Metabolism of glyceryl 1-14C-trilinoleate in rat testis

Animals of the Sprague-Dawley strain were injected intratesticularly with radioactive glyceryl 1-¹⁴C-trilinoleate in a sequential experiment and killed at 1/4, 1/2, 1, 3, 6, 12, 24, 36 and 48 hr. Distribution and concentration (specific activity) of radioactivity among the lipid classes and fatty acids were determined. The results showed that radioactive 1-¹⁴C-linoleic acid was released from the glyceryl trilinoleate and incorporated throghout the lipid classes. The pattern of the distribution of the radioactivity and specific activities showed that the transformation of linoleic acid between the triglyceride, diglyceride and fatty acid pools was an equilibrium process. Linoleic acid released from glyceryl 1-¹⁴C-trilinoleate was converted to higher polyunsaturated fatty acids which were incorporated throughout the lipid classes, and was catabolized as evidenced by the finding of radioactivity in palmitic acid. The main metabolic pools in the interconversion of linoleic acid were arachidonic and 22∶5 acids. Small amounts of 20∶3 and 22∶4 were also detected and had high specific activities indicative of their roles as precursors.     

Absorption and metabolism of 1-14C-hydroxy octadecadienoate in the rat

The metabolism of 1-¹⁴C-9(13)-hydroxy octadecadienoic acid methyl ester (1-¹⁴C-HAME) by the rat was investigated in vivo and in liver slices. A 1.5 mg dose of 1-¹⁴C-HAME administered by stomach tube was efficiently hydrolyzed and absorbed from the intestinal tract. In comparison with 1-¹⁴C-methyl linoleate (1-¹⁴C-ML), 1-¹⁴C-HMAE was more extensively oxidized to¹⁴CO₂ in vivo and in vitro. After 1-¹⁴C-HAME administration as much as 50% of the radioactivity in the adipose tissue triglycerides was associated with¹⁴C-hydroxy fatty acids. The remaining activity was present in randomly labeled normal fatty acids. No evidence was obtained for the incorporation of¹⁴C-hydroxy acids into liver lipids; most of the radioactivity from 1-¹⁴C-HAME in this organ was recovered in saturated and monoenoic fatty acids. About 10% of the radioactivity 24 hr after 1-¹⁴C-HAME administration was associated with triglyceride trienoic acids, indicating that at least a portion of this acid was dehydrated in the liver. An unidentified polar acid was detected in the urine of the 1-¹⁴C-HAME-treated animals.     

Effect of heated fat upon metabolism of 1-14C-acetate in the rat

Groups of rats were prefed for 18 weeks with fresh or heated corn oil (≈5% nonurea adduct) at 10, 20 and 30% protein levels and at 10% protein+2% cellulose; 1-¹⁴C-sodium acetate was injected intraperitonially into each animal and the radioactivity was measured in expelled CO₂ and also in the lipid fractions of liver. In the groups of rats fed 10% protein, one-half of the administered radioactivity was found 50 min after acetate injection into rats fed fresh oil, whereas 90 min were required to reach the same level in rats fed heated oil. The same trend was found in all the groups of rats receiving different protein levels. The conversion of acetate to CO₂ was significantly higher at a 10% dietary protein level than at the 20 or 30%, but there appeared to be no significant difference in the conversions at the 20 and 30% protein levels. However addition of 2% cellulose to the 10% dietary protein level significantly increased the conversion of the acetate to CO₂ in rats fed heated oil. The livers of animals receiving heated fat diets had a higher lipid content, mainly triglyceride. When the liver lipids from rats fed fresh corn oil were separated by argentation thin layer chromatography, the bulk of the radioactivity was found in the saturated fractions. Monoene, diene and triene fractions from the liver lipid of rats fed heated oil had almost twice the radioactivity of those from fresh oil, suggesting the preferential utilization of acetate in the synthesis of these unsaturated fatty acids. One of seven papers presented in the symposium “Biological Significance of Autoxidized and Polymerized Oils”, JOCS-AOCS Joint Meeting, Los Angeles, April 1972.     

