The biological origin of ketotic dicarboxylic aciduria. In vivo and in vitro investigations of the omega-oxidation of C6-C16-monocarboxylic acids in unstarved, starved and diabetic rats

The conversion of radioactive C6-C16-monocarboxylic acids to urinary adipic, suberic, sebacic and 3-hydroxybutyric acids was investigated in vivo in unstarved, starved and diabetic ketotic rats. Hexanoic, octanoic and decanoic acids were converted to C6-, C6-C8- and C6-C10-dicarboxylic acids, respectively, in fed and 72-h-starved rats. Lauric acid was converted to C6-C8-dicarboxylic acids in starved rats but not in unstarved rats. Decanoic and lauric acids were converted to relatively high amounts of C6-C8-dicarboxylic acids compared with myristic acid in myristic acid in ketotic diabetic rats, while radioactivity from [1-14C]-and [16-(14)] palmitic acid was not incorporated into C6-C8-dicarboxylic acids in diabetic ketotic rats. C6-C12-monocarboxylic acids in hydrolysed rat adipose tissue wee determined by gas-liquid chromatography-mass spectrometry (selected ion monitoring). Decanoic and lauric acids were found in amounts of 7.6-9.1 and 85.9-137.5 micrometers/100 mg tissue, respectively, whereas the amounts of hexanoic and octanoic acids were negligible. It is concluded that the biological origin of the C6-C8-dicarboxylic aciduria seen in ketotic rats are C10-C14-monocarboxylic acids, which are initially omega-oxidised solely or partly as free acids and subsequently beta-oxidised to adipic and suberic acids. The in vitro omega-oxidation of C6-C16-monocarboxylic acids to corresponding dicarboxylic acids in the 100,000 Xg supernatant fraction of rat liver homogenate was measured by selected ion monitoring. 0.09, 0.14, 16.1, 5.8, 7.0 and -6.9% of, respectively, hexanoic, octanoic, decanoic, lauric, myristic and palmitic acid were omega-oxidised to dicarboxylic acids of corresponding chain lengths after 90 min of incubation, when correction for the production of dicarboxylic acids in control assays was made. An in vitro production of C12-C16-dicarboxylic acids was detected in all assays ()including control assays), probably formed from"endogenous' monocarboxylic acids preexistent in the homogenate. Ths "endogenous' production of dicarboxylic acids was inhibited by C10-C16-monocarboxylic acids, where palmitic acid had the strongest effect. In fact, palmitic acid inhibited its own omega-oxidation when added in concentrations above 0.6 mM. Starvation of rats for 72 h did not alter the "endogenous' in vitro production of hexadecanedioic acid.     

Regulation of triacylglycerol biosynthesis in embryos and microsomal preparations from the developing seeds of Cuphea lanceolata.

