Changes in phosphatidylethanolamine metabolism in regenerating rat liver as measured by 31P-NMR

9id~hhnica t~ Biop/;t~icaActa, 1135(1992)27-34 ~; 1992ElsevierSo~ncc PublLsh=x~B.V.An tightsrcser.'ed tnl~7-dssg/92/$os.tg)

27

Changes in phosphatidylethanolamine metabolism in regenerating rat liver as measured by 31P-NMR E l i z a b e t h J. M u r p h y , Kevin M . B r i n d l e , C a r o l i n e J. R o r i s o n , R u t h M . DLxon, B h e e s h m a R a j a g o p a l a n a n d G e o r g e K. R a d d a MRC Cluucal aud Bi~hemicd Magm,oc Resonant(, L~lit. Depurtmwtt o) IluwhanLWo" gttlh.t.~ar of O~furd. Oxfard (UK/

(Received 311Dccemllert~}91) Key words: liver regeneration: NMR, ~1P-: Phosphalidylethanolaminc m=lal~flism: tRat liver) 31P .NMR :pcctra of regenerating rat liver in vivo show increases in resonance intensities in the phosphomonoester (PME) reginn

and decreases in the phosphodiester (PDE) region as early as 12 h post parlia[ hepatcctoray, which return to normal by 8 da~,s. The cor.lp.~unds primarily rL~[mnsible fur these changes have bccn identified in pcrchlmic acid extracts as the phosphomoIlOeSter pho~phoethanolamine and the phosphodiester glycePophosphucthantdamine (GPE), indicating altered phosphatidylethannlaraine metabolism. A ¢xlrrcspzmdingincrease in diacy[glycertd(DAG) levels during regeneration indicates a possible role for a phosphatidglethanolamine-speciiic phospholipase C in cellular proliferalitm. These results suggest that changes in phospholipid metabolites previously associated with neoplastic tissue can also be induced by normal tissue undergoing rapid c.¢llular proliferation. The spectral changes observed in the regenerating rat liver arc similar to changes seen in spectra from the livers of human patients in several disease states, indicting th::t 3lP-NMR may allow non-imasive study of cell turno~cr in liver disease. Introdnction The regenerating liver is one of the most rapidly growing tissues available for study and as such provides a good model for investigating homogeneous normal tissue growth. Almost immediately after partial hepatectomy there is an increase in RNA and protein synthesis, while glycogen stores become depleted. The cells hypertrophy until the onset of mitosis. DNA s'ynthesis begins 14-18 h after the operation and is at a maximum at 2 0 - 2 6 h [1,2]. Cellular proliferation follows close behind with the number of mitoses reaching a peak 6 - 8 h after maxJaalal DNA synthesis [3]. Total liver mass is restored by 7-10 days at which point growth stops [1], ATP concentrations are unchanged in the regenerating liver though there is an indication of greater ATP b r e a k d o ~ than in control liver when subjected to aooxia [4].

Ahhr~'.Aa1iops: PML phosphomonoestcr, PDE.phosphodiester;, ER, endaplasmlc reticulura; GPC, gl~rophc~ph~holine: GPE. gb~:eroph~lahoethanoiamine: PCA. perchIodc acid: PCr. nhosphncXealine:DAG.dia~lgly~rot:FID. free induction deca~ PEP. ph~sp h o ~ o / p ) ~ l e : ,N1Tp,nucl~ide tr~nhasphatc. Correspondent: EJ. Murphy, MR("Oinlca! and Bmch=micalM~g netm Resonanc~ Unix. I)epar~ment of niochg~istff. Unlve~itTof Oxford. $Oulh Parks Road, oxgord. OXI 3QU. UK.

