising in view of the fact that fatty acid esters of these compounds const,it.ute major com- ponents of the liver and muscle lipides of several groups of lower elasmo- branchs and are found to some extent throughout the teleosts and other phyla, including mammals (1, 3).

In the present work an attempt was made to establish a convenient method for the determination of a-glycerol ethers and to study the pattern of incorporation of Cl4 into glycerol ethers from usual lipide precursors, such as glycerol and acetate. In particular, the incorporation into glycerol ethers of Cl4 from these precursors has been compared with that into fatty acids.

EXPERIMENTAL

Biological Mater&&-All marine material was obtained alive or, in the case of some sharks very shortly after death, at the Woods Hole Marine Biological Laboratory. Starfish were also shipped alive in sea water and utilized in Boston.

* This work was supported in part by the Atomic Energy Commission and by a grant from the Eugene Higgins Trust through Harvard University. Some of the work was carried out during the tenure by one of us (M. L. K.) of a Lalor Foundation Fellowship at the Marine Biological Laboratory, Woods Hole, 1951.

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690 GLYCEROL ETHER.3

Extraction of Futs-Fats were extracted by the methods of Bloor (13) and of Folch et al. (14) and by extraction with chloroform in a Soxhlet apparatus after thorough grinding with a 3-fold amount of MgSO* by weight. In the case of some elasmobranchs examined, results were ob- tained by all three methods and were found to be closely comparable.

Isolation and Determinution of Umaponifiabk Fraction-The method of the Society of Public Analysts (15) was used. When only small amounts of fat were available, quantities were suitably adjusted throughout.

Detmminatim of a-Glycerol Ethers--Glycerol ethers were determined by selective oxidation of the unsaponifiable fraction in a suitable medium with periodic acid, destruction of excess reagent, removal of the fatty material by precipitation with water and filtration, and estimation of formaldehyde generated by the standard calorimetric techniques of Mac- Fadyen (16) and Tannenbaum and Bricker (17). The former method has been found more suitable and has been in routine use in this laboratory. Details of the determination, follow.

Reagents--Periodic acid (250 mg. per 25 ml. of aldehyde-free 95 per cent EtOH, freshly prepared daily).

HCl, 1 N.

Sodium-amen&, 1.2 N. Celite. Chromotropic acid reagent according to MacFadyen (16). The unsaponifiable fraction isolated as above was dissolved in alcohol

(95 per cent) or 50 per cent ethanol-50 per cent ethyl acetate. The optimal concentration of glycerol ethers, as batyl alcohol, was 1.74 mg. per ml. (5 gmoles per ml.).

1 ml. of the solution under test was pipetted into a tube graduated at 10 ml., and 1 ml. of periodic acid solution was added. The solutions were mixed with a thin, mushroom-tipped glass rod, which was left in the tube. After 1 hour the oxidation was terminated by adding in succession 3 ml. of distilled water, 1.5 ml. of N HCl, and 0.5 ml. of 1.2 N sodium arsenite. The solution was thoroughly mixed and allowed to stand until the yellow color had discharged. The stirring rod was then rinsed with distilled water and removed from the tube. The total volume was now made to 10 ml. with distilled water, and 0.5 gm. of Celite wti added. The mixture was stirred thoroughly with a clean, dry, mushroom-ended glass rod and poured through a small fluted filter (Whatman No. 42) into a clean, dry tube.

0.5 ml. aliquots were then removed for formaldehyde determination by the MacFadyen method (16). In this laboratory, readings were made in a Klett calorimeter with a No. 56 filter. Color development was carried out exactly as recommended by MacFadyen. Blanks were performed on the complete system without the unsaponifiable fraction. The concentra-

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M. L. KARNOVSKY AND A. F. BRUMM 691

tion of iodide was sufficiently low to obviate any interference from this source.

