Characteristics of fatty acid esterification by homogenates of bovine mammary tissue

Fatty acid esterifying activity of homogenates of bovine mammary tissue was associated with the particulate fraction of the cell, was strongly dependent upon ATP, CoA,d,l-glycerol-3-phosphate, and Mg²⁺, and was stimulated by NaF, dithiothreitol and bovine serum albumin. The system made phospholipids, mono-, di- and triglycerides but did not esterify butyrate. The inability to form greater than 58% triglyceride suggested some factor(s) was limiting the acylation of di-to triglyceride. The results were consistent with glyceride synthesis by the α-glycerophosphate pathway. This work is part of a Ph.D. Thesis submitted by E.W. Askew to the Dairy Department and Institute of Nutrition, Michigan State University. Published with approval of the Director of the Michigan Agricultural Experiment Station as Journal Article No. 5101.     

Acyl specificity in glyceride synthesis by lactating rat mammary gland

We have investigated the possibility that the nonrandom association of fatty acids in rat milk triglycerides results from specificity of the acyl transferases in the glycerolphosphate pathway. Subcellular fractionation of lactating rat mammary gland revealed that the microsomal fraction was the most active in acylation of 3-sn-[U-¹⁴C] glycerolphosphate with various acyl-CoA's. The major products were diacylglycerolphosphate and diglyceride; no monoacylglycerolphosphate was detected. Maximum rate of acylation occurred at or below the critical micelle concentration for each acyl-CoA, indicating that only the monomeric substrate molecules were acceptable by the enzyme system. The observed acyl specificity, 16∶0>18∶0≏14∶0>12∶0>10∶0>8∶0 is consistent with the concept that, in general, milk triglycerides are synthesized by insertion of a short or medium chain fatty acid into a long chain diglyceride.     

Preferential labeling of phosphatidylcholine during phospholipid synthesis by bovine mammary tissue

Bovine mammary cells and tissue synthesize de novo the classes of phospholipids, found in mammary tissue and milk, from various precursor molecules. Several short term experiments were carried out in vitro, using labeled precursors, i.e., 1-¹⁴C-fatty acids; 2-¹⁴C-acetate; U-¹⁴C-glycerol; 1,2-¹⁴C-choline; 1,2-¹⁴C-ethanolamine; 2-¹⁴C-serine; and Me-¹⁴C-methionine. All the phospholipid classes were labeled. The specific activity of tissue phosphatidylcholine was consistently three to six-fold greater than that of phosphatidylethanolamine. The results indicated that stepwise methylation of phosphatidylethanolamine with labeled methyl group of methionine was occurring to a minor extent, as was a negligible amount of choline exchange. Serine was incorporated into phosphatidylserine and sphingomyelin. Significant quantities of labeled phosphatidylserine were decarboxylated to phosphatidylethanolamine. Apparently phosphatidylcholine was synthesized de novo from choline via phosphorylcholine and CDP-choline. Based on the present observations and other data, it is suggested that there may be two pools of phosphatidylcholine in lactating mammary cells.     

Fatty acid synthetase of bovine mammary: Properties and products

Fatty acid synthetase purified (20 times) from lactating bovine mammary tissue had an approximate mot wt of 485,000. The enzyme had a high content of acidic and hydrophobic amino acid residues; 62±4 sulhydryl groups and one 4′ phosphopantetheine residue/mole of enzyme. The enzyme was relatively stable when stored (3 mg/ml) in potassium phosphate buffer (250 mM), containing dithiothreitol (5 mM) at −5 C or at −30C or as a lyophilized powder at −30 C. Preincubation at 37 C in presence of dithioltreitol (5 mM) was necessary for obtaining maximum activity at the optimum pH of 6.8. Maximum specific activity of the isolated enzyme was 55 nmoles acetyl-coenzyme A min⁻¹mg⁻¹ incorporated A min⁻¹mg⁻¹ incorporated into fatty acids. Butyryl-coenzyme A or acetyl-coenzyme A (30μM), malonyl-coenzyme A (65 μM), and nicotinamide adenine dinucleotide phosphate, reduced form (300 μM) were required for optimum fatty acid synthesis. Malonyl-coenzyme A decarboxylase activity (5 nmoles min⁻¹mg⁻¹) associated with the enzyme permitted fatty acid synthesis in the presence of nicotinamide adenine dinucleotide phosphate, reduced form and malonyl-coenzyme A. The enzyme utilized acetyl-coenzyme A, butyryl-coenzyme A, and hexanoyl-coenzyme A as primers, with butyryl-coenzyme A giving the maximum rate of fatty acid synthesis. Apparent Km values of 22,6.7, 3, 22, and 20 μM were obtained for malonyl-coenzyme A, acetyl-coenzyme A, butyryl-coenzyme A, hexanoyl-coenzyme A, and nicotinamide adenine dinucleotide phosphate, reduced form, The fatty acid synthetase was inhibited by N-ethylmaleimide, iodoacetamide, and progressively inhibited by increasing concentrations of long chain acyl-coenzyme A, i.e. palmityl-coenzyme A and myristyl-coenzyme A. This inhibition was relieved by bovine serum albumin or β-lactoglobulin (3 mg/ml). Palmitic acid was the major product of bovine mammary fatty acid synthetase. However, small amounts of fatty acids, 4∶0–14∶0 inclusive, also were synthesized. The pattern of fatty acids was altered by varying malonyl-coenzyme A to acetyl-coenzyme A ratios and by increasing the enzyme levels in the assays. At high concentrations of enzyme (0.5 mg/ml), greater amounts of short and medium chain fatty acids were generated.     

