The distribution of labeled palmitic acid into the diglycerides and triglycerides of rat adipose tissue

After in vitro incubation of rat epididymal fat pads with radioactive palmitic acid, the distribution of the label in the different lipid classes and in different triglycerides was determined by silica gel and silver nitrate-silica gel thin-layer chromatography (TLC). The radioactivity of the diglycerides was approximately half of the triglycerides. This ratio did not change with alteration in the time of incubation. It remained unaltered even after a subsequent 10-min incubation in a nonradioactive medium. When the fat pads were incubated, first with¹⁴C-, then with³H-labeled palmitic acid, the³H/¹⁴C ratio was slightly lower in diglycerides than in triglycerides. The fully saturated molecules contained 38% of the radioactivity of triglycerides. Addition of oleic acid or norepinephrine to the labeled palmitic acid-containing medium decreased this value. Subsequent incubation with these compounds did not alter the distribution of radioactivity.     

The turnover time of dietary cholesterol in the lactating rat

The secretion of dietary 4-¹⁴C-cholesterol into milk of the rat was determined as a function of post-feeding time by a single dose technique. The time interval which elapsed before maximum specific radioactivity was reached in milk (17–20 hr after maximum activity in the serum) suggests a route through the mammary gland involving transport of the cholesterol by intracellular membranes. It also suggests that the exogenous cholesterol is incorporated into the milk fat globule membranes rather than into the fat globules during their synthesis within the cell. Paper No. 3978 in the Journal Series of the Pennsylvania Agricultural Experiment Station.     

Determination of hydroxyls in mono- and diglycerides

A method for the analysis of hydroxyls employing esterification with 3,5-dinitrobenzoyl chloride, followed by titration with tetrabutylammonium hydroxide, has been applied to mono- and diglycerides. Pure mono- and dipalmitins were recovered quantitatively. A commercial glyceryl dioleate was analyzed by this method and the A.O.C.S. pyridine-acetic anhydride procedure with similar results.     

The incorporation of14C-glycerol into different species of diglycerides and triglycerides in rat liver slices

The relative rates of de novo synthesis of species of diglycerides and triglycerides from¹⁴C-glycerol were examined in rat liver slices. Diglycerides containing one or two double bonds per molecule and triglycerides containing four or more double bonds per molecule represented 70% and 60% respectively of the newly synthesized diglycerides and triglycerides. The newly synthesized triglycerides were more unsaturated than the endogenous triglycerides. Our results suggest that a nonrandom synthesis of species of diglycerides occurred followed by an almost random utilization of the various diglyceride species for the biosynthesis of triglycerides.     

Synthesis of mono- and diglycerides in water-in-oil microemulsions

Enzyme-catalyzed esterification was carried out in single-phase, oil-continuous microemulsions. The lipozyme was solubilized, along with glycerol and water, in the aqueous core of water/diethylhexyl sodium sulfosuccinate/hydrocarbon microemulsion system. Upon addition of fatty acid, mono- and diglycerides were formed, due to the esterification reaction taking place at the interface of the droplets in the microemulsion. The initial rate of conversion of oleic acid increases with oil chainlength of the continuous phase whereas final conversion is maximum for hexane. The conversion of stearic acid is 30% whereas conversion of oleic acid is 70%. The percent conversion of various fatty acids in the same continuous medium increases with fatty acid chainlength. The oleic acid/glycerol ratio is an important parameter for optimum conversion of oleic acid into glycerides. The yield can be increased by subsequent addition of glycerol after equilibrium is reached. High-performance liquid chromatography analysis of samples from microemulsions shows the presence of mono- and diglycerides. Possible mechanisms for the abovementioned effects are discussed.     