Standards-When pure batyl .alcohol.is available, it is undoubtedly the most suitable standard. Mannitol, glycerol, or formaldehyde, which are readily available, may also be used as standards, as follows: 1 ml. of a solution of the substance in distilled water (2.5 pmoles per ml. for mannitol or glycerol; 5 pmoles per ml. for formaldehyde) was added to 1 ml. of the solvent used for the lipide. 1 ml. of periodic acid (44 pmoles) was then added and the procedure carried out exactly as above. Mannitol, which may be obtained in a high degree of purity, is preferable to glycerol or formaldehyde since it constitutes a primary standard, whereas glycerol and formaldehyde solutions must themselves be standardized by an inde- pendent method.’

Preparation of Specimens of PuriJied Saturated Glycerol Ethers-Samples were prepared from two sources: (a) the unsaponifiable fraction of starfish (Asterias forbesi) diverticulum fat; (b) the unsaponifiable fraction of tiger shark (Galeocerdo cuwier) liver fat.

Starfish unsaponifiable fractions were treated by the method of Schlenk and Holman (19) to form a crystalline urea inclusion product of long chain aliphatic compounds. This was removed by filtration and twice recrystal- lized from isopropyl alcohol. It was then decomposed with water, the lipide extracted with ether, and twice recrystallized from ethyl acetate. The process was carried out on two different batches of starfish diverticu- lum fat, and the products melted at 68” and 70”, respectively. Further recrystallization did not raise the melting point, and some chimyl or other solid glycerol ether of chain length less than batyl alcohol was probably present. The urea inclusion product contained approximately 16 moles of urea per mole of glycerol ether (19).

A crystalline cu-glycerol ether was prepared from tiger shark liver fat by methods similar to those previously described (2, 9), involving separation of steroids from the non-saponifiable fraction, hydrogenation of the steroid- free residue, and frequent recrystallization of the product. The product

1 The method described has also been used for the determination of glycerol and 1,2-propanediol in experiments on blood preservation, of glycerol in metabolic ex- periments in vitro, and of mannitol in determinations of body fluid compartments. In these cases, aqueous solutions were used throughout, and the periodate oxidation was carried out in the presence of 1 ml. of N NaHCOs. The addition of Celite and filtration after dilution of the reaction mixture to 10 ml. were unnecessary. Solutions deproteinized by the barium hydroxide-zinc sulfate (18) method were found suitable for these determinations. When glucose was present, this was determined inde- pendently and the glycol results adjusted, since 1 mole of glucose yields 1 mole of formaldehyde under the conditions described. The method is suitable when inter- fering substances are minimal or could be accurately determined, or, as in the cases cited above, when the added a-glycol is present in relatively overwhelming amounts.

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692 GLYCEROL ETHERS

obtained here melted at 70.2-70.6”. (R p t, d e or e melting point, for batyl alcohol, 70-71” (9).)

Respiration of Fragments of Starfish Divert&&a-The arms were removed from starfish 6 inches in maximal diameter, and a cut was made along the entire lengt,h of the arm, between the rows of t,ube feet. The arm was opened, and the two diverticula were carefully removed, entire, from each arm. The tissue from one arm was more than sufficient for an average experiment on the Warburg machine.

The diverticula were thoroughly rinsed in filtered sea water which had been pasteurized at 70-80” for 30 minutes, cooled, and thoroughly oxygen- ated. Fragments of the tissue weighing approximately 100 mg. were cut, blotted, and accurately weighed on a Roller-Smit.h t.orsion balance before being introduced into t,he Warburg flasks, which contained pasteurized sea water. Measurements of the respiration of these fragments were made conventionally at 25’, the fat-free dry weights determined, and Qo, values calculated. Substrates were added to give a final concentration of 40 pmoles per ml.

h’xperiments with C14-Labeled Glycerol and Acetate-a-CY4-Glycerol was prepared by the met,hod of Gidez and Karnovsky (20) and l-C14-acetate by a standard procedure (Sakami et al. (21)). hpproximat.ely 3 gm. (accu- rately determined) of a st.arfish diverticulum cut into three to five pieces were pla.ced in each flask, which contained 15 ml. of pasteurized oxygenated sea water and the C14-substrate at a concentration of 40 pmoles per ml. The specific activities for glycerol and acetat,e were 2.20 X lo6 and 3.45 X 106 c.p.m. per mmole, respectively. When both substrates were used, one radioactive and the other not, each was at a concentration of 40 pmoles per ml. The flasks were put in a water bath at 25”, oxygen was passed over the surface of the medium for 15 minutes wit,h gentle shaking, and the flasks were then clamped. The incubation was carried out for 4 hours. pH determinations were made before and after the incubation and were 7.6 f 0.2.

