Phospholipids and their metabolism

Transcription

1 Phospholipids and their metabolism D. GOMPERTZ J. clin. Path., 26, suppl. (Ass. Clin. Path.), 5, -16 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 hydrophilic head groups. These chemical groupings make them particularly suitable compounds to serve as major constituents for biological interfaces. The structure of hydrophobic paraffin chains varies from phospholipid to phospholipid and greatly influences their physicochemical behaviour. There is now a vast literature concerning the chemistry of phospholipids in relation to biological membranes (see van Deenen, 171). However, while studies of artificial phospholipid membrane systems have been pursued with vigour, the role of phospholipids in other systems, more related to clinical problems, has also attracted attention. For example, the solubilizing power of bile for cholesterol depends on the combined phospholipid and bile salt composition of the bile, and lithogenic bile is characterized by an altered ratio of cholesterol, phospholipid, and bile salts; this is discussed by Dr Dowling later in this Symposium. The separate pathways for the synthesis of biliary lecithins and liver structural lecithins have been studied in detail. Phospholipids 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 classes of plasma lipoproteins all carry phospholipids and are characterized by varying proportions of phospholipids, cholesterol, and triglycerides; this too is discussed elsewhere in this Symposium (Dr B. Lewis). An important new aspect of phospholipid metabolism is the production of dipalmitoyl lecithin in the lung as an essential constituent of surfactant. Dipalmitoyl lecithin is synthesized from the 35th week of gestation onwards. Babies delivered before this date have inadequate surfactant production and a high proportion ofthem develop the respiratory distress syndrome. The measurement of the lecithinsphingomyelin ratio in amniotic fluid gives an indication of the maturity of the foetus and is proving useful in deciding when to induce delivery (Gluck, 171). The glycerides are both structurally and biosynthetically the parent compounds of the glycero- GLYCEROL CH2 OH 1C2 CHOH IH2 OH DIGLYCERI DE CH2OC -AMAMANW W CH3 I. CHOC WVMMMMMMAAA CH3 CH2OH CH2 PHOSPHATIDIC ACID WWAAAAMAMAAAW CH3 2 3 CH O C WAMIMNAW MM CH3 1 CH2-O-P- Fig. 1 Structural relationships ofglycerol, diglyceride, and phosphatidic acid. phospholipids. In fig. 1, the structural relationships between glycerol, diglyceride, and phosphatidic acid are shown. Both diglyceride and phosphatidic acid are important intermediates in phospholipid biosynthesis. Phosphatidic acid is chemically the parent of the glycerophosphatides, and the relationship between this compound and lecithin is shown in figure 2. Here choline is esterified via its hydroxyl group onto phosphatidic acid to give phosphatidyl choline. Some of the major phosphatides are illustrated in figure 3. Phosphatidyl choline and phosphatidyl ethanolamine are related biosynthetically and are referred to as the neutral glycerophosphatides; the other glycerophosphatides are acidic, having a net excess of negatively charged groups. J Clin Pathol: first published as 1.36/jcp.s on 1 January 173. Downloaded from on 17 September 218 by guest. Protected by