The metabolism of 1-14C arachidonic acid in pyridoxine-deficient and pair-fed control rats

The metabolism of 1-¹⁴C arachidonate was studied in pyridoxine-deficient and pair-fed pyridoxine-supplemented control rats. The studies included intestinal absorption, oxidation to¹⁴CO₂, organ uptakes and distribution of¹⁴C in the fatty acids of the various organs. Generally, pyridoxine deficiency resulted in little or no alteration of the metabolism of arachidonic acid 6 and 12 hrs after oral administration. A notable exception occurred in hearts of deficient animals in which the proportion of the incorporated¹⁴C activity found in fatty acids other than 20:4 was larger than that observed in hearts of pyridoxine-supplemented animals. A significant amount of¹⁴C activity in a water-soluble form was observed in hydrolysates of intestinal contents and of intestines of both groups. Pyridoxine-deficient rats had larger quantities than their respective pair-fed pyridoxine-supplemented controls. Most of the¹⁴C activity of fatty acids of various organs was present as arachidonic acid, but significant activity was present both in fatty acids of shorter and of longer retention time than 20:4. In brain about 20% of the¹⁴C activity in fatty acids was in a fraction tentatively identified as an 18:2 isomer. In lungs about 10% of the¹⁴C activity was in a fraction tentatively identified as a 22:2 isomer and a similar quantity was observed in a polyene tentatively identified as a 22:4.     

Fatty acid synthesis from 2-14C-acetate in rat testis mitochondrial and cytosol fractions in vitro

An in vitro system for acetate incorporation into fatty acids by the mitochondrial and the cytosol fractions of rat testis is described. The rate of incorporation of acetate into fatty acids was twice as fast with the mitochondrial as with the cytosol fraction; both systems were stimulated in the presence of adenosine triphosphate, reduced nicotinamide adenine dinucleotide phosphate, coenzyme A, and MgC1(2). The optimum pH was between 7.0-7.5 for the mitochondrial fraction and between 6.5-8.0 for the cytosol fraction. Radio gas chromatography showed that palmitic acid was the most highly labeled acid, followed by stearic acid, in the mitochondrial fraction in accord with the pathway of de novo fatty acid synthesis. Some of the labeled acetate was also incorporated into the 16:1 and 18:1 fatty acids of this fraction. Distribution of radioactivity among the mitochondrial lipid classes was highest in the phospholipids and monoglycerides, followed by diglycerides and cholesterol; little radioactivity was present in the triglyceride fraction. These observations are in accord with studies of the incorporation of labeled metabolites into testicular lipids following intratesticular injection and indicate the validity of the in vitro system for studies of specific reactions occurring in vivo.     

The metabolism of linoleic and arachidonic acids in rat testis

Linoleic and arachidonic acids, labeled with¹⁴C and injected intratesticularly, were used to study with time the interconversion of polyunsaturated fatty acids in rat testis and their incorporation into the major lipid classes. With both substrates¹⁴C activity was readily incorporated into longer chain, more highly unsaturated fatty acids. After the injection of 1-¹⁴C-linoleic acid the major portion of the¹⁴C was found in palmitic, linoleic, 8,11,14-eicosatrienoic, 5,8,11,14-eicosatetraenoic, 7,10,13,16-docosatetraenoic and 4,7,10,13,16-docosapentaenoic acids. Hydrogenation of the total fatty acids isolated from rat testes after intratesticular injection of 1-¹⁴C-linoleate revealed that the polyenoic acids hydrogenating to lignoceric acid (previously characterized as 9,12,15,18-tetracosatetraenoate and 6,9,12,15,18-tetracosapentaenoate) had a relatively high specific activity. After the injection of 1-¹⁴C-arachidonate significant¹⁴C activity was found in palmitate, 7,10,13,16-docosatetraenoate, 4,7,10,13,16-docosapentaenoate, 9,12,15,18-tetracosatetraenoate and 6,9,12,15,18-tetracosapentaenoate. The biosynthesis of the ω⁶ polyunsaturated fatty acids in rat testis is discussed in relation to these data. Investigation of the distribution of label in the complex lipid fractions demonstrated the majority of the¹⁴C activity to be present in phosphatides and triglycerides after injection of either of these¹⁴C substrates with only small quantities being present as nonesterified acids. At the time periods studied the polyenoic acids of triglycerides had a higher specific activity than the corresponding acids of phosphatides with the exception of linoleate. Presented in part at the Meeting of the American Institute of Nutrition, Atlantic City, April 1968 and at the AOCS Meeting in New York, April 1969. These data were taken from a thesis submitted by R. B. Bridges in partial fulfillment of the requirements for the Ph.D. degree, Vanderbilt University.     

