John Edmond

Fatty Acid Oxidation and Ketogenesis by Astrocytes in Primary Culture

Abstract: The oxidation of the fatty acids octanoate and palmitate to CO2 and the ketone bodies acetoacetate and D‐(–)‐3‐hydroxybutyrate was examined in astrocytes that were prepared from cortex of 2‐day‐old rat brain and grown in primary culture to confluence. Accumulation of acetoacetate (by mass) in the culture medium of astrocytes incubated with octanoate (0.3–0.5 mM) was 50–90 nmol C2 units h−1 mg of protein−1. A similar rate was obtained using radiolabeled tracer methodology with [1‐14C]octanoate as labeled substrate. The results from the radiolabeled tracer studies using [1‐14C]‐ and [7‐14C]octanoate and [1‐14C]‐, [13‐14C]‐, and [15‐14C]palmitate indicated that a substantial proportion of the ω‐terminal fourcarbon unit of these fatty acids bypassed the β‐ketothiolase step of the β‐oxidation pathway and the 3‐hydroxy‐3‐methylglutaryl (HMG)‐CoA cycle of the classic ketogenic pathway. The [14C]acetoacetate formed from the 1‐14C‐labeled fatty acids, obligated to pass through the acetyl‐CoA pool, contained 50% of the label at carbon 3 and 50% at carbon 1. By contrast, the [14C]acetoacetate formed from (ω‐1)‐labeled fatty acids contained 90% of the label at carbon 3 and 10% at carbon 1, whereas that formed from the (ω‐3)‐labeled fatty acid contained 20% of the label at carbon 3 and 80% at carbon 1. These results indicate that acetoacetate is primarily formed either by the action of 3‐oxo‐acid‐CoA transferase (EC 2.8.3.5) or acetoacetyl‐CoA deacylase (EC 3.1.2.11) or both on acetoacetyl‐CoA and not by the action of the mitochondrial HMG‐CoA cycle involving HMG‐CoA lyase (EC 4.1.3.4), which was readily detected, and HMG‐CoA synthase (EC 4.1.3.5), which was barely measurable.

Essential polyunsaturated fatty acids and the barrier to the brain: the components of a model for transport.

Several areas of research have contributed to the establishment of a paradigm that meets the requirements for the selective uptake of essential polyunsaturated fatty acids (EPUFA) into brain. First, discrete studies have demonstrated that cholesterol and the nonessential fatty acids, (palmitic, oleic, stearic) do not enter the brain parenchyma. These studies demonstrated that the 18 carbon-monocarboxylic fatty acids, linoleic acid with two cis-double bonds entered brain, whereas oleic acid, with one cis-double bond, did not enter brain. It was concluded the entry of essential fatty acids into brain is accomplished in a highly selective and discrete manner. Further, the typical blood-borne lipoproteins do not traverse the endothelial cells of the capillary network and enter into the brain, otherwise cholesterol, palmitic, oleic, and stearic acids from blood would be located within brain. Second, several investigators have shown that the endothelial cells of the capillary network contain lipoprotein receptors, yet one conclusion is that the brain does not utilize low-density lipoprotein (LDL)-cholesterol. Third, recently, the existence and function of a significant number of distinctive trans-membrane monocarboxylic acid transporters, (MCTs) and fatty acid transport proteins (FATPs) have been described. No transporters have been described to date with the specificity necessary to transfer only EPUFA into brain. A blueprint with the minimal elements for delivery and selectivity is proposed. Lipoproteins enter the endothelial cells because the lipoprotein receptors are positioned on their luminal membrane. Essential fatty acid transporter(s) are positioned on the abluminal membrane of these endothelial cells to allow for the entry of EPUFA into brain. Within the endothelial cell there is opportunity for lipid management and transformation such that EPUFAs are selectively culled for delivery to the essential fatty acid transporter(s), which facilitates their transfer into brain.