Neutral lipid content increases rapidly after partial hepatectomy reaching levels 2-5-times normal at 1-2 days and returns to control values by 3 days [3.5-7]. Unlike the neutral lipids, total and individual phospholipid contents remain constant throughout the regenerative process keeping pace with the increase in liver mass [7-11 ]. The total phospholipid content is restored by 7 days for all the phospholipids with the possible exception of cardiolipin [12]. Studies of subcellular organdies indicate that there may be small changes within the various subcollular membranes. While it has been shown that the phospholipid patterns of the plaspw membrane, mitochondria and microsomes i-cmain unchanged after partial hepatectomg [9,10], it has also been shown that there are decreased sphingomyelin levels in the plasma membranes and decreased sphingomyelin and increased phosphatidylcholine levels in the microsomes of regenerating liver [13]. There is also evidence of increased phospholipid content in the mierosomes post partial hepatectomy [13], which, combined with evidence of increased synthesis of m.;crnsomal phospholipids [14], could indicate a coordination between accelerdted synthesis and the formation of extra rough endoplasmic reticulum (ER) in preparation for increased ribosomal RNA and protein synthesis. There is some disagreement as to the effect of regeneration on rates o f phospholipid turnover in the

28 liver. Increased incorporation of "~"P into membrane pbospholipids during regeneration was reported a.s early as 1953 by Dawson and co-workers [15] and has since been confirmed by otber:~ [8,14,16 18]. This increased uptake, which is evident by i8 h after partial hepateetomy, remains elevated at two to three days and returns to normal by II days [g,17,18]. However, one study has found no significant difference between the rate of incorporation of 32p into phospholinids in normal and regenerating rat liver [19[. Attempts to investigate changes in turnover rates of indMdual phospholipids have produced inconsistent results; however most phospholipids studied showed increased turnover and all the studies showed increased phosphatidylethanolarnine turnover [gA7,18]. Analysis of phospho[iplds from subcellular fractions showed elevated phospholipid turnover in all the fractions studied: nuclei, mitochondria, microsomes and more specifically rough ER, smooth ER and free polyn'bosomes [14.18]. 3tP-NMR provides an opportunity for further studying phospholipid metabolism during liver zegeneration. "~tP-NMR has been used to investigate isolated rat bepatocyies [20], perfused rat livers [21-23], rat livers in vivo [24,25] and human livers [26,27]. ~tP-NMR investigations ill vivo of human liver in various d i s u s e states such as [ymphoma, alcoholic hepatitis, and viral h~patitis revealed changes in the PME and PDE reginns of the spectrum. Specifically. a relationship was established between an increase in the PME peak and the severity of disea~ [ 2 ~ 7 ] . ~I"ne PME region, as well as containing resonances from sugar phosphates, also has peaks from phospholipid metabolites such as phosphocholine and phosphocthanolamine. The PDE re# o n is composed primarily of the phospholipid m-.tabolites glyceropbosphocholine and g]yceruphOSphoethanolamine. At fields less than 4 T there is an additional contribution to the p D E region from phospholipid bilayer [28L A fundamental question is whether the metabolic changes observed by N M R in human livers could bc a result of rapid cell proliferation of novma[ tissue induced by the disease. Also, what role, if any, does phospholipid metabolism, as observed by changes in the compounds seen in the PME and POE spectral regions, have in cellular prolift:ration. In this paper we present work aimed at answering these questions using 3tP-NMR to study the phospholipid metabolism of the liver during regeneration following partial bepatectomy.

thetized with diethyl ether. The partial hepatoctomies, performed ~¢ordiug to the published procedure |29J, involved removal of 70% of tbe liver. The sham operation consiste¢* of a laparotomy and gentle palpati m of the liver. Animals were allowed to eat and drink ad libitum until the time of N M R study. Fasted animals were allowed water but no food for the 30 h prior to N M R investigation, For N M R experiments the animals were anaesthetized with 1-2% halothane in 50% N 2 0 / 5 0 % 0 2 and the liver was surgically exposed. Perchlodc acid extracts

