Fatty acid synthetase from the Harderian gland of guinea pig: biosynthesis of methyl-branched fatty acids

Fatty acid synthetase was isolated from guinea pig Harderian gland. This enzyme complex exhibited a unique character as compared with the fatty acid synthetase from the liver of the same animal. The former enzyme produced many odd-numbered and methyl-branched fatty acids in the presence of methylmalonyl-CoA. These fatty acids are characteristic components of the lipid secreted from this gland. The chemical structure of this lipid has been identified as 1-O-alkyl-2,3-diacylglycerol by previous work from this laboratory (Yamazaki, T., Seyama, Y., Otsuka, H., Ogawa, H., & Yamakawa, T. (1981) J. Biochem. 89, 683-691). Apparent Km values (5 X 10(-6) M) for acetyl-CoA and propionyl-CoA were the same, but the Vmax for propionyl-CoA was much higher than that for acetyl-CoA. The pI value of the fatty acid synthetase from Harderian gland was 5.3, and the molecular weight of the enzyme was 9 X 10(5) daltons. The beta-ketoacyl reductase had pro-S stereospecificity and the enoly reductase had pro-R stereospecificity for NADPH. The results presented in this paper indicate that the fatty acid synthetase from guinea pig Harderian gland can produce a set of fatty acids needed for the synthesis of the lipid secreted from this gland, and that the fatty acid synthetase has a characteristic organ specificity.     

Methods for measurement of fatty acid and cholesterol metabolism

The measurement of dynamic fluxes of lipids (biosynthesis, oxidation, and intermediary metabolism) poses difficult challenges. Two fundamental advances have been made recently for measuring the biosynthesis of lipids, namely mass isotopomer distribution analysis and labeled water incorporation. These techniques have resolved the central methodologic problem in biosynthesis by establishing the true precursor isotope enrichment. Mass isotopomer distribution analysis also permits uncontaminated pulse-chase decay curves and intracellular precursor fluxes to be measured. In contrast, the isotopic measurement of lipid oxidation rates remains unreliable because of the wide variability in the recovery of labeled carbon dioxide.     

New insights into the cytodynamics of the hamster Harderian gland as provided by the bromodeoxyuridine-labelling method

The fourth week of postnatal life is a critical point in the development of the hamster Harderian gland. During this week, cells with large lipid vacuoles (type-II cells) appear in the male gland, marking a morphological sex difference that is notorious in adult animals. The origin and fate of type-II cells are controversial. To gain insight into the mechanisms by which type-II cells become a major cell type in the gland of adult male hamsters, bromodeoxyuridine (BrdU) labelling was used to assess the proliferative activity of both types of glandular cells in 28-day-old animals. To search for possible sex differences in the proliferative activity of this gland, female animals of the same age as the males were also studied. No difference was found in the overall labelling index (BrdU-labelled cells/100 cells) between males (1.8 +/- 0.1%) and females (1.5 +/- 0.1%). In the gland of the males, the specific labelling index of type-II cells (3.4 +/- 0.4%) was significantly higher than that of type-I cells (0.9 +/- 0.2%). Interestingly, the proportion of type-II cells present in the male glands at this age (36.6%) was significantly lower than that of type-I cells. Our results strongly suggest that the proliferation of type-II cells, rather than a continuous differentiation of these cells from preexisting type-I cells, is a major event in the achievement of the mature form of this gland. The results reported here counsel a reappraisal of current theories about the cytodynamics of the hamster Harderian gland.     

Presence of a fatty acid-binding protein in the armadillo Harderian gland

Clinical measurements on gingival indices and morphologic observations were performed in this study to verify the defending mechanism of gingival soft tissue against foreign invasions from the perspective of epithelial adhesion/attachment to implant surfaces in the monkey mandible. The following zones were observed using scanning electron microscopy: (1) plaque zone, suggesting susceptibility of the gingival tissue to bacterial invasion; (2) nude zone, demonstrating indirect adhesion of epithelial cells to the implant surface through the mucous layer and preventing bacterial invasion; and (3) epithelial cell attached zone, having greater bond strength of epithelial cells at the cell-implant interface as compared to cell-cell bonding within the epithelial cell layer. This study suggested that epithelial cell attachment/adhesion may play a dominant role in retaining the successful condition of a dental implant.     