Embryos of Cuphea lanceolata have more than 80 mol% of decanoic acid ('capric acid') in their triacylglycerols, while this fatty acid is virtually absent in phosphatidylcholine (PtdCho). Seed development was complete 25-27 days after pollination, with rapid triacylglycerol deposition occurring between 9 and 24 days. PtdCho amounts increased until day 15 after pollination. Analysis of embryo lipids showed that the diacylglycerol (DAG) pool consisted of mainly long-chain molecular species, with a very small amount of mixed medium-chain/long-chain glycerols. Almost 100% of the fatty acid at position sn-2 in triacylglycerols (TAG) was decanoic acid. When equimolar mixtures of [14C]decanoic and [14C]oleic acid were fed to whole detached embryos, over half of the radioactivity in the DAG resided in [14C]oleate, whereas [14C]decanoic acid accounted for 93% of the label in the TAG. Microsomal preparations from developing embryos at the mid-stage of TAG accumulation catalysed the acylation of [14C]glycerol 3-phosphate with either decanoyl-CoA or oleoyl-CoA, resulting in the formation of phosphatidic acid (PtdOH), DAG and TAG. Very little [14C]glycerol entered PtdCho. In combined incubations, with an equimolar supply of [14C]oleoyl-CoA and [14C]decanoyl-CoA in the presence of glycerol 3-phosphate, the synthesized PtdCho species consisted to 95% of didecanoic and dioleic species. The didecanoyl-glycerols were very selectively utilized over the dioleoylglycerols in the production of TAG. Substantial amounts of [14C]oleate, but not [14C]decanoate, entered PtdCho. The microsomal preparations of developing embryos were used to assess the acyl specificities of the acyl-CoA:sn-glycerol-3-phosphate acyltransferase (GPAT, EC 2.3.1.15) and the acyl-CoA:sn-1-acyl-glycerol-3-phosphate acyltransferase (LPAAT, EC 2.3.1.51) in Cuphea lanceolata embryos. The efficiency of acyl-CoA utilization by the GPAT was in the order decanoyl = dodecanoyl greater than linoleoyl greater than myristoyl = oleoyl greater than palmitoyl. Decanoyl-CoA was the only acyl donor to be utilized to any extent by the LPAAT when sn-decanoylglycerol 3-phosphate was the acyl acceptor. sn-1-Acylglycerol 3-phosphates with acyl groups shorter than 16 carbon atoms did not serve as acyl acceptors for long-chain (greater than or equal to 16 carbon atoms) acyl-CoA species. On the basis of the results obtained, we propose a schematic model for triacylglycerol assembly and PtdCho synthesis in a tissue specialized in the synthesis of high amounts of medium-chain fatty acids.     

Lipid metabolism. Evidence of a δ-oxidation pathway for saturated fatty acids

1. Specific radioactivities of milk triglyceride fatty acids and gamma- and delta-hydroxy fatty acids were measured after the intramammary infusion of [1-(14)C]acetate, delta-hydroxy[1-(14)C]laurate and [1-(14)C]laurate as their sodium salts into fed lactating goats. 2. Net incorporations of the radioactive tracer into the total milk lipids were comparable, being 16, 17 and 21% of the label infused respectively. 3. The specific radioactivities of the C(4)-C(8) fatty acids after [1-(14)C]acetate infusion were lower than those of the C(10)-C(14) fatty acids. 4. After delta-hydroxy[1-(14)C]laurate administration the milk triglyceride fatty acids were labelled and their specific radioactivities were characterized by decreasing values with increasing chain length of the fatty acids, implicating C(4) unit incorporation. 5. The gamma- and delta-hydroxy fatty acids isolated after [1-(14)C]laurate infusion were highly labelled and the milk triglyceride fatty acids, other than laurate, exhibited a labelling pattern similar to that of the fatty acids derived from the radioactive delta-hydroxy fatty acid. 6. Evidence is presented for the existence of saturated fatty acid delta-oxidation in the mammary gland, in which the gamma- and delta-hydroxy fatty acids are active intermediates.     

Sex-related differences in desaturation and chain elongation of essential fatty acids studied in isolated rat hepatocytes

When [14C]linoleic acid (18:2(n-6)) or [14C]dihomogammalinolenic acid (20:3(n-6)) was incubated with isolated liver cells from rats fed an essential fatty acid deficient diet, delta 6- and delta 5-desaturation, chain elongation and synthesis of 14C-labelled C14-C18 fatty acids (from [14C]acetate) were enhanced in female cells compared with male ones. No sex difference in total secretion of very low density lipoproteins (VLDL) was observed. However, VLDL secreted from female cells contained significantly more C16-C18 fatty acids than male cells. It is suggested that the observed sex differences, at least in part, may be related to the different content of fatty acid binding proteins in female cells compared with males.     