After collection of spectra in vivo, the animals were remtwed from the magnet, the surface coil removed from between the lobes and the exposed livers were freeze clamped with liquid-nitrogen-cooled tongs while the animals were still anaesthetized. The frozen samples were stored in liquid nitrogen until the time of extraction. Livers were ground to a powder in a liquidnitrogen-cooled t~lortar and pestle. The powder was added to a 4% perehlorie acid (PCA) solution, using approx. I g tissue/4 ml PCA. This mixture was then homogenized with an electric homogenizer for 15 s and centrifuged for 5 rain. The supernatants were neutralized with 3 M K2CO 3. allowed to equilibrate for 5 rain and then respun. The resulting superaatants were then I~ophilized to dryness. Dried samples were stored at - 2 0 ° C until N M R study, at which time t h ~ were tesuspended in 3 ml of a solution of 15 mM EDTA, 50 mM Hepes buffer and 2 mM phosphocreatinc (PCr). The PCr was added as a reference. The final pH was between 8 and 9_ N M R in vivo

Materials and Methods

A e.vo~turn 2 em diameter surface coil was inserted between the lobes of the surgically exp~-~d liver. 3tp. N M R spoctra were collected at 32-5 MHz in a 30 cm bore, 1.89 T Oxford Instruments magnet interfaced to a Bruker Binspec I spectrometer. Spectra were acquired with a 60~ pulse into 2048 data points wilh a s~veep width of 4 kHz and an interpuise delay of 2.3 s. A total of 512 free induction decays (FIDs) were accumulated in just under 20 rain, Chemical shifts are given with a-ATP at 7~7 ppm, which corresponds to PCr at approx. 0 ppm. Spectra were processed with a profile correction followed by gaussian multiplication. This data processing helped eliminate the broad PDE component and provided the resolution necessa~ for peak integration which was achieved by fitting the entire sr,ectrum to Gaussian peaks using the Bruker computer integration routine GLINFIT. Each spectrum was integrated twice and the results were averaged.

Animals

NMR in vitro

Partial bepatectomies and sham operations were performed on 150-g male Wistar rats (OLAC) anaes-

~IP-NMR spectra were obtained from PCA liver extracts in a 7.05 T Oxford Instruments magnet inter-

29 faced to a Broker AM 300 spectrometer. The homebuilt 10-ram probe consisted of a Helmholtz phosphorus coil surrounded by a separate saddle coil tuned to the proton frequency. The spectra were acquired at a 31P-NMR frequency of 12L5 MHz using a 60 ° pulse. 4.7 s interpulse delay, 8192 data points and 6 kHz spectral width. Proton decoupling was achieved using WAL'I'Z-16 composite pulse [30] decoupling during acquisition at a power level of 3 W. Under these conditions th¢ resonances were partially saturated and therefore quantification of the spectra was not possible. As well as P e r in the buffer, a capillary with methylene diphusphonic acid was used as a reference. Chemical shifts are given relative to @PC at 2.85 which corresponds to PCr at 0 ppm at pHs greater than 7. Peak identifications in the PME and PDE regions were made by comparing titration curves of the unknown and the suspected compound produced under the same conditions. Also, extracts ,,~ere spiked with the suspected compound. Identifications of GPE, GPC, phosphoethanolantine and phosphocholinc agree with those given by Cohen [211. Spectra were processed using exponential multi#ication with 2 Hz line broadening. P M E / P D E ratios were determined using a computer integration routine. Other ratios were obtained by fitting the entire PME and PDE regions to Lorenztian lincshapes using GLINFIT. Again, all spectra were integrated twice and the results averaged. Diacylglycerol assay DiacTIglycerol levels in the liver were determined after partial hepatectomy or sham operation. Lipid extraction of the llvel was carried out by the chlorof o r m / methanol extraction procedure of [31]. The assay was done using Amersham's sn-t,2
Results N M R in rico Fig. 1 shows a typical control spectrum and a spectrum from a rat liver 48 h post partial hepatectomy. The PDE peak contains a broad component arising primarily from phospholipid bilayer with a small contri-

(m)

NTP

(b)