Studies on metabolism of linoleic-1-14C acid in testes of hypophysectomized rats

Studies are reported on the influence of hypophysectomy and pituitary gonadotrophins on the interconversion and incorporation of linoleic-1-¹⁴C acid into lipid classes of mature rat testis. Linoleic 1-¹⁴C acid was injected into the testes of mature rats 3 weeks after hypophysectomy. Groups of animals were killed at 0.25, 0.50, 1, 3, 12, 24, 36 and 48 hr after injection of the radioactive linoleic acid, and the incorporation of radio-activity into the fatty acids and lipid classes of the testicular lipids was determined. Similar experiments were carried out with hypophysectomized animals injected intramuscularly with luteinizing or follicle stimulating hormones, or both. The specific activitties of the triglyverides and phospholipids of the testicular lipid increased to a maximum ca. 1 hr after the injection of the radioactive linoleic acid, then decreased sharply. The general pattern of changes in the specific activities of these compounds indicated that the rate of fatty acid catabolism was greatly increased by hypophysectomy. In contrast, the specific activities of the cholesteryl esters and glyceryl ether diesters changed slowly after reaching a maximum at approximately the same time. This pattern, their accumulation in the lipid, and an increase in the constituent 22∶5 indicated that the turnover of these compounds was impaired by hypophysectomy. The conversion of linoleic acid to arachidonic acid was also impaired by hypophysectomy, as evidenced by an increase in the percentage and the high specific activity of 20∶3 compared to 20∶4. The administration of gonadotrophins partially prevented the effects of hypophysectomy, indicating that some of the enzymes in the testes involved in the metabolism of the lipids are hormone sensitive.     

Hepatic metabolism of 1-14C octanoic and 1-14C palmitic acids

The hepatic metabolism of 1⁻¹⁴C octanoic acid was compared with that of 1⁻¹⁴C palmitic acid in male rats which were fed. After intraportal injection only 1/6 to 1/18 as much octanoic acid as palmitic acid was incorporated into hepatic lipids. In contrast, octanoic acid yielded two to four times as much water-soluble product as did palmitic acid. Similar, but even more impressive, differences between the incorporation of these fatty acids into hepatic lipids were observed in liver slices incubated with¹⁴C octanoate and¹⁴C palmitate. The oxidation of octanoate to CO₂ was more than 10 times as great as that of palmitate. With both substrates, triglycerides comprised almost half the labeled lipid recovered. However octanoate yielded a higher proportion of labeled, unesterified fatty acids and a lower proportion of labeled phospholipid and monoglycerides than did palmitate. Most of the¹⁴C recovered in hepatic lipids after incubation with 1⁻¹⁴C octanoate was found in the carboxyl groups of long-chain fatty acids, suggesting that the latter had been synthesized from 2-carbon fragments formed from the oxidation of octanoate. In contrast, only a small fraction of the palmitate was elongated. The similarities and differences between the metabolism of octanoic and palmitic acid in liver and intestine, and the possible nutritional significance of octanoic acid are discussed.     

Hepatic metabolism of 1-14C octanoic and 1-14C margaric acids

The hepatic metabolism of 1-¹⁴C margaric acid, a 17 carbon long chain saturated fatty acid which is present in the liver in trace amounts, was compared with 1-¹⁴C octanoic acid and 1-¹⁴C palmitic acid to determine if the enhanced oxidation of medium chain fatty acids to CO₂ was dependent on fatty acid chain length or the endogenous pool size of the fatty acid substrate. Despite the fact that endogenous margarate is present in trace amounts, there was no significant difference in the oxidation of margarate and palmitate to CO₂, while the oxidation of octanoate to CO₂ was significantly more rapid. Both margarate and palmitate were more readily incorporated into lipid soluble products in contrast to the low rate of incorporation of octanoate. However, margarate was less readily incorporated into triglyceride, phospholipid and monoglyceride than palmitate. These studies suggest that the chain length rather than hepatic content of the fatty acid determines whether the carboxyl group of equimolar amounts of a 1-¹⁴C-carboxyl labeled fatty acid will be preferentially oxidized to CO₂ or incorporated into tissue lipid in the liver.     