Evidence for peroxisomal retroconversion of adrenic acid (22:4(n-6)) and docosahexaenoic acids (22:6(n-3)) in isolated liver cells

The intracellular localization of the oxidation of [2-14C]adrenic acid (22:4(n-6)) and [1-14C]docosahexaenoic acid (22:6(n-3)) was studied in isolated liver cells. The oxidation of 22:4(n-6) was 2-3-times more rapid than the oxidation of 22:6(n-3), [1-14C]arachidonic acid (20:4(n-6)) or [1-14C]oleic acid (18:1). (+)-Decanoylcarnitine and lactate, both known to inhibit mitochondrial beta-oxidation, reduced the oxidation of 18:1 distinctly more efficiently than with 22:4(n-6) and 22:6(n-3). In liver cells from rats fed a diet containing partially hydrogenated fish oil, the oxidation of 22:6(n-6) and 22:6(n-3) was increased by 30-40% compared with cells from rats fed a standard pellet diet. With 18:1 as substrate, the amount of fatty acid oxidized was very similar in cells from animals fed standard pellets or partially hydrogenated fish oil. Shortened fatty acids were not produced from [5,6,8,9,11,12,14,15-3H]arachidonic acid. In hepatocytes from rats starved and refed 20% fructose, a large fraction of 14C from 22:4 was recovered in 14C-labelled C14-C18 fatty acids. Oxidation of 22:4 thus caused a high specific activity of the extramitochondrial pool of acetyl-CoA. The results suggest that 22:4(n-6) and to some extent 22:6(n-3) are oxidized by peroxisomal beta-oxidation and by this are retroconverted to arachidonic acid and eicosapentaenoic acid.     

Beta-oxidation of medium chain (C8-C14) fatty acids studied in isolated liver cells

The beta-oxidation and esterification of medium-chain fatty acids were studied in hepatocytes from fasted, fed and fructose-refed rats. The beta-oxidation of lauric acid (12:0) was less inhibited by fructose refeeding and by (+)-decanoyl-carnitine than the oxidation of oleic acid was, suggesting a peroxisomal beta-oxidation of lauric acid. Little lauric acid was esterified in triacylglycerol fraction, except at high substrate concentrations or in the fructose-refed state. With [1-14C]myristic acid (14:0), [1-14C]lauric acid (12:0), [1-14C]octanoic acid (8:0) and [2-14C]adrenic acid (22:4(n - 6] as substrate for hepatocytes from carbohydrate-refed rats, a large fraction of the 14C-labelled esterified fatty acids consisted of newly synthesized palmitic acid (16:0), stearic acid (18:0) and oleic acid (18:1) while intact [1-14C]oleic acid substrate was esterified directly. With [9,10-3H]myristic acid as the substrate, small amounts of shortened 3H-labelled beta-oxidation intermediates were found. With [U-14C]palmitic acid, no shortened fatty acids were detected. It was concluded that when the mitochondrial fatty acid oxidation is down-regulated such as in the carbohydrate-refed state, medium-chain fatty acids can partly be retailored to long-chain fatty acids by peroxisomal beta-oxidation followed by synthesis of C16 and C16 fatty acids which can then stored as triacylglycerol.     

Biosynthesis of the polyoxins, nucleoside peptide antibiotics: a new metabolic role for L-isoleucine as a precursor for 3-ethylidene-L-azetidine-2-carboxylic acid (polyoximic acid).

The biosynthetic origin of the carbon skeleton of 3-ethylidene-L-azetidine-2-carboxylic acid (polyoximic acid) is described. This unique cyclic amino acid is the C terminus of the nucleoside peptide antibiotics, the polyoxins, elaborated by Streptomyces cacaoi var, asoensis. In vivo experiments show that 14-C from [1-14-C]isoleucine, [U-14-C]isoleucine, [1-14-C]methionine, [U-14-C]methionine, [U-14-C]threonine, and [1-14-C]glutamate is incorporated into polyoximic acid; however, 14-C from [5-14-C]glutamate and [methyl-14-C]methionine is not incorporated. The distribution of 14-C in polyoximic acid clearly shows that the intact carbon skeleton of L-isoleucine is utilized directly. The incorporation of 14-C from [U-14-C]methionine, [U-14-C]threonine, and [1-14-CA1glutamate into polyoximic acid occurred only after their conversion to isoleucine via 2-ketobutyrate. A scheme is presented in which either of the two beta-unsaturated amino acids isolated from Bankera fuligineoalba, L-2-amino-3-hydroxymethyl-3-pentenoic acid or L-2-amino-3-formyl-3-penetenoic acid, is regarded as a possible intermediate amino acid between isoleucine and polyoximic acid.     