Differential incorporation of acetate and glucose in maturing douglas fir seeds

In order to discern the synthetic pathways of lipids in coniferous seeds, decoated maturing Douglas fir seeds were incubated with 2-¹⁴C-acetate and ul-¹⁴C-glucose for 3 hr in phosphate buffer at pH 6.0. About 52% of incorporated acetate was found in lipids, but only 9% of the absorbed glucose was converted to lipids. Distribution of incorporated radioactivity in lipid classes was similar for both substrates, 45% in polar lipids, 22% in diglycerides, 15% in triglcerides, 7% in sterol esters, 4% in each of fatty acids and monoglycerides, and 3% in sterols. High specific activity was found in free fatty acids, diglycerides, monoglycerides and polar lipids indicating a rapid turnover of the intermediates for reserve triglycerides and structural polar lipids. Degradation analyses showed that 50% of incorporated acetate and glucose in lipids were in fatty acid moiety. Acetate contributed more in sterols and other unsaponifiables than in glycerol, and the reverse was true for glucose. All the data indicated that acetate is the direct precursor of fatty acids and sterols. General synthetic pathways prevail in fir seeds. Methods for complete analysis of chemical and radio-chemical composition were presented and results discussed. Technical paper 2346 Oregon Agricultural Experiment Station.     

Fatty acid and cholesterol synthesis from specifically labeled leucine by isolated rat hepatocytes

Hepatocytes isolated from female rats meal-fed a high-glucose diet were incubated in Krebs-Henseleit bicarbonate medium containing 16.5 mM glucose,³H₂O, and¹⁴C-labeled amino acids (−)-Hydroxycitrate depressed the incorporation of³H₂O and [¹⁴C] alanine into fatty acids and cholesterol. Incorporation of [U-¹⁴C] leucine into lipids was not affected but incorporation of³H₂O into lipids was decreased significantly by (−)-hydroxycitrate. (−)-Hydroxycitrate depressed the incorporation of radioactivity from [2-¹⁴C]leucine into fatty acids and cholesterol by 61 and 38%, respectively, and stimulated the incorporation of radioactivity from [4,5-³H]leucine 35 and 28%. As [2-¹⁴C]leucine labels the acetyl-CoA pool and [4,5-³H]leucine labels the acetoacetate pool, it was concluded that mitochondrial 3-hydroxy-3-methylglutaryl-CoA is not incorporated intact into cholesterol, and that acetoacetate can be activated effectively in the liver cytosol for support of cholesterol and fatty acid synthesis.     

Isolation and purification of perfluorodecanoic and perfluorooctanoic acids from rat tissues

A procedure for the extraction, separation, and isolation of perfluorodecanoic and perfluorooctanoic acids from biological samples is described. The use of conventional lipid extraction procedures leads to substantial loss of the perfluorinated fatty acids added to tissue. The presence of sulfuric acid in aqueous saline during phase partitioning is essential for the recovery of perfluorodecanoic and perfluorooctanoic acids in the organic phase following their extraction from tissue. The perfluorinated fatty acids are co-eluted with simple lipids from silica gel columns using diethyl ether/trifluoroacetic acid (100∶1, v/v). Simple lipids are separated by thin layer chromatography. By substituting trifluoroacetic acid for acetic acid in the developing solvents, perfluorodecanoic and perfluorooctanoic acids migrate with other free fatty acids.     

Studies of differential turnover of palmitoyl and stearoyl species of glycerophosphatides using labeled unsaturated acids

Normal and bile fistula rats were injected with 1-¹⁴C-linoleate and arachidonate as albumin complex and the glycerides and glycerophosphatides of the liver and bile were isolated at various time intervals. The distribution of radioactivity among the individual molecular species was determined by thin layer and radio gas chromatography and specific enzymic hydrolyses. At 30 min after administration of linoleate 30% of the radioactivity in liver was in lecithins and 8% in cephalins, while at 120 min 48% was in lecithins and 16% in cephalins. After arachidonate, 58% and 64% of the counts were in lecithins and 12% to 13% in the cephalins at the above periods of sampling. The specific activity of the palmitoyl linoleoyl lecithins and cephalins was two to three times higher than that of the corresponding stearoyl linoleoyl species, which was of the same order but much lower magnitude than found previously for lecithins using labeled phosphate and choline. The palmitoyl and stearoyl species of arachidonoyl lecithins possessed equal specific activities, in sharp contrast to previous findings with radioactive phosphate, which showed a 12 times higher specific activity for the palmitoyl arachidonate. The palmitoyl arachidonoyl cephalins had two to three times greater specific activity than the corresponding stearoyl species in agreement with previous work using labeled phosphate. The distribution of radioactivity suggests that the arachidonate was incorporated into the lecithins largely via acyl transfer, while the linoleate contributed to both acyl transfer and de novo synthesis. Interpretation of the mechanism of uptake of these acids into the cephalins awaits further studies. Presented in part at the Federation of American Societies for Experimental Biology Meeting, Atlantic City, April 1970.     