Influence of dietary fat on metabolism of (14-14C)erucic acid in the perfused rat liver. Distribution of metabolites in lipid classes

Two groups of rats were fed diets containing 20% by weight of either partially hydrogenated marine oil supplemented with sunflower seed oil (PHMO) or palm oil (PO) for 8 wk. Using a liver perfusion system, the effect of dietary long chain monoenoic fatty acids on the uptake and metabolism of [14-¹⁴C]erucic acid was studied. The perfusion times were 15 and 60 min, respectively. The two groups showed equal ability for erucic acid uptake in the liver but differed in the channeling of the fatty acids into various metabolic pathways. A higher metabolic turnover of 22∶1 in the PHMO livers relative to the PO livers was demonstrated by an increased recovery of total [¹⁴C]labeling in the triglyceride (TG) and phospholipid (PL) fractions, already evident after 15 min of perfusion. The chainshortening capacity was highest in the PHMO group, reflected by a higher [¹⁴C]18∶1 incorporation in both TG and PL, and increasing from 15 to 60 min of perfusion. The amount of [¹⁴C]18∶1 found in PL and TG after 60 min of perfusion of livers from rats fed PO corresponded to that shown for the PHMO group after 15 min. The PL demonstrated a discrimination against 22∶1 compared to TG, and, when available, 18∶1 was highly preferred for PL-synthesis. The total fatty acid distribution in the TG, as determined by gas liquid chromatography (GLC), reflected the composition of the dietary fats. In the total liver PL, 22∶1 and 20∶1 were present in negligible amounts, although the PHMO diet contained 12–13% of both 22∶1 and 20∶1. In the free fatty acid fraction (FFA), the major part of the radioactivity (≈80%) was [14-¹⁴C]erucic acid, and only small amounts of [¹⁴C]18∶1(<2%) were presents, even after 60 min of perfusion. The shortened-chain 18∶1 was readily removed from the FFA pool and preferentially used for lipid esterification.     

Absorption and metabolism of 1-14C-methyl linoleate hydroperoxide

The absorption and metabolism of 1-¹⁴C-methyl linoleate hydroperoxide by rats was investigated. After intubation with 2 mg of peroxide, peak¹⁴CO₂ production occurred at 90 min and 25% of the dose was expired in 24 hr. Fortyfive per cent stil remained in the gastrointestinal tract after 24 hr, most of it bound to the stomach epithelium in the form of intact peroxide. Lymph was collected from the thoracic duct 2 hr after intubation and examined for labeled metabolites. Seven per cent of the radioactivity in the lymph was present in a free 1-¹⁴C-hydroxy fatty acid and 31% in its methyl ester. Fifty-seven per cent occurred in lymph triglycerides where it was equally distributed between a 1-¹⁴C-trienoic fatty acid and an unidentified 1-¹⁴C-oxy acid. The radioactivity in liver lipids was associated mainly with randomly labeled normal fatty acids. No¹⁴C-hydroxy acids were detected in liver lipids and no evidence was obtained for the absorption of unchanged peroxide. The hydroxy and trienoic acids appear to be formed during absorption by a reduction-dehydration reaction sequence.     