Fatty acid biosynthesis in rabbit mammary gland during pregnancy and early lactation

The pattern of fatty acids synthesized by mammary-gland explants from rabbits during pregnancy and early lactation has been studied. From day 12 to day 18 of pregnancy, long-chain (C(14:0)-C(18:1)) fatty acids were the major products. From day 18 to day 21 of pregnancy there was an increase of up to 12-fold in the rate of fatty acid synthesis per unit wet weight of tissue that was almost exclusively caused by the synthesis of octanoic fatty acid and decanoic fatty acid, which are characteristic of rabbit milk. These medium-chain fatty acids were mainly incorporated into triglycerides. From day 22 to day 27 of pregnancy there was little change in the rate of fatty acid synthesis and the proportions of fatty acids synthesized were essentially the same as those synthesized by the lactating gland, i.e. 80-90% octanoic acid plus decanoic acid. About 2-4 days before parturition a second lipogenic stimulus occurred, although the pattern of fatty acids synthesized did not change.     

The fractionation of the fatty acid synthetase activities of avocado mesocarp plastids.

1. The range of fatty acids formed by preparations of ultrasonically ruptured avocado mesocarp plastids was dependent on the substrate. Whereas [1-14C]palmitate and [14C]oleate were the major products obtained from [-14C]acetate and [1-14C]acetyl-CoA, the principal product from [2-14C]malonyl-CoA was [14-C]stearate. 2. Ultracentrifugation of the ruptured plastids at 105000g gave a supernatant that formed mainly stearate from [2-14C]malonyl-CoA and to a lesser extent from [1-14C]acetate. The incorporation of [1-14C]acetate into stearate by this fraction was inhibited by avidin. 3. The 105000g precipitate of the disrupted plastids incorporated [1-14C]acetate into a mixture of fatty acids that contained largely [14C]plamitate and [14C]oleate. The formation of [14C]palmitate and [14C]oleate by disrupted plastids was unaffected by avidin. 4. The soluble fatty acid synthetase was precipitated from the 105000g supernatant in the 35-65%-saturated-(NH4)2SO4 fraction and showed an absolute requirement for acyl-carrier protein. 5. Both fractions synthesized fatty acids de novo.     

The effect of feeding rats with partially hydrogenated marine oil or rapeseed oil on the chain shortening of erucic acid in perfused heart.

1. The metabolism of [14(-14)C]erucic acid and [U-14C]palmitic acid was studied in perfused hearts from rats fed diets containing hydrogenated marine oil, rapeseed oil or peanut oil for three weeks. 2. [14C]Erucic acid was shortened to [14C]eicosenoic acid (20 : 1, n -- 9) and [14C]oleic acid (18 : 1, n -- 9) in perfused rat hearts from all diet groups. The rapeseed oil diet caused a three-fold increase and the marine oil diet a four-fold increase in the amount of chain-shortened products recovered in heart lipids at the end of perfusion, compared to peanut oil diet. 3. The content of C16:1, C18:1 and C20:1 fatty acids was increased in heart lipids of rats fed hydrogenated marine oil or rapseed oil diet, compared to peanut oil diet. 4. Feeding hydrogenated marine oil or rapeseed oil to the rats induced a 85% increase in catalase activity, a 20% increase in the activity of cytochrome oxidase and a 30--40% increase in the content of total CoA in the heart compared to rats fed peanut oil diet. 5. It is suggested that [14(-14)C]erucic acid is shortened by the beta-oxidation system of peroxisomes in the heart. The increased chain shortening in the hearts from animals fed rapeseed oil or partially hydrogenated marine oil for three weeks may be an important part of an adaptation process.     