Relative incorporation of acetate and glucose into glycerides ofGlomerella cingulata

Cultures of the fungusGlomerella cingulata were pulse-labeled for 30 min with 1-¹⁴C-acetate at ages from 2 to 12 days old. The greatest incorporation into triglycerides and phosphoglycerides occurred in the youngest cultures, but more of the radioactive acetate was diverted to the phosphoglycerides than to the triglycerides in these cultures. In another series of experiments the incorporation of 1-¹⁴C-acetate or 2-¹⁴C-glucose into the triglycerides and phosphoglycerides of the fungus was investigated at 15-minute intervals for 1 hr. Hydrolysis of the two classes of glycerides revealed some labeling of glycerol from acetate; but, not surprisingly, acetate was incorporated into the fatty acid moieties to a greater extent than into the glycerol moieties, and there was relatively greater incorporation of 2-¹⁴C-glucose into glycerol than into fatty acids. Some relationships of these results to the growth and development ofG. cingulata are suggested, and implications relative to control mechanisms are pointed out.     

Level of dietary protein intake affects glucose turnover in endurance-trained men

Background

To examine the effects of higher-protein diets on endogenous glucose metabolism in healthy, physically active adults, glucose turnover was assessed in five endurance-trained men (age 21.3 ± 0.3 y, VO_2peak 70.6 ± 0.1 mL kg^-1 min^-1) who consumed dietary protein intakes spanning the current dietary reference intakes.

Findings

Using a randomized, crossover design, volunteers consumed 4 week eucaloric diets providing either a low (0.8 g kg^-1 d^-1; LP), moderate (1.8 g kg^-1 d^-1; MP), or high (3.6 g kg^-1 d^-1; HP) level of dietary protein. Glucose turnover (Ra, glucose rate of appearance; and Rd glucose rate of disappearance) was assessed under fasted, resting conditions using primed, constant infusions of [6,6-²H₂] glucose. Glucose Ra and Rd (mg kg^-1 min^-1) were higher for MP (2.8 ± 0.1 and 2.7 ± 0.1) compared to HP (2.4 ± 0.1 and 2.3 ± 0.2, P < 0.05) and LP (2.3 ± 0.1 and 2.2 ± 0.1, P < 0.01) diets. Glucose levels (mmol/L) were not different (P > 0.05) between LP (4.6 ± 0.1), MP (4.8 ± 0.1), and HP (4.7 ± 0.1) diets.

Conclusions

Level of protein consumption influenced resting glucose turnover in endurance athletes in a state of energy balance with a higher rate of turnover noted for a protein intake of 1.8 g kg^-1 d^-1. Findings suggest that consumption of protein in excess of the recommended dietary allowance but within the current acceptable macronutrient distribution range may contribute to the regulation of blood glucose when carbohydrate intake is reduced by serving as a gluconeogenic substrate in endurance-trained men.     

Stimulatory effect of glucose upon triglyceride synthesis from acetate,decanoate, and palmitate by mammary gland slices from lactating mice

Slices prepared from the mammary glands of lactating mice incorporate only small amounts of (1--14C) acetate, (1--14C) decanoate, or (1--14C) palmitate into lipids. However, when glucose is added to the incubation medium, fatty acid incorporation is stimulated-13-fold from acetate, 17-fold from decanoate, and 2-fold from palmitate. Over 90% of the -14C activity in the lipid fraction is in triglycerides. Analysis of fatty acids in the triglycerides showed that almost all of the decanoate and the palmate were incorporated as intact molecules, while acetate yielded acids of varying chain lengths. The glucose stimulation of triglyceride synthesis is not solely due to its effect upon chain elongation but also could involve glyceride-glycerol availability, as well as other unknown factors. However, from measurements of the amounts of glycerol 3-phosphate in the tissue incubated in the presence of palmitate, it would appear that glyceride-glycerol availability is not rate limiting in triglyceride synthesis.     