At the end of the incubation period, the tissue was removed from each flask, blotted dry, and washed for 2 minute periods in each of the following: 10 ml. of sea water, 10 ml. of sea water, 10 ml. of distilled water. The tissue was again blotted, ground with anhydrous magnesium sulfate, and extracted with about 25 ml. of chloroform in a small Soxhlet extractor for 24 hours. The total chloroform extract was then transferred to a glass- stoppered Erlenmeyer flask, and 15 ml. of cold distilled water were added. The layers of chloroform and water were very gently rotated with a mag- netic stirrer for 45 minutes and allowed to stand still for 15 minutes, and the water was carefully withdrawn with a syringe. The process was re- peated four times more. This washing had previously been shown to

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M. L. KARNOVSKY AND A. F. BRUMM 693

remove all traces of C14-glycerol or CY4-acetate added to non-radioactive starfish diverticulum fat in chloroform.

The chloroform solut.ion was finally dried over anhydrous sodium sulfate overnight and filtered. The solids were thoroughly washed with fresh chloroform, and the washings were added to the filtrate. The chloroform was removed by distillation in vacua and the fat recovered and maintained in a desiccator over paraffin wax, in racuo, overnight. The t.otal lipide was weighed, and a sample was counted.

The remaining fat was separated into neutral fat and phosphatides by acetone precipitation of t.he latter overnight in the ice box, with the aid of MgCl, (13). The phosphatides were centrifuged and washed with cold acetone, and t,he neut,ral fat was treated again as above to insure maximal phosphatide removal.

Saponifiation of Neutral Fat and Recovery of Unsaponi$able Fraction and Fatty Acids-The acetone was removed in va.cw from the neutral fat frac- tion and the fat weighed. When constant weight was attained, the fat was saponified in 5 ml. of 0.5 ?u’ KaOH (alcoholic) and the unsaponifiable fraction isolated essentially according to the Society of Public Analysts method (15). The alcoholic alkaline fraction, after removal of t,he un- saponifiable fraction, was made to pH 1 and exhaustively extracted with petroleum ether (b.p. 30-60’). The combined extracts containing fatty acids were washed with small volumes of water, and t.he petroleum ether was removed.

Treatment qf Unsaponifiable Fraction-This fraction was dissolved in chloroform and heated wit,h alcoholic digitonin to remove the sterols (22). After filtering the sterol digitonides and washing thoroughly with alcoholic digitonin, the filtrate and washings were combined and evaporated to dry- ness with gentle warming in a stream of nitrogen. The residue was ex- tracted with dry ether and filtered. Excess digitonin remained in the residue, and the sterol-free unsaponifiable fraction passed into t.he filtrate. The ethereal filtrate was evaporated in a stream of N2, and the substance obtained was oxidized in a 50 ml. non-protein nitrogen tube in ethyl alcohol with periodate, according to t,he method previously described (2), on a reduced scale. After dest.roying excess periodate with arsenit,e and ad- justing the pH to 4.6, the solut.ion was filtered through a small Celite cake to remove fatty substances. The cake was thoroughly washed and the formaldehyde in the combined filtrate and washings precipitated with dimedon. The formaldimedon was recrystallized and counted (melt,ing point and mixed melting point with authentic formaldimedon, 189”). This represented t$hc a-carbon of glycerol-ether glycerol. The Celite cake was sucked dry and extract.ed with 6 ml. of hot ethanol poured slowly through the pad. The clear extract was collected in a 15 ml. centrifuge

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694 GLYCEROL ETHERS

tube and cooled. Dinitrophenylhydrazone reagent (23) was added and the mixture stirred, warmed gently, and allowed to stand for several hours. The 2,4-dinitrophenylhydrazone of the fatty alcohol ether of glycolalde- hyde precipitated as crystals and was centrifuged and recrystallized twice from diluted alcohol. The product was separated and dried, m.p. 74.6- 75.2”. The melting point of the compound obtained as above from pure batyl alcohol was 75”. A mixed melting point was sharp and showed no depression. The reported melting point of the 2,4dinitrophenylhydra- zone of glycolaldehyde octadecyl ether is 73” (9).

