Lipids of cultured hepatoma cells: VIII. Utilization of D-[1-14C] glucose for lipid biosynthesis

Minimal deviation hepatoma 7288C cells (HTC) were incubated in serum-supplemented and serum-free Swim’s 77 medium in the presence of D-[1-¹⁴C] glucose for 1, 2, 4, 8, 12 and 24 hr. Glucose oxidation to CO₂, incorporation into total cell mass, and incorporation into cell and medium lipids were determined. The percentage distribution of total cell lipid radioactivity in individual neutral and polar lipid classes was followed as a function of time. Degradation studies of individual lipid classes were performed to ascertain the percentage of radioactivity in acyl and glycerol moieties. The percentage of D-[1-¹⁴C] glucose oxidized to¹⁴CO₂, incorporated into cell matter and cell lipids was elevated in cells incubated in serum-free medium as opposed to serum-supplemented medium. The percentage distribution of total cell lipid radioactivity into individual neutral lipid classes from both serum-free and serum-supplemented cultures was as follows: sterols > triglycerides > free fatty acids > sterol esters. The percentage distribution of total cell lipid radioactivity into individual polar lipid classes of serum-supplemented cultures was as follows: phosphatidylcholine > phosphatidylinositol > sphingomyelin > phosphatidylethanolamine > phosphatidylserine. The distribution of glucose radiolabel into individual polar lipid classes of serum-free HTC cells was different from their serum-supplemented counterparts: sphingomyelin > phosphatidylcholine > phosphatidylinositol > phosphatidylethanolamine > phosphatidylserine. Glycerol from glyceride classes contained a higher percentage of radioactivity than the acyl moieties, with this percentage significantly elevated in serum-free cultures. The data indicate that, although glucose is a substrate for HTC cell lipids, other precursors present in the culture system also contribute to the lipid constituency of this hepatoma cell line.     

Utilization of l-serine in the in vivo biosynthesis of glycerophospholipids by rat liver

The incorporation of L-serine-U-¹⁴C, L-serine-3-¹⁴C, and D,L-serine-1-¹⁴C into the glycerophospholipids of rat liver in vivo was determined over a period of 3 min to 13 hr following intravenous injection. The radioactivity from these serines was transferred to variable extent into the glycerol, fatty acid, and nitrogenous base parts of all the glycerophospholipids and neutral lipids. The half-lives and turnover rates of phosphatidylserine calculated from the precursor-product specific activity curves obtained with L-serine-U-¹⁴C were 14 min and 0.28 μmol/min/liver, respectively. The half-lives and turnover rates of phosphatidylserine as measured from the decay data of lipid serine from all markers averaged, respectively, 8.2 hr and 0.008 μmol/min/liver. The discrepancy between these turnover rates was attributed to an underestimation of degradation of phosphatidylserine due to its continued biosynthesis and/or an extensive reutilization of L-serine. By monitoring the formation of radioactive lipid ethanolamine, it was found that phosphatidylserine was decarboxylated at one-half the rate of lipid serine biosynthesis. It is suggested that as much as one-half of total phosphatidylserine may be degraded by other mechanisms, such as base exchange with choline, ethanolamine, and serine, as already demonstrated in vitro by other workers. The time course and nature of labeling of phosphatidylcholine was consistent with an extensive conversion of radioactive L-serine to 1-carbon fragments and a rapid methylation of phosphatidylethanolamine to phosphatidylcholine. Presented in part at the AOCS Fall Meeting in Ottawa, September 1972.     

Lipids of cultured hepatoma cells: III. Triglyceride and phosphoglyceride biosynthesis in minimal deviation hepatoma 7288C