Fractionation and analysis of rat liver14CH3-lecithins labeled in vivo

The¹⁴CH₃-lecithins were biosynthesized by normal adult rats injected with¹⁴CH₃-methionine. About 20% of the dose was incorporated into liver lecithins. The¹⁴CH₃-lecithins were isolated by thin-layer chromatography. Separation of lecithins on AgNO₃-treated silica gel yielded lecithins containing a saturated fatty acid in combination with mainly one unsaturated fatty acid, namely, oleic, linoleic, eicosatrienoic, or arachidonic acid. These fractions were eluted with methanolic choline chloride, which prevented elution of AgNO₃. The lecithins, after extraction into petroleum ether, were analyzed for radioactivity and for fatty acid composition. Yields were about 75%, based upon fatty acids or radioactivity applied to the plate. Specific activities differed sharply between the fractions, and arachidonoyllecithins had the highest specific activity. The sum of the activities contributed by each of the fractions agreed well with the specific activity of total lecithins, indicating the recovery of intact lecithin molecules. The recovery of intact molecules allows this procedure to be used with lecithins containing any isotopic labels. The high specific activity of arachidonoyl-lecithins relative to the other fractions indicates a high degree of specificity in the metabolic reactions which lead to the formation of rat liver lecithins.     

Behavior of diglycerides and conjugated fatty acid triglycerides in reverse-phase chromatography

The behavior of conjugated fatty acid triglycerides and diglycerides on reverse-phase chromatography was studied. Trieleostearin is a geometric isomer of trilinolenin. The conjugated double bond arrangement in trieleostearin enhances its hydrophobic interaction with the stationary phase and causes it to be eluted later than trilinolenin. In separation of “critical pairs” of tri- and diglycerides, diglycerides elute later than triglycerides due to the longer fatty acid constituent. Position isomers of 1,2- and 1,3-diglycerides can be separated by reverse-phase high-performance liquid chromatography.     

Determination of mono- and diglycerides in palm oil,olein and stearin

Partial glycerides are important constituents of palm oil and can have significant effects on the physical properties of products containing palm oil or on the fractionation of palm oil. A method is described for their routine determination in palm oil. By analysis of 28 weekly composite samples of crude palm oil the following results were obtained: free fatty acids, mean=3.76%, range 2.4 to 4.5%; monoglycerides, mean=0.28%, range 0.21 to 0.34%; diglycerides, mean=6.30%, range 5.3 to 7.7%. During detergent fractionation of palm oil, diglycerides concentrate in the palm olein, but monoglycerides concentrate in the palm stearin. Palm fatty acid distillate was found to contain approximately 3% each of mono- and diglycerides. Because the refining and fractionation processes are continuous in the refinery, it is not possible to follow a single identifiable batch of crude palm oil through the refinery. To circumvent this problem, crude palm oil, stearin and olein from the refinery were bleached and steam refined in the laboratory and the partial glyceride contents determined at each stage of processing. Except for fractionation, the content of glycerides did not change during processing. For oil, olein and stearin, monoglycerides were reduced significantly both after bleaching and after steam refining.     

Studies on long chaincis- andTrans-acyl-CoA esters and acyl-CoA dehydrogenase from rat heart mitochondria

The β-oxidation of long chain fatty acids was investigated in a preparation of rat heart mitochondria. The acyl-CoA esters of thecis andtrans isomers of Δ9-hexadecenoic, Δ9-octadecenoic, Δ11-eicosenoic, and Δ13-docosenoic acids were prepared. Rates of the acyl-CoA reaction were determined with an extract from rat heart mitochondria. The apparent Michaelis constant (K_m) and maximum velocity (V_max) were calculated for each substrate. In general, apparent V_max values decreased with increasing chain length of the monoenoic substrates. Reduced activity of acyl-CoA dehydrogenase with long chain acyl-CoA esters could have contributed to accumulation of lipids in hearts of rats fed diets containing long chain fatty acids. Presented in part at the 19th Annual Meeting of the Canadian Federation of Biological Societies, Halifax, Canada, June 1976.     