Fig- i. ~IP-NMR spectra at 32.5 Mllz of rat liver in viva. Spect~ represent the sum of 512 FIDs processed with profiletx~cction and Gaussian muitiplicalion.(a) $pect~m of a ~nt~l liver whh no opnmtion. (b) Spectrum of a liver 48 h post partial hepatec~:my. Peak asslanraems: PME, phosphomonoester~: P,, inorgan~ phosphate; PDE. phosphodiL~lc~NTp, nucleolide tripho~phat~,largely ATP with contribution from GTP.

button from a motionally averaged macromolecule [28]. However, processing the spectra with convolution difference removes most of this broad component. Fig. I shows a clear decrease in the PDE peak and an increase in the PME peak with respect to the N I P peak. The ratio of the integrated areas of these two peaks for several different time points are presented in Fig. 2. At 12 h, the cattiest time point studied, there is already a significant increase in the ratio. This increase continues, reaching a maximum at approx. 2 days and then returns towards normal at 8 oays, by which time liver mass is rcstoced. At 25 days, the P M E / P D E ratio is still slightly above controls but not significantly so. Livers from the sham-operated animals, when compared to non-operated controls, show an initial decrease in the P M E / P D E ratio but this is not statistically significant. This is followed by a slight, hut not statistically significant, increase with time after surgery. Rats fasted for 30 h gave a P M E / P D E ratio of 0.38 _+

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F/g. 2- Ratio of PME/PDE peak are~ (mean +_S.E.) from Ji P-NMR spectra of rat Iber in v h u after sham operation ( o ) or partial hepatectoray ( D )- Number of animah studied is indicated in parentheses. 0.0/, below the ratio found for n o n - o p e r a t e d controls (0.48 _+ 0.12) a n d slightly below that found for shamo p e r a t e d animals at 12 h (0.43 + 0.06).

NMR in t~lro T h e P M E / P D E ratios for the s p e c t r a from acid extracts are p r e s e n t e d in Fig- 3. T h e same t r e n d s s e e n in the spectra in vivo are observed here. Differences in the r a n g e of the ratios, 0 to 1 in the spectra in vivo ~ , 0 to l0 in the spectra in vitro, are due to the p r e s e n c e of

.,-,-,.,.,-,-,.,.,.,.,.,., 2 4 6 8 1o i2 14 16 18 20 22 24 26 T I ~ (OaSs} ~g. 3. Ratio of PME/PDE pc-dkar¢~ (mean _+S.E.)from ~IP-NMR s~ctra r r ~ PCA e~racts of rat lk~r after partial hcpaI¢clc~al~,( [ ] ) or sham ogc~tlon ( • k All resuas are based on a 3 except for 2.5 da)~ partial hcpatectomywhere n 2. o

a b r o a d , n o n - a d d - e x t r a c t a b l e c o m p o n e n t in t h e P D E region of spectra obtained in vivo. Typical 3 t P - N M R s p e c t r a of extracts from the liver of a s h a m - o p e r a t e d control a n d a rat 12 h after partial h e p a t e c t o m y a r e shown in Fig. 4. T h e r e is a d e c r e a s e in G P E a n d a dra,natie increase in the signal in the P M E region 12 h post bepatectomy. T h e metabolite p r i m a n i y responsible for this increase was identified as p h o s p h o e t h a n o l a m i n e . While p h o s p h o e t h a n o l a m i n e increases, phosphocholiae initially decreases.

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Fig. 4. ~nP-NMR SlX'clraat 121-gMHz of PCA extracts of rat liver 12 h after (a) sham operation and (b) partial hepatectomy.