Hydrolysis of Phosphutides and Recovery of Phosphatide Fatty Acids- The phosphatide fractions obtained above were dissolved in 1 ml. of EtOH, and 5 ml. of 6 N HCl were added with rotation of the flask. The mixture was heated 2 hours under reflux in a boiling water bath with frequent shaking. At the end of this time, 4 ml. of water were added, and the solu- tion was transferred to a small separatory funnel and extracted four times, with a total of 69 ml. of petroleum ether. The petroleum ether extract was washed twice with 4 ml. of distilled water, which was added to the aqueous fraction. The petroleum ether solution was then shaken three times with 5 ml. of aqueous 0.5 N KOH solution each time. The alkaline extracts were combined and made to pH 1, and the fatty acids were re- extracted with ethyl ether, which was then removed under nitrogen, and the fatty acids were obtained.

Periodate Oxidation of Aqueous Fractions Obtained after Hydrolysis of Neutral Fats and, Phosphatides. Neutral Fat-The aqueous fraction ob- tained after removal of the non-saponifiable fraction and fatty acids above was chilled, filtered, and oxidized with periodate after adjusting to pH 8 with bicarbonate. After 1 hour the excess periodate was destroyed by the addition of arsenite and HCl. The formaldehyde formed was distilled and the pH of the distillate brought to 4.6 by the addition of sodium acetate and HCl. ‘I’he formaldehyde was precipitated with dimedon, centrifuged, twice reprecipitated from acetone by addition of water, and counted.

Phoaphatides--The aqueous fraction after removal of fatty acids was chilled, filtered, made to pH 1, and shaken with 2 gm. of Dowex 1 in the sodium cycle (24) for 10 to 15 minutes. The mixture was centrifuged and poured through a fluted filter into a clean centrifuge tube and carefully neutralized with alkali. This treatment had been shown in exhaustive tests to remove serine and ethanolamine from the hydrolysate, which was ninhydrin-negative. The neutral aqueous fraction was then treated with NaHCOa and periodate as above. After the reaction was stopped, the pH was brought to 8, and the formaldehyde was distilled into a chilled centrifuge tube, where it was precipitated and reprecipitated as described.

Counting--All counting was carried out in a gas flow counter with high

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M. L. KARNOVSKY AND A. F. BRUMM 695

e5ciency (about 45 per cent) and low background (less than 10 c.p.m.) (25). Fats and fatty acids were counted as such on lens paper disks in the planchets. Formaldimedon and the 2,4-dinitrophenylhydraaone of the aldehyde-ether produced by periodate oxidation of glycerol ethers were plated directly. Under the conditions of counting in these laboratories, counts obtained on organic substances and on the BaC03 obtained by com- bustion of those substances agree well. These data will be presented else- where (26).

IJM BATYL ALGO

0

OXIDATION TIME IN MINUTES

Fro. 1. Characteristics of the formation and determination of formaldehyde derived from a-glycerol ethers by oxidation with periodic acid. Conditions as de- scribed in the experimental section. The oxidation of batyl alcohol (0) and a star- fish unsaponifiable fraction (0) was allowed to proceed for varying times, and the formaldehyde determined. Oxidation for 1 hour and with varying amounts of batyl alcohol (A).

