Model microemulsions containing vegetable oils part 1: Nonionic surfactant systems

Nonionic microemulsions containing triglycerides and fatty acid esters as lipophilic components have been studied. The phase inversion temperature (PIT) of the systems was determined by a conductometric method. Partial phase diagrams were constructed in the phase inversion temperature range. Water solubilization capacity of the nonionic surfactant systems studied was dependent on surfactant and oil types in analogy to ordinary hydrocarbon systems. The PIT:s increased with increased molecular weight for both esters and triglycerides.     

A comparison between lipase-catalyzed esterification of oleic acid with glycerol in monolayer and microemulsion systems

Lipase-catalyzed synthesis-esterification of oleic acid with glycerol-was carried out in L2 microemulsions and in monolayers. The microemulsions were based on isooctane as a nonpolar component and various water-glycerol mixtures as polar component. The substrate, oleic acid/sodium oleate, constituted the microemulsion surfactant. The lipase resides mainly in the water pools. Monolayers of oleic acid/sodium oleate were formed on subsolutions of glycerol and water, and the enzyme solution was injected under the compressed monolayers. Thus, the arrangement of the reactants at the oil-water interface of the microemulsion can be regarded as analogous to that at the airwater interface of the monolayer. The microemulsion structure was characterized by self-diffusion nuclear magnetic resonance. It was found that the higher the glycerol-to-water ratio, the lower are the water D-values. The reactions in microemulsions generally gave a low degree of oleic acid conversion. The yield increased with increasing glycerolto-water ratio. Monoglycerides were the main product, and no triglyceride could be detected. The monolayer experiments gave a somewhat higher degree of conversion, with tri- and diglycerides being the major reaction products. The reason why triglycerides are formed in monolayer experiments but not in microemulsions is believed to be due to an unfavorable partitioning of the diglyceride in the microemulsion systems. Once formed, the diglyceride will partition into the hydrocarbon domain and become inaccessible for reaction with the enzyme-O-acyl intermediate at the oil-water interface. In addition, the interfaces in the two systems are different. The monolayer interface is static, whereas the microemulsion interface is highly dynamic, and this difference may also influence the product patterns.     

Radical chlorinations of triglycerides

Several triglycerides were synthesized with an iodoaroyl group. Intramolecular radical chain transfer chlorinations were conducted that resulted in the associated pair of fatty acids of the triglyceride becoming chlorinated. The distribution of monochlorinated species was similar to that obtained by direct radical chlorination of the relevant fatty acid methyl esters. Functionalization was, as expected, away from the carboxylate group but gave no indication that either alignment of chains or the constraints of an intramolecular process could limit the manifold of products of the reaction. To gauge the effect on halogenation of a structure, bearing more than one electron-withdrawing group, methyl oleate was converted to thebis trifluoroacetate of methyl 9,10-dihydroxystearate. No products of chlorination on carbons 2–8 were observed.     

A study of fatty acid methyl esters with epoxy or alkyne functionalities

Described are the physical and chemical properties of the methyl esters of two uncommon fatty acids: vernolic acid, containing an epoxy group, and crepenynic acid, containing a triple bond. The incorporation of an epoxy or alkyne group into the fatty acid structure is shown to greatly affect the properties compared to conventional unsaturated fatty acids. The methyl esters have been characterized and compared with ordinary fatty acid methyl esters (i.e., methyl oleate and linoleate) with respect to spectroscopic characterization [¹H nuclear magnetic resonance (NMR)], ¹³C NMR, and Fourier transform infrared), rheological properties, and oxidative reactivity (using chemiluminescence). Both methyl vernoleate and methyl crepenynate could successfully be produced by transesterification under basic conditions without reaction of the epoxy or alkyne groups. Rheological measurements showed that the methyl esters had a significantly lower viscosity compared to their triglyceride analogs. Smaller differences were seen when comparing the different methyl esters where methyl vernoleate had the highest viscosity due to the presence of the more polar oxirane group. Very large differences were found with respect to the oxidation rate of the different methyl esters. Methyl crepenynate was shown to oxidize extremely rapidly, whereas methyl vernoleate was very stable toward oxidation.     

