Evidence of accumulation of ceramides containing [14C] nervonic acid in the rat lung following injection of [14C] erucic acid

A mixture of albumin-bound [¹⁴C]erucate and [³H]oleate was injected into rats fed a stock pellet diet containing 4% by weight of lipid. Chylomicrons containing the same labelled fatty acids were also injected into rats fed diets containing 15% by weight of rapeseed oil (48% of erucic acid), canbra oil (< 5% of erucic acid) or ground nut oil (no erucic acid). Lung lipids were analyzed at various times after injection.In all cases, except in the rapeseed oil diet group, ¹⁴C radioactivity of lung ‘monoacylglycerol’ was ten times higher than ³H radioactivity. More than 85% of this ¹⁴C radioactivity was found in nervonic acid (24:1). It was shown by TLC and GLC analysis that 85–90% of the ¹⁴C radioactivity of this fraction was in ceramides (N-acyl-4-sphingenine).Ceramides containing [¹⁴C] nervonic acid disappeared from the lung with time and their incorporation with time into sphingomyelin was also observed. The absence of accumulation of ³H and ¹⁴C (18:1) in ceramides showed that oleic acid was not incorporated into sphingomyelin in the same way as nervonic acid.In the rapeseed oil diet group, there was no accumulation of ¹⁴C radioactivity in ceramides and conversion of erucic acid into nervonic acid was less, and into oleic acid more, than in other diet groups indicating a possible enzyme adaptation.     

Prostaglandin and fatty acid omega- and (omega-1)-oxidation in rabbit lung. Acetylenic fatty acid mechanism-based inactivators as specific inhibitors

Terminal acetylenic fatty acid mechanism-based inhibitors (Ortiz de Montellano, P. R., and Reich, N. O. (1984) J. Biol. Chem. 259, 4136-4141) were used as probes in determining the substrate specificity of rabbit lung cytochrome P-450 isozymes of pregnant animals in both microsomes and reconstituted systems. Lung microsomal and reconstituted P-450 form 5-catalyzed lauric acid omega- and (omega-1)-hydroxylase activities were inhibited by a 12-carbon terminal acetylenic fatty acid, 11-dodecynoic acid (11-DDYA), and an 18-carbon terminal acetylenic fatty acid, 17-octadecynoic acid (17-ODYA). Rabbit lung microsomal lauric acid omega-hydroxylase activity was more sensitive to inhibition by 11-DDYA than was (omega-1)-hydroxylase activity. In reconstituted systems containing purified P-450 form 5, both omega- and (omega-1)-hydroxylation of lauric acid were inhibited in parallel when either 11-DDYA or 17-ODYA was used. These data suggest the presence of at least two P-450 isozymes in rabbit lung microsomes capable of lauric acid omega-hydroxylation. This is the first report indicating the multiplicity of lauric acid hydroxylases in lung microsomes. Lung microsomal prostaglandin omega-hydroxylation, mediated by the pregnancy-inducible P-450PG-omega (Williams, D. E., Hale, S. E., Okita, R. T., and Masters, B. S. S. (1984) J. Biol. Chem. 259, 14600-14608) was subject to inhibition by 17-ODYA only, whereas 11-DDYA acid was not an effective inhibitor of this hydroxylase. We have recently developed a new terminal acetylenic fatty acid, 12-hydroxy-16-heptadecynoic acid (12-HHDYA), that contains a hydroxyl group at the omega-6 position. We show that 12-HHDYA possesses a high degree of selectivity for the inactivation of rabbit lung microsomal prostaglandin omega-hydroxylase activity which cannot be obtained with the long chain acetylenic inhibitor, 17-ODYA. In addition, 12-HHDYA has no effect on lauric acid omega- or omega-1-hydroxylation or on benzphetamine N-demethylation. The development of this new terminal acetylenic fatty acid inhibitor provides us with a useful tool with which to study the physiological role of prostaglandin omega-hydroxylation in the rabbit lung during pregnancy.     

Oxidation of D-[U-(14)C] glucose and [1,12-(14)C] dodecanedioic acid by pancreatic islets from Goto-Kakizaki rats.