Fatty acid metabolism in Atlantic salmon (Salmo salar L.) hepatocytes and influence of dietary vegetable oil

Isolated hepatocytes from Atlantic salmon (Salmo salar), fed diets containing either 100% fish oil or a vegetable oil blend replacing 75% of the fish oil, were incubated with a range of seven (14)C-labelled fatty acids. The fatty acids were [1-(14)C]16:0, [1-(14)C]18:1n-9, 91-(14)C]18:2n-6, [1-(14)C]18:3n-3, [1-(14)C]20:4n-6, [1-(14)C]20:5n-3, and [1-(14)C]22:6n-3. After 2 h of incubation, the hepatocytes and medium were analysed for acid soluble products, incorporation into lipid classes, and hepatocytes for desaturation and elongation. Uptake into hepatocytes was highest with [1-(14)C]18:2n-6 and [1-(14)C]20:5n-3 and lowest with [1-(14)C]16:0. The highest recovery of radioactivity in the cells was found in triacylglycerols. Of the phospholipids, the highest recovery was found in phosphatidylcholine, with [1-(14)C]16:0 and [1-(14)C]22:6n-3 being the most prominent fatty acids. The rates of beta-oxidation were as follows: 20:4n-6>18:2n-6=16:0>18:1n-9>22:6n-3=18:3n-3=20:5n-3. Of the fatty acids taken up by the hepatocytes, [1-(14)C]16:0 and [1-(14)C]18:1n-9 were subsequently exported the most, with the majority of radioactivity recovered in phospholipids and triacylglycerols, respectively. The major products from desaturation and elongation were generally one cycle of elongation of the fatty acids. Diet had a clear effect on the overall lipid metabolism, with replacing 75% of the fish oil with vegetable oil resulting in decreased uptake of all fatty acids and reduced incorporation of fatty acids into cellular lipids, but increased beta-oxidation activity and higher recovery in products of desaturation and elongation of [1-(14)C]18:2n-6 and [1-(14)C]18:3n-3.     

In vivo biosynthesis of -linolenic acid in plants

[1-¹⁴C]acetate was readily incorporated into unsaturated fatty acids by leaf slices of spinach, barley and whole cells of $\text{Chlorella}\text{pyrenoidosa}$ and $\text{Candida}\text{bogoriensis}$. In these systems the [¹⁴C] label in newly synthesized oleate and linoleate was approximately equally distributed in the C_1–9 and the C_10–18 fragments obtained by reductive ozonolysis of these acids, whereas in a-linolenic acid over 90% of the total [¹⁴C] was localized in the C_1–9 fragment. While [1-¹⁴C]oleic acid was converted by whole cells of $\text{Chlorella}$ to [1-¹⁴C]linoleic and [1-¹⁴C]linolenic acids, [U-¹⁴C]oleic acid yielded [U-¹⁴C]linoleic acid but a-linolenic acid was labeled only in the carboxyl terminal carbon atoms. When spinach leaf slices were supplied with carboxyl labeled octanoic, decanoic, dodecanoic, tetradecanoic and octadecanoic acids, only the first three acids were converted to a-linolenic acids while the last two acids were ineffective. Thus we suggest that (a) linoleic acid is not the precursor of a-linolenic acid and (b) 12:3(3, 6, 9) is the earliest permissible trienoic acid which is then elongated to a-linolenic acid.     

The effects of alkylthioacetic acids (3-thia fatty acids) on fatty acid metabolism in isolated hepatocytes

Long-chain alkylthioacetic acids (3-thia fatty acids) inhibit fatty acid synthesis from [1-14C]acetate in isolated hepatocytes, while fatty acid oxidation is nearly unaffected or even stimulated. Desaturation of [1-14C]stearate (delta 9-desaturase) is also unaffected. [1-14C]Dodecylthioacetic acid (a 3-thia fatty acid) is incorporated in triacylglycerol and in phospholipids more efficiently than [1-14C]palmitate in isolated hepatocytes. The metabolism of [1-14C]dodecylthioacetic acid to acid-soluble products (by omega-oxidation) is slow compared to the oxidation of [1-14C]palmitate. In hepatocytes from adapted rats (rats fed tetradecylthioacetic acid for 4 days) the rate of [1-14C]palmitate oxidation is increased and its rate of esterification is decreased. Stearate desaturation is also decreased. The rate of cyanide-insensitive peroxisomal fatty acid beta-oxidation is several-fold increased. The metabolic effects of long-chain 3-thia fatty acids are discussed and it is concluded that they behave essentially like normal fatty acids except for their slow breakdown due to the sulfur atom in the 3 position, which blocks normal beta-oxidation.     

