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J Clin Pathol: first published as 10.1136/jcp.s1-5.1.11 on 1 January 1973. Downloaded from

J. clin. Path., 26, suppl. (Ass. Clin. Path.), 5, 11-16

Phospholipids and their metabolism

D. GOMPERTZ From the Departments of Medicine and Chemical Pathology, Royal Postgraduate Medical School, London

Phospholipids are characterized by the presence of non-polar hydrophobic side chains and polar CH2 OH hydrophilic head groups. These chemical groupings 1C2 make them particularly suitable compounds to CHOH serve as major constituents for biological interfaces. The structure of hydrophobic paraffin chains varies IH2 OH from to phospholipid and greatly influences their physicochemical behaviour. There is now a vast literature concerning the chemistry of DIGLYCERI DE phospholipids in relation to biological membranes 9 (see van Deenen, 1971). However, while studies of CH2OC -AMAMANW W CH3 artificial phospholipid membrane systems have been I .9 pursued with vigour, the role of phospholipids in CHOC WVMMMMMMAAA CH3 other systems, more related to clinical problems, has also attracted attention. For example, the CH2OH copyright. solubilizing power of bile for cholesterol depends on the combined phospholipid and bile salt com- PHOSPHATIDIC ACID position of the bile, and lithogenic bile is charac- terized by an altered ratio of cholesterol, phospho- CH20 WWAAAAMAMAAAW CH3 , and bile salts; this is discussed by Dr Dowling 2 3 later in this Symposium. The separate pathways for CH O C WAMIMNAW MM CH3 the synthesis of biliary and liver structural http://jcp.bmj.com/ lecithins have been studied in detail. Phospholipids CH2-O-P-01 9 are also involved in a later stage of fat absorption, ie, the assembly of chylomicrons during the exit of dietary triglyceride from the enterocyte. The various Fig. 1 Structural relationships ofglycerol, , classes ofplasma lipoproteins all carry phospholipids andphosphatidic acid. and are characterized by varying proportions of phospholipids, cholesterol, and triglycerides; this too is discussed elsewhere in this Symposium (Dr B. phospholipids. In fig. 1, the structural relation- on September 30, 2021 by guest. Protected Lewis). ships between glycerol, diglyceride, and phos- An important new aspect of phospholipid metab- phatidic acid are shown. Both diglyceride and olism is the production of dipalmitoyl in phosphatidic acid are important intermediates in the lung as an essential constituent of surfactant. phospholipid . Phosphatidic acid is Dipalmitoyl lecithin is synthesized from the 35th chemically the parent of the glycerophosphatides, week of gestation onwards. Babies delivered before and the relationship between this compound and this date have inadequate surfactant production lecithin is shown in figure 2. Here is esterified and a high proportion ofthem develop the respiratory via its hydroxyl group onto phosphatidic acid to distress syndrome. The measurement of the lecithin- give phosphatidyl choline. Some of the major ratio in amniotic fluid gives an phosphatides are illustrated in figure 3. Phosphatidyl indication ofthe maturity of the foetus and is proving choline and phosphatidyl ethanolamine are related useful in deciding when to induce delivery (Gluck, biosynthetically and are referred to as the neutral 1971). glycerophosphatides; the other glycerophosphatides The glycerides are both structurally and bio- are acidic, having a net excess of negatively charged synthetically the parent compounds of the glycero- groups. 11 J Clin Pathol: first published as 10.1136/jcp.s1-5.1.11 on 1 January 1973. Downloaded from

12 D. Gompertz

PHOSPHATIDIC ACID LECITHIN

9 0 1 CH2O C AMMMMMNWM N SATURATED 11 CH3 CH2 0 C MWMAAM CH3 19 0~~~~~~~ 2CHOC 3 UNSATURATED 11 WIMNWANVWCH CH C 0 VVWW/MMAMM CH3 19 + 3 CH2O-P,-O-CH2CH2N ( CH3)3 CH2 O-P-O-

8- 1S SATURATED 2 - UNSATURATED LECITHIN 0 16:0 -48% 18:1*12% 18:0= 47% 18:2 -35% CH2 0 C CMMMMMMMM.WCH3 18:1 5%o 20:4=47%

