Biosynthesis of rat brain phosphatidylethanolamines from intracerebrally injected ethanolamine

Brain Research, 124 (1977) 317-329

317

© Elsevier/North-Holland Biomedical Press, Amsterdam - Printed in The Netherlands

BIOSYNTHESIS OF RAT BRAIN P H O S P H A T I D Y L E T H A N O L A M I N E S FROM INTRACEREBRALLY INJECTED ETHANOLAMINE*

GIUSEPPE ARIENTI, LANFRANCO CORAZZI, HELMUT WOELK** and GIUSEPPE PORCELLATI Department of Biochemistry, The Medical School, University of Perugia, 06100 Perugia (Italy)

(Accepted July 23rd, 1976)

SUMMARY [2-aH]Ethanolamine was injected intracerebrally into male rats and the brains of the animals immediately removed by particular procedures at regular intervals over the first 1200 sec. The incorporation of radioactivity into brain phosphorylethanolamine, cytidine-5'-diphosphate (CDP) ethanolamine and phosphatidylethanolamines was examined and quantitated. The nature of phosphatidylethanolamine molecular subspecies, which became labelled, was also investigated after isotope administration. Phosphorylethanolamine, CDP-ethanolamine and phosphatidylethanolamines were all labelled already 5 sec after the administration of labelled ethanolamine. The specific radioactivities of different phosphatidylethanolamine molecular subspecies varied according to the time elapsed from the injection to the sacrifice of the animals. This last result, together with the data on time course of labelling of ethanolamine phosphoglycerides and their precursors, provides indications that this base may be incorporated into lipids not only by net synthesis pathway, but also by base-exchange reaction.

INTRODUCTION In mammalian brain, phosphatidylethanolamines are synthesized from ethanolamine via the intermediate formation of phosphorylethanolamine and cytidine-5'diphosphate ethanolamine (CDP-ethanolamine)3. These in vivo findings have * Part of this work has been presented at the 20th Meeting of the National Society of Biochemistry, Riva del Garda, 25-28 September, 1974 and at the 5th Meeting of the International Society for Neurochemistry, Barcelona, Spain, 2-6 September, 1975. ** Present address: Universit~its Nervenklinik mit Poliklinik, Einheit fiJr Neurochemie, 8520 Erlangen, Schwabachanlage 10, G.F.R.

318 received strong support from in vitro experiments2,20,'zl, which indicated that the base is transformed into brain lipid through the cytidine coenzyme-dependent pathway. The incorporation of ethanolamine into brain phosphatidylethanolamines may take place, however, also through a base-exchange reaction between free ethanolamine and glycerophospholipid-bound base(s)L q-he existence of this enzymic reaction has been amply demonstrated in vitro for nervous tissue 9,15,17A9, but not in vivo. In this work the utilization in vivo of intracerebrally injected [2-3H]ethanolamine after very short time intervals has been examined, in order to assess in some detail the mechanism of its incorporation into phosphatidylethanolamines and the possible importance of base-exchange reactions. Part of this work has been briefly reported elsewhere4, 5. MATERIALS AND METHODS

Materials [2-3H]Ethanolamine (2.3 Ci/mmole) was obtained from New England Nuclear Corp. (Frankfurt, G.F.R.), and its radioactive purity verified as reported previouslylL The product was dried and dissolved in 0.9 ~ sodium chloride to obtain a final concentration of 1/~Ci//A. This solution was used to inject animals. [l,2-x4C]Ethanol amine phosphate was obtained from the same commercial source mentioned above. Labelled CDP-ethanolamine (CMP-[1,2-14C]ethanolamine phosphate) was prepared and purified as reported elsewhere 10. Before use, all labelled compounds were tested by TLC on cellulose layers for their radiochemical purity and isotope content. Lipid reference standards were obtained from Pierce Chemical Company (Rockford, I11., U.S.A.) and purified, if necessary, by silicic acid column chromatography. AGI-x4 (200--400 mesh, chloride form) and AG50W-x8 (H + form) resins were from Bio-Rad Laboratories (Richmond, Calif., U.S.A.). Organic solvents were all freshly distilled over calcium chloride and stored under nitrogen. Scintillation chemicals were products of Packard S.A. (Ziirich, Switzerland).