Fatty acid synthesis in rat testes injected intratesticularly or incubated with 1-14C acetate

Fatty acid synthesis was studied in testes of young and adult rats either injected intratesticularly or incubated with 1-¹⁴C acetate. The pattern of¹⁴C incorporation into lipids and individual fatty acids in the two age groups was similar but results obtained with intratesticular injection differed considerably from those obtained in the in vitro studies. In the former more than 70% of the¹⁴C incorporated in total lipids was in phosphatides, with about 15% in triglycerides and only minor amounts in cholesteryl esters and free fatty acids. Most of the¹⁴C incorporated into total fatty acids was in saturated acids (predominantly 16∶0). A small amount of¹⁴C was in the higher polyenes and there was a progressive increase with time after acetate injection in the¹⁴C content of 22∶5 W6. In testes incubated with 1-¹⁴C acetate, the phosphatide, triglyceride, and free fatty acid fractions had similar amounts of¹⁴C. In the total fatty acid fraction about 40% of the incorporated¹⁴C was in saturated acids (predominantly 14∶0 and 16∶0) and about 50% in the higher polyenes. Twenty carbon polyenes and 22∶5 W6 had significant¹⁴C incorporation, but most of the¹⁴C was in 22∶4 W6. About 80% of the¹⁴C in the latter compound was in the carboxyl carbon, indicating its origin from endogenous 20∶4 W6 elongated by the added 1-¹⁴C acetate used as substrate. The¹⁴C 22∶4 was present predominantly in the triglyceride and phosphatide fractions with minor amounts in other lipids.¹⁴C-labeled compounds of retention time greater than 22∶5 were also present in all lipid fractions. Presented at the ISF-AOCS World Congress Chicago, September 1970.     

2-Aminoethylphosphonic acid metabolism in the rat

Time course studies of the incorporation of radioactive 2-aminoethylphosphonic acid (AEP) into the tissues of rats demonstrated that maximum incorporation into the liver lipids occurred within 12 to 30 hr after injection, compared to 2 to 3 hr for the incorporation of phosphorylethanolamine. Little incorporation of AEP was observed in the other tissues investigated (heart, lung, spleen, adipose, kidney). The AEP was incorporated to the greatest extent into 1,2-diacylglyceryl-aminoethylphosphonate (diacylglyceryl-AEP), the phosphonate analogue of phosphatidylethanolamine, with some incorporation into the lyso derivative. Diacylglycerol-AEP apparently was not further metabolized by the rat; no methylation of diacylglyceryl-AEP to phosphonolecithin was observed. Subcellular fractionation was performed on the livers of rats who received³H-AEP 12, 30, 36, and 48 hr prior to sacrifice. The greatest amount of radioactivity was recovered in the soluble fractions. Lipid extraction was performed on the subcellular fractions, and most of the radioactivity present in the lipids was found in the microsomal fraction, with the next highest recovery in the mitochondrial and nuclear fractions. This work is based upon a thesis submitted by J.C.-J. to the Graduate College of the University of Illinois at the Medical Center in partial fulfillment of the requirements for the Ph.D. degree in Biological Chemistry. Present Address: Department of Biochemistry, Northwestern University Medical Center, Chicago, Illinois, 60611.     

The metabolism of C14-α-tocopheryl quinone and C14-α-tocopheryl hydroquinone

The metabolism of C¹⁴-d-α-tocopheryl quinone (α-TQ) and its hydroquinone (αTHQ) was investigated. Forty-six hours after intraperitoneal administration of either compound to rats the radioactivity isolated from the liver was present almost exclusively in C¹⁴-α-TQ. The results indicated, however, that in situ this compound was present primarily in the reduced form. No conversion to C¹⁴-α-tocopherol or other liver metabolites was observed. α-THQ was eliminated from the tissues more rapidly than α-TQ. The main metabolite excreted in the urine was a conjugate of α-tocopheronic acid and the main metabolite excreted in the feces was a conjugate of α-TQ. Free C¹⁴-α-TQ was present in the feces after administration of this compound but not after C¹⁴-α-THQ administration.     

Influence of diet on conversion of14C1-linolenic acid to docosahexaenoic acid in the rat