Minimal deviation hepatoma 7288 C cells were cultured on media containing 25% serum to the confluent stage. The growth media was replaced with serumfree media containing 1-¹⁴C-palmitate, and incubations were continued for 0.75, 1.5, 3, 6, 12, and 24 hr. The distribution of radioactivity among the major neutral lipids and phosphoglycerides was determined for cells and culture media. Radioactivity in individual fatty acids of cellular triglyceride, phosphatidylcholine, and phosphatidylethanolamine also was determined. After 24 hr, more than 95% of the administered radioactivity was recovered in neutral and phosphoglycerides, indicating that only a small amount of the fatty acid was oxidized. At any time period examined, over 80% of the incorporated radioactivity was found in triglyceride, phosphatidylcholine, and phosphatidylethanolamine. Incorporation of the label into cellular triglyceride and phosphatidylcholine plateaued at 12 hr, whereas incorporation of radioactivity into phosphatidylethanolamine still was increasing at 24 hr. In contrast, during the entire incubation period the relative distribution of¹⁴C among esterified lipid classes in the culture media remained constant. Elongation of palmitic acid to stearic acid and its subsequent desaturation to oleic acid suggests that these cells possess an active elongation and monoenoic desaturation system. Labeled glycerol ether diesters were not detected in the cells or culture media. Positional distribution of the¹⁴C label in the triglyceride and phosphatidylcholine suggests that minimal deviation hepatoma cells do not exhibit diglyceride selectivity in the biosynthesis of these two lipid classes.     

Incorporation of arachidonic acid and eicosapentaenoic acid into phospholipids by polymorphonuclear leukocytes in vitro

The present study demonstrated that the patterns of the incorporation of [1-¹⁴C] arachidonic acid and [1-¹⁴C] eicosapentaenoic acid into individual phospholipids by polymorphonuclear leukocytes were similar. However, human leukocytes exhibited higher activity than guinea pig periotoneal leukocytes in the formation of arachidonoyl- and eicosapentaenoyl-phosphatidic acid. Cells from both origins showed a decrease of label in phosphatidylcholine accompanied by an increase of label in phosphatidylethanolamine after a longer period (30–120 min) of incubation, suggesting that part of the arachidonoyl or eicosapentaenoyl moiety in phosphatidylethanolamine may be derived from that of phosphatidylcholine. The observed difference between human cells and elicited cells in the time-course of the incorporation of both fatty acids into phosphatidylcholine and phosphatidylethanolamine appears to be due to different contents of the diacyl and ether-linked class compositions of these phospholipids in cells from different origins. Both labeled fatty acids were incorporated more rapidly into the diacyl-linked class, but were retained to a greater extent in alkylacyl-phosphatidyl-choline and alkenylacyl-phosphatidylethanolamine. The data suggest that, in addition to alkylacyl-phosphatidylcholine and phosphatidylinositol, alkenylacyl-phosphatidylethanolamine may be an important endogenous source of arachidonic acid and eicosapentaenoic acid in stimulated human leukocytes.     

Synthesis of Phosphatidylcholine Through Phosphatidylethanolamine <Emphasis Type="Italic">N</Emphasis>-Methylation in Tissues of the Mussel <Emphasis Type="Italic">Mytilus galloprovincialis</Emphasis>

We previously demonstrated the importance of upregulation of phosphatidylethanolamine N-methylation pathway in euryhaline fish and crustaceans facing hyperosmotic conditions. In marine molluscs phosphatidylcholine synthesis through N-methylation of phosphatidylethanolamine has not been described until now. In vivo labeling of the mussel Mytilus galloprovincialis with [1-³H]-ethanolamine showed that the digestive gland is the tissue expressing the highest incorporation into lipids. A sustained increase in lipid labeling was observed up to 72 h following label injection with 79–92% of radioactivity concentrated into phosphatidylethanolamine and phosphatidylcholine. A direct correlation (r = 0.47, p < 0.01) between the specific radioactivities of phosphatidylcholine in plasma and the digestive gland was observed. Moreover, the phosphatidylcholine fatty acid compositions of plasma and the digestive gland were similar but differed from those of phosphatidylcholine purified from other tissues. In vitro incubation of tissues with [1-³H]-ethanolamine or L-[3-³H]-serine showed that a significant labeling of the choline moiety of phosphatidylcholine was observed in the digestive gland and hemocytes. Pulse-chase experiments with [1-³H]-ethanolamine also demonstrated that hemocytes are exchanging the newly formed phospholipids with plasma. Finally, phosphatidylethanolamine N-methyltransferase assays demonstrated salinity-dependent activities in the digestive gland and hemocytes. We conclude that in M. galloprovincialis an active phosphatidylcholine synthesis through N-methylation of phosphatidylethanolamine occurs in the digestive gland and hemocytes and that this newly formed phosphatidylcholine is partly exchanged with plasma.     