Effects of fatty acids and hormones on fatty acid metabolism and gluconeogenesis in bovine hepatocytes

Primary cultures of hepatocytes were used to study the effects of extracellular oleate concentration and hormones on fatty acid metabolism and gluconeogenesis. Rates of oleate uptake and oxidation to acid-soluble products varied linearly as oleate concentrations increased (0.1 to 2 mM), but rates of triglyceride accumulation varied quadratically. Insulin increased the proportion of oleate that was esterified by 22% without affecting the formation of acid-soluble products. Cells incubated with 2 mM [1-(14)C]oleate for 24 h eliminated 9.6% of the labeled intracellular lipid as acid-soluble products in the following 24 h when no oleate was present during depletion and eliminated 7.7% when 2 mM oleate was present. Insulin reduced labeled triglyceride depletion by 49%. Gluconeogenesis from [2-(14)C] propionate was depressed by 24%, and formation of acid-soluble products was increased by 46% in cells infiltrated with lipid because of previous exposure to 2 mM oleate for 45 h. Rates of gluconeogenesis from propionate were reduced 23% when 2 mM oleate was present during the 3-h period that gluconeogenesis was measured, and the effect was not modified by lipid infiltration. Lipid infiltration influenced hepatic function, and insulin regulated hepatic triglyceride concentration.     

Microparticles of novel branched copolymers of lactic acid and amino acids: preparation and characterization

Macrophage inflammatory protein (MIP)-1alpha and MIP-1beta regulate leukocyte activation and trafficking. To assess the role of MIP-1alpha and MIP-1beta in human inflammation, healthy subjects were studied during experimental endotoxemia with prior administration of ibuprofen, a cyclooxygenase inhibitor, or dimeric p75 tumor necrosis factor (TNF)-alpha receptor, a TNF antagonist; septic patients were also studied. Following endotoxin, blood levels of both MIP-1 molecules rose acutely and fell to baseline by 6 h (P=. 001). While MIP-1 mediates fever in animals independent of cyclooxygenase blockade, in subjects given endotoxin and ibuprofen, MIP-1 levels increased and fever was suppressed. MIP-1 levels were not diminished by inhibiting circulating TNF-alpha in humans. In septic patients, elevated levels of MIP-1alpha and MIP-1beta were detected within 24 h of sepsis and fell in parallel with TNF-alpha and interleukin-6 (P<.01). MIP-1alpha and MIP-1beta increase during acute inflammation but are not associated with fever in endotoxemic humans during cyclooxygenase blockade.     

Medium chain fatty acid metabolism and energy expenditure: obesity treatment implications

Fatty acids undergo different metabolic fates depending on their chain length and degree of saturation. The purpose of this review is to examine the metabolic handling of medium chain fatty acids (MCFA) with specific reference to intermediary metabolism and postprandial and total energy expenditure. The metabolic discrimination between varying fatty acids begins in the GI tract, with MCFA being absorbed more efficiently than long chain fatty acids (LFCA). Subsequently, MCFA are transported in the portal blood directly to the liver, unlike LCFA which are incorporated into chylomicrons and transported through lymph. These structure based differences continue through the processes of fat utilization; MCFA enter the mitochondria independently of the carnitine transport system and undergo preferential oxidation. Variations in ketogenic and lipogenic capacity also exist. Such metabolic discrimination is supported by data in animals and humans showing increases in postprandial energy expenditure after short term feeding with MCFA. In long term MCFA feeding in animals, weight accretion has been attenuated. These differences in metabolic handling of MCFA versus LCFA are considered with the conclusion that MCFA hold potential as weight loss agents.     