NEONATAL POLYUNSATURATED FATTY ACID METABOLISM

The importance of n−6 and n−3 polyunsaturated fatty acids (PUFA) in neonatal development, particularly with respect to the developing brain and retina, is well known. This review combines recent information from basic science and clinical studies to highlight recent advances in knowledge on PUFA metabolism and areas where research is still needed on infant n−6 and n−3 fatty acid requirements. Animal, cell culture, and infant studies are consistent in demonstrating that synthesis of 22∶6n−3 involves C24 PUFA and that the amounts of 18∶2n−6 and 18∶3n−3 influence PUFA metabolism. Studies to show that addition of n−6 fatty acids beyond Δ6-desaturase alters n−6 fatty acid metabolism with no marked increase in tissue 20∶4n−6 illustrate the limitations of analyses of tissue fatty acid compositions as an approach to study the effects of diet on fatty acid metabolism. New information to show highly selective pathways for n−6 and n−3 fatty acid uptake in brain, and efficient path-ways for conservation of 22∶6n−3 in retina emphasizes the differences in PUFA metabolism among different tissues and the unique features which allow the brain and retina to accumulate and maintain high concentrations of n−3 fatty acids. Further elucidation of the Δ6-desaturases involved in 24∶5n−6 and 22∶6n−3 synthesis; the regulation of fatty acid movement between the endoplasmic reticulum and peroxisomes; partitioning to acylation, desaturation and oxidation; and the effects of dietary and hormonal factors on these pathways is needed for greater understanding of neonatal PUFA metabolism.

Milk-substitutes comparable to rat's milk; their preparation, composition and impact on development and metabolism in the artificially reared rat

1. Procedures are described to prepare nutritionally adequate rat milk-substitutes by modifying commercially available processed cows milk, rich in carbohydrate and low in protein and fat compared with rats milk. 2. Premilk formulas, prepared as intermediates in the preparation of rat milk-substitutes, are rich in protein but low in their concentration of fat, carbohydrate, and minerals when compared with rats milk. 3. Premilks were supplemented with lactose, vitamins, minerals, fat as oil mixtures, certain amino acids and other constituents to yield rat milk-substitutes which resemble the known composition of rats milk in their properties and composition. 4. Detailed analyses of the milk-substitutes show them to be comparable to rats milk in energy content, pH, osmolarity, the concentration of the macronutrients, fat, protein and carbohydrate, and the major minerals. 5. Rat pups were artificially reared from postnatal day 4 or 5 until days 16-18 by fitting them with gastric cannulas through which the milk-substitutes could be infused automatically. 6. The nutritional impact of the milk-substitutes was assessed by a comparison of growth and metabolic characteristics for artificially reared rats with age-matched sucking rats reared by their mother. 7. Indices which were taken to be appropriate included (a) body-weight gain; (b) the concentration in blood of protein, amino acids, ketone bodies, carnitine, glucose, galactose, lactate, insulin, and the electrolytes calcium, sodium, potassium and chloride; (c) the turnover of glucose and 3-hydroxybutyrate; (d) the concentration in brain of protein, cholesterol, cerebroside sulphate and the activities of the enzymes pyruvate dehydrogenase (EC 1.2.4.1), 3-oxo-acid-CoA transferase (EC 2.8.3.5) and acetoacetyl-CoA ligase (EC 6.2.1.16). 8. The studies suggest that milk-substitutes approximating to rats milk in composition promote acceptable metabolism in the artificially reared rat pup.

A chemically-defined medium for organotypic slice cultures.

Organotypic slice cultures provide an excellent system for the analysis of study of the molecular mechanisms of this development necessitates the use of a chemically defined culture medium. We report here the development of a medium, EOL1 defined medium, designed specifically for this purpose. Cultures of both cerebral cortex and basal forebrain demonstrate that this defined medium allows a high degree of cytoarchitectural maintenance while promoting neural metabolism and process outgrowth.

