Variation in molecular species of polar lipids from Thermoplasma acidophilum depends on growth temperature

Five types of molecular species of C₄₀ isoprenoid chains, having different numbers of cyclopentane rings, were detected in the ether core lipid of Thermoplasma acidophilum. The average cyclization number of the hydrocarbon chains in the lipids increased with increasing growth temperatures.     

Solubilization of phospholipid vesicular structures into mixed micelles of zwitterionic surfactants

Interactions between the binary combination of dimethyltetradecylammoniopropanesulfonate (TPS) and l-α-phosphatidylcholine (PC), 1,2-didecanoyl-sn-glycero-3-phosphocholine, and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine in the aqueous bulk phase were evaluated with the help of pyrene fluorescence (l ₁/l ₃) measurements by studying the aggregation processes of TPS in pure water and in the presence of 7–36 μM of fixed concentrations of each lipid. The fluorescence measurements showed that TPS monomers undergo two kinds of aggregation process, which were identified by the three breaks. The first break, C₁, and the second, C₂, indicated the onset and completion of bilayer solubilization, respectively, on the incorporation of TPS monomers into the bilayer assemblies, which led to bilayer solubilization in the form of mixed micelles. This process was not clearly visible in the presence of PC, whereas some kinds of structure transitions were observed upon the incorporation of surfactant monomers. The partition coefficient (K), which defines the degree of partitioning of surfactant monomers into the bilayers with respect to the aqueous medium, was evaluated. A high K value of TPS-lipid aggregates indicated stronger interactions between surfactant and bilayer assemblies of lipid. The K values determined for the three phospholipids are close to each other, which indicates that K values do not depend on the hydrocarbon chain length of the phospholipid but of the surfactant used.     

Cell membrane localization of long chain C24−C30 fatty acids in two marine demosponges

Subcellular fractionation by differential centrifugation was performed on two previously unstudied marine sponges (Reniera sp. andPseudaxinyssa sp.) that represent both major subclasses of the Demospongiae. Long chain fatty acids (LCFA) with 24–30 carbon units were found as major constituents of cell membrane isolates of both sponges. Most LCFA were polyunsaturated and were constituents of the phospholipids, which are typical membrane lipids, and in particular the amino-phospholipids. The LCFA composition of phospholipids from whole sponge tissue was shown to provide a reliable indication of the LCFA composition of cell membrane phospholipids in the sponges studied. An unusual triply branched C₁₆ isoprenoid fatty acid, 4,8,12-trimethyltridecanoic acid, also was identified as a cell membrane acid in the spongePseudaxinyssa sp. Part 16 of “Phospholipid Studies of Marine Organisms.” For Part 15 in this series see Dasgupta, A., Ayanoglu, E., Tomer, K.B., and Djerassi, C. (1987)Chem. Phys. Lipids 43, 101–111.     

Sponge fatty acids. 3. Occurrence of series of n−7 monoenoic andiso-5,9 dienoic long-chain fatty acids in the phospholipids of the marine spongeCinachyrella aff.schulzei keller

The fatty acid composition of phospholipids from the New Caledonian spongeCinachyrella aff.schulzei Keller was studied. More than 60 fatty acids were identified as methyl esters andN-acyl pyrrolidides by gas chromatography and gas chromatography/mass spectrometry. Two isoprenoid fatty acids also were shown to be present, namely 4,8,12-trimethyltridecanoic and 5,9,13-trimethyltetradecanoic acids. The unusual 6-tetradecenoic, 6-pentadecenoic, 12-nonadecenoic and 26-methylheptacosanoic (iso-28∶0) acids were found for the first time in sponge phospholipids. A series of six n−7 monoenoic long-chain fatty acids (C₂₃ to C₂₈) were identified, including the rare 16-tricosenoic, 18-pentacosenoic and 21-octacosenoic acids. Fifteen fatty acids possessing the typical 5,9 dienoic moiety accounted for 30% of the total fatty acid mixture. Two new fatty acids were identified, namely 5(Z)-octacosenoic and 27-methyl-5(Z),9(Z)-octacosadienoic (iso-5,9-29∶2). Based on gas chromatography/Fourier transform infrared experiments, the double bonds were assigned the (Z) configuration. For part 2 of this series, see Reference 1.     