Transaminase of branched chain amino acids. XI. Leucine (methionine) transaminase of rat liver mitochondria

An aminotransferase (transaminase) which is active for leucine and methionine, but not for valine or isoleucine, was purified from rat liver mitochondria. The purified preparation appeared homogeneous on polyacrylamide disc gel electrophoresis. Its molecular weight was shown to be 55 000 by gel filtration. It differed from enzyme II (leucine aminotransferase, EC 2.6.1.6) in the supernatant fraction, another transaminase which is also specific for leucine and methionine, in molecular weight, Km values for substrates, electrophoretic mobility, chromatographic behavior and heat stability. From comparison with related transaminases it was concluded to be a new enzyme and named mitochondrial leucine (methionine) transaminase.     

Current concepts of amino acid and protein metabolism in the mammary gland of the lactating ruminant

Milk protein responses to protein nutrition are typically poor and, in part, may be due to the low efficiency (approximately 25 to 30%) of converting dietary N into milk. Posthepatic availability of amino acids (AA) is not limited, yet only approximately 30% is converted into milk. The poor capture of AA by the mammary gland may relate to the imbalanced and uncoordinated timing of nutrient delivery to the gland. The infusion of essential AA improves the efficiency of utilization (0.31); however, further catabolism of AA within the mammary gland suggests that AA transport is not a major limitation. These losses may serve ancillary or functional roles, but mammary oxidation of some AA occurs only when AA extraction exceeds the stoichiometric requirements for milk protein synthesis. Intracellular substrate supply may be more limiting than is the appartus for protein synthesis. Studies utilizing isotope labeling and conducted in vitro and in vivo now suggest that circulating peptides and proteins can serve as sources of perhaps all AA for casein synthesis, but the source of these remains elusive. Constitutive protein and casein turnover contribute significantly (42 to 72%) to mammary protein synthesis. All AA are extensively channeled through an intermediary protein pool or pools that have rapid turnover rates. The AA are then incorporated into casein, which appears to be fixed in association with protein turnover. The mammary gland is a major controller of its metabolism, and the mechanisms of AA extraction and conversion into milk protein are linked to secretion events. Blood flow may be a key point of regulation whereby mechanisms sense and respond to nutrient supply and balance to the gland via alterations in hemodynamics.     

Effects of dietary protein or amino acids in the perfusion medium on amino acid metabolism in perfused adult rat liver

To elucidate the response of amino acid metabolism in the liver to dietary protein and plasma amino acids, the livers of adult rats fed on diet containing 10% (control) or 3% (low-protein) egg protein for 3 weeks were perfused for 120 min with amino acid-free medium in Experiment 1 or medium containing an amino acid mixture simulating that in plasma in Experiment 2. During perfusion about 40% of the free amino acids were lost from the liver in Exp. 1, and about 30% in Exp. 2. During this period, in Exp. 1 the releases of free amino acids and urea into the medium were 140 mumol and 2.52 mg, respectively, in the control group and 207 mumol and 1.10 mg respectively, in the low-protein group. Thus release was greater than decrease in free amino acids in the liver. Essential amino acids, particularly lysine and branched chain amino acids, were released preferentially. The results suggest that the amount of breakdown of liver protein in the two groups was similar, but that the nitrogen was mainly released as free amino acids in the low-protein group, and as urea in the control group. On the contrary, in Exp. 2 the amount of nitrogen released from the liver was comparable to the decrease in amino acids in the liver, and the releases of urea were also less, being 1.83 mg in the control group and 0.54 mg in low-protein group. The results show that amino acid metabolism in the liver is greatly affected by the nutritional state of the animal and the amino acid content of the perfusion fluid.     

Ursodeoxycholic acid improves the hepatic metabolism of essential fatty acids and retinol in children with cystic fibrosis

An ELISA sandwich for the detection of Giardia lamblia antigens in human faeces was standardized. 175 samples were studied: 77 positive, 61 negative to cysts and/or trophozoites by direct faeces test, and 19 positive to other parasite different from G. lamblia. The sensitivity of the technique was 94.8% and the specificity 98.3%. The method detects an antigen concentration of 31 ng. The procedure is simple, sensitive and specific so, it may be useful for diagnosis and in epidemiological studies.     