Lipids of cultured hepatoma cells: IV. Effect of serum and lipid upon cellular and media neutral lipids

Minimal deviation hepatoma 7288C cells were cultured in a modified Swim's medium supplemented with decreasing levels of serum, lipid-free serum, lipid-free serum plus fatty acids, and other additives. Cellular and media neutral lipid classes were quantitated, the fatty acids of triglycerides and sterol esters analyzed, and the carbon number distribution of triglycerides determined. Cellular triglyceride biosynthesis virtually was inhibited when the medium was supplemented with bovine serum alone. This inhibition was not observed when the medium was supplemented with fetal calf serum alone or mixtures of fetal calf serum and bovine serum. Cells cultivated on medium supplemented with lipid-free serum plus palmitic or linoleic acids had much lower levels of free and esterified cholesterol. The fatty acid composition of cellular triglycerides and cholesterol esters differed dramatically from the corresponding media lipid classes. Except when linoleic acid was added to the medium, changes in the media serum and lipid levels had only marginal effects upon the fatty acid composition of cellular triglycerides and cholesterol esters. These data, in conjunction with earlier data that showed the media neutral lipid levels did not decrease during cell growth, indicate that these hepatoma cells utilize little or no serum triglycerides and cholesterol esters. Linoleic acid added to the medium dramatically reduced the level of 18∶1 acids in cellular triglycerides and cholesterol esters. Palmitic acid added to the medium did not change the fatty acid compositions significantly. Comparison of experimentally determined and calculated triglyceride carbon number percentages indicated a random distribution of fatty acids in this glyceride. The fatty acid composition of cellular triglycerides was similar to the composition of the cholesterol esters. The lack of characteristic and distinguishable compositions of these two classes that occur in most normal tissues suggests a loss of specificity in the lipid metabolism of this neoplasm at the class level.     

Quantitative and qualitative analyses of isolated lipid droplets from interstitial cells in renal papillae from various species

The lipid droplets of renal papillae homogenates from four different species were obtained by ultracentrifugation. Ca. 80–98% of the lipids (triglycerides, phospholipids, free fatty acids, and cholesterol esters) consist of triglycerides. The triglycerides were fractionated by argentation thin layer chromatography and each fraction characterized by gas liquid chromatography. No fraction contained any unique triglyceride. The fatty acid composition of the total triglycerides, as analyzed by gas liquid chromatography and ozonolysis, differed markedly from the fatty acid composition of the corresponding plasma triglycerides. The papillary triglycerides were characterized by higher concentrations of stearic acid, arachidic acid, and polyunsaturated acids with 20 or more carbon atoms. Particularly interesting was the presence in the lipid droplets of docosa-7,10,13,16-tetraenoic acid. This acid has been shown to be a major component in the cholesterol ester fraction of rat and canine adrenal lipids. In the papillary triglycerides, this acid accounted for 7%, 15%, and more than 20% of the total fatty acids in the dog, rat, and rabbit, respectively. The pig differs from these three species in having only ca. 1% of this acid. These observations suggest that the interstitial cells produce these triglycerides. This production could occur either by a transacylation from phospholipids and cholesterol esters and by a de novo synthesis from locally produced fatty acids. The possibility that the triglyceride production may be involved in a control of the prostaglandin production of the renal medulla is discussed.     

Solubility behavior of ternary systems of lipids in supercritical carbon dioxide

In this review, the phase behavior of lipids in ternary and higher systems was analyzed to determine the effect of operating parameters and feed composition on solubility behavior and separation efficiency. The ternary systems studied included two triglycerides (TG) (trilaurin (LLL)/tripalmitin (PPP), trimyristin (MMM)/PPP and LLL/MMM), two fatty acids (FA) (oleic acid (OA)/linoleic acid (LA)), two fatty acid methyl esters (FAME) (methyl myristate (MeM)/methyl palmitate (MeP) and methyl oleate (MeO)/methyl linoleate (MeL)), FA/TG (OA/triolein (OOO)) and FAME/FA (MeO/OA) in SCCO₂. The quaternary and quinary systems analyzed contained three TG (LLL/MMM/PPP), three glycerides (monoolein (MO)/diolein (DO)/OOO) and four TG (PPP/palmitoyl-dioleoylglycerol (POO)/oleoyl-dipalmitoylglycerol (PPO)/OOO), respectively, in SCCO₂. While solubility of the less soluble component increased in ternary systems of solid triglycerides in SCCO₂, that of the more soluble component decreased or was not affected. Solubility diminution was observed for both solutes in some liquid mixtures such as fatty acid (oleic acid/linoleic acid) and fatty acid ester (methyl oleate/methyl linoleate) mixtures. Solubility enhancement for one of the mixture components was also observed (for example, for oleic acid in the presence of methyl oleate). Separation efficiency was lower than that predicted by binary data when the solubility of the less soluble solute was enhanced in the mixture, whereas it was improved if the solubility of the more soluble component was enhanced as observed in quaternary glyceride mixtures.     