Detailed structure of lipid A isolated from lipopolysaccharide from the marine proteobacterium Marinomonas vaga ATCC 27119.

The chemical structure of a novel lipid A, the major component of the lipopolysaccharide from the marine gamma-proteobacterium Marinomonas vaga ATCC 27119(T), was determined by compositional analysis, NMR spectroscopy, and MS. It was found to be beta-1,6-glucosaminobiose 1-phosphate acylated with (R)-3-[dodecanoyl(dodecenoyl)oxy]decanoic acid [C10 : 0 (3O-C12 : 0 [3O-C12 : 1])] or (R)-3-(decanoyloxy)decanoic acid [C10 : 0 (3O-C10 : 0)], (R)-3-hydroxydecanoic acid [C10 : 0 (3OH)], and (R)-3-[(R)-3-hydroxydecanoyloxy]decanoic acid (C10 : 0 [3O-[C10 : 0 (3OH)]]) at the 2, 3, and 2' positions, respectively. It showed low lethal toxicity, which is probably related to specific structural attributes. The absence of a fatty acid at the 3' position and a phosphoryl group at the 4' position and also the presence of an amide-linked (R)-3-hydroxyalkanoic acid that is further O-acylated with another (R)-3-hydroxyalkanoic acid, distinguish M. vaga lipid A from other such molecules.     

Studies in Tetrahymena membranes substrates for desaturation of fatty acyl chains in Tetrahymena pyriformis microsomes

[1-14-C]Palmitoyl-Co A was incubated with Tetrahymena microsomes containing the complete enzyme system for desaturation during various time periods. The level of [1-14C]palmitoleoyl-CoA increased to a maximum during the 1--3 min incubation time, while [1-14C]palmitoleic acid in the phospholipid reached a maximum level during 6--7 min incubation time. The radioactivity of [1-14C]palmitoleic acid in free fatty acid and the triglyceride fraction was not significantly observed upon 3 min incubation. Incubation of [1-14C]palmitoyl-CoA with microsomes in the absence of NADH produced [1-14C]palmitoyl lipid without desaturation. Radioactive palmitic acids in the microsomal lipids were not converted to palmitoleic acids after addition of NADH by the complete enzyme system. When microsomes prepared from cells labeled with [1-14C]palmitic acid or [1-14C]stearic acid were incubated alone in the presence of O2 and NADH, no significant increase in [1-14C]palmitoleic acid in the phospholipid was observed, wherease an increase in [1-14C]linoleic acid and gamma-[1-14C]linolenic acid did occur at the expense of [1-14C]oleic acid in the phospholipid. From these results it can be concluded that the enzyme involving desaturation of palmitic acid to palmitoleic acid requires palmitoyl-CoA as the substrate. However, the possibility of oleoyl and linoleoyl phospholipids being substrates in the desaturation of Tetrahymena microsomes was suggested.     

Phospholipid molecular species, beta-oxidation, desaturation and elongation of fatty acids in Atlantic salmon hepatocytes: effects of temperature and 3-thia fatty acids