2 12 Fig. 2 PHOSPHATIDIC ACID CH2 C MWMAAM CH C CH2 ~~~~~~~ CH3 VVW W/MMAMM CH3 O-P-O- 8- LECITHIN CH2 C CMMMMMMMM.WCH 3 CHOC H 3WAMMMMMMMWCH3 O + CH2 PO CH2 CH2N (CH3)3 O- CHOLI NE Structure ofphosphatidic acid and lecithin. CHOCCH2 CH CH2 CH2 CH2 CH2 CH3 HOCC2 CH2CH2 CH2 CH2 CH2--- CH2-PF-O HO CH2 CH2 N (CH3 )3 HO CH2 CH2 N H3 HO CH2 CH N H3 2COO-3 OH OH HO OH HO CH 2 CHOH. CH OH H Fig. 3 PHOSPHATIDYL CHOLINE ETHANOLAMINE SERINE I NOS ITOL GLYCEROL --CH3 Some important mammalian glycerophosphatides. LECITHIN 1 CH2O C AMMMMMNWM N CH3 SATURATED 1 2CHOC D. Gompertz WIMNWANVWCH 3 UNSATURATED CH2O-P,-O-CH2CH2N ( CH3)3 1S SATURATED -48% 18:= 47% 18:1 5%o 2 - UNSATURATED 18:1*12% 18:2-35% 2:4=47% Fig. 4 A typical distribution offatty acids between the 1- and 2-positions of lecithin. By convention fatty acids are represented by the number of carbon atoms followed by a colon and then the number ofdouble bonds. Thus = palmitic acid; 18: = stearic acid; 18:1 = oleic acid; 18:2 = linoleic acid; 2:4 = arachidonic acid. Lecithin appears to be quantitatively the most 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- and 2-positions of the glycerol backbone of rat liver lecithins is shown in figure 4. The data for rat liver lecithins are similar to those from other tissues and to data from other mammalian species that have been investigated. There are two important characteristics of the fatty acid esterification pattern that have concerned investigators in the field: (1) the tendency for thefattyacids in the 1 -position to bepredominantly saturated, and for those in the 2-position to be unsaturated or polyunsaturated, and (2) the much higher proportion of polyunsaturated longer chain fatty acids, eg, arachidonic acid, 2:4, in the 2- position of lecithin (fig. 4) than in diglycerides or triglycerides. Investigation of the positional distribution of the fatty acids in various lipids has drawn attention to the range of molecular species of each individual lipid present in each tissue. A representative series of the molecular species of rat liver lecithins is shown in figure 5. Each of these species has its individual turnover rate and their proportions vary from tissue to tissue. The mechanisms by which this specificity of esterification is established are best discussed in J Clin Pathol: first published as 1.36/jcp.s on 1 January 173. Downloaded from on 17 September 218 by guest. Protected by

3 Phospholipids and their metabolism PC TRACE 18: 18:2 8% 18:1 14% 18:1 18:2 4% FATTY ACIDS + CoA + ATP - FATTY ACYL CoA fh2 OH CHOH CH -O-P-O- 2 18: 18:1 PC 5% 2:4 17%h 18:2 1% 18: 2:4 31% 2H2 C CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH3 + 2 FATTY ACYL CoA --- CHO C CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH3 l o CH 2 -P-- A - PHOSPHATIDIC ACID DIGLYCERI DE CDP CDP CHOL I NE ETHANOL > [LECIIN PwE G±P PHOSPHATIDIC ACID OCH2CH2N (CH3 )3 -OCH H NH ~ C1C N3 S ~~~COO~ 3xMe +CO2 CDP-DIGLYCERI DE,SERI NE -INOSITOL GLYCEROL-P [Hs p, P[G1 Fig. 5 Some molecular species of rat liver lecithins. 13 Fig. 6 The first two reactions in the synthesis of glycerophospholipids by the Kennedy pathway. Fig. 7 The major pathways ofglycerophospholipid biosynthesis. PE, phosphatidyl ethanolamine; PS, phosphatidyl serine; PI, phosphatidyl inositol; PG, phosphatidyl glycerol; and CDP, cytidine diphosphate. J Clin Pathol: first published as 1.36/jcp.s on 1 January 173. Downloaded from on 17 September 218 by guest. Protected by