Dietary fatty acids: Their metabolic fate and influence on fatty acid biosynthesis

Adult rats fed a low-fat diet or diets containing 15% of either tripalmitin, triolein or trilinolein were injected intraperitoneally with H³-labeled acetate. Those which received fat were also given by mouth, simultaneously with acetate, the 1-C¹⁴-labeled sodium salt of the respective dietary fatty acid. The fate of the tagged material was followed by time-spaced biopsies of subcutaneous adipose tissue and by collection of the expired C¹⁴O₂. After 72 hr, 51, 64, and 52% of the dietary palmitate, oleate, and linoleate, respectively, were catabolized, as indicated by the corresponding percentages of the label having been excreted as C¹⁴O₂. Dietary linoleate was relatively less incorporated into body triglycerides than palmitate and oleate. Animals ingesting diets of 15% triolein had only about one-half the amount of phospholipids in their tissues as had the other groups. The distribution of both the C¹⁴ and H³ labels in the tissue triglycerides showed that all diets containing fats decreased fatty acid synthesis but did not inhibit conversion of palmitate to oleate. Conversions of oleate or linoleate appeared to be through acetate. As a result of these factors, the fatty acid composition of the tissue triglycerides after 3 months’ ingestion of tripalmitin was essentially the same as that of the low-fat group, whereas the ingestion of triolein produced triglycerides with a very high content of oleic acid. Trilinolein ingestion produced effects similar to triolein but to a less pronounced degree. Both the respiratory C¹⁴O₂ and the C¹⁴- and H³-labeled fatty acids in subcutaneous adipose tissue exhibited a second rise in specific activity 12 to 24 hours after the administration of the label. Presented at the AOCS meeting, Chicago, 1964.     

Über trans-Lipoide: Die Lipoide des Hühnerei-Dotters

Trans Lipids: The Egg Yolk Lipids of the Hen The feeding of hens with a trans-containing edible fat resulted in an increase in the trans fatty acids of the egg yolk lipids to the extent of 10%, which completely disappeared within 14 days after stopping the trans-containing feed. The fatty acids in the triglyceride fractions contained in each case more trans unsaturated fatty acids than those in the corresponding phosphatide fractions. The gas chromatographic analysis of the fatty acid methyl esters showed that the triglycerides contained more oleic acid than the phosphatide fatty acids whereas stearic as well as polyunsaturated components were concentrated predominently in the phosphatides. The analysis of the fatty acid methyl esters from triglyceride and phosphatide fractions after feeding of trans-containing fat showed distinct changes in both the groups, thus for example a reduction of oleic acid in the neutral fat analogous to that in the total lipids as against its increase in phosphatides. The amount of linoleic acid in the phosphatide fatty acids increased while it remained constant in triglyceride fatty acids.     

Preparation of sucrose esters by interesterification

Reactions between sucrose and esters of long chain fatty acids customarily have been conducted in a mutual solvent, such as dimethylformamide. The solvent-free interesterification of molten sucrose and fatty acid esters at temperatures between 170–187 C has now been performed with the aid of lithium, sodium and potassium soaps as catalysts and solubilizers. When the reactants were heated rapidly and then subjected to reduced pressure, the interesterifications could be brought to equilibrium in 12 min or less, including the time necessary to melt the sucrose. The several soaps and combinations of soaps employed differed markedly in their performance. No sucrose esters were obtained with lithium palmitate, while the yield with lithium oleate was among the best, but consisted of over 90% tetra- and higher esters of sucrose. Lower esters were best produced with combinations of lithium oleate with sodium or potassium oleate employed at a level of about 25% total soaps, based on the weight of sucrose. The type of fatty acid ester employed also markedly affected the yield of sucrose esters. Among the esters tested, methyl carbitol palmitate (which could be formed in situ), monopalmitin, distearin and technical grade diglycerides (48% diglycerides) prepared from completely hydrogenated cottonseed oil, interesterified readily. Presented at the AOCS Meeting, Minneapolis, October 1969. So. Utiliz. Res. Dev. Div., ARS, USDA.     