Phospholipid metabolism in cells in culture

The proportions of the different lecithin fractions have been determined in HeLa and KB tissue culture cells and Ehrlich Ascites tumor. 82.8% of the total phosphatidyl choline phosphorus is found in fraction 3 of HeLa cells. The major phosphatidyl cholines found in KB cells and Ehrlich Ascites tumor are in fractions 3 and 4 and representing 66.6% and 88.7% of the total phosphatidyl choline P, respectively. The incorporation of 1,2-¹⁴C-choline and 1,2-¹⁴C-ethanolamine into the various phosphatidyl choline fractions has been assayed to determine their biosynthesis in Ehrlich Ascites tumor. The incorporation of 1,2-¹⁴C-choline into fractions 3 and 4 is 100 times the 1,2-¹⁴C-ethanolamine. This evidence indicates that the methylation pathway of phosphatidyl choline synthesis is very low in HeLa, KB and Ehrlich Ascites cells. One of 13 papers presented at the symposium “Lipid Metabolism in Cells in Culture,” AOCS Meeting, Houston, May 1971. Part of a thesis submitted to the Graduate School of the University of North Dakota in partial fulfillment of the Degree of Master of Science.     

Influence of dietary cholesterol on the relative synthesis of hepatic glycerides and molecular classes of 1,2-diglycerides and phospholipids in the gerbil in vivo

The influence of dietary cholesterol on the relative rates of synthesis of hepatic lipids in the male Mongolian gerbil,Meriones unguiculatus, was studied. The semi-purified starch-based diet used lard as the dietary fat and was fed with or without a 0.5% (by wt.) cholesterol supplement. Each animal received 300 μCi [2-³H]-glycerol i.p. after 3 or 7 days on the dietary regimens. Relative rates of [2-³H]-glycerol incorporation into the major hepatic glycerides in vivo was not affected significantly by dietary cholesterol (0.5% level), suggesting that alteration in the relative biosynthesis of these lipids could not readily account for the higher triglyceride (TG) to phospholipid (PL) mass ratio in liver with cholesterol feeding. However, there was evidence for an increased formation of 1,2-diglyceride (1,2-DG). The complement of molecular species of hepatic 1,2-DG, phosphatidylcholine (PC), and phosphatidylethanolamine (PE) formed de novo, as measured using isotopic glycerol, was not influenced greatly by dietary cholesterol, although lower mean rates of synthesis of tetraenoic relative to dienoic species of phospholipids were indicated in cholesterol-fed gerbils.     

Studies on phospholipid biosynthesis in hepatocytes from alcoholic rats by using radiolabeled exogenous precursors

We have studied the synthesis of phospholipids in hepatocytes isolated from chronically ethanol-treated rats by using isotopically labelled serine, ethanolamine, and choline as exogenous precursors. Our results demonstrate that ethanol induces specific effects on the biosynthesis of phosphatidyl-ethanolamine and phosphatidylcholinevia CDP-derivatives and also on the synthesis of phosphatidylserinevia the Ca⁺⁺-dependent base-exchange reaction. Thus, the synthesis of phosphatidylethanolamine from [³-H]ethanolamine and the incorporation of [³H]serine into phosphatidylserine were clearly higher in hepatocytes from ethanol-treated rats compared to controls. The synthesis of phosphatidylcholine from [methyl-¹⁴C] choline, on the other hand, decreased markedly, suggesting a specific inhibition of cholinephosphotransferase activity. We have also demonstrated that the phosphatidylcholine levels are markedly decreased in hepatocytes isolated from chronically ethanol-treated rats as a consequence of the lower phosphatidylcholine biosynthesis. The decrease in the incorporation of radioactivity from choline to betaine, which we also found, is interpreted as being the result of a higher use of betaine as methyl donor instead of methionine to maintain the hepaticS-adenosylmethionine levels in chronic alcoholism.     