In pancreatic islets from hereditarily diabetic GK rats, [1,12 -(14)C] dodecanedioic acid (5.0 mM) was oxidized at a rate representing about 5 % of that of D-[U - (14)C] glucose (8.3 mM). Dioic acid and hexose failed to exert any significant reciprocal effects on their respective oxidation. The production of (14)CO(2) from [1,12 -(14)C] dodecanedioic acid was proportional to its concentration in the 0.2 - 5.0 mM range. These results were essentially comparable to those obtained in islets from control rats. They extend, therefore, to GK rats the knowledge that dodecanedioic acid acts as a nutrient in pancreatic islet cells.     

The turnover of phosphoglycerides in the subcellular fractions of mouse brain: a study using (1-14C)oleic acid as precursor

Abstract— The turnover of phosphoglycerides in subcellular fractions of adult mouse brain was examined after intracerebral injection of [1-¹⁴C]oleic acid. Radioactivity of the total brain homogenate decreased rapidly thereafter, with only 4 per cent of the radioactivity remaining at the end of 3 months. The rate of decrease of radioactivity in the subcellular fractions was in the order: cytosol, microsomes, synaptosomes and myelin. Increasing amounts of radioactivity were detected in the alkenyl groups and cerebrosides, but metabolic conversions were not as extensive as found previously with the palmitoyl group. The specific radioactivities for diacyl sn-glycero-3-phosphorylcholine and diacyl sn-glycero-3-phosphorylethanolamine were highest in the microsomal fraction and decreased with time. The apparent half-lives for the diacyl sn-glycero-3-phosphorylcholine and the diacyl sn-glycero-3-phosphorylethanolamine in the microsome and synaptosome-rich fractions were 1-3.5 days when estimated between 1 and 7 days after injection. The rate of decay for the brain membrane phosphoglycerides was not linear with time, probably because of the extensive amount of recycling occurring within the system. Radioactivity was incorporated into the phosphoglycerides of the myelin but equilibration of radioactivity between microsomes and myelin required 7–14 days.     

The early and late effects of a radiation lesion by 14C(14C-stearic acid)

In experiments with mice a study was made of the biological effect of radiocarbon (14C-stearic acid) injected intraperitoneally in doses of 2.2, 1.2 and 0.5 MBq/g. The doses absorbed within the body made up an average of 13.4 and 1.7 Gy respectively. The animals of the 1st group exhibited a severe degree of radiation affection and those of the 2nd and 3d groups median and mild degrees. Metabolism peculiarities and formation of absorbed 14C doses, as well as early and remote effects of radiation were investigated.     

Solid-liquid phase behavior of binary fatty acid mixtures 3. Mixtures of oleic acid with capric acid (decanoic acid) and caprylic acid (octanoic acid)

Solid-liquid phase behavior of binary mixtures of oleic acid (OA)/capric acid (C10A) and OA/caprylic acid (C8A) were investigated by means of differential scanning calorimetry (DSC), Fourier transform infrared spectroscopy (FT-IR), and X-ray diffraction. The phase diagram of OA/C10A mixture constructed from the DSC results suggested that a molecular compound with the composition of OA:C10A = 3:2 is formed in a solid phase, and OA and the molecular compound are miscible, while C10A and the molecular compound are completely immiscible. The formation of the molecular compound was supported by the IR spectroscopic observation, and a possible model of the structure was proposed on the basis of X-ray diffraction spectrum in small angle region. This compound formation is characteristic of the OA/C10A mixture, and may be attributed to the similarity of the acyl chain length of C10A to the lengths of Delta- and omega-chains of OA (i.e., the chain segments divided by cis-double bond). The mixture of OA and C8A, whose chain length is close to but shorter than the two chain segments of OA, provided a eutectic-type phase diagram showing a partial mixing of the two components in OA-rich region. Thermodynamic analysis of the liquidus line in the phase diagram exhibits a systematic trend for the non-ideality parameter of mixing with the variation of the chain length difference between OA and saturated fatty acid species.     

Metabolism of L-(U-14C)valine, L-(U-14C)leucine, L-(U-14C)histidine and L-(U-14C) phenylalanine by the isolated perfused lactating guinea-pig mammary gland.