Purification, molecular weight, amino acid, and subunit composition of arylsulfatase A from human liver

Abstract Arylsulfatase A (EC 3.1.6.1) from human liver self-associates at pH 5.0. This behavior was used to purify the enzyme to a specific activity of 2872 μmol nitrocatechol sulfate hydrolyzed per hour per milligram of protein, a 7000-fold increase in specific activity over the crude homogenate. No evidence of heterogeneity can be detected by acrylamide gel electrophoresis at pH 8.9 and 6.8 or by sedimentation equilibrium centrifugation at pH 8.1 and 5.0. The purified enzyme also exhibits one precipitin line on immunodiffusion and immunoelectrophoresis against antibody prepared to an impure enzyme sample. The amino acid analysis of the enzyme reveals a high content of proline and the presence of carbohydrate. The molecular weight, measured by sedimentation equilibrium centrifugation, is 104,500 at pH 8.1 and 413,000 at pH 5.0. Centrifugation in 6 m guanidine hydrochloride indicated the presence of two components of molecular weights 53,000 and 66,000. The presence of two components was verified by acrylamide gel electrophoresis of denatured enzyme in 6 m urea and in sodium dodecyl sulfate. The molecular weights of the components, determined from protein standards, by sodium dodecyl sulfate acrylamide gel electrophoresis are 49,000 and 59,000.

The origin of palmitic acid in brain of the developing rat

A rat milk substitute containing lower amounts of palmitic and oleic acid in the triacylglycerols in comparison to natural rat milk was fed to artificially reared rat pups from day 7 after birth to day 14. Pups reared by their mother served as controls. Free trideuterated (D3) palmitic acid [(C2H3)(CH2)14COOH, 98 atom % D] and free perdeuterated (D31) palmitic acid [C152H31COOH, 99 atom % D] in equal quantity were mixed into the triacylglycerols of the milk substitute in an amount equal to 100% of the palmitic acid in the triacylglycerols. A control milk substitute contained unlabeled free palmitic acid in an amount equal to 100% of the palmitic acid in the triacylglycerols of the milk substitute. The objective was to determine if palmitic acid in the diet contributed significantly to the palmitic acid content of developing brain and other organs. The methyl esters of the fatty acids were analyzed by gas chromatography and the palmitic acid methyl ester was examined by fast atom bombardment mass spectrometry. The proportion of deuterated methyl palmitate as a percentage of total palmitate was determined; 32% of the palmitic acid in liver and 12% of the palmitic acid in lung were trideuterated and perdeuterated palmitic acid in approximately equal amounts. The brain, by contrast, did not contain the deuterated palmitic acid moiety. Quantitation of palmitic acid and total fatty acids revealed a significant accumulation in organs in the interval from 7 to 14 days of age. Under our experimental conditions, labeled palmitic acid does not enter the brain. Consequently, we conclude that the developing brain produces all required palmitic acid byde novo synthesis.

Selective specification of CNS stem cells into oligodendroglial or neuronal cell lineage: Cell culture and transplant studies

Neural stem cells (NSCs) were isolated from embryonic day 16 Sprague–Dawley rats and cultured in a novel serum‐free stem cell medium that selected for the growth of NSCs and against the growth of GFAP+ cells (astrocytes). NSCs maintained in culture for extended periods of time retained immunoreactivity for both nestin and PSA‐NCAM, two markers characteristic of the stem cell phenotype. Moreover, using an oligodendrocyte (OL) specification medium, NSCs differentiated into OL as evidenced by their morphology and expression of multiple oligodendrocyte/myelin‐specific markers. In addition, NSCs are capable of acquiring a neuronal phenotype as evidenced by expressing neuronal markers, such as neurofilament (NF) and NeuN when cultured in a defined medium for neurons indicating that these cells are also a good source of neuroblasts, which could be used to replace neuronal populations in the brain. We also showed successful propagation and differentiation of NSCs into OL after cryostorage, allowing for the later use of stored NSCs. The long‐term goal of culturing NSCs and committed oligodendrocyte progenitors (OLP) is to obtain homogeneous populations for transplantation with the goal of remyelinating the myelin‐deficient CNS. Our preliminary experiments carried out on normal and myelin deficient rats demonstrate that these cells survive and migrate extensively in both types of hosts. NSCs grafted as such, as well as cells derived from NSCs exposed to selective specification before grafting, are able to differentiate within the host brain. As expected, NSCs are capable of giving rise to astrocytes in a medium favoring this phenotype.