Fatty acid metabolism of the calanoid copepodParacalanus parvus: 2. Palmitate,stearate, oleate and acetate

The de novo biosynthesis of fatty acids in the wild, calanoid copepodParacalanus parvus was studied. The incubation of labeled acetate proved the de novo biosynthesis of saturated and monounsaturated even fatty acids from 14 to 20 carbons and the 22∶1 acid. Saturated and monounsaturated uneven fatty acids from 15 to 21 carbons were also synthesized. The copepod could not synthesize linoleic and α-linolenic acids. By administration of [1-¹⁴C]palmitate, [1-¹⁴C] stearate and [1-¹⁴C]oleate, it was possible to elucidate the general pattern of the de novo biosynthesis of fatty acids in the wildP. parvus.     

The effect of 3-methylindole on the uptake and incorporation of14C-choline into phospholipids in lung tissue slices

3-Methylindole (3MI) is the causative agent in the development of acute bovine pulmonary edema. Microscopic studies revealed a structural disruption in the lamellar bodies of type II cells, indicating an abnormal metabolism of phospholipid in the lung of 3MI treated animals. In the present study, lung slices from 4 goats were used to investigate the changes in phosphatidylcholine metabolism induced by 3MI. Eighteen slices were cut from each healthy lung and divided into control and 3MI groups. After a 4-hr pretreatment with 3MI (.19 or .57 mM) or carrier, the level of incorporation of¹⁴C-choline into phosphatidylcholine, sphingomyelin and their water soluble intermediates was studied. The uptake of¹⁴C-choline and its incorporation into phosphatidylcholine and sphingomyelin was depressed by 3MI treatment. In the water soluble fractions, the radioactivity increased in free choline and CDP-choline while it decreased in P-choline. This suggests that choline kinase and the P-choline transferases have become relatively more rate limiting and may play a role in the depressed de novo synthesis of phosphatidylcholine induced by 3MI.     

Incorporation of [1-14C] acetate into phospholipids by soybean seedlings

Incorporation of [1-¹⁴C] acetate into lipids by slices of soybean seedlings was studied. The results were as follows: (a) the greatest amount of radioactivity was detected in the phospholipid fraction prepared from the main axis; (b) in the cotyledons, radioactivity was about the same in pigment, phosphatidylethanolamine, and phosphatidylglycerol; (c) phosphatidylcholine was the main phospholipid labeled in the axis; (d) the distribution of radioactive fatty acids in the axis suggested that this tissue has the capacity for both phospholipid synthesis and fatty acid desaturation.     

Transfer of arachidonate from phosphatidylcholine to phosphatidylethanolamine and triacylglycerol in guinea pig alveolar macrophages

Guinea pig alveolar macrophages were labeled by incubation with either arachidonate or linoleate. Arachidonate labeled phosphatidylcholine (PC), phosphatidylethanolamine (PE) and triglycerides (TG) equally well, with each lipid containing about 30% of total cellular radioactivity. In comparison to arachidonate, linoleate was recovered significantly less in PE (7%) and more in TG (47%). To investigate whether redistributions of acyl chains among lipid classes took place, the macrophages were incubated with 1-acyl-2-[1-¹⁴C]arachidonoyl PC or 1-acyl-2-[1-¹⁴C]linoleoyl PC. After harvesting, the cells incubated with 1-acyl-2-[1-¹⁴C]linoleoyl PC contained 86% of the recovered cellular radioactivity in PC, with only small amounts of label being transferred to PE and TG (3 and 6%, respectively). More extensive redistributions were observed with arachidonate-labeled PC. In this case, only 60% of cellular radioactivity was still associated with PC, while 22 and 12%, respectively, had been transferred to PE and TG. Arachidonate transfer from PC to PE was unaffected by an excess of free arachidonate which inhibited this transfer to TG for over 90%, indicating that different mechanisms or arachidonoyl CoA pools were involved in the transfer of arachidonate from PC to PE and TG. Cells prelabeled with 1-acyl-2-[1-¹⁴C]arachidonoyl PC released¹⁴C-label into the medium upon further incubation. This release was slightly stimulated by zymosan and threefold higher in the presence of the Ca²⁺-ionophore A₂₃₁₈₇. Labeling of macrophages with intact phospholipid molecules appears to be a suitable method for studying acyl chain redistribution and release reactions.     