Injection procedure Male Sprague-Dawley rats (150-180 g body wt.) were used throughout. Procedures for the dietary conditions of the animals and their successive treatment have been given elsewhere 8. The injection of [2-3H]ethanolamine (10 #1, 10 #Ci, 4.34 nmole) was made intracerebrally6 in a hole whose distance from the coronaric and the sagittal sutures was 3 mm posterior and 3 mm lateral, respectively. The solution was injected over about 1 sec at a depth of 3 mm from the external surface of the skull.

Extraction of brain lipids At fixed time intervals from injection the brain material was rapidly removed~, by means of a vacuum-driven suction apparatus which splashed the cerebral material in about 1 sec against a metal plate precooled with liquid N2. Only about half the brain was collected by this procedure, but it included the part closer to the site of injection. The time from the end of the administration and the freezing of the tissue was recorded.

319 The frozen brain material was subsequently transferred into a precooled glassteflon homogenizer, homogenized with 20 vol. of chloroform-methanol (2:1, v/v), and extracted at room temperature for 1 h ~2. After the addition of 5 vol. of water, the phases were separated by centrifugation. The water-methanol phase was collected and the chloroform washed first with 10 vol. of methanol-10 mM HCI (1:1, v/v) and successively with an equal volume ot methanol-I ~ NaC1 (1:1, v/v). The chloroform, which contained lipids, was collected and stored at --20 °C until use.

Extraction of the water-soluble components The methanolic phases recovered from the washings were pooled together with the first methanol phase (see above). The lipid-free brain residue was extracted for 20 min with 20 ~ ethanol, while keeping the extraction flask in hot water and repeating the procedure three times. The final water-soluble extract, prepared as reported previously 6, gave a recovery of the water-soluble ethanolamine-containing compounds of nearly 100 ~o, as checked by the use of radioactive reference standards. Analysis of the hydrosoluble extract Ethanolamine phosphorylethanolamine and CDP-ethanolamine were separated on AG l-x4 resin columns, as described in detail by Arienti et al. n. Peaks were detected by counting for radioactivity small amounts of each eluted fraction (7 ml each), which, were successively identified by the use of reference standards, q-he radioactivity of CDP-ethanolamine was usually too small to be detected directly by this procedure. Cold CDP-ethanolamine (5 mg) was therefore added to the extracts soon before chromatography and the CDP-ethanolamine-containing fractions detected by ultraviolet absorption at 280 nm ~4. The fractions containing ethanolamine, phosphorylethanolamine and CDP-ethanolamine, which were well separated among each other, were collected and counted for radioactivity. Normally, the CDPethanolamine-containing fractions were reduced in vacuo at 40 °C before counting, because the small radioactivity content did not allow a direct determination. Recovery values calculated by ultraviolet absorption, before and after evaporation, were usually greater than 95 ~o. Glycero-3-phosphorylethanolamine was not retained by the column and therefore it was eluted with free ethanolamine. The concentration of the pool sizes for the ethanolamine water-soluble compounds was determined following the techniques reported elsewhere 8. The corresponding levels (/~mole/g wet wt. ± S.E.M.) were 1.26 ± 0.10, 0.89 :~ 0.13, 0.043 i 0.008 and 17.77 ± 0.86 for ethanolamine, phosphorylethanolamine, CDP-ethanolamine and ethanolamine phosphoglycerides, respectively (see ref. 6). Lipid analysis The methanol-chloroform extract, representing the total lipid material, was evaporated nearly to dryness under N~ and chromatographed on a silica gel G TLC plate, using chloroform-methanol-water (65:25:4, v/v/v), as the solvent. Labelled or unlabelled marker phospholipids were used to identify the well-separated spots, which were normally detected by iodine vapour. Spots were successively scraped off the

320 plates, extracted 3-5 times with chloroform-methanol-water (45:45:10, v/v/v), and the pooled concentrated extracts counted for radioactivity. Nearly all the radioactivity of the original lipid extract was found in the phosphatidylethanolamine spot, which contains both the diacyl and alkenylacyl forms of ethanolamine phosphoglycerides. Radioactivity was never detected in the choline-containing phosphoglycerides, thus confirming previous observations (see ref. 1). The phosphatidylethanolamine spot was resolved into the diacyl and alkenyl forms of ethanolamine phosphoglycerides by treating the TI_C plate with gaseous HCI between developments6,11,16. Phospholipid P content was determined as reported elsewhere 19, in order to determine the pool sizes of phosphatidylethanolamines.