We have investigated the effects of a 3-thia fatty acid (TTA) and of temperature on the fatty acid (FA) metabolism of Atlantic salmon (Salmo salar). One experiment investigated the activity of the peroxisomal beta-oxidation enzyme, acyl-CoA oxidase (ACO), and the incorporation of TTA into phospholipid (PL) molecular species. Salmon hepatocytes in culture were incubated either without TTA (control(spades)) or with 0.8 mM TTA (TTA(spades)) in a short term (48 h) temperature study at 5 degrees C and at 12 degrees C. TTA was incorporated into the four PL classes studied: phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI) and phosphatidylserine (PS). TTA was preferentially esterified with 18:1, 16:1, 20:4 and 22:6 in the PLs. Hepatocytes incubated with TTA had higher ACO activity at 5 degrees C than at 12 degrees C. In a second experiment salmon were fed a diet based on fish meal-fish oil without any TTA added (control) or a fish meal-fish oil diet supplemented with 0.6% TTA for 8 weeks at 12 degrees C and 20 weeks at 5 degrees C. At the end of the feeding trial, hepatocytes from fish acclimated to high or low temperatures were isolated from both dietary groups and incubated with either [1-(14)C]18:1 n-9 or [1-(14)C]20:4 n-3 at 5 degrees C or 12 degrees C. Radiolabelled 18:1 n-9 was mainly esterified into neutral lipids (NL), whereas [1-(14)C]20:4 n-3 was mainly esterified into PL at both temperatures. The rate of elongation of [1-(14)C]18:1 n-9 to 20:1 n-9 was twice as high in hepatocytes from fish fed the control diet than it was in hepatocytes from fish fed the TTA diet, at both temperatures. The amount of [1-(14)C]20:4 n-3 converted to 22:6 n-3 was approximately the same in hepatocytes from the two dietary groups, but there was a tendency to higher production of 22:6 n-3 at the lower temperature. Oxidation of [1-(14)C]18:1 n-9 to acid soluble products (ASP) and CO(2) was approximately 10-fold greater in hepatocytes kept at 5 degrees C than in those kept at 12 degrees C and the main oxidation products formed were acetate, oxaloacetate and malate.     

Biochemical effects of the hypoglycaemic compound pent-4-enoic acid and related non-hypoglycaemic fatty acids. Fatty acid oxidation

1. The effects of the hypoglycaemic compound, pent-4-enoic acid, and of four structurally related non-hypoglycaemic compounds (pentanoic acid, pent-2-enoic acid, cyclopropanecarboxylic acid and cyclobutanecarboxylic acid), on the oxidation of saturated fatty acids by rat liver mitochondria were determined. 2. The formation of (14)CO(2) from [1-(14)C]palmitate was strongly inhibited by 0.01mm-pent-4-enoic acid. 3. The inhibition of oxygen uptake was less than that of (14)CO(2) formation, presumably because fumarate was used as a sparker. 4. The oxidation of [1-(14)C]-butyrate, -octanoate or -laurate was not strongly inhibited by 0.01mm-pent-4-enoic acid. 5. The other four non-hypoglycaemic compounds did not inhibit the oxidation of any saturated fatty acid when tested at 0.01mm concentration, though they all inhibited strongly at 10mm. 6. The oxidation of [1-(14)C]-myristate and -stearate, but not of [1-(14)C]decanoate, was strongly inhibited by 0.01mm-pent-4-enoic acid. 7. The oxidation of [1-(14)C]palmitate was about 50% carnitine-dependent under the experimental conditions used. 8. The percentage inhibition of [1-(14)C]palmitate oxidation by pent-4-enoic acid was the same whether carnitine was present or not. 9. Acetoacetate formation from saturated fatty acids was inhibited by 0.1mm-cyclopropanecarboxylic acid to a greater extent than their oxidation. 10. The other compounds tested inhibited acetoacetate formation from saturated fatty acids proportionately to the inhibition of oxidation. 11. Possible mechanisms for the inhibition of long-chain fatty acid oxidation by pent-4-enoic acid are discussed. 12. There was a correlation between the ability to inhibit long-chain fatty acid oxidation and hypoglycaemic activity in this series of compounds.     

omega-Oxidation of fatty acids and the acetylation p-aminobenzoic acid.