Role of hepatic fatty acid:coenzyme A ligases in the metabolism of xenobiotic carboxylic acids

1. Formation of acyl-coenzymes (Co)A occurs as an obligatory step in the metabolism of a variety of endogenous substrates, including fatty acids. The reaction is catalysed by ATP-dependent acid:CoA ligases (EC 6.2.1.1-2.1.3; AMP forming), classified on the basis of their ability to conjugate saturated fatty acids of differing chain lengths, short (C2-C4), medium (C4-C12) and long (C10-C22). The enzymes are located in various cell compartments (cytosol, smooth endoplasmic reticulum, mitochondria and peroxisomes) and exhibit wide tissue distribution, with highest activity associated with liver and adipose tissue. 2. Formation of acyl-CoA is not unique to endogenous substrates, but also occurs as an obligatory step in the metabolism of some xenobiotic carboxylic acids. The mitochondrial medium-chain CoA ligase is principally associated with metabolism via amino acid conjugation and activates substrates such as benzoic and salicylic acids. Although amino acid conjugation was previously considered an a priori route of metabolism for xenobiotic-CoA, it is now recognized that these highly reactive and potentially toxic intermediates function as alternative substrates in pathways of intermediary metabolism, particularly those associated with lipid biosyntheses. 3. In addition to a role in fatty acid metabolism, the hepatic microsomal and peroxisomal long-chain-CoA-ligases have been implicated in the formation of the acyl-CoA thioesters of a variety of hypolipidaemic and peroxisome proliferating agents (e.g. clofibric acid) and of the R(-)-enantiomers of the commonly used 2-arylpropionic acid non-steroidal anti-inflammatory drugs (e.g. ibuprofen). In vitro kinetic studies using rat hepatic microsomes and peroxisomes have alluded to the possibility of xenobiotic-CoA ligase multiplicity. Although cDNA encoding a long-chain ligase have been isolated from rat and human liver, there is currently no molecular evidence of multiple isoforms. The gene has been localized to chromosome 4 and homology searches have revealed a significant similarity with enzymes of the luciferase family. 4. Increasing recognition that formation of a CoA conjugate increases chemical reactivity of xenobiotic carboxylic acids has led to an awareness that the relative activity, substrate specificity and intracellular location of the xenobiotic-CoA ligases may explain differences in toxicity. 5. Continued characterization of the human xenobiotic-CoA ligases in terms of substrate/inhibitor profiles and regulation, will allow a greater understanding of the role of these enzymes in the metabolism of carboxylic acids.     

Effects of medium chain fatty acids (MCFA), myristic acid, and oleic acid on serum lipoproteins in healthy subjects

In this study we investigated the effects on lipoproteins of medium chain fatty acids (MCFA) and myristic acid relative to those of oleic acid. Thirty-seven women and 23 men consumed a 3-wk run-in diet enriched in oleic acid followed by a 6-wk test diet rich in MCFA (n = 21), myristic (n = 20), or oleic acid (n = 19). Experimental fats were incorporated into solid foods. Total fat intake was 40 En% fat. The dietary compositions were the same except for 10 En%, which was provided by MCFA, myristic, or oleic acids, respectively. With the myristic acid diet, low density lipoprotein (LDL) cholesterol was 0.37 mmol/L higher compared with the oleic acid diet (P = 0.0064 for difference in changes). The MCFA diet increased LDL cholesterol, though not significantly, with 0.23 mmol/L relative to the oleic acid diet (P = 0.0752). Compared with the oleic acid diet, HDL cholesterol concentrations increased with the myristic acid diet by 0.10 mmol/L (P = 0.0273) but not with the MCFA diet. The MCFA diet slightly elevated triacylglycerol concentrations, but responses did not significantly differ between the diets. The MCFA diet significantly decreased the apoA-I to apoB ratio compared with both other diets (P < 0.02). We conclude that MCFA raise LDL cholesterol concentrations slightly and affect the apoA-I to apoB ratio unfavorably compared with oleic acid. Myristic acid is hypercholesterolemic, although less than predicted earlier, and raises both LDL and HDL cholesterol concentrations compared with oleic acid.     