Influence of Individual Phospholipids on the Physical Properties of Oil-Based Suspensions

The impact of soybean lecithin and three individual phospholipids at different concentration (C_PL) on rheology and sedimentation behavior of sugar/soybean oil suspensions (? = 0.31) was studied and compared with attraction and retraction forces between sugar surfaces in soybean oil as measured by atomic force microscopy (AFM). In general, a surfactant‐induced reduction of yield stress, apparent viscosity and sediment volume of the suspensions coincides with a decrease of adhesive interactions between sugar particles in soybean oil. Although the general influence of individual phospholipids and soybean lecithin is comparable, it is concluded from investigations at low C_PL that individual phospholipids exhibit a less pronounced impact on the analyzed parameters. Furthermore, at low C_PL, binary mixtures of the phospholipids are more efficient than individual phospholipids as regards the reduction of yield stress and sediment volume. While the same tendency was detectable for AFM results, these differences were not statistically verified. Slight differences were also evident when comparing individual phospholipids and their influence on rheology and sedimentation which are, however, not in line with the results of AFM. A general understanding of these interrelations between surfactant composition and possible synergistic or antagonistic effects in mixtures of individual phospholipids contributes to optimizing lecithin composition with respect to functionality.     

Neutral lipids from the temporal gland of the African elephant,Loxodonta africana

Lipids from inactive and active temporal glands of the African elephant,Loxodonta africana, were isolated and fractionated. The inactive gland had a much higher total lipid content per gram of fresh tissue than the active gland. Lipids from the inactive gland consisted predominantly of neutral lipids while the active gland contained large quantities of phospholipids. Neutral lipids from the active gland contained much more hydrocarbons, cholesterol, and alkoxy glycerides than neutral lipids from an inactive gland. The alkoxy glyceride fraction did not contain any alkenyl glycerides. The hydrocarbons consisted of a mixture containing predominantly straight chain even numbered saturated and unsaturated hydrocarbons from C₁₈–C₃₀. Fatty acids from various fractions were investigated by gas liquid chromatography. Those from the active gland were characterized by a higher percentage of unsaturated acids. The change, from inactive to the active state involves mainly a reduction in palmitic and an increase in oleic acid content.     

Studies on the Biosynthesis of the Antibiotic Moenomycin A

Feeding experiments ( ¹³C and ¹⁵N‐labeled precursors) shed light on the biosynthetic origin of the chromophore (unit A of 1 ), the N‐acetyl groups, the 4‐C‐methyl group of the moenuronamide unit (part F of 1 ), the sugar units, and the lipid part (unit I of 1 ) of the antibiotic moenomycin A( 1 ). The lipid part is completely isoprenoid and is constructed via the non‐mevalonate pathway. The central C ₁₀ part originates from a precursor like geranyl or linalyl diphosphate and is formed by a route involving ring formation between C‐2 and C‐6 of the monoterpene unit, two successive rearrangements to give a 7‐membered ring intermediate and cleavage of the ring between C‐5 and C‐11 (moenocinol numbering).     

Lipid composition of commercially canned single-strength orange juice

The lipid composition of commercially canned single-strength orange juice ranged from 84–101 mg/100 ml juice (overall mean 95 ± 6). Phospholipid phosphorus, expressed as mg/100 ml juice, showed a range of from 1.56–1.95, while phospholipid phosphorus/lipid values (as µg-P/mg lipid) were within a very narrow range, 18.9 ± 1.1. The percentage distribution of lipid classes in these juices was 24–35% neutral lipids, 18–23% resin acids and glycolipids, and 43–53% phospholipids and other polar lipids. Five fatty acids, i.e. C₁₆, C_16:1, C_18:1, C_18:2, and C_18:3, accounted for over 93% of all fatty acids. The relative percentages of C_18:2 and C_18:3 differed between seasonal juices. The lipid composition does not warrant inclusion in nutritional labeling; however, lipid levels may be useful in detecting adulteration.     

Base-catalyzed derivatization methodology for FA analysis. Application to milk fat and celery seed lipid TAG

In this paper, an alternative base-catalyzed methodology for the facile derivatization in mild conditions of lipid TAG prior to FA analysis is proposed. Reagents were prepared by proton exchange between potassium tert-butoxide and either ethanol, n-propanol, n-butanol, or 2-methoxyethanol and used for the synthesis, at 40°C for 15 min, of the corresponding derivatives, which were directly analyzed by GC. This methodology can be used on a routine basis and has been applied to standard and complex natural lipid samples. Tripalmitin was used to determine optimal reaction conditions; and bovine milk fat, containing C₄ to C₂₂ acids, and celery (Apium graveolens) seed oil, characterized by a high level of petroselinic acid, were comparatively analyzed as their ethyl, n-propyl, n-butyl, and 2-methoxyethyl esters.     