Inhibition of acylcoenzyme A:Cholesterol acyltransferase activity by PD128O42: Effect on cholesterol metabolism and secretion in CaCo-2 cells

The regulation of cholesterol uptake and secretion by acylcoenzyme A:cholesterol acyltransferase (ACAT) was investigated in the human intestinal cell line, CaCo-2. A new ACAT inhibitor, PD128042 (CI-976), was first characterized. The addition of the fatty acid anilide to membranes prepared from CaCo-2 cells inhibited ACAT activity without altering the activities of HMG-CoA reductase, fatty acid Co-A hydrolase, or triglyceride synthetase. PD128042 was a competitive inhibitor of ACTA with 50% inhibition occurring at a concentration of 0.2 μg/mL. When added to the medium of CaCo-2 cells at a concentration of 5 μg/mL, PD128042 inhibited oleate incorporation into cholesteryl oleate by 92% and increased oleate incorporation into triglycerides and phospholipids by 51% and 38%, respectively. After incubating CaCo-2 cells with the ACAT inhibitor, the rate of newly synthesized cholesterol decreased by 75% and membranes prepared from these cells contained significantly less HMG-CoA reductase activity. PD128042 significantly decreased the basolateral secretion of newly synthesized cholesteryl esters without affecting the secretion of newly synthesized triglycerides or phospholipids. The inhibitor decreased the esterification of labeled exogenous cholesterol which was taken up by the cell from bile salt micelles. Moreover, after 16 hr of ACAT inhibition, less labeled unesterified micellar cholesterol was associated with the cell. The esterification of cholesterol in CaCo-2 cells plays an integral role in the uptake of cholesterol through the apical membrane and its eventual secretion at the basolateral membrane.     

Physical Properties of Triglycerides IV. Dielectric Constant

Dielectric constants at 20° and at 40° C of a number of triglycerides in the liquid state have been measured. A molar additive function of the dielectric constant, based on a relation derived by J. van Elk, was used in combination with a previously derived equation for triglycerides to give an equation which relates the dielectric constant of a triglyceride or triglyceride mixture to its refractive index, density, and iodine value. Quantification of this relation was based on prime data obtained from these model triglycerides and from previously published data on fatty acid methyl esters.     

Effect of dietary fat supplementation on the composition and positional distribution of fatty acids in ruminant and porcine glycerides

Dietary fats which were protected from ruminal metabolism were fed to ruminants, and the constituent fatty acids subsequently appeared in the glycerides of tissues and secretory products. These dietary fat induced alterations in tissue lipid composition were particularly apparent when the fat source was enriched with linoleic acid. Similarly, when pigs were fed linoleic-enriched fats, the linoleic acid was incorporated into the adipose tissue triglycerides. Stereospecific analyses were carried out on triglycerides from various tissues and secretory products obtained from animals fed control or linoleate-enriched diets. The analysis of adipose tissue triglycerides showed that linoleate and oleate were preferentially esterified to positions 2 and 3 (cattle and sheep), and positions 1 and 3 (pigs). Of the other major adipose tissue fatty acids, palmitate was preferentially esterified at position 1 (ruminants) and position 2 (pigs), and stearate was preferentially esterified at positions 1 and 3 (ruminants), and position 1 (pigs). Stereospecific analysis of high mol wt milk triglycerides showed that linoleate was either evenly distributed on all three positions (goats), or predominantly on position 3 (cows). Furthermore, the incorporation of this linoleate did not markedly alter the positional specificity of the other major milk triglyceride fatty acids. Of these fatty acids, the short and medium chain length acids (butyratelaurate) were mainly on position 3, myristate and palmitate on positions 1 and 2, and stearate and oleate evenly distributed. Thoracic duct lymph triglycerides from sheep tended to show preferential incorporation of linoleate at position 3, palmitate at position 2, and stearate at position 1 and 3; oleate, on the other hand, tended to be evenly distributed on all three positions of the lymph triglyceride. The stereospecific arrangement of fatty acids in sheep liver triglycerides was similar to that of lymph triglycerides, and this may reflect the uptake of intact or partially hydrolysed chylomicron and/or very low density lipoprotein triglycerides by the liver. There were also some analogies in the stereospecific arrangement of fatty acids on ruminant lymph and milk triglycerides and this may reflect an incomplete hydrolysis of chylomicron and/or very low density lipoprotein triglycerides prior to uptake by the mammary gland. An unusual feature of lymph from sheep fed linoleate was the presence of phospholipids which contained large amounts of linoleate in ca. equal proportion at both positions 1 and 2 of the phospholipid molecule.     