Analysis of phosphatidylethanolamines A procedure will be now described which enables the separation of the ethanolamine lipids according to the degree of unsaturation of their fatty acyl residues. For this purpose, the lipids of 1 g of cerebral matter were extracted with 5 ml of chloroform-methanol (2:1, v/v) with freshly distilled solvents, q-he extraction mixture contained 0.1 ~ of 2,6-di-tert-butyl-4-methyl phenol (Aldrich Chemical Co., Inc., Milwaukee, Wisc., U.S.A.) as antioxidant, and 3.3~o of water, q-he residue obtained after centrifugation of the homogenate was again extracted with the same solvent (6 ml) and the extracts pooled together. Chloroform (4 ml) and 4 ml of water were then added to the mixture, which was successively stirred and centrifuged in order to separate phases. The aqueous phase was discarded and the organic phase washed two times with 8 ml of methanol-2.5 ~ NaCI (containing 1 ~ of cold ethanolamine) (1:1, v/v). The lipid extract was evaporated almost to dryness under Nz and chromatographed on silica gel G, using chloroform-methanol-water (65:25:4, v/v/v), as developing solvent. The plates were dried under N2 and treated with 2,7-dichlorofluorescein. The spot corresponding to phosphatidylethanolamines was scraped off the plate and eluted with chloroform-methanol-acetic acid-water (50:39:1:10, v/v/v/v). The dye was eliminated by washing with 4 Nammonia and successively with methanolwater (1:1, v/v). Chloroform was evaporated to dryness under N2 and 1 ml of chloroform was then added to the lipids. In order to acetylate the polar groups of phosphatidylethanolamines, 0.2 ml of triethylamine and 0.2 ml of acetic anhydride were added to this solution z3 and the mixture, which was allowed to stand at room temperature for 2 h, was then washed three times with 2 ml of methanol-0.1 N HC1 (1:3, v/v) and three times with 3 ml of methanol-water (1:1, v/v). The N-acetyl derivatives of the phosphatidylethanolamines were thus formed. The chloroform was evaporated to dryness under N2 and 2 ml of methanol-ethyl ether (5:95, v/v) were added. Freshly prepared diazomethane (1 ml) was then added to this solution 23, and the reaction mixture kept 30 min at room temperature in order to prepare the Omethyl-derivatives of N-acetylphosphatidylethanolamines at the level of the phosphate residue. To eliminate unreacted diazomethane, samples were evaporated to dryness under N2 and dissolved into 1 ml of chloroform. Lipids were purified on

321 silica gel G plates, using chloroform-methanol-acetic acid (96:4:0.5, v/v/v), as solvent. Spots were revealed by staining with dichlorofluorescein, scraped off the plate and eluted with chloroform-methanol-acetic acid-water (50:39:1:10, v/v/v/v). Samples were freed from dichlorofluorescein by washing three times with 3 ml of 4 N ammonia and three times with methanol-water (45:10, v/v). The chloroform, which contained lipid material, in the form of the N-acetyl-O-methyl derivative, was evaporated almost to dryness under N2 and subsequently chromatographed on AgNO3-impregnated silica gel G TLC plates, which were developed first with chloroform-methanol-water (80:15:2, v/v/v) for 7 cm of length and then, after having been dried under N2, again with chloroform-methanol (97:3, v/v) for the full length of the plate, in the same direction. Spots were visualized by staining with dichlorofluorescein and scraped from the plate. Elutions were performed as described before. The eluates, corresponding to differently unsaturated phosphatidylethanolamines, were divided into two portions: one was evaporated under N2 and used for radioactivity counting, while the other was used for GLC analysis. For this purpose, the eluate was evaporated and methanolysed with 1 ml of a solution containing 3 H2SOa in methanol at 80 °C for 1 h. The methylated fatty acids were extracted with n-hexane and purified1. GLC was performed at 195 °C with a Fractovap Model-GV (Carlo Erba, Milan, Italy), equipped with 150 cm × 0.3 cm (internal diameter) stainless steel columns. The stationary phase was 20 ~ EGA on sylanized Chromosorb-P (100-200 mesh) and the carrier gas was N2. Calculation was made according to Akesson et al. 1.