RESULTS AND DISCUSSION

Determination of cr-Glycerol Ethers-Fig. 1 illustrates the relationship of formaldehyde produced by an a-glycerol ether to time of reaction with periodate, when conditions are as described in the experimental section. It is clear that the reaction is complete within 30 minutes and a stable situation is attained. 1 hour has been adopted as the standard oxidation period. At this time the determination has been repeatedly found to con- form to Beer’s law, as is also shown in Fig. 1. With mannitol as the stand- ard, samples of purified a-glycerol ether described in the experimental sec- tion were analyzed with the following results (calculated as batyl alcohol): starfish product (m.p. 68’), 106.2 per cent; (m.p. 709, 100.0 per cent; tiger shark product (m.p. 70.4-70.6“), 108.1 per cent.

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696 GLYCEROL ETHERS

The mannitol used a.s standard assayed at 190.5 per cent by the gravi- met.ric method of Reeves (27). When this mannitol was analyzed by the calorimetric method described above against a formaldehyde standard which had been independently assayed by the method of Yoe and Reid (28), a result of 98.6 per cent purity was obtained.

A number of organisms or &sues have been assayed for glycerol ethers by the calorimetric method described and by the more tedious gravimetric method, requiring large quantities, that had previously been employed

TABLE I

Glycerol Ether Contents of Some Unsaponijiable Fractions (Expressed As Batyl Alcohol)

I Species Common name Source of fat

____~_,~ -_-- -~ -_.

Loligo peulei Ommsstrephes

1 Squid 1 Sorthern squid

( Digestive gland 1 “

~illecibrossus !

Asterias forbesi ’ Starfish Arbacia punctulata’ Sea-urchin E’ch.inarachnius Sand-dollar

SP. l’hyone briareus Sea-cucumber Ciona sp. Sea-squirt Mustelus canis Smooth dogfish Carcharinus ob- Dusky shark

scurus Galeocerdo cuvier Tiger “

Clemmys guttata : Sea-turtle

Diverticulum Viscera Whole animal

“ I‘

Liver “

“ ‘I

T

1

Fat*

-

t 1

UlLW- ponili- ablet

-._ Per cm

10.5 18.4

-.- ier cm

3.9 10.0

9.5 1.8

9.5 21.3 19.0

1.1 10.1 2.5 30.2

30 5.8 28 4.3

56 13.7 13.2 7.6

Glycerol ethers

New netha -- w cm

6.3 7.3

01.8 3.8 2.2

11.0 4.1

42.1 11.9

96.8 3.7

-.

Old nethod

>cr cent 0.0

11.0

63.9

11.5

43.8 10.6

91.5

* Based on fresh weight of material in preceding column. t Of fat in preceding column.

(2, 3). Table I summarizes the results. It may be seen that results by the two methods are in reasonable agreement.

By the method described here it is possible to obtain replicates that agree to within about 2 per cent,; the accuracy is estimated to be between 3 and 5 per cent. Further, the method will give positive results with all cu-glycols in which a hydroxyl is in a primary carbinol group. With the exception of some steroid compounds of mammalian adrenals and some long chain 1,2-glycols recently isolated from sheep wool fat (29), no inter- fering substances are known. The operations leading to t,he isolation of bhe fat and of the unsaponifiable fraction remove any water-soluble glycols, and t,he method is regarded as reliable, part,icularly in cases in which

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M. L. KARNOVSKY AND A. F. BRUYM 697

u-glycerol ethers constitute a major component of the unsaponifiable frac- tion.

Experiments with Starfish-The starfish appeared to be a suitable animal for studies on the metabolism of a-glycerol ethers, since it is easily available and has a high content of these compounds occurring almost entirely as batyl alcohol (octadecyl Lu-glyceryl ether).

It is of interest to note that no “etherase” (catalyzing the hydrolytic split of the ether bond) could be detected in homogenates of starfish di- verticula when a variety of glycol ethers and phenol ethers was employed. This was true also of shark pancreas and rat liver. This absence of hydro- lytic splitting is in accord with thermodynamic expectation. Further, starfish maintained at a low oxygen tension for 2 weeks appeared no dif- ferent with respect to their glycerol ether content. It would thus appear that these compounds are not formed in response to an environment of low oxygen tension.