4 14, relation to the known pathways for glycerophosphatide biosynthesis. The first reaction leading to synthesis of phospholipid molecules de novo is the acylation of glycerol 3-phosphate (fig. 6), giving rise to phosphatidic acid. After phosphatidic acid is formed, there are separate pathways for the synthesis of neutral and acidic glycerophosphatides. The neutral glycerophosphatides, phosphatidyl choline and phosphatidyl ethanolamine, are synthesized from diglycerides formed by the action of phosphatidate phosphatase (EC ) on phosphatidic acid (fig. 7). However, the acidic glycerophosphatides are synthesized from cytidine diphosphate-diglyceride (CDP-diglyceride); this intermediate, half lipid, half nucleotide, can be regarded as a nucleotideactivated diglyceride. Thus synthesis of the neutral phosphatides is accomplished by activating the bases, choline and ethanolamine, with cytidine triphosphate, while the acidic phosphatides are synthesized from an activated diglyceride without the further activation of the polar head group (fig..7). The pathway of lecithin biosynthesis from glycerol 3-phosphate via diglyceride and CDPcholine was the first pathway of phospholipid biosynthesis to be established in detail and is referred to as the 'Kennedy pathway'. Two other reactions are indicated in figure 7. The first is the tri-methylation of the ethanolamine residue of phosphatidyl ethanolamine with S-adenosyl methionine to give phosphatidyl choline. The other is the decarboxylation of phosphatidyl serine to give phosphatidyl ethanolamine. The former of these two reactions (the methylation pathway) appears to be quantitatively important in several mammalian tissues. However, this outline of the biosynthetic pathways leading to the glycerophosphatides gives no indication of the mechanisms involved in establishing the 1-saturated, 2-unsaturated distribution of fatty acid esterification in lecithin and other phosphatides. In the 15s, Lands noticed that the fatty acids of lecithin turn over faster in relation to the glycerol backbone than the fatty acids of triglycerides. Lands postulated that there were tissue enzymes that could remove fatty acids independently from the 1- and 2- positions of lecithin, and other enzymes that could re-esterify new ones back in their place. This was soon demonstrated experimentally (fig. 8). Lands demonstrated that the two isomeric lysolecithins could be re-acylated by enzyme systems present in the microsomal fraction of the cell. The enzyme system acylating a free hydroxyl at the 1-position was shown to be specific for saturated acyl CoAs, while the enzyme acylating a free hydroxyl at the 2-position was specific for unsaturated and poly- PHOSPHOLI PASE AI 1 OH 2 3 SATURAT ACYL CoA Fig. 8 D. Gompertz 331. PC ~~~~~A2 2 PHOS PHOLI PASE fta :. ;, 1 i ll 2 -OH 1 SATURATED 2PUNSATURATED 2~iATURATED 3- PC 3 L PC The Lands deacylation-reacylation cycle. OLYUNSATURATED ACYL CoA unsaturated fatty acids. Van Deenen (171) and his colleagues then demonstrated the presence of tissue phospholipases (A1 and A2), and this completed the Lands/van Deenen cycle. This cycle will obviously take a lecithin molecule synthesized de novo by the Kennedy pathway and remould it to form a lecithin with a 1-saturated, 2-unsaturated distribution. This mechanism of deacylation and reacylation was considered for some time to account entirely for the positional specificity of fatty acid esterification in phosphatides. Subsequently the specificities of other steps in phospholipid biosynthesis were investigated in more detail (fig. ), and one of the new pieces of information that had to be explained was that phosphatidic acid and diglyceride also have a 1-saturated, 2-unsaturated distribution. The other important observation was that there is a much higher proportion of arachidonic acid (2:4) in lecithin than in di- and triglycerides and phosphatidic acid (fig. ). This extra arachidonic acid in lecithin could be introduced by three separate mechanisms: (1) selection of arachidonyl diglycerides specifically for lecithin biosynthesis at the cholinephosphotransferase (EC ) step (ie, CDP-choline + diglyceride); (2) synthesis of arachidonyl lecithins by methylation of arachidonyl-phosphatidyl ethanolamines; and (3) introduction of arachidonic acid by the Lands deacylation-reacylation cycle. Experimental work has shown that the cholinephosphotransferase step does not select arachidonyl diglycerides specifically, and now the general weight J Clin Pathol: first published as 1.36/jcp.s on 1 January 173. Downloaded from on 17 September 218 by guest. Protected by