Incorporation of linoleic acid and its conversion to λ-linolenic acid in fungi

The incorporation of [1-¹⁴C]linoleic acid (LA) into lipids ofMortierella ramanniana var.angulispora was studied to determine which lipid classes participated in the δ6-desaturation of [1-¹⁴C]LA. [1-¹⁴C]LA was rapidly taken up into fungal cells and esterified into various lipids. Comparison of the profile of [1-¹⁴C]LA incorporation between fungal cells at the exponential growth phase and the stationary growth phase showed that [1-¹⁴C]LA incorporation into most lipids—except for triacylglycerol (TG) and phosphatidylcholine (PC)—were greatly reduced at the stationary growth phase. Desaturation of [1-¹⁴C]LA into λ-linolenic acid (GLA) readily occurred at the exponential growth phase, but was greatly decreased at the stationary growth phase. Moreover, pulse-chase experiments revealed that the radiolabel incorporated into phosphatidylserine (PS) and PC rapidly turned over, while that in TG and diacylglycerol (DG) accumulated after the 4 hr chase. In addition to the change of the radiolabel in individual lipids, the content of radiolabeled GLA converted from [1-¹⁴C]LA varied with individual lipids. In phospholipids such as PC, phosphatidylethanolamine (PE) and PS, radiolabeled GLA rapidly increased after 1 hr and then decreased after 4 hr. On the other hand, a gradual increase in radiolabeled GLA until 4 hr was observed in TG. These results suggest that LA, which has been esterified into phospholipids such as PC, PE and PS, is readily desaturated to GLA, which is then transferred to TG. These differences in the fate of GLA derived from LA between phospholipids and neutral lipids may be reflected in the GLA content in the individual lipids.     

Lipid synthesis by rat lung in vitro

Oxidation and lipogenesis in isolated rat lung tissue were studied in vitro. The minced tissue was incubated in a Krebs-Ringer bicarbonate buffer (pH 7.4) with 1-¹⁴C-acetate, 2-¹⁴C-pyruvate, U-¹⁴C-D-glucose, 1,5-¹⁴C-citrate, 1-¹⁴C-laurate, 1-¹⁴C-palmitate, 1-¹⁴C-stearate, 1-¹⁴C-oleate, 1-¹⁴C-linoleate. The lung tissue readily oxidized all of these substrates to¹⁴CO₂ and incorporated them into¹⁴C-lipids with the exception of 1,5-¹⁴C-citrate, for which there was no significant incorporation into¹⁴C-lipids. Most of the lipid¹⁴C was recovered in phospholipids, more specifically phosphatidyl choline. Twenty-eight per cent of glucose carbons was incorporated into the fatty acid moiety of phospholipids, while more than 90% of the carbons of other substrates was found in phospholipid fatty acids. The main fatty acid of the phospholipid fraction synthesized from acetate, pyruvate or glucose was palmitic acid. The oxidation of fatty acids was apparently influenced both by the carbon chain length and number of double bonds. Accumulation of¹⁴C-fatty acids in the tissue was observed when fatty acids were used as substrates; this finding suggests that the rate limiting step was not in the uptake of fatty acids. Chemical degradation of¹⁴C-myristic and palmitic acids obtained by hydrolysis of phospholipids biosynthesized from 1-¹⁴C-laurate indicated that the phospholipid fatty acids were synthesized via the de novo synthesis pathway. Presented at the AOCS-ISF World Congress, Chicago, September 1970.     

Modulation of fatty acid incorporation and desaturation by trifluoperazine in fungi

The effects of trifluoperazine (TFP) on [1-¹⁴C]fatty acid incorporation into the lipids ofMortierella ramanniana var.angulispora were studied. TFP decreased [1-¹⁴C]-fatty acid incorporation into phosphatidylcholine, phosphatidylethanolamine and triacylglycerol, but greatly increased¹⁴C-labeling in phosphatidic acid. These changes in [1-¹⁴C]fatty acid incorporation induced by TFP were accompanied by a decrease in desaturation of some [1-¹⁴C]fatty acids taken up by the fungal cells. When [1-¹⁴C]lioleic acid (LA) was incubated with the fungal cells, total γ-linolenic acid (GLA) formation from incorporated [1-¹⁴C]LA decreased, but the¹⁴C-labeled GLA conent in individual lipid classes was essentially unchanged. This suggests that the site of the TFP effect on GLA formation from [1-¹⁴C]LA taken up from the medium is not the desaturase acting on LA linked to complex lipids. On the other hand, GLA formation from [1-¹⁴C]oleic acid was much less susceptile to TFP, which suggests that in this fungus Δ6 desaturation to GLA has at least two different pathways with different degrees of susceptibility to TFP.     