¹⁴C₁-Linolenic acid was incorporated into lipids of hearts, livers, and carcasses of male rats. We studied the influence of diet composition on extent and distribution of radioactivity. A CHOW diet, a purified, essential fatty acid (EFA)-deficient diet, a purified control diet, and EFA-deficient diets with four fatty acid supplements were used. Supplements of 18∶2n−6, 20∶4n−6, 18∶3n−3, and 22∶6n−3 were given as single doses. Radioactivities in liver phosphatidyl ethanolamines (PE), phosphatidyl cholines, and neutral lipids were measured. The distribution of radioactivity among the fatty acids in liver phospholipids was determined. Rats on CHOW diet incorporated far less radioactivity than any other group into lipids of hearts and livers. Most of the activity in livers was recovered as 20∶5n−3 and 22∶6n−3 in all rats. In EFA-deficient rats, the radioactivity in 22∶6n−3 of liver PE was still increasing 36 hr after¹⁴C₁-linolenic acid had been administered. The n−6 supplements (18∶2n−6 and 20∶4n−6) seemed to reduce the conversion of 20∶4n−3 to 20∶5n−3 (desaturation), whereas the n−3 supplements (18∶3n−3 and 22∶6n−3) reduced the conversion of 20∶5n−3 to 22∶5n−3 (elongation). Formation of 22∶6n−3 may be controlled by 22∶6n−3 itself at the elongation of 20∶5n−3 to 22∶5n−3.     

Perfluorodecanoic acid and lipid metabolism in the rat

Alterations in lipid metabolism were axamined in adult male Sprague-Dawley rats seven days after a single intraperitoneal injection of perfluorodecanoic acid (PFDA; 20, 40 or 80 mg/kg). Because PFDA treatment caused a dose-related reduction in feed intake, the response of vehicle-treated rats pair-fed to those receiving PFDA was monitored to distinguish direct effects of the perfluorinated fatty acid from those secondary to hypophagia. Carcass content of lipid phosphorus and free cholesterol decreased in dose-dependent fashion in both PFDA-treated and pair-fed rats. Carcass triacylglycerols diminished in a similar manner, yet PFDA-treated rats at each dose had a higher concentration of neutral acylglycerols than their vehicle-treated, pair-fed counterparts. In vehicle-treated, pair-fed rats at the 80 mg/kg dose level, lipid phosphorus and free cholesterol as a proportion of carcass fat increased, whereas the share of the triacyl-glycerols declined. Because of the higher concentration of triacylglycerols in the carcass of rats treated with 80 mg/kg PFDA, enrichment of lipid phosphorus and free cholesterol in carcass fat was less than in their pair-fed partners. The amount of lipid phosphorus and free cholesterol per hepatocyte was similar in both PFDA-treated rats and their pair-fed partners. Liver triacyl-glycerols were markedly increased in PFDA-treated rats. A similar but less extensive augmentary effect of PFDA on hepatic esterified cholesterol was found. Concentration of triacylglycerols in plasma was not elevated in PFDA-treated rats, in spite of hepatic accumulation of esterified compounds. Also, the plasma level of free fatty acids and 3-hydroxybutyrate was similar in all treatment groups, including those receiving PFDA. Thus, the administration of PFDA appears to divert fatty acids from oxidation toward esterification in the liver.     

Partial synthesis of [1-14C]phytanic acid

[1-¹⁴C] Phytanic acid has been prepared in good yield from the unlabeled acid. Pristanyl iodide, prepared from the latter by a modified Hunsdieker reaction, is converted to the corresponding [¹⁴C]-nitrile by reaction with sodium [¹⁴C] cyanide in dimethyl sulphoxide; hydrolysis of the [¹⁴C] nitrile yields [1-¹⁴C]phytanic acid. The labeled acid should prove to be a useful substrate for the diagnosis of Refsum's disease.     

The synthesis of 1-14C-arachidonate and 3-14C-docosa-7,10,13,16-tetraenoate

Methyl 1-¹⁴C-arachidonate was prepared by coupling 1-bromotetradeca-2,5,8-triyne with 4-pentyn-1-ol to yield nonadeca-4,7,10,13-tetrayn-1-ol. This compound was reduced with Lindlars catalyst. The resulting alcohol was converted to the mesylate and then to the nitrile which in turn was converted to methyl 1-¹⁴C-arachidonate by hydrolysis with anhydrous HCl in methanol. The methyl 1-¹⁴C-arachidonate was reduced to the alcohol with LiA1H₄ and converted to the mesylate which in turn was treated with diethyl malonate. Following saponification and decarboxylation 3-¹⁴C-docosa-7,10,13,16-tetraenoic acid was obtained. Presented at the AOCS-ISF Meeting, Chicago, September 1970.