Triacylglycerol assembly from binary mixtures of fatty acids byApiotrichum curvatum

To observe how the stereospecific distribution of acyl groups in triglycerides is affected by the composition of fatty acids available for esterification, the oleaginous yeastApiotrichum curvatum was grown on various binary mixture of palmitic, stearic, oleic and linoleic acids as carbon sources, and the yeast triglycerides were analyzed. When oleic acid-linoleic acid mixtures in various ratios were used as substrates, the yeast grew well, and the composition of the intracellular triglycerides reflected the substrate composition, but more linoleate than oleate was deposited in the triglycerides. Oleate was favored over linoleate at thesn-2 position of the glycerol. With substrates containing palmitic and stearic acids, the yeast accumulated less oil, and incorporation of stearic acid into the triglycerides also was very limited. When mixtures of palmitic acid-oleic acid and palmitic acid-linoleic acid were used as substrates, the yeast triglyceride composition did not reflect that of the substrate, and the accumulation in the yeast of the unsaturated acid in the substrate was favored. Possibly, the yeast had more limited access to solid than to liquid substrates. For oleic acid-linoleic acid substrates, when the percentages of oleate and linoleate at the three glycerol positions were plottedvs. the percentage of these acyl groups in the total triglyceride, apparent linear relations were observed for most of the range, and the sums of the intercepts and slopes of the three lines for each acyl group were 0 and 3, respectively. Two mathematical models of triglyceride assembly are proposed, both of which fit the experimental data. One model assumes that for a certain proportion of the glycerol molecules, the acyl composition of the threesn positions is rigidly controlled independently of the substrate concentration. The other assumes that the various acyl groups are distributed on the threesn positions of glycerol with different affinities.     

The simultaneous determination of both the quantity and the fatty acid composition of the triglycerides in three to ten microliters of plasma

An integrated system using chromatography on silica gel-impregnated glass paper, an internal standard and gas-liquid chromatography (GLC) makes possible the isolation of triglycerides, the determination of triglyceride, fatty acid composition, and the quantitative determination of triglyceride. Recoveries are good and values for total triglyceride obtained by this system compare favorably with values obtained by application of the hydroxamate colorimetric method to the triglyceride eluate from silicic acid columns. The fatty acid composition of the cholesteryl ester (CE) fraction is obtainable at the same time by GLC of methyl esters prepared from the CE fractions which also appears as a distinct spot on these chromatograms. Also, a densitometric method is described for triglyceride determination by photometry of the charred spots obtained after chromatography on the gel-paper. Presented at the AOCS Meeting, Houston, April, 1965     

The effect of mono-enoic fatty acid esters on the growth and fecal lipides of rats

Summary The growth rates of rats fed a purified diet containing 5% corn oil or the methyl esters of oleic, eicosenoic, orerucic acid have been compared. At this level none of the esters significantly depressed the growth of male rats, but all three reduced the growth of females below that of those fed corn oil. Food efficiency and food consumption were not significantly reduced by any of the esters. Symptoms of essential fatty acid deficiency occurred only among rats fed the fatty acid esters and not among rats fed the fat-free diet. Methyl erucate increased the excretion of true fatty acids, acidic material not soluble in hexane and unsaponifiable matter, as compared to methyl oleate. The true fatty acid excreted was largely erucic. Methyl eicosenoate gave results not appreciably different from methyl oleate except for a slight increase in the excretion of mono-unsaturated fatty acid from rats fed eicosenoate. Both corn oil and methyl erucate increased the excretion of unsaponi-fiable matter. The coefficient of digestibility of methyl eicosenoate and erucate were 96% and 90%, respectively, which are higher values than have been previously reported.     