AnGlyses Phospholipid P of TLC spots was determined as reported previously19, while the phosphorus content of phosphorylated water-soluble compounds was assayed according to Ernster et al. la. Protein was determined according to Lowry et al. 18. Radioactivity measurements were performed with a Packard Tri-Carb liquid scintillation spectrometer model 3375 using previously described scintillation mixtures10. Correction for quenching was made with the channels ratio method. Quantitation of mass in phosphatidylethanolamines was based on phosphorus determination of "I'LC spots, as described before. RESULTS

Ethanolamine utilization The recovery of administered ethanolamine radioactivity was 65.4 ± 15.3~o, as calculated on 30 samples, and it decreased slightly with time up to 20 min. No definite dependency of the recoveries upon time could be found during the experiments. This contrasts with the results of Ansell and Spanner 2 which, however, reflected much longer intervals after administration. In our experimental conditions recoveries are expected to be very much dependent upon leakage which could have occurred during the injection which was performed in 1 sec. In addition, further variation is to be expected because only a part of the brain was taken after ethanolamine ad-

322 ministration, as reported in Methods. The results of this work will be best expressed, therefore, as the percentage of the recovered radioactivity present in each lipid or water-soluble precursor over the total recovered radioactivity. After the injection, the label disappeared from free ethanolamine, and, although the brain could have been damaged by the injection procedure, there was a rapid transfer of labelled ethanolamine into phospholipids and their precursors, phosphorylethanolamine and CDP-ethanolamine, so that 1.5~ of the recovered radioactivity was already observed as phosphorylethanolamine 2 min after the injection of the base. Even at times as short as 5 sec after the injection, the examined ethanolamine-containing compounds were all labelled. Radioactive glycero-3-phosphorylethanolamine was not found. Choline-containing hydrosoluble compounds and choline lipids were not appreciably labelled during the time intervals examined. The rate of disappearance of label from ethanolamine, although a little greater, was comparable to that already reported for choline6, being of the order of 30 ~ of recovered radioactivity 20 min after the injection. The specific radioactivity of free ethanolamine was greater than that of any of its products, as may easily be calculated (see later).

Phosphorylethanolamine production Part of the radioactivity which had disappeared from free ethanolamine was 100

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0.1

I

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time ( ~ )

Fig. 1. Percentages of recovered radioactivity injected as [2-aH]ethanolamine and determined in brain as residual ethanolamine ( O - - O - - O ) , phosphorylethanolamine (E]--Tq--Vt) and CDPethanolamine ( A - - A - - A ) at various time intervals (sec) from administration. Vertical bars represent the S.D. for each point. Each point is the mean value calculated from at least 4 experiments. Data are reported on a logarithmic scale. See the text for experimental details.

323 found in phosphorylethanolamine. The time-course of this labelling was almost linear with time and represented some 30 ~o of the total recovered radioactivity after 20 min (Fig. 1). As far as phosphorylethanolamine production is concerned, the results are comparable with those reported elsewhere6 for choline incorporation into phosphorylcholine under similar experimental conditions. If we assume that the concentration of ethanolamine compounds in the area which has been injected does not differ substantially from the average content of the same compounds in the whole brain, then it would be possible, in order to obtain values of specific radioactivity, to divide the percentage of the recovered radioactivity present in each ethanolamine-containing compound by the amount of the same compound present in 1 g of fresh tissue. Of course, this would probably not be a true specific radioactivity since, during the time periods examined, labelled ethanolamine compounds would not have diffused evenly into the whole brain particularly for the shortest times. The specific radioactivities calculated in this way, h~wever, would give an idea of the relative specific radioactivities of ethanolamine compounds. In terms of specific radioactivities, the labelling of phosphorylethanolamine after the injection of ethanolamine increases much more slowly with time in our experiments than in those reported by Sundler for liver 2~. Moreover, the specific radioactivity increased linearly with time in our experiments (Fig. 2), contrary to the results of the cited authors. The work of Sundler, however, was made with liver tissue by using an intravenous administration of the base. 100

