Incorporation of radioactive bases into calvaria of the new-born rat, Rattus norvegicus

Comp. Biochem. Physiol., 1975, Vol. 50B, pp. 345 to 349. Pergamon Press. Printed in Great Britain

INCORPORATION OF RADIOACTIVE BASES INTO CALVARIA OF THE NEW-BORN RAT, RATTUS NORVEGICUS TrIOMAS R. DIRKSEN Departments of Oral Biology and Cell and Molecular Biology, Schools of Dentistry and Medicine, Medical College of Georgia, Augusta, Georgia 30902, U.S.A. (Received 6 November 1973)

Abstract--1. Calvaria of new-born Rattus norvegicus was incubated with choline-X4C and the lipids extracted. Lecithin (90 per cent of incorporated counts), lysolecithin, sphingomyelin and occasionally phosphatidyl ethanolamine became labeled. 2. Phosphatidyl ethanolarnine and lysophosphatidyl ethanolamine were labeled with ethanolamine~'C but lecithin was not. 3. Inositol-SH was incorporated into only a single lipid, phosphatidyl inositol. 4. Bone did not use (methyl-14C) methionine for lipid biosynthesis, thus demonstrating the lack of the methylation pathway for lecithin synthesis. 5. Incubation of bones, prelabeled with choline-X4C, in 0.01 M nonradioactive choline or serine did not alter lipid radioactivity. 6. Radioactivity of ethanolamine-~4C labeled bones, incubated in 0.01 M ethanolamine or serine, was significantly less than control bones, thus suggesting significant base exchange.

INTRODUCTION PREVIOUS studies have shown that calvaria of the

new-born rat, R a t t u s norvegicus, can synthesize a variety of lipids under in vitro conditions (Dirksen, 1969; Dirksen et al., 1970a, b). Following incubation with orthophosphate-8~P, glucose-x4C or acetatex4C, lecithin was heavily labeled, phosphatidyl ethanolamine and phosphatidyl inositol were moderately labeled, and phosphatidyl serine and sphingomyelin contained little or no radioactivity. Incubations conducted with serine-x4C, however, demonstrated heavy labeling of phosphatidyl serine, phosphatidyl ethanolamine and sphingomyelin. Therefore, it became apparent that different lipids show different degrees of radioactivity, depending upon the choice of labeled precursor. These results advocated using a variety of labeled precursors to more fully explore lipid synthetic pathways within calcifying tissues. To this end, the studies reported here were conducted employing choline-uC, ethanolamine-x4C, (methyl-~4C) methionine and inositol-SH. MATERIALS AND METHODS The execution of experiments was similar to that previously described (Dirksen, 1969; Dirksen et al., 1970a, b). To study lipid synthesis from different substrates, groups of ten to fifteen calvaria from new-born, DC strain rats, Rattus noroegicus, from Charles River Breeding Laboratories, Wilmington, Massachusetts, were incubated for 4 In" in 4 ml Krebs--Ringer phosphate buffer containing 18 nag ~o glucose and 5 pCi chollne-X4C,

cthanolamine~X4C, (methyl-14C) methionlne or inositol8H. A second series of experiments was conducted to determine whether non-radioactive choline, ethanolamine or serine could affect radioactivity of bones previously labeled with cholineJ*C or ethanolaminex4C. For this purpose, calvaria were incubated for 2 hr with isotope, washed in non-radioactive buffer and incubated for an additional 2 hr in buffer with or without non-radioactive 0.01 M choline, ethanolamine or serine. Following incubation, bones were washed, weighed, demineralized with 15~0 EDTA and the lipids extracted. Extracts were purified (Folch et al., 1957) and chromatographed on sillcic acid impregnated papers (Marinetti, 1964; Wuthier, 1966). Lipid phosphorous was determined by the micro-Bartlett procedure (Marinetti, 1962a) and the valves used for comparison of results (Dirksen et aL, 1970a). To assay lipid radioactivity, lipids were located on chromatographs with the aid of autoradiograms and/or Rhodamine 6G staining. The spots were removed from the chromatograms and counted in a Model 3375 Packard Tri-Carb Liquid Scintillation Spectrometer using Bray's solution (Bray, 1960). (Methyl-uC) choline chloride (7.6 pCi/mM), ethanolamine-l-2-uC (11.0 tzCi/mM), inositol myo-2-aH (1 Ci/ raM) and glycerol-2-SH (152-6 mCi/mM) were purchased from New England Nuclear Corporation, Boston, Massachusetts. ~S~TS The identification of lipid classes on chromatograms depended upon various chemical and enzymatic tests (Marinetti, 1960, 1962a, b; Marinetti et