5 Phospholipids and their metabolism S GLYCEROL-3-P _PHOSPHATIDIC ACID (. 2:4) DIGLYCERI DE TRIGLYCERIDE / N (Low Content / N LEC ITH I N 1, Methylation PHOS PHAT I DYL / 24% 2:4 5 ETHANOLAMINE (High Content 242:41 Lysolecithin 1 or 2 FATTY ACIDS of evidence favours the third explanation, ie, that it is the deacylation-reacylation cycle that is responsible for the high proportion of arachidonic acid in lecithin. The pathways that have been discussed so far indicate that there are three mechanisms for introducing fatty acids into lecithin: (1) the acylation of glycerol 3-P; (2) methylation of phosphatidyl ethanolamine; and (3) fatty acid exchange reactions. There are also three mechanisms for introducing a choline residue on to the glycerol backbone: (1) cholinephosphotransferase, (2) synthesis ofcholineby methylation of phosphatidylethanolamine, and (3) ClH2OH C=O CH2 O FA CoA DHAP CH2 C WWMMMAMAMMWW CH3 C=O CH2 2H M1 SATURATED CH2C VIW-WMMW)WAWWWCH3 SHOH CH2 ( CHO CHOH CH2 I FA Pn CHO ra O CHOC CH2O C2H CH2H Fig. Specificity of esterification in the synthesis of lecithin. S = reactions shown to have specificity in relation to fatty acid composition. another pathway not mentioned so far, a lecithinfree choline exchange. It was originally thought that, although these parts of the lecithin molecule could originate in several different ways, the glycerol backbone was always derived from glycerol 3- phosphate. Recent work has shown that there are two other possible sources, namely, the triose phosphates of the glycolytic pathway (fig. 1). Both dihydroxyacetone phosphate and glyceraldehyde 3- phosphate can be acylated first and reduced afterwards. The reduction products are isomeric lysophosphatidic acids. The product from dihydroxyacetone phosphate contains a saturated fatty acid GA3P VAMAMMMMWCH3 UNSATURATED ClMHOC&AAMVWw CH3 CH2( Fig. 1 Acylation of triose phosphates with their subsequent reduction to form lyso-phosphatidic acids. 1 5 J Clin Pathol: first published as 1.36/jcp.s on 1 January 173. Downloaded from on 17 September 218 by guest. Protected by

6 16 D. Gompertz CH2 C W MAMWWM CH3 3 Acylation of G3-P Acylation of DHAP LANDS enzyme from PE CHO c WWMMMAPWAW C 3 Acylation of G-3P O Acylation of GA3P C1H o-p l O CH N (CH LANDS enzyme 2 2 ~~~33 from PE Glycerol-3-P CDP choline DHAP methylation of PE GA3P choline exchange Fig. Various pathways giving rise to different functional groups of the lecithin molecule. G3-P, glycerol 3-phosphate; DHAP, dihydroxyacetons phosphate; PE, phosphatidyl ethanolamine; GA3P, glyceraldehyde 3-phosphate, CDP, cytidine diphosphate. and that from glyceraldehyde 3-phosphate contains an unsaturated fatty acid. These lysophosphatidic acids can be converted to phosphatidic acid in the same manner as lysolecithins are reacylated in the Lands cycle. These phosphatidic acids can enter the pathways of phosphatide and triglyceride synthesis (fig. 7). Figure summarizes the possible sources of the individual parts of the lecithin molecule, and demonstrates the complexity involved in the biosynthesis and turnover of just one phospholipid. It is important to realize that, although these pathways have in part been worked out using isotope data, their complexity makes it difficult to establish the quantitative importance of each pathway using isotopic methods alone. These separate pathways ensure that lecithin molecules can be made, and remodelled even after synthesis, in response to different metabolic requirements. Increased synthesis de novo might be required for an increase in lipid-transporting activity, while remodelling might be a consequence of or a response to a change in membrane permeability. Full details of the experimental studies giving rise to this outline of phospholipid metabolism and the important contributions of the major groups working in this field are well described in the reviews listed below. Reviews J Clin Pathol: first published as 1.36/jcp.s on 1 January 173. Downloaded from van Deenen, L. L. M. (171). Chemistry of phospholipids in relation to biological membranes. Pure appl. Chem., 25, Gluck, L. (171). Biochemical development of the lung: clinical aspects of surfactant development. R.D.S. and the intrauterine assessment of lung maturity. Clin. Obstet. Gynec., 14, Hill, E. E., and Lands, W. E. M. (17). Phospholipid metabolism. In Lipid Metabolism, edited by S. J. Wakil, ch. VI, pp Academic Press, New York. McMurray, W. C., and Magee, W. L. (172). Phospholipid metabolism. Ann. Rev. Biochem., 41, on 17 September 218 by guest. Protected by