Mifepristone Treatment Results in Differential Regulation of Glycerolipid Biosynthesis in Baby Hamster Kidney Cells Expressing a Mifepristone-Inducible ABCA1

ATP binding cassette A1 (ABCA1) transports cholesterol, phospholipids and lipophilic molecules to and across cellular membranes. We examined if ABCA1 expression altered cellular de novo glycerolipid biosynthesis in growing Baby hamster kidney (BHK) cells. Mock BHK cells or cells expressing a mifepristone-inducible ABCA1 (ABCA1) were incubated plus or minus mifepristone and then with [³H]serine or [³H]inositol or [³H]ethanolamine or [methyl-³H]choline or [³H]glycerol or [¹⁴C]oleate and radioactivity incorporated into glycerolipids determined. Mifepristone did not affect [1,3-³H]glycerol or [¹⁴C]oleate or [³H]ethanolamine or [methyl-³H]choline uptake in BHK cells. In contrast, [³H]glycerol and [¹⁴C]oleate incorporated into phosphatidylserine (PtdSer) were elevated 2.4-fold (p < 0.05) and 54% (p < 0.05), respectively, upon ABCA1 induction confirming increased PtdSer biosynthesis from these precursors. However, mifepristone inhibited [³H]serine uptake and incorporation into PtdSer indicating that PtdSer synthesis from serine in BHK cells is dependent on serine uptake. Mifepristone stimulated [³H]inositol uptake in mock and ABCA1 cells but not its incorporation into phosphatidylinositol indicating that its synthesis from inositol is independent of inositol uptake in BHK cells. [³H]glycerol and [¹⁴C]oleate incorporated into triacylglycerol were reduced and into diacylglycerol elevated only in mifepristone-induced ABCA1 expressing cells due to a decrease in diacylglycerol acyltransferase-1 (DGAT-1) activity. The presence of trichostatin A, a class I and II histone deacetylase inhibitor, reversed the ABCA1-mediated reduction in DGAT-1 activity but did not affect DGAT-1 mRNA expression. Thus, mifepristone has diverse effects on de novo glycerolipid synthesis. We suggest that caution should be exercised when using mifepristone-inducible systems for studies of glycerolipid metabolism in cells expressing glucocorticoid responsive receptors.     

Relationships between base-exchange reaction and the microsomal phospholipid pool in the rat brain in vitro

The calcium-stimulated incorporation of ethanolamine, choline and L-serine into rat brain microsomal phospholipids has been investigated. The membranes were prelabeled in vitro in their choline or serine phosphoglycerides by base-exchange and then chasing experiments were done by displacing the lipid-bound base by ethanolamine, choline, or L-serine labeled with a different isotope. The results indicate that membrane phosphatidylcholine is presumably a substrate for the exchange with all the three bases, whereas phosphatidylserine exchanges only with ethanolamine and L-serine but not with choline. A small phospholipid pool (3–7% of the total available pool) is active in the calcium-dependent exchange with choline, ethanolamine, and L-serine. When the microsomal membranes are prelabeled in vitro in their phosphatidylcholine moiety through the cytidine-dependent pathway and then chasing experiments are performed with the three nitrogenous bases, as above, the small phospholipid pool is hardly detectable. In view of these and other results (Gaiti et al., FEBS Letters 49:361 1975), it is suggested that at least two different pools of phosphatidylethanolamine, phosphatidylserine, and phosphatidylcholine might exist in rat brain microsomes.     