Composition of the surface lipids of pea leaves (Pisum sativum)

Surface lipid of pea leaves (Pisum sativum var. Frosty) was analyzed with column, thin layer and gas liquid chromatography in conjunction with mass spectrometry and infrared spectroscopy. It contained 42%n-hentriacontane and 7.3%n-hentriacontan-16-ol. About 5% was wax esters, C₄₀–C₅₀ consisting of primarily C₂₆ and C₂₈ alcohols and C₁₆–C₂₂ acids. Almost 5% was aldehydes, mainly C₂₆ and C₂₈. Primary alcohols, chiefly C₂₆ and C₂₈, made up 20% of the surface lipid.     

Changes in flax (Linum usitatissimum L.) seed lipids during germination

Flax (Linum usitatissimum L.) seeds were germinated for 8 d under laboratory conditions, and changes in their lipid fraction were studied by various chemical and chromatographic methods. Total lipid content of the seeds was reduced fourfold at the end of the 8-d germination period as compared to ungerminated seeds on a fresh weight basis. The neutral lipids comprised the major fraction of seed lipids, and triacylglycerols predominated over all other lipid components even during the germination period. Both the spectrophotometric and thin-layer chromatography-flame-ionization detection methods of quantification showed a considerable increase in the content of free fatty acids. The glycolipid fraction of lipids increased, but the phospholipid fraction exhibited only minor changes. Lipase activity of flaxseed increased at the beginning of germination and then remained constant until the fifth day. Phosphatidylcholine was the major phospholipid of flaxseed lipids, and its content was reduced during the germination. The contents of lysophosphatidylcholine and phosphatidic acid increased from negligible amounts to 46% of the total phospholipids. Linolenic, linoleic, and oleic acids, respectively, were the predominant fatty acids of all the lipid fractions of flaxseed, and remained unchanged during the germination period. The glycolipid fraction had the lowest content of polyunsaturated fatty acids. Fatty acids C_14:0, C_20:0, C_24:0, C_20:1, C_22:1, and C_20:5 appeared after d 2 of germination in neutral, glyco- and phospholipid fractions.     

Acetate and mevalonate labeling studies with developingCuphea lutea seeds

Cuphea seeds contain large amounts of medium chain (C₈ to C₁₄) fatty acids, mainly as triacylglycerols. The biosynthesis of these lipids was studied in vivo by incubating developingCuphea lutea seeds with labeled acetate. Incorporation of label into triacylglycerols and into medium chain fatty acids occurred principally during the period of endogenous lipid deposition, but some label was encountered in these products even during seed dehydration. At this later stage palmitate and oleate were the dominant labeled fatty acids. During the period of rapid endogenous lipid deposition acyl lipids other than triacylglycerols were minor labeled components. The labeling patterns were consistent with the Kennedy pathway for triacylglycerol biosynthesis. The fatty acid composition of the acyl-CoA pool was similar to the total lipid fatty acid composition, but the acyl-ACP pool contained relatively more short chain acyl groups. Squalene was labeled from acetate throughout the period of seed development, but labeled sterols were not detected. Using [2-¹⁴C]mevalonic acid lactone as substrate, squalene was the principal labeled product. Small amounts of label were found in free sterols. However, in terms of mass, free sterol dominated over squalene. The possibility of two independent sites of isoprenoid biosynthesis in the developing embryo is discussed.     

Phospholipid studies of marine organisms: New branched fatty acids fromStrongylophora durissima

The phospholipids of the spongeStrongylophora durissima were analyzed. The major phospholipids present were phosphatidylethanolamine (PE), phosphatidylcholine (PC), phosphatidylserine (PS), phosphatidylglycerol (PG) and phosphatidylinositol (PI). The major fatty acid components of the phospholipids consisted of short chain (C₁₄−C₁₉) and very long chain (C₂₅−C₃₀) “Demospongic” acids. Three novel branched Δ⁵ monounsaturated acids,Z-19-methyl-5-pentacosenoic,Z-19-methyl-5-hexacosenoic andZ-19-methyl-5-heptacosenoic acids were encountered in the sponge. The 3-saturated counterparts of these compounds, 19-methylpentacosanoic, 19-methylhexacosanoic and 19-methylheptacosanoic acids, as well as 19-methylpentacosanoic and 20-methyloctacosanoic acids also are hitherto undescribed acids present in the sponge. Trace amounts of 2 very long chain acids also were detected and their structures tentatively assigned as 19,21-dimethylheptacosanoic and 20,22-dimethyloctacosanoic acids. The distribution of these fatty acids according to phospholipid head groups also was described.     