345

346

THOMASR. DIRKSEN

al., 1960) and the chromatography of known standards. The results of these tests were consistent with the identifications used in the text.

Into Lipids

Incorporation of radioactive bases into calvaria lipids Choline-~4C. Radioactive choline was readily incorporated into lipids by rat calvaria. All radioactivity was observed to remain at the origin during neutral lipid chromatography. However, following treatment with sodium methoxide or phospholipase A, some radioactive material (much less than 1 per cent of total incorporated counts) was observed to migrate with fatty acids. A graph demonstrating incorporation of choline~4C into lipids of rat calvaria is shown in Fig. 1. The radioactivity of lecithin, lyso-lecithin and sphingomyelin increased throughout the entire 3-hr incubation period. Virtually all radioactivity was found in lecithin. In this experiment, lyso-lecithin contained less than 2 per cent and sphingomyelin contained less than 1 per cent of total incorporated counts. In Incorporation

Into Lipid= of R o t

105 .

Calvaria

of

Rat Calvario

y

Phosphatidyl Ethanolamine

t,i I--

~ 103-

i0 z

of 14C-Choline

of 14C-Ethonolamine

Incorporation

io

io

i

,o

i

,20

,;o

TIME (minutes) iO s -:

Fig. 2. Incorporation of ethanolamine-14C into lipids of rat calvaria over a 3-hr incubation period. Calvaria from the 30-, 60-, 90- and 180-min samples contained 41.1, 35.8, 40.2 and 35.0 gamma phospholipid phosphorus respectively. The counts for each time interval were compared on the basis of phospholipid phosphorus, according to the legend of Fig. 1. The 30-min values represent true incorporated counts.

10 4 Z

i w Iz o o

tOa

~

!0z io

~

h

i

n ehn

i

i

¢"

i

!

60

90

120

150

180

T I M E (minutes)

Fig. 1. Incorporation of choline-X4C into lipids of rat calvaria over a 3-hr incubation period. Calvaria from the 30-, 60-, 90- and 180-min samples contained 29.9, 32"0, 30.5 and 30-6 gamma phospholipid phosphorus respectively. The counts for each time interval were compared on the basis of phospholipid phosphorus. The 60-min values represent true counts/rain incorporated into the different lipid classes. Values for the other time intervals were obtained by multiplying true counts/rain by a normalizing factor. As an example, 32.0 gamma P/29.9 gamma P = 1-07 which is the normalizing factor used to calculate the 30-rain values.

some experiments, phosphatidyl ethanolamine was found to become slightly labeled. Ethanolamine-14C. In experiments employing ethanolamine-a4C, all radioactivity remained at the origin of neutral lipid chromatograms. During phospholipid chromatography, greater than 90 per cent of incorporated counts migrated with phosphatidyl ethanolamine. No radioactivity was observed to migrate with lecithin. Some minor and unidentified radioactive materials were occasionally observed to migrate near phosphatidyl ethanolamine. Their lipid nature was suggested by the inability to remove them from chromatograms during repeated water washes. Snake venom enzymes had no effect on these unknown lipids and the results of sodium methoxide treatment were inconclusive. Figure 2 shows the incorporation of ethanolamine14C into lipids of rat calvaria over a 3-hr incubation period. As can be seen, total incorporated counts increased with time. Lyso-phosphatidyl ethanolamine did not exceed 6 per cent of total incorporated counts at any time interval and the most frequently encountered unknown component never exceeded 1 per cent.