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Fig. 2. Specific activity (% incorporated radioactivity//tmole; see the text) of phosphorylethanolamine (I-]--I-q--[]) and CDP-ethanolamine ( A - - A - - A ) after the intracerebral injection of 12-aHkthanolamine. Vertical bars indicate the S.D. Each point represents the mean value calculated from at least 4 experiments. Data are presented on a logarithmic scale.

324 In our experiments the specific radioactivity of phosphorylethanolamine was smaller than that of CDP-ethanolamine, especially at the shortest time intervals (Fig. 2). This finding has also been reported by Sundler for liver tissue2L In order to explain this finding, it can be postulated that the distribution of phosphorylethanolamine in the brain is not even or, in other words, that the assumptions made for the calculation of specific radioactivities are not met by nervous tissue. If this is not the case, then it should be admitted that phosphorylethanolamine, which is formed from the injected ethanolamine, does not mix readily with its endogenous pool.

CDP-ethanolamine formation The incorporation of radioactivity into CDP-ethanolamine was low, but well evident, also at the shortest time intervals after the injection of ethanotamine (Fig. 1). It increased over the time period considered, and never exceeded the major part of the injected dose. Curiously enough, there is already a peak of incorporation of radioactivity into CDP-ethanolamine 5 sec after the injection (Fig. 2). This peak has been constantly observed, and may be due to the same reasons which were outlined above to explain the higher specific radioactivity of CDP-ethanolamine compared to that of phosphorylethanolamine. From 2 min onwards the specific radioactivity of CDP-ethanolamine becomes similar to that of phosphorylethanolamine (Fig. 2), as would be expected if a large pool of phosphorylethanolamine (see Methods and ref. 9) is being transferred to a small pool of CDP-ethanolamine. It is worth mentioning that at all time intervals examined the specific radioactivity of ethanolamine phosphoglycerides was much lower than that of CDP-ethanolamine, as will be shown later. This result probably shows that, under our conditions, phosphorylethanolamine is not being efficiently transferred from ethanolamine phosphoglycerides back to CDP-ethanolamine. It is worth mentioning also that, in the course of the present work, phosphorylcholine or cytidine-5'-diphosphate choline (CDP-choline) have never been found labelled after ethanolamine administration, thus excluding any methylation of water-soluble ethanolamine-containing intermediates in brain tissue under our conditions.

Production of ethanolamine phosphoglycerides Brain ethanolamine phosphoglycerides contained a small but detectable radioactivity, already 5-10 sec after the injection of labelled ethanolamine (Figs. 3 and 4). The slope of the graph of the time-course of lipid labelling (Fig. 3) seems to be higher at longer time intervals. Moreover, as a parallel check of this observation, the curve of lipid labelling versus time does not meet the zero point at zero time, as is shown in Fig. 4. The incorporation of radioactivity into lipids then increased steadily with time, reaching values of about 1 ~o of the total recovered radioactivity after 20 min (Fig. 3). This value is less than that reported for choline incorporation into lipids (5.4 ~ ) in similar experimental conditions 6. The specific activity of ethanolamine phosphoglycerides is at any time much lower than that of CDP-ethanolamine, on comparing Fig. 3 with Fig. 2, due to the high content of these lipids in the brain (see Methods and ref. 6). The peak noticed

325 10

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Fig. 3. The conversion of intracerebrally injected [2-aH]ethanolamine into brain ethanolamine phosphoglycerides. On the left the values are shown as per cent of total recovered radioactivity; on the right as specific radioactivity (% total recovered radioactivity//~mole; see the text). Vertical bars indicate the S.D. Each point represents the mean value calculated from at least 4 experiments. Data are represented on a logarithmic scale.

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Fig. 4. Formation of ethanolamine phosphoglycerides from intracerebrally injected [2-all]ethanolamine at very short time intervals (5-120 sec) from administration. Data are expressed as per cent of the total recovered radioactivity and represent mean values calculated from at least 4 experiments. Verticals bars represent the S.D. for each point.