CHOCH 3WAMMMMMMMWCH3 Fig. 4 A typical distribution offatty acids between the O 1- and 2-positions oflecithin. By convention fatty acids 11 + 0PO are represented by the number ofcarbon atomsfollowed CH2 CH2 CH2N (CH3)3 by a colon and then the number ofdouble bonds. Thus O- 16:0 = palmitic acid; 18:0 = stearic acid; 18:1 = oleic acid; 18:2 = linoleic acid; 20:4 = . CHOLI NE Fig. 2 Structure ofphosphatidic acid and lecithin. Lecithin appears to be quantitatively the mostcopyright. important glycerophosphatide both in membrane systems and in lipid-transporting mechanisms, and much of the biosynthetic work has been concerned with its synthesis. This work has been reviewed in detail several times recently (see bibliography) and only an outline of the major findings will be given

here. The distribution of fatty acids between the 1- http://jcp.bmj.com/ 0 and 2-positions of the glycerol backbone of rat liver

CH lecithins is shown in figure 4. The data for rat liver CHOCCH2110 CH2 CH2 CH2 CH2 CH3 lecithins are similar to those from other tissues and to data from other mammalian species that have been

HOCC2 CH2CH2 CH2 CH2 investigated. There are two important characteristics CH2--- --CH3 of the esterification pattern that have CH20-PF-O PHOSPHATIDYL concerned investigators in the field: (1) the tendency for thefattyacidsinthe 1-position tobepredominantly on September 30, 2021 by guest. Protected saturated, and for those in the 2-position to be and the much HO N CHOLINE unsaturated or polyunsaturated, (2) CH2 CH2 (CH3 )3 higher proportion of polyunsaturated longer chain fatty acids, eg, arachidonic acid, 20:4, in the 2- HO CH2 CH2 N H3 ETHANOLAMINE position of lecithin (fig. 4) than in or triglycerides. HO CH2 CH N H3 SERINE Investigation of the positional distribution of the 2COO-3 fatty acids in various has drawn attention to OH OH the range of molecular species of each individual HO I NOS ITOL lipid present in each tissue. A representative series of the molecular species of rat liver lecithins is shown OH in figure 5. Each of these species has its individual CHOH. CH OH HOH- CH22. 2 GLYCEROL turnover rate and their proportions vary from tissue to tissue. The mechanisms by which this specificity Fig. 3 Some important mammalian glycerophosphatides. of esterification is established are best discussed in J Clin Pathol: first published as 10.1136/jcp.s1-5.1.11 on 1 January 1973. Downloaded from

Phospholipids and their metabolism 13

16:0 16:0 18:0 16:0 16:0 18:1 18:1 18:2 Fig. 5 Some molecular species of rat liver lecithins. PC -PC PC -PC TRACE 14% 5% 10%

18:0 18:1 16:0 18:0 18:2 18:2 20:4 20:4

-PC -PC -PC -PC 8% 4% 17%h 31%

FATTY ACIDS + CoA + ATP - FATTY ACYL CoA

0 Fig. 6 The first two reactions fH2 OH 11 in the synthesis of copyright. 2H20 C CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH3 CHOH by the + 2 FATTY ACYL CoA --- CHO C CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH3 Kennedy pathway. l o CH -O-P-O- 2 CH20-P-0- A 0 0- PHOSPHATIDIC ACID http://jcp.bmj.com/

G±P

PHOSPHATIDIC ACID

Fig. 7 The major pathways on September 30, 2021 by guest. Protected ofglycerophospholipid biosynthesis. PE, phosphatidyl ethanolamine; PS, phosphatidyl serine; PI, phosphatidyl ; PG, DIGLYCERI DE CDP-DIGLYCERI DE phosphatidyl glycerol; and CDP CDP CDP, cytidine diphosphate. CHOL I NE ETHANOL > ,SERINE -INOSITOL GLYCEROL-P

[LECIIN PwE [Hs p, P[G1

OCH2CH2N )3 22(CH33 3 ~2.2 3 -OCH2C1C H NH3N3 S ~~~COO~

3xMe +CO2 J Clin Pathol: first published as 10.1136/jcp.s1-5.1.11 on 1 January 1973. Downloaded from