Incorporation of radioactive bases into calvaria of new-born rat

(Methyl-l~C) methionine. The pathway for lecithin formation through progressive methylation of phosphatidyl ethanolamine by methionine has been well documented for liver. In the present study, however, the methyl-14C group of methionine was not incorporated into lipid by bone. While some radioactivity was observed at the origin of neutral lipid chromatograms, the radioactivity of cholinecontaining phospholipids was only slightly above that of background and it is doubtful whether origin radioactivity represented true lipid synthesis. To test the suitability of reagents and (methylx4C) methionine for lipid synthesis, a comparison study was conducted with new-born rat liver slices. The methyl group of methionine was readily incorporated into lipid with 84-4 per cent of incorporated counts being present in lecithin. Lyso-lecithin contained 7.6 per cent of total incorporated counts and other radioactive lipids were present. One of these had the chromatographic characteristics of either mono-methyl or di-methyl phosphatidyl ethanolamine. lnositol-3H. Inositol-labeled lipids were located on chromatograms by Rhodamine 6G staining since the low energy of tritium precluded autoradiographic procedures. All radioactivity remained at the origin of neutral lipid chromatograms. During phospholipid chromatography, virtually all radioactivity migrated to an area which in previous studies had been designated as phosphatidyl inositol. Table 1 shows the incorporation of inositol-SH into phosphatidyl inositol throughout a 4-hr incubation period. Table 1. Incorporation of inositol-SH into phosphatidyl inositol by rat calvaria Time (min)

Counts/min

30 60 120 240

425 1645 3221 6977

347

Table 2. Incubation of choline-UC labeled calvaria with or without 0.01 M choline Lipid

No choline counts/rain* S.D.

Lecithin Lysoleeithin and sphingomyelin Phosphatidyl ethanolamine

88,519 6856 1170

0.01 M choline counts/rain* S.D.

14,438 1490

72,843 4255

138

1063

5880 969 32

Six groups of calvaria were incubated with (14C) choline, washed and incubated with or without 0.01 M non-radioactive choline. Experimental calvaria contained less radioactivity than the control calvaria, although the results were not significant (0.5
Lipid

No ethanolamine counts/min* S.D.

Phosphatidyl 72,904 ethanolamine Lyso-phosphatidyl 39,222 ethanolamine

0"01 M ethanolamine countslrnin* S.D.

2907

45,904

2614

400

26,427

1312

Incubation of labeled calvaria in non-radioactive bases

Six groups of calvaria were incubated with (1%-) ethanolamine, washed and incubated with or without 0.01 M non-radioactive ethanolamine. Experimental calvaria had less radioactivity (P<0.01). Control lipid extracts contained 37.0, 32.0 and 34.1 gamma phospholipid phosphorus. The 0.01 M ethanolamine-incubated samples contained 36.6, 35.6 and 37.0 gamma phospholipid phosphorus. Values have been normalized for comparison purposes according to the legend of Fig. 1. * Mean counts/rain of three samples.

Choline-14C. Three experiments were conducted during which choline-14C labeled calvaria were incubated for an additional 2 hr in buffer with or without 0.01 M non-radioactive choline. The results of one experiment are given in Table 2. Greater than 90 per cent of incorporated counts were found in lecithin. While lower lipid radioactivity was observed after incubation in 0-01 M non-radioactive choline, the differences were not significant (0.5
ethanolamine. As can be seen, radioactivity of bones incubated in media supplemented with 0.01 M ethanolamine during the post-label period was significantly less than that of control bones ( P < 0.01). Incubation of ethanolamine-a4C labeled calvaria for 2 hr with or without 0.01 M serine also indicated differences between the control and experimental groups. As shown in Table 4, 0-01 M serine in the

Calvaria for the 30-, 60-, 120- and 240-min samples contained 41.8, 35.2, 26-4 and 30.1 gamma phospholipid phosphorus respectively. The counts for each time interval were compared on the basis of phospholipid phosphorus. See legend of Fig. 1 for determination of normalization factor.

THOMASR. DIRKsEN

348

Table 4. Incubation of ethanolamine-lac labeled calvaria with or without 0'01 M serine Lipid

No serine counts/min* S.D.

Phosphatidyl 75,682 ethanolamine Lyso-phosphatidyl 28,795 ethanolamine

0-01 M serine counts/min* S.D.