326 for CDP-ethanolamine at 5 sec from injection (Fig. 2) was not noticed during phospholipid labelling (Fig. 4). Phospholipid labelling in molecular subspecies o f phosphatidylethanolamines The rate of incorporation of ethanolamine into differently unsaturated ethanolamine phosphoglycerides was examined at different time intervals after isotope administration. The pattern of the fatty acids of phospholipids which became labelled at various times after the administration of labelled ethanolamine varied markedly according to the time elapsed from isotope administration. At 5 sec from injection of the labelled base, the hexaenoic species predominated, reaching about 74 ~ of the ethanolamine phosphoglyceride labelling. On the contrary, after 5 min, the saturated species were the most labelled, whereas the per cent of labelling was intermediate at intermediate times. Moreover, the ethanolamine moiety of the tetraenoic species had its maximum of incorporation at intermediate times. Therefore it seems that the molecular species of ethanolamine lipids became labelled as a function of time. It must be mentioned that the labelling of the molecular subspecies refers here chiefly to the diacyl derivatives of phosphatidylethanolamines, which are the most heavily labelled ethanolamine lipids at these time intervals. The specific activity of the lipid molecular species which became labelled, calculated after the determination of the differently unsaturated ethanolamine phosphoglycerides, increased with time, as expected; this increase was mainly due to the less unsaturated species (Table I). Indeed, saturated and monoenoic species increase their specific activity some 20 times from 5 sec to 5 min, whilst the hexaenoic species only double their specific radioactivity. This means that ethanolamine is very rapidly incorporated into hexaenoic species, and less rapidly in other molecular species; on the other hand, the incorporation into hexaenoic species lasts for very limited periods of time. This may mean that hexaenoic diacyl glycerols are utilized very rapidly, and

TABLE I Specific radioactivities of different ethanolamine phosphoglycerides after the injection of labelled ethanolamine into rat brain* Fraction

Saturated Monoenoic Dienoic*** Tetraenoic**** Hexaenoic

Content**

1.24 1.88 1.41 4.54 8.68

Time after injection (sec) 5

20

60

300

0.26 0.057 -0.048 0.213

0.602 0.12 0.066 0.165 0.147

0.650 0.428 0.404 0.265 0.182

5.10 1.31 1.63 0.363 0.418

* Values are expressed as per cent of the total recovered radioactivity in each fraction/ffmoles x 102, and represent mean values from 5 to 10 rats. ** #moles P/g fresh tissue. Mean values of 4 estimations each. *** Plus trienoic. **** Plus pentaenoic.

327 quickly consumed, or that two reactions are involved in phospholipid biosynthesis when ethanolamine is injected in vivo. On this last assumption, the base has to be transformed in the brain during the initial periods into a lipid form by a pathway not involving CDP-ethanolamine, which, on the contrary, certainly takes place in nervous tissue at longer time intervalsL The base-exchange pathway~5 represents the only pathway described which bypasses CDP-ethanolamine, although the present data cannot confirm that the radioactivity incorporated into ethanolamine lipids at short time intervals represents only base-exchange. DISCUSSION In this work the utilization in vivo of intracerebrally injected [2-aH]ethanolamine has been examined in order to assess in some detail the mechanism of its incorporation into ethanolamine phosphoglycerides and the possible importance of base-exchange reactionsg,15,17,19. From previous data15,x9 it has been ascertained that base-exchange in vitro is faster than 'net synthesis' reactions, and, for these reasons, the incorporation of radioactive ethanolamine into lipid and water-soluble compounds of rat brain has been examined at very short time intervals after its intracerebral injection, to minimize the contribution of 'net synthesis' for ethanolamine incorporation into lipids. Following these precautions, it has been shown in previous work 6 that injected choline may enter choline phosphoglycerides at very short time intervals from its administration, probably by a metabolic route which bypasses CDP-choline, namely by base-exchange. Further work has confirmed these views8. q-he results of the present paper seem to also extend these findings to ethanolamine incorporation into ethanolamine phosphoglycerides. It must be admitted, however, that with our experimental conditions the utilization of ethanolamine was investigated in a small brain area, due to the procedure of injection; for this reason, the diffusion of ethanolamine, which is supposed to have been very small at very short time intervals, did not allow calculation of true, but only relative specific radioactivities (see Results). The experimental findings of the present work provide some indications that the base-exchange system may be operating in vivo. Brain tissue is able to convert ethanolamine in a rather efficient way to phosphorylethanolamine and CDP-ethanolamine, at very short time intervals, q-he time-course of labelling of these two watersoluble ethanolamine-containing compounds (Fig. 1), if extrapolated to much longer time intervals, is in rather good agreement with Ansell and Spanner's findingsz, obtained at longer periods. However, although the specific radioactivity of CDPethanolamine constantly decreases at short time intervals between 5 and 20 sec (Fig 2), as observed in various experiments, ethanolamine phosphoglyceride labelling continues to increase in a very linear manner throughout the same interval (Fig 4). Moreover, on carefully examining the slope of lipid labelling in Fig. 3, and on taking into account the data of Fig. 4, a different rate of incorporation of label into lipid seems to take place at shorter and at longer time intervals, the curve at the shortest time intervals (Fig. 4) not meeting the zero point at zero time. These findings seem