Application of a U-13C-labeled amino acid tracer in lactating dairy goats for simultaneous measurements of the flux of amino acids in plasma and the partition of amino acids to the mammary gland

A preliminary study was conducted using lactating British Saanen goats (n = 5) at 109 to 213 d in milk that yielded 1.67 to 3.68 kg of milk/d to examine the application of a U-13C-labeled amino acid (AA) mixture obtained from hydrolyzed algal proteins as a tracer for measuring plasma flux (n = 5) and partition to the mammary gland (n = 3; arteriovenous difference) of 13 AA simultaneously. Except for Ile and Ser, there was incomplete (6 to 54%) equilibration of the tracer with AA from packed blood cells (> 90% erythrocytes) during the 6-h infusions. This result agreed with the large ratio of packed cells to gradients for plasma AA concentration that was also observed. However, net mass and isotope removals by the mammary gland were predominantly from plasma, indicating that the erythrocytes did not participate in kinetic exchanges. Plasma AA fluxes (millimoles per kilogram of metabolizable protein intake per kilogram of body weight 0.75) differed among goats that consumed different protein sources; however, overall rates were lowest for Met (5 to 14) and His (8 to 17) and highest for Leu (48 to 70) and Ala (53 to 88). On average, 25% of plasma flux was partitioned to the mammary gland. Less than 20% of His, Ser, Phe, and Ala were directed to the mammary gland; 20 to 30% of Arg, Thr, Tyr, and Leu were directed to the mammary gland; and 30 to 40% of Pro, Ile, Lys, and Val were directed to the mammary gland. The unidirectional AA flux in the mammary gland (AA apparently available for protein syntheses, oxidation, and metabolite formation) did not match the pattern that is required for casein synthesis, suggesting differences in the metabolic requirements of AA for nonmilk protein synthesis.     

Suppression of fatty acids synthesis in brown adipose tissue of mice fed diets rich in long chain fatty acids

The influence of dietary lipid on fatty acid synthesis in brown adipose tissue has been investigated by feeding different high-fat diets to cold-acclimated mice for a period of 2 weeks. Fatty acid synthesis was measured in vivo with 3H2O, and the fats used in the study were maize oil, beef tallow and medium chain triacylglycerol oil. In the mice fed the maize oil and the beef tallow diets fatty acids synthesis was inhibited in all tissue examined--interscapular brown adipose tissue, epididymal white adipose tissue, the liver and the carcass. Synthesis was more inhibited, however, in brown adipose tissue than in other tissues, and the inhibition was greater on the maize oil diet than on the beef tallow. The medium chain triacylglycerol oil had no inhibitory effect on fatty acid synthesis in any tissue, and hepatic synthesis was even elevated on this diet. It is concluded that fatty acid synthesis in brown adipose tissue, as in other lipogenic tissues, is subject to strong suppression by dietary long chain fatty acids, and particularly by linoleic acid.     

Differences in tissue fatty acids and cholesterol of swine from different genetic backgrounds

A review of 1,000 consecutive coronary angiograms, most of them performed for evaluation of angina pectoris, yielded 9 examples of congenital anomalies of the coronary arteries. In 2 cases the angina may have been due to malposition of the left coronary artery or one of its branches. There were 2 cases of aberrant origin of the circumflex artery from the right coronary artery, 2 cases of aberrant left anterior descending artery, 3 cases in which all three major coronary branches arose from the right aortic sinus, and 2 cases of coronary artery fistulas. Malposition of the coronary artery should be considered as a possible cause of angina.