1151

60,227

2775

1008

24,494

1033

Six groups of calvaria were incubated with ethanolamine-~4C, washed and incubated with or without 0"01 M non-radioactive serine. Experimental calvaria had less radioactivity (P < 0"01). Control lipid extracts contained 28.7, 25"9 and 27'1 gamma phospholipid phosphorus. The 0"01 M serine-incubated samples contained 30.8, 29.2 and 27.1 gamma phospholipid phosphorus. Values have been normalized for comparison purposes according to the legend of Fig. 1. * Mean counts/min of three samples. media reduced experimental counts to only 90 per cent of control values ( P < 0.01). DISCUSSION Until recently little information has been available regarding lipid synthesis by bone, particularly under hi vitro conditions. It is chiefly due to the lack of information that bone lipid synthesis has been presumed to follow pathways similar to those described for other tissues. The studies reported here, as well as in previous work, have demonstrated the ability of bone to synthesize lipids from a variety of radioactive precursors and their analysis allows one to speculate on the synthetic mechanism. There are at least four pathways to consider for incorporation of radioactive precursors into lecithin. They are: (1) acylation of pre-existing lyso-lecithin (Erbland et al., 1965); (2) transfer of methyl groups from methionine to pre-formed phosphatidyl ethanolamine (Wilson et al., 1960); (3) reaction of CDPcholine by base exchange (Dils et al., 1961; Treble et al., 1970; Bjerve, 1973). While the present studies do not lend themselves to assess importance of the first mechanism, lyso-lecithin was never observed to become heavily labeled in these experiments. The second pathway, that of phosphatidyl ethanolamine methylation by S-adenosyl-methionine can also be ignored in newborn calvaria since lecithin labeling was non-existent in experiments employing either ethanolamine-~4C or (methyl-14C) methionine. By comparison, new-born liver was quite active in incorporation of methyl-14C groups into lipids. Previous studies (Dirksen et al., 1970a), employing radioactive glucose and glycerol, acetate or phosphate have shown that bone rapidly incorporates these labeled substrates into the glycerol moiety, the fatty acid moiety or the phosphate moiety of lecithin. The present work with choline-~4C confirms the previous observations and de novo synthesis is

certainly implied. Since CTP has been shown to stimulate glycerol-14C incorporation into lecithin (Dirksen et al., 1970a), it is suggested that the major pathway for lecithin synthesis within bone involves the reaction of CDP-choline with D-1,2,diglyceride (Kennedy & Weiss, 1956). A factor which must also be considered is the exchange of lecithin-bound choline with free choline. While this mechanism might operate within bone, it seems of minor importance in view of the results obtained where choline-~4C labeled bones were incubated with or without non-radioactive choline. The best defined pathway for phsophatidyl ethanolamine synthesis is that involving CDPethanolamine and diglyceride (Kennedy & Weiss, 1956). Previous experiments with bone and radioactive acetate, glucose, glycerol or phosphate showed only a small percentage of total incorporated counts in this lipid which probably represents de novo synthesis (Dirksen et al., 1970a, b). With both serine-3-14C and ethanolamine-14C, however, phosphatidyl ethanolamine became heavily labeled. It is suggested that much of this intense labeling was due to base exchange with pre-existing phosphatidyl ethanolamine, or possibly, some other glycerol phospholipid. The significant decrease in radioactivity observed when ethanolamine-~4C labeled bones were incubated in non-radioactive 0.01 M ethanolamine supports the operation of base exchange in addition to the possible CDP-pathway. The incorporation of inositol-aH by rat calvaria into a single lipid aids in more positive identification of phosphatidyl inositol on chromatograms. Previous identification was made on the basis of chromatographic mobility and Rhodamine staining since no specific chemical tests have been developed for this lipid. SUMMARY

1. Calvaria from new-born R. norvegicus were incubated with various radioactive substrates to study lipid biosynthesis. 2. Lecithin was the most heavily labeled lipid synthesized by calvaria from choline-14C. Radioactive lyso-lecithin and sphingomyelin were also synthesized as was phosphatidyl ethanolamine on occasion. 3. Phosphatidyl ethanolamine and lyso-phosphatidyl ethanolamine were synthesized from ethanolamineJ4C, but lecithin was not labeled by this substrate. 4. Bone did not utilize (methyl-X~C) methionine for lipid synthesis although new-bom rat liver slices synthesized lecithin, lyso-lecithin and several lipids including a substance which had the chromatographic mobility of either mono-methyl or dimethyl phosphatidyl ethanolamine. 5. Inositol-3H was incorporated into only a single lipid, phosphatidyl inositol.