Factors affecting fatty acid oxidation in bovine mammary tissue

Oxidation of fatty acids was studied in bovine mammary tissue slices in order to evaluate their potential contribution to energy metabolism. Rates of fatty acid oxidation decreased with increasing chain length: acetate>octanoate>palmitate or oleate. Rates of oxidation of long chain, but not short chain, fatty acids increased over time, which could not be explained by carnitine palmitoyltransferase (CPT) activity. This phenomenon is not an artifact of the incubation system or caused by substrate solubility, as rates of palmitate oxidation were constant in rat kidney cortex slices. Preincubating mammary tissue with or without unlabeled palmitate showed that increasing rates of palmitate oxidation is not caused by use of endogenous fatty acids. Palmitate at 0.26 mM, equivalent to arterial fatty acid concentration, gave maximal rates of oxidation. The β-oxidation enzymes may restrict fatty acid oxidation as oxidation of [1-¹⁴C] palmitate exceeded that of [U-¹⁴C] palmitate. Acetate inhibited palmitate oxidation (75%) but not esterification, suggesting that acetate inhibits palmitate oxidation by substrate competition at the mitochondrial level or via malonyl-CoA inhibition of CPT. Glucose inhibited palmitate oxidation (67%) and stimulated esterification. Low palmitoyl-CoA levels would favor glyceride synthesis over oxidation, since the apparent K_m for palmitoyl-CoA, of the glycerol-3-phosphate acyltransferases is lower than that for CPT. Thus, glucose presumably diverts palmitate from oxidation to glycerolipids. Clofenapate, a glyceride synthesis inhibitor, decreased triacylglycerol formation, and marginally increased palmitate oxidation. We estimated that long chain fatty acids can potentially account for 6–10% of the oxidative metabolism of mammary tissue. Published with approval of the Director of the Agricultural Experiment Station as Journal Article No. 9292.     

Fatty acid and glyceride composition of jubaea spectabilis palm oil

Determination of the fatty acid distribution in three samples of oil from the kernels of the palmJubaea spectabilis shows that the uniquely low melting point of the oil is due to a higher content of shorter chain fatty acids than other members of the subclass Cocoideae.     

Composition and synthesis of three higher ganglioside homologs in bovine mammary tissue

Three higher gangliosides were identified as constituents of bovine mammary gland. The structures of these three gangliosides were shown to be ceramide-glucose-galactose-(sialic acid)-N-acetylgalactosamine-galactose, ceramide-glucose-galactose-(sialic acid)₂-N-acetylgalactosamine, and ceramide-glucose-galactose-(sialic acid)₂-N-acetylgalactosamine-galactose These gangliosides accounted for only a small fraction (<20%) of the lipid-bound sialic acid in mammary gland. While fatty acids with even carbon numbers from C₁₄ to C₂₆ were predominant in these gangliosides, they also contained C₂₃ and C₂₅ fatty acids. Mammary gland Golgi apparatus-rich fractions had all glycosyltransferases required for synthesis of these gangliosides starting with ceramide.     

Fatty acid desaturase specificity in tetrahymena

The ciliate,Tetrahymena, was provided a supplement of the fatty acid [1-¹⁴C] 18∶2Δ6,9. After a period of growth the cells were claimed, the lipids extracted, the polar lipids recovered and the mild alkali-labile fatty acid methyl esters generated. The fatty acids were resolved by high pressure liquid chromatography (HPLC), the 18∶3Δ6,9,12 (γ-linolenic acid) was recovered and its identity verified by high pressure liquid chromatography (HPLC), gas liquid chromatography (GLC), hydrogenation and oxidation. Fifty-three percent of the cell-associated label was found in γ-linolenic acid; thus, a Δ12 fatty acid desaturase converts the 6,9 octadecadienoic acid to the 6,9,12 derivative. No carboxyl or methyl terminus restriction appears on Δ9 monoenoic or dienoic fatty acid desaturation in this cell as is found in higher plants and animals.     