Geometrical and positional isomer content of the monounsaturated fatty acids from various rat tissues

The percentage distribution of the geometrical and positional isomers in the hexadecenoates and octadecenoates isolated from triglycerides, phosphatidylch olines, and phosphatidylethanolamines of brain, heart, kidney, liver, lung, muscle, spleen, and adipose tissues from normal rats maintained on a laboratory diet has been determined. All of the octadecenoates and most of the hexadecenoates from the lipid classes of all the tissues consisted of more than 95%cis isomers. Generally, palmitoleic was the predominant hexadecenoate, but many of the tissue phospholipids contained relatively high percentages of the Δ6 and Δ7 isomers. Oleate and vaccenate were the predominant octadecenoates in all tissues. Except for brain and adipose tissues, the oleate to vaccenate ratios differed for each lipid class, as well as between most tissues. In contrast to the monoenes of the phospholipids, the triglyceride monoenes exhibited the same approximate: percentage composition; percentage of geometrical isomers; and percentage distribution of hexadecenoate and octadecenoate positional isomers. These data add to our basic information about the percentage distribution of geometrical and positional isomers of naturally occurring unsaturated fatty acids in the major lipid classes of various normal tissues. Some new concepts were advanced as possible explanations to some of the observed positional isomer distributions.     

Incorporation of conjugated linoleic acid and vaccenic acid into lipids from rat tissues and plasma

The objective of this study was to determine the incorporation of conjugated linoleic acid (CLA) into triacylglycerols (TAG) and phospholipids (PL) of tissues and plasma, and to interpret the role of dietary‐derived vaccenic acid (VA) in increasing the tissue content of CLA (c9,t11) and the influence on the fatty acid profile. We fed five groups of rats semi‐purified diets with varying levels of CLA and VA: control butter with low CLA (c9,t11) and VA; control butter added 5% CLA (c9,t11); control butter added 5% Tonalin [equal amount of CLA (c9,t11) and CLA (t10,c12)]; control butter added 5% VA; butter with high CLA (c9,t11) and VA (H‐CLA), for 3 weeks. The highest incorporation of CLA (c9,t11) was found in adipose tissue, and the lowest was observed in liver. Low intake of CLA (c9,t11) combined with high intake of VA resulted in a higher incorporation of CLA (c9,t11) in tissues due to the conversion of VA to CLA (c9,t11), compared to feeding CLA (c9,t11) without VA. However, in enterocytes, the proportion of CLA (c9,t11) was low after feeding VA, indicating no or only a minor conversion of VA to CLA (c9,t11) in the intestine. The incorporation of CLA (t10,c12) into TAG from plasma and tissues was generally much lower than that of the CLA (c9,t11) isomer, except in the enterocyte TAG, which had similar proportions of the two isomers.     

Occurrence of long and very long polyenoic fatty acids of the n-9 series in rat spermatozoa

Dietary deficiency of essential fatty acids of the n−3 and n−6 series is known to promote a compensatory increase in polyenoic fatty acids of the n−9 series in the lipids of mammalian tissues. In the present study long-chain n−9 polyenes were found to be normal components of the epididymis and especially of sperm isolated from that tissue, in healthy, well-fed, fertile rats maintained on essential fatty acid-sufficient diets. The n−9 polyenes occurred in large concentrations in the choline glycerophospholipids (CGP), the major phospholipid class of spermatozoa in epididymalcauda, and were highly concentrated in plasmenylcholine, the major subclass of CGP. The uncommon polyene 22∶4n−9 was found in the highest proportion, followed in order of relative abundance by 22∶3n−9, 20∶3n−9 and 24∶4n−9. These polyenes were probably derived from oleate (18∶1n−9) in much the same way as long-chain polyenes of the n−6 and n−3 series are derived from linoleate (18∶2n−6) and linolenate (18∶3n−3), respectively.     

Effects of oxidative stress on glycerolipid acyl turnover in rat hepatocytes

Lipid peroxidation was induced in freshly isolated rat hepatocytes by incubation in the presence of Fe³⁺, resulting in accumulation of thiobarbituric acid reactive substances. Analysis of lipid classes revealed that the levels and fatty acid compositions of the two major phospholipids, phosphatidylcholine (PC) and phosphatidylethanolamine (PE), remained unchanged but the levels of triacylglycerols (TAG) were significantly reduced, and some of their polyunsaturated fatty acids were selectively lost as the result of oxidant treatment. Acyl turnover in PC and PE as determined by ¹⁸O incorporation from H₂ ¹⁸O-containing media remained largely unchanged during oxidant treatment, while some increased turnover of the saturated fatty acids in TAG was observed. We hypothesize that constitutive recycling of membrane phospholipids rather than selective in situ repair eliminates peroxidized species of PC and PE, TAG could serve as an expendable fatty acid reserve, providing a limited but very dynamic pool of polyunsaturated fatty acids for the resynthesis of phospholipids.