GLC analysis ofHeliothis virescens triglycerides at various metamorphic stages

The triglycerides ofHeliothis virescens (F.), the tobacco budworm, reared on a semidefined diet, were examined at different stages of development. The distribution of triglyceride carbon numbers and the composition of triglyceride fatty acids were determined by gas-liquid chromatographic analysis of intact triglycerides and methyl esters. Triglycerides of carbon number 50 decreased in the unfed adults with age; the decrease corresponded with a decrease in palmitic acid. Linoleic acid and carbon number 50 exhibited fluctuations in concentrations that were correlated with metamorphosis. The observed changes in specific triglyceride carbon numbers with a simultaneous change in specific triglyceride fatty acids indicate a nonrandom fatty acid distribution in the triglycerides of this insect. Under contract with the U. S. Atomic Energy Commission. Oak Ridge Associated Universities Research Participant.     

Carboxylate surfactant systems exhibiting phase behavior suitable for enhanced oil recovery

A large variety of aliphatic and aromatic carboxylate surfactants with alcohols as cosurfactants displayed phase behavior with n-decane that indicated they could be useful in enhanced oil recovery. Three-phase systems with large volume fraction middle-phase microemulsions were observed with sodium isostearate and sodium oleate. The interfacial tension between top and bottom phases was at a minimum (∼0.001 dyne/cm) at the optimal salinity where the uptake of oil and water in the middle phase was equal. The phase behavior of carboxylates was affected by pH and added bases. In cases where a bicarbonate-carbonate buffer was used to control pH, the optimal salinity decreased as the pH decreased. Use of ethoxylated alcohols as cosurfactants increased optimal salinities markedly and caused formation of very large volume fraction middle phases. Neutralized tall oils and tall oil fatty acids with alcohols as cosurfactants gave 3-phase behavior with n-decane with large volume fraction middle-phase microemulsions.     

Changes in the structure of soybean triglycerides during maturation

Soybeans of the Hawkeye variety were picked at eleven periods from 30 to 111 days after flowering and extracted with chloroform-methanol. The triglyceride fraction of five pickings, selected 35 to 91 days after flowering (when synthesis of lipid was most active), were isolated by silicic acid thin layer chromatography (TLC) and species composition determined using argentation TLC and lipase hydrolysis. The triglyceride content of the total lipid increased from 6.5% at 30 days after flowering to 85% in the mature bean (111 days). The major changes in fatty acid composition of the triglycerides occurred during the first 52 days after flowering. During this period linolenic acid decreased from 34.2% to 11.7%, the percentages of linoleic and oleic acids increased, stearic remained fairly constant and palmitic decreased slightly. Large quantitative changes occurred in the molecular species of the triglycerides of the bean during maturation; some triglycerides containing linolenic acid could not be detected approximately 66 days after flowering. Although changes occurred in the percentage and amount of each triglyceride species, the positional distribution of fatty acids remained virtually unchanged throughout maturation. Linolenic acid was distributed fairly uniformly between the β-position and the α-positions, linoleate favored esterification in the β-position, and oleate the α-positions. Most of the stearic and palmitic acids were esterified in the α-positions. The consistency of the positional arrangement of the fatty acids indicated that the mode of glyceride synthesis was established very early during maturation and molecular species composition was controlled by the fatty acids available for synthesis.     

Adipose hormone-sensitive lipase preferentially releases polyunsaturated fatty acids from triglycerides

Rat adipose hormone-sensitive lipase-mediated release of fatty acids from triglycerides was studied in three model systems: i) cultured preadipocytes containing polyunsaturated fatty acid-enriched triglyceride; ii) perfused epididymal fat pads; and iii)in vitro incubations of crude preparations of hormone-sensitive lipase with synthetic triglyceride-analogues as substrates. We found that cultured preadipocytes challenged with 10μM norepinephrine tended to release more ω6 and ω3 polyunsaturated fatty acids than saturated fatty acids. Fat pads perfused with 10 μM norepinephrine preferentially released arachidonate and α-linolenate but tended to retain oleate and linoleate. Finally, crude preparations of hormonesensitive lipase released from the triglyceride-analogue substrates α-linolenate twice as fast as oleate. We conclude that rat adipose hormone-sensitive lipase preferentially releases polyunsaturated fatty acids from triglycerides. We suggest that this may be a mechanism by which these fatty acids are kept from being trapped in fat depots and maintained in the circulation.