Incorporation of radioactive bases into calvaria of new-born rat Acknowledgements--This investigation was supported by Public Health Research Grant AM-15800-01, National Institute of Arthritis and Metabolic Diseases. The author would like to acknowledge the technical assistance of Miss Anne Cannady and Mrs. Nancy Smith and the secretarial assistance of Mrs. Marilyn Hutto. REFERENCES

BJERVE K. S. (1973) The phospholipid substrates in the Ca~+-stimulated incorporation of nitrogen bases into microsomal phospholipids. Biochim. biophys. Acta 306, 396-402. DtLs R. R. & HOBSCrmRG. (1961) The effect of calcium ions on the incorporation of labeled choline into rat liver microsomes. Biochim. biophys. Acta 46, 505-513. DmKSEN, T. R. (1969) The in vitro incorporation of acetate-l*C into the lipids of new born rat calvaria. Archs Biochem. Biophys. 134, 603-709. DmKSENT. R., M ~ G. V. & PECKW. A. (1970a) The in vitro incorporation of (14C) glycerol and (14C) glucose into lipids of bone and bone cell cultures. Biochim. biophys. Acta 202, 67-79. DmKSENT. R., MARrNETrlG. V. & PECKW. A. (1970b) The in vitro incorporation of (a~p) orthophosphate and (t4C) serine into lipids of bone and bone cell cultures. Biochim. biophys. Acta 202, 80--90. ERBLANDJ. F. & MAmNETrIG. V. (1965) The enzymatic acylation and hydrolysis of lysolecithin. Biochim. biophys. Acta 106, 128-138. FOLCH J., LEES M. & SLOAN~ STANLEYG. H. (1957) A simple method for the isolation and purification of total lipids from animal tissues. J. bioL Chem. 226, 497-509.

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GIBSON K. D., WILSONJ. D. & UDENFRIENDS. (1961) The enzymatic conversion of phospholipid ethanolamine to phospholipid choline in rat liver. J. bioL Chem. 236, 673-679. KENNEDY S. P. & WEISS S. B. (1956) The function of cytidine coenzymes in the biosynthesis of phospholipids. J. bioL Chem. 222, 193-214. MAmNETTIG. V. (1962a) Chromatographic separation, identification and analysis of phosphatides. J. Lipid Res. 3, 1-80. MARINETrI G. V. (1962b) Hydrolysis of lecithin with sodium methoxide. Biochemistry 1, 350-353. MARINETTIG. V., ERBLANDJ. F. & BROSSARDM. (1964) Biosynthesis of phosphelipids and glycerides. In Metabolism and Physiological Significance of Lipids (Edited by DAWSONR. M. C. & RHODESD. N.), pp. 71-93. Wiley, London. MARINETEIG. V., ERBLANDJ. F., TEMPLEK. & STOTZE. (1960) Hydrolysis of lecithins by venom phospholipase A--I. Structure of the enzymically formed lysolecithins. Biochim. biophys. Acta 38, 524-534. TREBLED. H., FRUMKINS., BALINTJ. A. & BEELERn . A. (1970) The entry of choline into lecithin, in vivo, by base exchange. Biochim. biophys. Acta 202, 163-171. WILSON J. D., GIBSON K. D. & UDENFIELDS. (1960) Studies on the precursors of the methyl groups of choline in rat liver. J. biol. Chem. 235, 3213-3217. WUTHIERR. E. (1966) Two dimensional chromatography on silica gel-loaded paper for microanalysis of polar lipids. J. Lipid Res., 7, 544--550. Key Word Index--Lipid biosynthesis; choline-14C; ethanolamine-X4C;(methyl-l~C) methionine; inositol-SH; calcification; bone; Rattus norvegicus.