Lipids and lipophilic pollutants in three species of migratory shorebirds and their food in shepody bay (Bay of Fundy,New Brunswick)

To study food web transfers of lipids in the intertidal zone, three specimens of each of the semipalmated sandpiper (Calidris pusilla [L.]), the semipalmated plover (Charadrius semipalmatus Bonaparte) and the short-billed dowitcher (Limnodromus griseus [Gmelin]) were examined after capture during the summer of 1989. They were feeding intensively, prior to migration, on the amphipod,Corophium volutator, on the mudflats of Dorchester Cape, New Brunswick, Canada. The ragworm,Nereis diversicolor, and samples of sea foam were also collected from the mudflats at the same location in 1989 and 1990. Total body lipids of semipalmated sandpipers and short-billed dowitchers were about 20% of the wet weights, while those of the semipalmated plovers ranged from 35 to 40%. Adipose tissue fatty acids showed a trend from marine fatty acids (20∶5n−3, 22∶6n−3) at 10% ofC. pusilla acids through 5% inC. semipalmatus to 1% inL. griseus. These proportions confirm differences in feeding habits proposed from analyses of gut contents. Short-billed dowitcher adipose fat included the lowest values of polychlorinated biphenyls (0.078 to 0.266 ppm). The other two species of shore-birds showed levels as high as 1.64 ppm. Concentrations of 1,1-bis(4-chlorophenyl)-2,2,2-trichloroethane and 2,2-bis(4-chlorophenyl)-1,1-dichloroethylene were also variable, but with three exceptions were ≤0.1 ppm of adipose fat. Sea foam collected in the tidal channels and at the edge of the water during the rising tides was found to include diverse lipophilic materials, capable of transfer to higher trophic levels. Triacylglycerols, phospholipids, sterols, hydrocarbons and pigments were typically present at a concentration of 65×10³ μg/L. The long-chain isoprenoid squalene, of endogenous origin and apparently derived from featherwax, was the only hydrocarbon present in the depot fats of the three species of birds analyzed. This indicates that the birds basically inhabited a pristine environment free of hydrocarbon pollutants. The hydrocarbon present in the sea foam consisted of a series of normal alkanes, from C₂₀ to C₃₉, with no odd/even chain-length preference (carbon preference index=1.008). These hydrocarbons probably originated in detritus from decaying vascular plants but could possibly reflect a very low level of petroleum contamination. The lack of local pollution appears to favor continued success of the migratory pattern of these birds.     

Lipid composition of yoshida ascites hepatoma and of livers and blood plasma from host and normal rats

The lipid composition of Yoshida ascites hepatoma cells was analyzed together with that of ascitic plasma and of livers and blood plasma from host and normal rats. In comparison to normal livers, host livers showed no significant differences in the content of the various lipid classes, but contained a higher percentage of palmitic acid and a lower proportion of arachidonic acid in the major phospholipid classes. In addition, tumor growth induced a marked hypertriglyceridemia in host animals; changes in the concentration of other plasma lipid classes were not statistically significant. The ascitic plasma contained small amounts of lipids mainly constituted by cholesteryl esters and phospholipids. Yoshida hepatoma cells contained less phospholipids in comparison to both host and normal liver, while the increased level of triglycerides and the decrease of free fatty acids were not statistically significant. Hepatoma cells showed appreciable amounts of ether-linked lipids associated in part to neutral lipids (as glyceryl ether diesters) and, in part, to ethanolamine and choline phosphoglycerides. The alkyl groups in GEDE as well as in ethanolamine and choline phosphoglycerides were mainly constituted by C_16∶0 and C_18∶0 followed by C_18∶1. The alk-1-enyl groups in ethanolamine and choline phosphoglycerides were C_16∶0 and C_18∶0 with only a minor proportion of C_18∶1. In comparison to both host and normal liver, Yoshida hepatoma cells showed significant changes in the fatty acid composition of neutral lipids and phospholipids. Some of the major changes consisted of an increase of monoenoic acids associated with a decrease of arachidonic and docosahexaenoic acids in phosphatidylethanolamine, phosphatidylcholine, and phosphatidylinositol.     