p-Aminobenzoic acid was fed to normal and alloxan-induced diabetic rats injected with [omega-14C]labeled and [2-14C]labeled fatty acids. The p-acetamidobenzoic acid that was excreted was hydrolyzed to yield acetate which was degraded. The distribution of 14C in the acetates formed when an [omega-14C]labeled fatty acid was injected was similar to that when a [2-14C]labeled fatty acid was injected. This contrasts with the finding that in acetates from 2-acetamido-4-phenylbutyric acid excreted when 2-amino-4-phenylbutyric acid was fed, there was a difference in the distributions of 14C, a difference attributable to omega-oxidation of the fatty acid. Acetylation of p-aminobenzoic acid is then concluded to occur in a different cellular environment than that of 2-amino-4-phenylbutyric acid, one in which omega-oxidation is not functional. When 2-amino-4-phenylbutyric acid was fed and [6-14C]palmitic acid injected, rather than [16-14C]palmitic acid, the distribution of 14C in acetate was the same as when [2-14C]palmitic acid was injected. This indicates that the dicarboxylic acid formed on omega-oxidation of palmitic acid does not undergo beta-oxidation to form succinyl-CoA. Thus, glucose is not formed via omega-oxidation of long-chain fatty acid.     

The irreversible disposal rate of free fatty acids in the plasma of fed and starved rats

1. The irreversible disposal rate coefficient for free fatty acids in the plasma of fed and starved rats was determined after a single intravenous injection of [1-(14)C]palmitic acid into each rat. The dose of labelled palmitic acid was given as a complex with (131)I-labelled albumin in rat serum. The total amount of [1-(14)C]palmitic acid remaining in the plasma was determined at short times after injection from the (14)C/(131)I ratio in the injected serum and in the collected plasma. The rate coefficient was determined from the area under the curve that describes the disappearance of [1-(14)C]palmitic acid with time from the plasma. Possible sources of error in these determinations are discussed. 2. The irreversible disposal rate coefficient was significantly higher in fed rats (2.07min(-1)) than in rats which had been starved for 24h (1.53min(-1)). The possible relationship between this difference and the processes whereby free fatty acids are removed from the plasma is discussed briefly. 3. An estimate of the irreversible disposal rate for free fatty acids in plasma was made from the concentration of free fatty acids in plasma and from the volume of distribution of (131)I-labelled albumin. The irreversible disposal rate was significantly lower in the fed state than in the starved.     

Metabolism of sialic acids from exogenously administered sialyllactose and mucin in mouse and rat

A mixture of N-acetyl-[4,5,6,7,8,9-14C]neuraminosyl-alpha (2-3(6]-galactosyl-beta (1-4-glucose[( 14C]sialyl-lactose) and N-acetylneuraminosyl-alpha (2-3(6]-galactosyl-beta(1-4)-glucit-1-[3H]ol(sialyl-[3H]lactitol) as well as porcine submandibular gland mucin labeled with N-acetyl- and N-glycoloyl-[9-(3)H]neuraminic acid were administered orally to mice. The distribution of the different isotopes was followed in blood, tissues and excretion products of the animals. One half of the [14C]sialyl-lactose/sialyl-[3H]lactitol mixture given orally was excreted unchanged in the urine. The other half was hydrolysed by sialidase and partly metabolized further, followed by the excretion of 30% of the 14C-radioactivity as free N-acetyl-[4,5,6,7,8,9-14C]neuraminic acid and 60% of this radioactivity in the form of non-anionic compounds including expired 14CO2 within 24 h. The 14C-radioactivity derived from the [14C]sialyl-lactose/sialyl-[3H]lactitol mixture which remained in the bodies of fasted mice after 24 h was less than 1%. In the case of well-fed mice, a higher amount of the sialic acid residues was metabolized. The bulk of radioactivity of the mucin was resorbed within 24 h. About 40% of the radioactivity administered was excreted by the urine within 48 h; 30% of this radioactivity represented sialic acid and 70% other anionic and non-anionic metabolic products. 60% of the radioactivity administered remained in the body, and bound 3H-labeled sialic acids were isolated from liver. Sialyl-alpha (2-3)-[3H]lactitol was injected intravenously into rats; the substance was rapidly excreted in the urine without decomposition. These studies show that part of the sialic acids bound to oligosaccharides and glycoproteins can be hydrolysed in intestine by sialidase and be resorbed. This is followed either by excretion as free sialic acid or by metabolization at variable degrees, which apparently depends on the compound fed and on the retention time in the digestive tract.