14, D. Gompertz relation to the known pathways for glycerophos- phatide biosynthesis. The first reaction leading to synthesis of PHOSPHOLI PASE 2 PHOS PHOLI PASE phospholipid molecules de novo is the acylation AI 31. ~~~~~A2 of glycerol 3-phosphate (fig. 6), giving rise to 3 PC phosphatidic acid. After phosphatidic acid is formed, ftA there are separate pathways for the synthesis of : . neutral and acidic glycerophosphatides. The neutral glycerophosphatides, phosphatidyl choline and 1 OH ;, 1 phosphatidyl ethanolamine, are synthesized from 2 ll 2 -OH diglycerides formed by the action of phosphatidate phosphatase (EC 3.1.3.4) on phosphatidic acid 3 -PC i 9 3 L PC (fig. 7). However, the acidic glycerophosphatides are synthesized from cytidine diphosphate-digly- ceride (CDP-diglyceride); this intermediate, half SATURATED lipid, halfnucleotide, can be regarded as a nucleotide- 1 SATURAT OLYUNSATURATED activated diglyceride. Thus synthesis of the neutral 2~iATURATED2PUNSATURATED ACYL phosphatides is accomplished by activating the ACYL CoA bases, choline and ethanolamine, with cytidine CoA 3- PC triphosphate, while the acidic phosphatides are synthesized from an activated diglyceride without Fig. 8 The Lands deacylation-reacylation cycle. the further activation of the polar head group (fig. .7). The pathway of lecithin biosynthesis from glycerol 3-phosphate via diglyceride and CDP- unsaturated fatty acids. Van Deenen (1971) and choline was the first pathway of phospholipid bio- his colleagues then demonstrated the presence synthesis to be established in detail and is referred to of tissue (A1 and A2), and this com-copyright. as the 'Kennedy pathway'. pleted the Lands/van Deenen cycle. This cycle will Two other reactions are indicated in figure 7. The obviously take a lecithin molecule synthesized de first is the tri-methylation of the ethanolamine novo by the Kennedy pathway and remould it to residue of phosphatidyl ethanolamine with S-aden- form a lecithin with a 1-saturated, 2-unsaturated osyl methionine to give phosphatidyl choline. The distribution. other is the decarboxylation of phosphatidyl serine This mechanism of deacylation and reacylation

to give phosphatidyl ethanolamine. The former of was considered for some time to account entirely http://jcp.bmj.com/ these two reactions (the methylation pathway) for the positional specificity of fatty acid esteri- appears to be quantitatively important in several fication in phosphatides. Subsequently the speci- mammalian tissues. ficities of other steps in phospholipid biosynthesis However, this outline of the biosynthetic pathways were investigated in more detail (fig. 9), and one leading to the glycerophosphatides gives no in- of the new pieces of information that had to be dication of the mechanisms involved in establishing explained was that phosphatidic acid and diglyceride the 1-saturated, 2-unsaturated distribution of fatty also have a 1-saturated, 2-unsaturated distribution. acid esterification in lecithin and other phosphatides. The other important observation was that there is a on September 30, 2021 by guest. Protected In the 1950s, Lands noticed that the fatty acids of much higher proportion of arachidonic acid (20:4) lecithin turn over faster in relation to the glycerol in lecithin than in di- and triglycerides and phos- backbone than the fatty acids of triglycerides. Lands phatidic acid (fig. 9). postulated that there were tissue enzymes that could This extra arachidonic acid in lecithin could be remove fatty acids independently from the 1- and 2- introduced by three separate mechanisms: (1) positions of lecithin, and other enzymes that could selection of arachidonyl diglycerides specifically for re-esterify new ones back in their place. This was lecithin biosynthesis at the cholinephosphotrans- soon demonstrated experimentally (fig. 8). Lands ferase (EC 2.7.8.2) step (ie, CDP-choline + digly- demonstrated that the two isomeric lysolecithins ceride); (2) synthesis of arachidonyl lecithins by could be re-acylated by enzyme systems present in methylation of arachidonyl-phosphatidyl ethano- the microsomal fraction of the cell. The enzyme lamines; and (3) introduction of arachidonic acid system acylating a free hydroxyl at the 1-position by the Lands deacylation-reacylation cycle. Experi- was shown to be specific for saturated acyl CoAs, mental work has shown that the cholinephospho- while the enzyme acylating a free hydroxyl at the transferase step does not select arachidonyl di- 2-position was specific for unsaturated and poly- glycerides specifically, and now the general weight J Clin Pathol: first published as 10.1136/jcp.s1-5.1.11 on 1 January 1973. Downloaded from

Phospholipids and their metabolism 15

S GLYCEROL-3-P _PHOSPHATIDIC ACID (9. 20:4)

DIGLYCERI DE Fig. 9 Specificity of TRIGLYCERIDE / N esterification in the synthesis (Low Content / N of lecithin. S = reactions shown to have specificity in relation to fatty acid composition. LEC ITH I N 1 , Methylation PHOS PHATI DYL /24% 20:4 5 ETHANOLAMINE20420:41(High Content