The lipid composition of microsomal preparations from lactating bovine mammary tissue

The microsomes isolated from lactating bovine mammary tissue contained 4.3 mg lipid per milligram nitrogen. Phospholipids comprised 83% of the lipids. The neutral lipids were composed of triglycerides (20–30%), diglycerides (5–10%), free fatty acids (15–30%, cholesterol (35–40% and cholesterol esters (10–12%, respectively. Phosphatidylcholine was the predominant phospholipid component (>50%), and the remainder consisted of phosphatidylethanolamine (21–13%), phosphatidylserine (4–6%), phosphatidylinositol (8%), sphingomyelin (9%) and lysophosphatidylcholine (2%) respectively. The composition of the microsomal phospholipids was similar to that of isolated mammary cells and tissue homogenates but quite different from milk and fat globule membrane phospholipids. The triglycerides contained short chain fatty acids but their relative concentrations were lower than in milk triglycerides. The various lipid fractions had a variable proportion of saturated fatty acids, i.e., triglycerides (47.7%), diglycerides (86.7%), free fatty acids (70.6%), phosphatidylcholine (50.6%), phosphatidylethanolamine (50.8%), phosphatidylserine (35.3%), phosphatidylinositol (40.5%) and sphingomyelin (82.3%), respectively. The molecular distribution of fatty acids in the microsomal triglycerides and phosphatidylcholine was similar to that occurring in milk, i.e., the short chain and unsaturated fatty acids were concentrated in the primary positions (sn1 andsn3) of the triglycerides, and the unsaturated acids were preferentially located in positionsn2 of the phosphatidylcholine. The compositional data indicate that mammary microsomes are not the direct source of the phospholipids of the milk fat globule.     

Pulmonary surfactant lipid synthesis from ketone bodies,lactate and glucose in newborn rats

The contribution of acetoacetate (AcAc), β-hydroxybutyrate (βOHB), lactate and glucose to pulmonary surfactant lipid synthesis in three-to five-day-old rats was measured. Minced lung tissue was incubated with³H₂O and [3-¹⁴C]AcAc, [3-¹⁴C]βOHB, [U-¹⁴C]lactate or [U-¹⁴C]glucose, and the radioactivity incorporated into surfactant lipids was measured. When expressed as nmol of substrate incorporated/g lung tissue per four hr, lactate was incorporated more rapidly than other substrates into total surfactant lipids and phosphatidylcholine (PC). There was no difference in the rates of incorporation of lactate, AcAc or glucose into disaturated PC (DSPC). Substrates other than glucose were incorporated almost exclusively into fatty acids, whereas 60–80% of glucose incorporated into surfactant phospholipids was found in fatty acids, with the remaining in glyceride-glycerol. When expressed as nmol acetyl units incorporated/g lung tissue per four hr, the rates of AcAc, lactate and glucose incorporation into total surfactant fatty acids were comparable. Glucose incorporation into DSPC and PC was greater than that of AcAc and lactate. When glucose was the only exogenous substrate added to the incubation medium, it contributed 37% of total surfactant fatty acids synthesized de novo. In the presence of other substrates, the contribution of glucose to de novo fatty acid synthesis dropped to 14–20%. In the presence of unlabeled glucose,¹⁴C-labeled AcAc, lactate and βOHB contributed 52%, 40% and 19%, respectively, of the total fatty acids synthesized de novo. The rate of βOHB incorporation into surfactant lipids was only about 50% that of other substrates and was accompanied by low activity of β-hydroxybutyrate dehydrogenase measured for newborn lung. These results demonstrate that AcAc and lactate are important precursors for surfactant lipids in neonatal rat lung.     

Compartmental study of rat renal phospholipid metabolism

Phospholipid content and metabolism were studied in rat renal papillary, medullary and cortical slices. The highest concentration of phospholipids was found in cortex and the lowest in papilla samples (ratio cortex/medulla, 1.3; cortex/papilla, 3.7). The profile of the various phospholipids was different depending on the zone. The most important difference was the relative concentrations of sphingomyelin (CerPCho) and phosphatidylinositol (PtdIns) with ratios for PtdIns/CerPCho of 5.0, 3.3 and 2.5 in papilla, medulla, and cortex, respectively. In the three zones, PtdIns showed the highest specific activity for [2-¹⁴C]glycerol and [1-¹⁴C]arachidonic acid incorporation. By contrast, a higher amount of [1-¹⁴C]palmitic acid was incorporated into phosphatidylcholine than into any other phospholipid. The various radioactive precursors were only poorly incorporated into phosphatidylethanolamine. No radioactivity was associated with phosphatidylserine. The papilla possesses the most active phospholipid metabolism of all the pathways studied.