328 to suggest that soon after administration ethanolamine is able to be converted rapidly in a lipid form by a mechanism which does not seem to depend strictly on CDPethanolamine labelling, namely by base-exchangeg,l~,17,19. Probably, the net synthesis mechanism would be the cause of the subsequent increased rate of ethanolamine incorporation into lipid (Fig. 3), which is taking place in the same time interval required for CDP-ethanolamine to become sufficiently labelled to drive the cytidinedependent net synthesis reaction of ethanolamine lipid production. These considerations seem to receive further support by the findings reported in this work on the labelling of the various ethanolamine phosphoglyceride molecular subspecies. Indeed, the results of Table I indicate that the pattern of labelling among the subspecies varied in a very different way at shorter and longer times after injection, with much greater incorporation into highly unsaturated species in the first seconds after administration and a much higher rate of labelling of saturated and monounsaturated species at longer time intervals; the latter certainly representing the periods when 'net synthesis' mechanisms take place. Moreover, it must be mentioned that in vitro the pattern of incorporation by base-exchange of ethanolamine into phosphatidylethanolamines of different unsaturation 13 is quite similar to that observed in vivo at short time intervals from administration (5-20 sec). This finding signifies that soon after ethanolamine administration a pattern of labelling of ethanolamine lipid molecular subspecies is found which is very similar to that observed for the same lipid in vitro, thus pointing again to the possibility that base-exchange reactions probably occur in vivo for ethanolamine incorporation at the initial stages. This result seems to be confirmed by recent findings s. ACKNOWLEDGEMENTS The skilful technical assistance of Mr. Antonio Boila is gratefully acknowledged. This work has been aided by a research grant from the Consiglio Nazionale delle Ricerche, Rome (Contract n.75.00676.04).

REFERENCES 1 Akesson, B., Elovson, J. and Arvidson, G., Initial incorporation into rat liver glycerolipids of intraportally injected [all]glycerol, Biochim. biophys. Acta (Amst.), 210 (1970) 15-27. 2 Ansell, G. B. and Metcalfe, R. F., Studies on the CDP-ethanolamine-l,2-diglyceride ethanolaminephosphotransferase of rat brain, J. Neurochem., 18 (1971) 647-655. 3 Ansell, G. B. and Spanner, S., The metabolism of labelled ethanolamine in the brain of the rat in vivo, J. Neurochem., 14 (1967) 873-885. 4 Arienti, G., Corazzi, L. e Porcellati, G., La reazione di scambio delle basi azotate quale meccanismo della sintesi fosfolipidica cerebrale in vivo. In XX Congresso Nazionale della Societd Italiana di Biochimica, Riva del Garda, 1974, Comm. 97.

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6 Arienti, G., Corazzi, L., Woelk, H. and Porcellati, G., Biosynthesis of rat brain phosphatidylcholines from intracerebrally injected choline, J. Neurochem., 27 (1976) 203-210.

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