1. The incorporation of L-[U-14C]leucine, L[U-14C]histidine and L-[U-14C]phenylalanine into casein secreted during perfusion of isolated guinea-pig mammary glands was demonstrated. 2. The extent of incorporation of label into casein residues was consistent with their being derived from free amino acids of the perfusate plasma. 3. The mean transit time of the amino acids from perfusate into secreted casein was approx. 100 min. 4. Whereas radioactive histidine and phenylalanine were incorporated solely into milk protein, radioactivity from [U-14C]valine was also transferred to CO2 and to an unidentified plasma component, and from [U-14C]leucine to plasma glutamic acid. 5. Evidence from experiments with [U-14C]phenylalanine suggests that, as in rats, but in contrast with ruminant species, guinea-pig mammary tissue does not possess phenyl alanine hydroxylase activity. 6. The results are discussed in relation to the possible role of essential amino acid catabolism in the control of milk-protein synthesis.     

The metabolism of (1-14C) stearic acid in rat testicular tissue.

(1) The metabolism of stearic acid was studied in vivo following intratesticular injection of [1-14C] stearate. Soon after injection 14C activity was found mainly in the free fatty acid pool. This was followed at later time periods by transfer of label primarily to the phosphatide pool. During each time period significant amounts of label were recovered at 14CO2. (2) Analysis of 14C-labeled fatty acids from the injected testes demonstrated an initial rapid rate of oxidation and desaturation of [1-14C] stearate followed by a slower steady state rate. It was concluded that the initial rate was due to the rapid turnover of the highly labeled free fatty acid pool followed by a much slower rate as [14C] stearate was esterified to the more metabolically stable phospholipids. Elongation of the labeled stearic or its desaturated derivative was not observed. (3) The rate of desaturation in vitro of stearic acid was measured in microsomal preparations from rat testes and found to be 12.0 +/- 0.5 pmol/min/mg compared to the estimated in vivo value of 22 pmol/min/mg and the value of 390 pmol/min/mg for hepatic microsomal desaturase.     

Formation of omega- and (omega-1)-hydroxyfatty acids and plausible formation of ketofatty acid from microsomal phospholipids

Rat liver microsomes labeled with spin-labeled phosphatidylcholine release the label into the aqueous phase during the aerobic incubation with NADPH (Biochem. Biophys. Res. Commun. (1979) 87, 300-307). To establish the chemical nature of the released moiety, microsomes were labeled with [14C]phosphatidylcholine. When the 14C-labeled microsomes were incubated with NADPH under aerobic conditions, a few percent of the radioactivity was liberated into the aqueous phase within 60 min. Thin-layer chromatographic analysis of the radioactive substance liberated showed the presence of hydroxylated fatty acids derived from the 2-position of glycerol moiety. About one-third of the fatty acids formed from [14C]phosphatidylcholine during the incubation were converted into hydroxy-derivatives. Gas chromatography/mass spectrometry analysis further confirmed an NADPH-dependent formation of 16-hydroxypalmitic acid, 15-hydroxypalmitic acid, and hydroxy-derivatives of other fatty acids from the phospholipids of the microsomal membrane. Evidence was also obtained indicating the formation of ketopalmitic acid.     

Fatty acid omega and (omega-1)-Hydroxylation in rabbit intestinal mucosa microsomes.

The microsomes from rabbit intestinal mucosa which had been washed quickly and thoroughly with phenylmethylsulfonyl fluoride were found to catalyze the hydroxylation of fatty acids in the presence of NADPH and molecular oxygen. Myristic and palmitic acids were converted to the corresponding omega-and (omega-1)-hydroxy fatty acids, whereas lauric acid was converted only to 12-hydroxylauric acid, and capric acid, to 9-and 10-hydroxycapric acids together with an unknown polar acid.Among these fatty acids, both myristic and lauric acids appeared to be the most efficient substrates. The inhibition of the hydroxylation by SKF 525-A and carbon monoxide suggested that the activity depended upon cytochrome P-450. The specific activity of the fatty acid hydroxylation was almost constant along the small intestine, while the aminopyrine N-demethylation activity and the cytochrome P-450 content were highest at the proximal end of the intestine and progressively declined toward the caudal end. The cytochrome P-450 was solubilized from the intestinal microsomes and purified by 6-amino-n-hexyl Sepharose 4B chromatography. The partially purified cytochrome P-450 was active in fatty acid hydroxylation in combination with intestinal NADPH-cytochrome c reductase and phosphatidylcholine.