Medium-chain triglycerides in infant formulas and their relation to plasma ketone body concentrations

ABSTRACT. A mild ketosis is known to prevail in the mother, fetus, and newborn infant during the 3rd trimester and in the early neonatal period. It has been shown that during an equivalent period in the rat ketone bodies are readily oxidized and serve as key substrates for lipogenesis in brain. Since medium-chain triglycerides are known to be ketogenic, preterm infants may benefit from dietary medium- chain triglycerides beyond the point of enhanced fat absorption. Our objective was to determine the ketogenic response in preterm infants (gestational age: 33 ± 0.8 wk) fed three different isocaloric formulas by measuring the concentrations of 3-hydroxybutyrate and acetoacetate in the plasma of these infants. At the time of entrance to the study the infants were receiving 110 kcal/kg/24 h. Study I (11 infants): the infants were fed sequentially in the order; PM 60/40 (PM), Special Care Formula (SCF), and Similac 20 (SIM). In SCF greater than 50% of the fat consists of medium-chain length fatty acids while PM and SIM contain about 25%. The concentration of 3-hydroxybutyrate in plasma was significantly higher when infants were fed SCF than PM and SIM [0.14 ± 0.03, 0.06 ± 0.01, and 0.05 ± 0.01 mM, respectively (p< 0.01)]. Study II (12 infants); the infants were fed SCF, then SIM, or the reverse. The concentration of acetoacetate in plasma was 0.05 ± 0.01 and 0.03 ± 0.01 mM when infants were fed SCF and SIM, respectively (0.1>p> 0.05). The concentrations of 3-hydroxybutyrate in plasma were similar to those measured in study I for the respective formulas. The increased plasma levels of 3-hydroxybutyrate and total ketone bodies when SCF was fed indicate that SCF promotes a mild ketosis in preterm infants similar to that reported in breast-fed term infants.

Fatty Acid Cycling in Human Hepatoma Cells and the Effects of Troglitazone

Fatty acid cycling by chain shortening/elongation in the peroxisomes is an important source of fatty acids for membrane lipid synthesis. Its role in the homeostasis of nonessential fatty acids is poorly understood. We report here a study on the cycling of saturated fatty acids and the effects of troglitazone in HepG2 cells in culture using [U-13C]stearate or [U-13C]oleate and mass isotopomer analysis. HepG2 cells were grown in the presence of 0.7 mmol/liter [U-13C]stearate or [U-13C]oleate, and in the presence and absence of 50 μm troglitazone for 72 h. Fatty acids extracted from cell pellets after saponification were analyzed by gas chromatography/mass spectrometry. Peroxisomal β-oxidation of uniformly 13C-labeled stearate (C18:0) and oleate (C18:1) resulted in chain shortening and produced uniformly labeled palmitate (C16:0) and palmitoleate (C16:1). In untreated cells, 16% of C16:0 was derived from C18:0 and 26% of C16:1 from C18:1 by chain shortening. Such contributions were significantly increased by troglitazone to 23.6 and 36.6%, respectively (p < 0.001). Desaturation of stearate contributed 67% of the oleate, while reduction of oleate contributed little to stearate (2%). The desaturation of C18:0 to C18:1 was not affected by troglitazone. Our results demonstrated a high degree of recycling of C18:0 and C18:1 to C16:0 and C16:1 through chain shortening and desaturation. Chain shortening was accompanied by chain elongation in the synthesis of other long chain fatty acids. Troglitazone specifically increased recycling by peroxisomal β-oxidation of C18 to C16 fatty acids, and the interconversion of long chain fatty acids was associated with reduced de novo lipogenesis.