TABLE[ Ratios o f itlJeg~ted 19eak ~

f r ~ l • tP slzt'ctra o f exlracted rat ]it er a~er p'drtial hefmteclomy or ~llum ofleralton

Time after surgel3' ~day0

PE/PC helralectomy

shamoperated

03 2 8

535_+038 ** 258_+0.57 * 0.30_+0.06

0.63_+0.11 0.19_+0.16** 0.74-+0.24 0.25+_0.03** 1.05_+0.31 1.48_+0.47

partial

GPE/PE partial hepatectomy

shamoperated

GPC/PC partial shamhepaleetomy operated

2.811_+0.20 2.37_+U.12* 2.16+_11.06 0.43_+{).10 1.69÷a.27 0.20+003

GPE/OPC partial

0.78+U.20 0.37~030 " 0.75_+11.34 1.46_+11.(19 (L94+u.36 1.98+0.39

sham2.45-b038 2.37_+0.30 2.05+0.13

Results are mean+S.E. All resultsare based on n = 3. ** P < 0.001. ~ P < it01. + P < 0.05compared 1o sham operated walues. To illustrate these changes better, ratios for phosphoethano!amine/phosphocholine, GPE/phosphoethanolamiue, GPC/phosphocholine and G P E / G P C were calculated and are shown in Table I. The most dramatic changes occur 12 h after partial hepatecaomy where, due to the increasing phosphoethanolamine and decreasing phosphoehofine, the phosphoethanolamine/phosphocholine ratio has increased over 8-fold. At 2 days this ratio begins to return to sham-operated values primarily due to a return to normal of phosphocholine levels as evidenced by the GPC/phosphocholine ratios. However, the GPE/phosphoethanolamine ratio is still significantly lower, indicating continued elevation of phosphoethanolamine. A decrease in GPE also contributes to this lowered ratio as evidenoed by the lowered G P E / G P C ratios at 12 h and 2 days. By 8 days, all ratios are comparable with those in the sham-operated controls. In summa~, the dma seem to indicate (!) .~ decrease in pht~-~hoehelL-;c, iu the first 12 h that returns to normal after t~a3 days, (2) an increase in phosphoethanolamine in the first 12 h that continues for 2 days, returning to normal after 8 days and (3) a decrease in GPE during the filst 12 h that has started to return to normal after 2 days but is not complete until 8 days. Diacylglycero! assay

DAG concentrations obtained from a group of livers at different time imints after partial hepatectomy and sham operation art; sh~::~ ~ Table II. As early as 3 h TABLEII DAG let'els m rat ht'er a]i~ Imrttal

Time after surger/(h)

3 12 24 48

#.ei~receamyor sham operation

DAGtnmal/g ~ t wt.) partial ~aam hePatetaorr6' o~¢ration 1558_+228** a59_+ 60 68i_+ 79 * 420± 32 83g_+i54 * 368_+ 61 095_+ 80 464_+10!

Results arc m~n-+S.E. All results arc based on n 4. =*P < 0.0I * P < 0.u3.

and

post partial hepatectomy liver DAG levels are highly significantly elevated when compared to levels in sham-operated animals. While significantly elevated at 6 h, DAG levels have already begun to return to normal. Though DAG levels were higher than in sham-operated animals at 48 h, this did not reach statistical significance. Discussion Two ~tP-NMR studies of the regenerating liver have been previously attempted. The firsL investigating the regenerating mouse liver [32] found a decrease in GPC and GPE after one day which persists for 4 days post partial bepateetomy. A slight increase in the 'sugar phosphate' region after | day followed by a decrease below normal after 2 days was also found. However, this study was done o n excised, nun-egtracted, tissue at 4°C and the levels of metabelites were expressed relative to what must certainly have been a rapidly changing P, peak. Another study, which looked at perchloric acid extracts of regenerating rat liver, showed 31p "high resolution" spectra with no separation of peaks in the PME or PDE regions and no resonances from the dior trlphosphates [33]. Therefore, one must hesitate to draw any conclusions from this work as well. In contrast to previous studies, the work presented here clearly shows that the regenerating rat liver produces changes in XtP-NMR spectra in vivo and in vitro. The changes in vivo are similar to changes seen in spectra from patients with liver disease and spectra in vitro indicate that the changes represent altered levels of phospholipid metabolites. Changes in levels of the phospholipid metabolites observed with 31P-NMR often have been attributed to neoplastic tissue or cell transformation. Elevation of phosphoethanolamine in a homogeneous tissue experiencing rapid cell growth indicates that the same spectral c~tanges can also arise from normal tissue undergoing cellular proliferation. The time-course of the change in phospholipid metaholites follows the time-course of the regeneration process itself. As early as 12 h, before DNA synthesis has begun, there are already significant