It was, however, found that a-glycerol et.hers accumulated in the starfish with age. The experiments were as follows:

The maximal diameter was used as an index of age, and batches of twenty to thirty starfish were accurately sorted into groups 4, 6 to 8, and 8 to 10 inches in maximal diameter. The diverticula were dissected, pooled in groups, and extracted. It was not found possible to remove the diverticula of animals 2 inches in diameter, and in this case the entire animal was ex- tracted. It should be noted that previous analyses had shown that the exoskeleton fat contained no a-glycerol ethers; in the case of the 2 inch starfish, no glycerol ethers at all were detected in the whole animal. Fig. 2 illustrates the results and demonstrates the increase of a-glycerol ether ester content of the diverticulum fat with age.

Respiration of Fragments of Sturjish Dive&&a-The diverticulum of the starfish has been shown to be a very thin walled tube, greatly folded. The wall of this tube consists essentially of a row of tall columnar epithelial cells resting on a basement membrane, outside of which is a serosal layer. The thickness of the wall is about 250 p (30). It was thought that frag- ments of such an organ might be suitable for metabolic experiments in vitro, since adequate diffusion of oxygen and nutrients into the tissue could occur.

Fig. 3 demonstrates the respiration of fragments of starfish diverticula under the conditions outlined in the experimental part. It is clear that the organ fragments continue to respire uniformly for lengthy periods, and that the respiration may be stimulated by the addition of glucose, glycerol, or acetate. The Qoz values were as follows: endogenous, 1.1; with glycerol, 1.7; with glucose, 2.2; with acetate, 1.9. These results indicated the feasi- bility of carrying out radio experiments in vitro with this system.

Incorporation of Cl4 into StarJish Lip&k Fractions in V&o--Fragments of

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698 GLYCEROL ETHERS

starfish diverticula were incubated with radioglycerol or radioacetate and the lipides extracted, fractionated, and counted as described in the experi- mental section.

DIAMETER IN INCHES

Fra. 2. Increase in a-glycerol ethers of starfish diverticuls with age. Diameter used aa an indication of age. 0, total fat, percentage of freeh tissue; X, unsaponifi- able fraction, percentage of total fat; 0, glycerol ethers@ batyl alcohol), percent- age of unsaponifiable fraction; q I, glycerol ether esters (as batyl dioleate), percentage of total fat.

40

SUBSTRATE ADDED

60 120 180 240 300

MINUTES

Wa. 3. Respiration of fragments of starfish diverticula. Conditions as described in the experimental section.

The following fractions were counted: (1) the fatty acids of the whole neutral fat fraction (including fatty acids originally esterified with glycerol ethers); (2) fatty acids of phosphatides; (3) the free primary carbinol group

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M. L. KARNOVSKY AND A. F. BRUMM 699

of a-glycerol ethers, Le. an a-carbon of the glycerol moiety, and conse- quently a measure of the activity of the glycerol moiety, since a-CY4-glycerol was employed as substrate; (4) the fatty alcohol ether of glycolaldehyde, obtained after the periodate oxidation of the glycerol ether fraction to obtain the fraction (3) above (by subtracting from the specific activity of this fraction the activity of the residual a-carbon of the glycerol moiety, the specific activity of the fatty alcohol moiety (calculated as octadec- anol) could be obtained) ; (5) formaldehyde produced by periodate oxida- tion of the aqueous hydrolysis fractions from both neutral fats and phos- phatides.

TABLE II Incorporation of Cl” into Lipide Fractions of Star$sh Diverticulum

Total fat set at 100 c.p.m. per mg. after normalization of the activity adminis- tered; results as counts per minute per millimole X 10-z, f the standard error of the mean.

Substrate No. of erperi- ments

Glycerol$ 6

‘( $ and acetate 1 Acetatet 4

“ # and glycerol 1

Fatty acids’ Formaldehyde

7.3 f1.5

3.6 26.0

f2.9 35.3

Phos ha- 3

Glycerol Ne$J Ph; ,““-

3 t1 e ethers

gz- hydro-

?lg;;;l

lysate -----

14.2 17.4 8.9 33.4 5.7 12.3 f4.5 f1.3 ~~6.1 a2.1

2.2 9.5 17.0 57.0 3.7 52.4 29.2 0.2 1.7 0.0

h4.0 fll.1 ZlZo.15 fl.1 20.0 15.9 0.0 0.0 0.0

* Calculated on the basis of stearic acid as the fatty acid. t Calculated as octadecanol (Le., the Cla-alkyl moiety of batyl alcohol). $ Denotes radioactive substrate.