Lysolecithin FATTY ACIDS 1 or 2 of evidence favours the third explanation, ie, that it another pathway not mentioned so far, a lecithin- is the deacylation-reacylation cycle that is respon- free choline exchange. It was originally thought sible for the high proportion of arachidonic acid in that, although these parts of the lecithin molecule lecithin. could originate in several different ways, the glycerol The pathways that have been discussed so far backbone was always derived from glycerol 3- indicate that there are three mechanisms for intro- phosphate. Recent work has shown that there are ducing fatty acids into lecithin: (1) the acylation of two other possible sources, namely, the triose phos- copyright. glycerol 3-P; (2) methylation of phosphatidyl phates of the glycolytic pathway (fig. 10). Both ethanolamine; and (3) fatty acid exchange reactions. dihydroxyacetone phosphate and glyceraldehyde 3- There are also three mechanisms for introducing a phosphate can be acylated first and reduced after- choline residue on to the glycerol backbone: (1) wards. The reduction products are isomeric lyso- cholinephosphotransferase, (2) synthesis ofcholineby phosphatidic acids. The product from dihydroxy- methylation of , and (3) acetone phosphate contains a saturated fatty acid http://jcp.bmj.com/

DHAP GA3P ClH2OH CHO C=O CHOH Fig. 10 Acylation of triose phosphates with their CH20 O CH200 subsequent reduction to form lyso-phosphatidic acids. on September 30, 2021 by guest. Protected FA CoA I rAFA PnO CH20 C WWMMMAMAMMWW CH3 CHO C=O CHOC VAMAMMMMWCH3 CH20 ® CH20O 2H C2H

M1 SATURATED CH20C VIW-WMMW)WAWWWCH3 CH20H UNSATURATED SHOH ClMHOC&AAMVWw CH3 CH20( CH20( J Clin Pathol: first published as 10.1136/jcp.s1-5.1.11 on 1 January 1973. Downloaded from

16 D. Gompertz demonstrates the complexity involved in the bio- Acylation of G3-P Acylation of DHAP synthesis and turnover of just one phospholipid. It is LANDS enzyme important to realize that, although these pathways from PE have in part been worked out using isotope data, CH20|C W MAMWWM CH3 their complexity makes it difficult to establish the 3 quantitative importance of each pathway using iso- CHO c WWMMMAPWAW C 3 Acylation of G-3P | O Acylation of GA3P topic methods alone. These separate pathways C1Ho-P l O CH N (CH LANDS enzyme ensure that lecithin molecules can be made, and 2 2 ~~~33 from PE remodelled even after synthesis, in response to Glycerol-3-P CDP choline different metabolic requirements. Increased syn- DHAP methylation of PE GA3P choline exchange thesis de novo might be required for an increase in lipid-transporting activity, while remodelling might Fig. 11 Various pathways giving rise to different be a consequence of or a response to a change in functional groups of the lecithin molecule. membrane permeability. G3-P, glycerol 3-phosphate; DHAP, dihydroxyacetons Full details of the experimental studies giving rise phosphate; PE, phosphatidyl ethanolamine; GA3P, to this outline of phospholipid metabolism and the glyceraldehyde 3-phosphate, CDP, cytidine diphosphate. important contributions of the major groups working in this field are well described in the reviews listed below. and that from glyceraldehyde 3-phosphate contains an unsaturated fatty acid. These lysophosphatidic Reviews acids can be converted to phosphatidic acid in the van Deenen, L. L. M. (1971). Chemistry of phospholipids in relation to biological membranes. Pure appl. Chem., 25, 25-56. same manner as lysolecithins are reacylated in the Gluck, L. (1971). Biochemical development of the lung: clinical Lands cycle. These phosphatidic acids can enter the aspects of surfactant development. R.D.S. and the intrauterine assessment of lung maturity. Clin. Obstet. Gynec., 14, 710-721. pathways of phosphatide and triglyceride synthesis Hill, E. E., and Lands, W. E. M. (1970). Phospholipid metabolism.copyright. (fig. 7). In , edited by S. J. Wakil, ch. VI, pp. 185-277. Academic Press, New York. Figure 11 summarizes the possible sources of the McMurray, W. C., and Magee, W. L. (1972). Phospholipid metab- individual parts of the lecithin molecule, and olism. Ann. Rev. Biochem., 41, 129-160. http://jcp.bmj.com/ on September 30, 2021 by guest. Protected