Fatty acid desaturation by mammary gland microsomes from lactating mice

Microsomal fractions obtained from lactating mouse mammary glands catalyze the desaturation of myristic, palmitic, and stearic acids in the presence of adenosine 5′-triphosphate, coenzyme A, and nicotinamide adenine dinucleotide, reduced form. The products of this reaction are the corresponding monoenoic fatty acids, myristoleic, palmitoleic, and oleic acids respectively. Desaturase, activity was greater with stearate as substrate than with either palmitate or myristate. Palmitate was desaturated at a rate ca. 50% greater than that of myristate. Although both nicotinamide adenine dinucleotide, reduced form and nicotinamide adenine dinucleotide phosphate, reduced form served as electron donors, the former was superior in this regard. Desaturase activity was not influenced by the addition of glycerol 3-phosphate to the incubation medium. However, the presence of this compound did affect the type of lipid formed. In the absence of the added glyceride-glycerol precursor, the major products were phospholipids, whereas in its presence they were triglycerides.     

Monoacyl-sn-glycerol 3-phosphate acyltransferase specificity in bovine mammary microsomes

The acyl-CoA∶:acyl-sn-glycerol 3-phosphate acyltransferases located in the microsomal fraction of lactating bovine mammary tissue show a preference for palmityl-CoA particularly above the apparent Km values of the acyl acceptors. Using saturating levels of monopalmityl-sn-glycerol 3-phosphate, the order of acylation was palmityl->myristyl-> oleyl->stearyl->linoleyl-CoA. Apparent Km values for monopalmityl- and monooleyl-sn-glycerol 3-phosphate with palmityl-CoA as donor were 16 and 13μM, respectively, while the Km values for palmityl-CoA with these two acyl acceptors were 5 and 5.2μM, respectively. The apparent Vmax values for the palmityl acceptor and donor were 25 and 30 nmol/min/mg protein. Phosphatidic acid was the principal product. The inclusion of magnesium in the assay depressed activity while the addition of ethylenediaminetetraacetate doubled the rate of acylation.     

Fatty acid composition and glyceride structure of piglet body fat from different sampling sites

Pigs were slaughtered at 16 weeks of age, and fat samples were obtained from outer and inner shoulder, outer and inner loin, and kidney. Fatty acid composition and glyceride type of distribution were determined. Glyceride structure was determined by pancreatic lipase hydrolysis. There were highly significant differences in fatty acid composition between sites. Fatty acids containing less than 18 carbon atoms were preferentially incorporated in the internal positions of the glycerides. The content of saturated fatty acids and fatty acids containing less than 18 carbon atoms at the 2-position of shoulder and loin glycerides was higher than in kidney glycerides. Differences in glyceride types were noted between sites.     

脂肪酸甘油酯磺酸盐的合成

以α-磺酸脂肪酸甲酯和甘油为原料,经酯交换及中和反应合成了脂肪酸甘油酯磺酸盐。得到较佳的反应条件为:四氯化碳作溶剂,n(α-磺酸脂肪酸甲酯)∶n(甘油)=1∶5,75℃反应4 h,高效液相色谱分析α-磺酸脂肪酸甲酯的转化率可达66.33%。采用红外光谱和质谱确证了脂肪酸甘油酯磺酸盐的结构,结果表明,反应产物中除了含有单脂肪酸甘油酯磺酸盐外,还含有脂肪酸甲酯磺酸盐和二脂肪酸甘油酯磺酸盐。     

酶催化合成碳酸甘油酯的研究

用脂肪酶溶剂相催化甘油和碳酸二甲酯反应合成了碳酸甘油酯,并以单因素分析法研究了各反应条件对此酯交换反应的影响。pH=7,60℃反应23h后,甘油的转化率最高达85％。     

Fatty acid and sterol synthesis by rat small intestine in vitro

Slices of rat jejunum were incubated with [2-¹⁴C]pyruvate, [1-¹⁴C]acetate, or [³H]H₂O to determine lipogenic activity. Under all conditions studied, pyruvate acted as a better precursor than acetate for fatty acid synthesis but not for the synthesis of sterol. Exogenous glucose significantly (P≤0.05) increased the conversion of both pyruvate and acetate to fatty acids. By contrast, fasting resulted in a decrease (p≤0.05) in lipogenic activity. The highest levels of lipogenesis were observed when [³H]H₂O + glucose at a concentration of 20 mM was used. From such experiments, the absolute rate of fatty acid synthesis in the tissue preparation was calculated: 734±54 nmoles acetyl units incorporated into fatty acids/g tissue/hr.     