The results are reported in Table II. For convenience, the activity of the total fat has been set at 106 c.p.m. per mg., and the other fractions are expressed proportionally. This minimizes differences among individual starfish due to dietary status and other factors.

The results show the following. (1) The carbon of both acetate and glycerol is incorporated into the fatty acids of neutral fat and phosphatide. The fatty acids of the latter were, when only one substrate was present, about twice as active as those of the neutral fat fraction. (2) Activity was significantly incorporated into the long chain alkyl moiety of the glycerol ethers when either radioglycerol or radioacetate was present. (3) When radioglycerol was the substrate, activity appeared in t,he a-carbon of the glycerol moiety of the glycerol ethers, but not when radioacetate was the

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700 GLYCEROL ETHERS

substrate. This is true also for the formaldehyde obtained by periodate oxidation of the aqueous hydrolysis fractions of neutral fat and phos- phatide, for which the slight activity observed in the case of experiments with radioacetate cannot be regarded as significant. This formaldehyde might represent with reasonable accuracy the a-carbons of triglyceride and phosphatide glycerol, since in the latter case serine and ethanolamine had been removed. This has been found to be true in the rat (31). However, in a fat as imperfectly characterized as t.hat examined here, such an as- sumption would not be warranted.

The specific activity of the fatty alcohol moiety of the glycerol ethers was calculated as mentioned above by correction of the measured specific activity of the glycolaldehyde alkyl ether for the activity of the remaining glycerol a-carbon. Such a calculation assumes equality of labeling of the 2 a-carbons of glycerol-ether glycerol (which is true of the administered a-CY4-glycerol) and low randomization of counts into the &carbon of glycerol-ether glycerol. Randomization is, in the case of the rat, very low in such short term experiments (31) and might be expected to be so in the much more slowly metabolizing system used here.

The incorporation of radiocarbon into a-glycerol ether moieties at rates comparable to those observed in the more commonly studied lipide frac- tions indicates that the st.arfish does not merely accumulate these com- pounds from its food. The mechanism of synthesis and splitting of the ether bond is as yet not understood, but the t.issue studied here appears a promising source of the enzymes involved.

SUMMARY

1. A method has been devised for the calorimetric det.ermination of a-glycerol ethers in the unsaponifiable fractions of fats, and a number of different fats have been analyzed by this method.

2. The starfish has been used as an experimental animal. h’o hydrolytic mechanism of splitting ethers was detected, nor was any response to oxygen tension of the environment discernible. Increasing age (as determined by size) could, however, be strikingly correlated with the magnitude of cy-glyc- erol ether occurrence.

3. Following satisfactory respiration experiments, exposure of pieces of starfish diverticula to C14-acetate or C14-glycerol indicated that both these substrates were incorporated into a-glycerol ethers to an extent comparable to that found for other lipide entities.

4. Although glycerol carbon was incorporated into both glycerol and fatty alcohol moieties of a-glycerol ethers, acetate carbon appeared only in the fatty alcohol moiety.

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M. L. KARNOVSKY AND A. F. BRU)rlM 701

BIBLIOGRAPHY

1. Deuel, H. J., Jr., The lipids, New York, 1, 392-396 (1951). 2. Karnovsky, M. L., and Rapson, W. S., J. Sot. Chem. Ind., 66, 138 (1946). 3. Karnovsky, M. I,., Rapson, W. S., and Black, M., J. Sot. Chem. Ind., 66, 426

(1946). 4. Karnovsky, M. L., Rapson, W. S., Schwartz, H. M., Black, M., and Van Rens-

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Manfred L. Karnovsky and Anne F. Brumm-GLYCEROL ETHERSαOCCURRING

STUDIES ON NATURALLY

1955, 216:689-702.J. Biol. Chem. 

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