32 been p r ~ e d that phosphatidylcboline metabolism may ~ produce second messengers such as D A G and amchidonic acid [37-39], Pborboi esters have been shown to enhance ph~phatidylcl~oline-specifie phosp h o ~ p ~ C activity and produce elevated levels of phosphocholine and choline in HeLa cells [40] and elevated p h ~ . h ~ b o l i n e and D A G levels in MadinDarby canine kidne'~' cells [39]. Phorbol diesters, fetal bovine sermn, and platelet-derivcd growth factor also sthmdate hydrol~sis of phosphat[dylcholine with a concomitant production of D/~G and phoxphocholine [38]. A phospholipase C which selectively hydrolyzes phosphatid~lcholinc and phosphatidylethanolamine but she~vs no at~,,ity for pl'a~sphatidylinositol has been partially pur[g-w.d from canine myocardinm [41]. Further results suggest that this phesphatidylcholinespecific phosphotipase C in rat fiver plasma membranes is coupled to putinergic receptors by a GTP-binding protein [42]. Perhaps phosohatidylethanolamine metabolism, like phosphati~'linositol and phosphatidylcholine, can also generate second mes~nge."s. ?Mi attlUCfivc explanation for the N M R results seen in this study is an altered route of phosphatidylethanolamine breakdown, as illustrated in Fig. 5. Rather than production of G F E from the action of phospholipase A2 followed by the appropriate lysopbospholipase (Route 1), the turnover cycle changes to favour Route 2 and the production of phosphoethanolamine and D A G via plmspholipase C action. This would account for the decrease in GPE with the concomitant ;ncrease in phosphcethanolamine and would also provide elevated levels of D A G for

changes in pbespholipid metabellte levels. A study of t/me points earlier than 12 h would reveal when these changes begin and help determine their possible relationship to the initiation of the lx~generative process. The changes p~rsi~.* through 2 days, indicating that they are poss~ly related not only to initiation of the regenerative process but to its maintenance as well. At 8 days, by which point growth has ceased, phospholipid metabolita levels have returned to control values. Phosphoethanolamine is generally thought of as a precursor to phosphatidylethanolamine, while GPE is one of its breakdown products which results from the action of phosphol/pase AI or A2 followed by action of the appropriate t,p~phospholipa~. GPE is then fur*.her hydrolyzed to produce ethano!amine and m glycerol 3-phcephate which then can go back into the s~athetie pathway to produce phosphatidate and eventually DAG. In the phosphatidylethanolamine biosynthetic pathway there is strong evidence indicating that the flux-controlling enzyme is CTP:phosphoethanolamine cytidylyl transferase [34]. Therefore, with an increased rate of phosphatidylethanolamine symhesis in the regenerating liver as evidenced ~, the 32p incort',o_ra_tion studies [8,14,16,18], one might expect a deerea~e, rather than increase, in the suostrate for this en2ym~, phosphoethanolamiue. Phosphoethanolamine can, however, also be produced by the action of phospholipase C on pbosphatidylethauolamine. The importance of phospholipase C in phosphatidylinositol metaboiism, D A G production and protein kinase C aet[vatinn has been well documented in the last 10 years (for reviews see Refs. 35,36). it has receutly

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R3 PE (FriacFIglycer°l) (GPE) Fig_5. Pt~.ible rout¢~Ofphosphatidyiethallolamine breakdown. Route 1 leads Io GPE ~'hile Route 2 leads to phosplmelhanolamhl¢.RL 17,2and R3. fatty a~l chains; F, phosphel~lgroup; E ¢lhanolamine.