Acyl-CoA acyl-sn glycerol-3 phosphorylcholine acyl transferase of bovine mammary tissue

Microsomal material from lactating bovine mammary tissue possess very active acyl-CoA:1-acyl-sn-glycerol-3-phosphorylcholine acyl transferase (EC2.3.1.-1). Oleyl-CoA was the preferred substrate and rates of 6.3, 6.4, 1.8, 21.0, and 0.1 nmoles acylated/min/mg were obtained for myristyl-, palmityl-, stearyl-, oleyl- and linoleyl-CoA, respectively, when 1-oleyl-sn-glycerol-3-phosphorycholine was used compared to 5.2, 4.1, 1.0, 3.5, and 0.2 nmoles/min/mg with 1-palmityl-sn-glycerol-3-phosphorylcholine as acyl acceptor. Apparent Km values of 4.5 and 5.2 μM for oleyl-CoA and 6.5 and 4.6 μM for palmityl-CoA were obtained with 1-oleyl- and 1-palmityl-sn-glycerol-3-phosphorylcholine, respectively. The Km values of 1-oleyl-sn-glycerol-3-phosphorylcholine were 20 and 50 μM using oleyl-CoA and palmityl-CoA as acyl donors. The possible involvement of this enzyme in membrane dynamics during lipid secretion is discussed.     

The lipid composition of microsomal preparations from lactating bovine mammary tissue

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

Isomerization of L-glyceraldehyde to dihydroxyacetone during glyceride synthesis by rat liver microsomes

L-glyceraldehyde is converted to phosphatidic acid by the action of rat liver microsomal enzymes and glycerol kinase in the presence of fatty acid, ATP, CoASH and NADH. L-glycerol 3-phosphate is not an intermediate in this synthesis since microsomes in the presence of NADH neither reduce L-glyceraldehyde nor, in the additional presence of glycerol kinase and ATP, convert it to L-glycerol 3-phosphate. However dihydroxyacetone is produced when L-glyceraldehyde is incubated with microsomes. This was shown enzymatically by the subsequent conversion to dihydroxyacetone phosphate which was confirmed by the oxidation of NADH in the presence of glycerol 3-phosphate dehydrogenase. Isomerization of L-glyceraldehyde and the synthesis of dihydroxyacetone phosphate may be one of several possible mechanisms in the conversion of the triose to either glucose or glycerideglycerol which has been reported to occur in tissue. Presented at the AOCS Meeting, Houston, May 1971.     

Fatty acid distribution in the bovine pre- and postpartum testis

Testes from fetuses, calves, bulls and recently castrated animals were analyzed for total lipids, lecithin, cephalin, triglycerides, diglycerides, cholesteryl esters and cholesterol. Total lipids increase somewhat with age, but in the castrated animal the increase is more marked. Phospholipid content increases with age, but decreases in the castrated animal. Cholesterol decreases and triglyceride increases after birth and in the castrated animal. Polyunsaturated acids increase with age in all lipid classes. Eicosatrienoic acid is more abundant in fetal testicular lipids than in testes removed after birth. In the castrated testis there is a general decrease in the unsaturated fatty acids. Acids of the ω6 family are the predominant polyunsaturated acids and increase somewhat with age in all lipids. The ω3 family of polyunsaturated acids appears mostly toward the end of fetal life and increases after birth. Acids of the linoleate family reach approximately 25% of total acids in most lipid classes at maturity whereas the ω3 acids range from 1 to 9%. Presented at the American Dairy Science Association Meeting, Lexington, Ky., June 1965.     

Hormonal regulation of medium chain fatty acid synthesis by mouse mammary gland explants

Explants of pregnant mouse mammary tissue were cultured in media supplemented with various hormones. During the last few hours of culture, explants were labeled with [¹⁴C] acetate. Fatty acid synthesis by the tissue was analyzed using reverse phase thin layer chromatography, and incorporation of radioactivity into the medium chain fatty acid (MCFA) fraction was calculated as total MCFA per mg tissue fresh weight and as a percentage of the total fatty acid radioactivity (%MCFA). After 48 hr of culture, explants had an elevated %MCFA synthesis only when exposed to media containing insulin, cortisol and prolactin, confirming previous reports. Increasing doses of prolactin (maximal response at 300 mg/ml) caused a coordinate rise in both %MCFA synthesis and total MCFA synthesis per mg fresh weight. Epithelial cells isolated from explants after culture for 48 hr with insulin, cortisol and prolactin showed an elevated %MCFA synthesis compared to cells from explants cultured with insulin and cortisol alone, indicating that hormonal stimulation of mammary explants causes a change in the chain length of fatty acids synthesized by mammary epithelial cells. The specificity of the explant response to prolactin was tested with other hormones: FSH and calcitonin had no effect, whereas bovine growth hormone showed activity only at high concentration. Progesterone and 17β-estradiol also had no effect. The analysis of MCFA synthesis provides another means of (a) assessing hormonal action upon mammary tissue, and (b) evaluating the biological activity of prolactin.