33 protein kinage C activation a n d cellular proliferation. It has b e e n s h o w n t h a t t h e r e is a n increase in p r o t e i n kinasc C associated with t h e particulate fraction, w h e r e it is in its active form, as o p p o s e d to the inactive form in t h e cylosolic fraction 30-61/rain after partial h e p a t e clomy [43]. T h e observed c h a n g e s in D A G levels after partial h e p a t e c t o m y a r e consistent w i t h this hypothesis. A previous study f o u n d elevated D A G levels t h r o u g h t h e first 2 h a f t e r partial h e p a t e c W m y [44]. T h e results p r e s e n t e d h e r e e x t e n d those findings showing elevated D A G levels t h r o u g h o n e a n d possibly two days after surgeP/. T h u s , D A G m a y play a n i m p o r t a n t role in cellular proliferation t h r o u g h o u t t h e r e g e n e r a t i o n process. C o n c e n t r a t i o n s o f p h o s p h o e t h a n o l a m i n e in t h e norm a i liver a r c appror,. 0.4 raM, a s s u m i n g 1 g / m l o f liver [45] a n d G P E c o n c e n t r a t i o n s at approximately twice t h a t l e v e l I f all t h e p h o x p h a t i d y l e t h a n o l a m i n e breakd o w n is via phospholipase C (i.e., R o u t e 2 in Fig. 5), c h a n g e s in D A G [e'.,e~ ~ ¢ a l d b e in the miliimolar range. This is i n d e e d t h e case for t h e levels seen 3 h after hepatectomy. T h e lower P M E / P D E ratio for fasting animals c a n be explained by k n o w n c h a n g e s in phospholipid metabolism w i t h fasting [46]. A -~-h fast produces a s h a r p decrease in phosphatldylcholine a n d its precursors, particularly p h o s p h o c h o l i n e , while t h e same conditions p r o d u c e only a m i n o r decrease in p h o s p h a t i d y l e t h a n o l a m i n e a n d its precursors. As animals used in this study w e r e all t a k e n f r o m different stages in t h e i r f e e d i n g cycle, this could also provide a n explanation f o r t h e g r e a t e r variation observod in phosp h o c h o l i n e vs. F h o s p h o e t h a n o l a m i n e levels in spectra from a n i m a l s within t h e s a m e study g r o u p ( d a t a n o t s h o w n L Dietary factors m i g h t also explain t h e reduction iu p h o s p h o e h o l i n e s e e n in t h e first day o f r e g e n e r ation w h e n t h e r e is a significant fall in food intake a n d loss in body weight as compared w i t h s h a m - o p e r a t e d anirc~Js [5]. As p h o s p h o c h o l i n e , like p h o s p h o e t h a n o l a m i n e , is the substrate for t h e flux-controlling e n z y m e in phospholipid s~a,tbesls, this fall in p h o s p b o c h o l i n e could also h e explained by t h e increased rate o f p h a s phatidyleholiBe synthesis f o u n d by s o m e [17,18[ a f t e r partial_ hepateetumy. F u r t h e r s u p p o r t f o r this has b e e n recently r e p o r t e d [47]. T h e r e g e n e r a t i n g 1K'er p r o d u c e s s o m e very m a r k e d c h a n g e s in ~ P - N M R spectra, b o t h in vivo a n d in vitro as early as 12 h post partial hepatectomy. Identification h e r e o f t h e c o ~ , , u n d s responsible for these c h a n g e s in tim r e g e n e r a t i n g liver as p h o s p h o e t h a n o l a m i n e , G P E a n d p h n s p h o c h o l i n e indicates s o m e role for p h o s p h o lipid metabollsre in t h e regenerative process. Aeknowledgemelats T h i s w o r k was s u p p o r t e d by t h e Medical Research Council o f G r e a t Britain. E.J.M. t h a n k s t h e R h o d e s

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