Composition of milk fat globules with increased linoleic acid

Comparisons were made of the composition and distribution of the lipids in fat globules from conventional milks and polyunsaturated milks produced by cows fed protected lipid supplement. Washed creams were prepared from the milks of three individual cows fed a conventional ration, and three fed a protected sunflower-soybean supplement rich in linoleic acid. The washed creams were fractionated by treatment with sodium deoxycholate and centrifugation. Each washed cream and four fractions (designated as outer globule membrane, inner membrane, pellet, and globule core) were analyzed for protein, lipid, phospholipid, cholesterol, tocopherols, carotenoids, and fatty acid composition. The outer and inner membrane fractions were further fractionated into neutral and polar (phospholipid) lipid classes by thin layer chromatography. For both types of washed cream the approximate weight distribution of total solids was: outer membrane, 1%; inner membrane, 2%; pellet, 0.1%; and core, 96%. The percentages of protein, phospholipid, cholesterol, and carotenoids were all lower in the polyunsaturated than in the conventional creams. In the polyunsaturated creams, the percentages of both saturated and unsaturated C₁₈ acids were higher, and of acids of C₁₆ and shorter chain length lower, than in the conventional creams. The phospholipids in the outer and inner membranes from the polyunsaturated milks had larger proportions of linoleic acid than did the phospholipids from the conventional milks. However, this increase in unsaturation was less than that of the core neutral lipids. Pancreatic lipase hydrolysis of the core fractions showed that the increased linoleic acid was introduced preferentially at the 2-position of the triglycerides. In general, the observed changes in physical properties and in susceptibility of polyunsaturated milk to the development of oxidized flavor are consistent with the differences in the relative proportions of the various classes of lipids in the conventional and polyunsaturated milks. Data from thesis of D.H. Bianco submitted in partial fulfillment of the requirements for the M.S. Degree in Food Science, University of California, Davis. Presented in part at the AOCS Meeting, New Orleans, April, 1976.     

Lipid molecular order in liver mitochondrial outer membranes, and sensitivity of carnitine palmitoyltransferase I to malonyl-CoA

Mitochondrial outer membranes were prepared from livers of rats that were in the normal fed state, starved for 48 h, or made diabetic by injection of streptozotocin. Membranes were also prepared from starved late-pregnant rats. The latter three conditions have previously been shown to induce varying degrees of desensitization of mitochondrial overt carnitine palmitoyltransferase (CPT I) to malonyl-CoA inhibition. We measured the fluorescence polarization anisotropy of two probes, 1,6-diphenyl-1,3,5-hexatriene (DPH) and 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene-p-toluenesulfonate (TMA-DPH) which, when incorporated into membranes, report on the hydrophobic core and on the peripheral regions of the bilayer, respectively. The corresponding polarization indices (r _DPH and r _TMA-DPH) were calculated. In membranes of all three conditions characterized by CPT I desensitization to malonyl-CoA, r _DPH was decreased, whereas there was no change in r _TMA-DPH, indicating that CPT I is sensitive to changes in membrane core, rather than peripheral, lipid order. The major lipid components of the membranes were analyzed. Although significant changes with physiological state were observed, there was no consistent pattern of changes in gross lipid composition accompanying the changes to membrane fluidity and CPT I sensitivity to malonyl-CoA. We conclude that CPT I kinetic characteristics are sensitive to changes in lipid composition that are localized to specific membrane microdomains.     

Distinctive medium chain wax esters,triglycerides, and diacyl glyceryl ethers in the head fats of the pacific beaked whale,Berardius bairdi

Lipids were extracted from the mandibular fat body (jaw), the fatty forehead (melon), and the dorsal blubber of a Pacific beaked whale (Berardius bairdi) and separated into lipid classes by preparative thin layer chromatography. The head fats were mixtures of wax esters and triglycerides with a very small amount of diacyl glyceryl ether. The blubber fat contained 97% was ester and 3% triglyceride. Gas liquid chromatography (GLC) of the intact lipid classes indicated an unusually low C₂₆–C₃₀ range for most of the jaw and melon wax esters compared to the more normal C₃₂–C₄₀ molecules found in the blubber. Distinctive lower molecular weight C₂₄–C₄₀ triglycerides occurred in the head fats vs. the usual C₄₄–C₅₈ range in the blubber. Most diacyl glyceryl ethers were in the C₃₅–C₄₆ range, below the molecular weight of hexadecyldipalmitoyl glyceryl ether (C₄₈). GLC of the derived fatty acid methyl esters showed that the lower molecular weight neutral lipids in the head fats were due to high levels of iso-10∶0, n−10∶0, iso-11∶0, iso-12∶0, n−12∶0, and iso-13∶0 acids. The wax ester fatty alcohols and the alkoxy chains of the glyceryl ethers were mostly the C₁₄–C₂₀ chain lengths commonly observed in marine organisms. The distinctive medium chain neutral lipids in the jaw and melon fats of this whale may be related to the postulated acoustical role of these tissues in echolocation.     

Effect of chlorpromazine on rat arterial lipid synthesis,in vitro

The effect of chlorpromazine, a major tranquilizer, on arterial lipid metabolism was studied in vitro in rat aortas incubated with [¹⁴C] acetate and [¹⁴C] mevalonate as lipid precursors. Chlorpromazine at a level of 0.25 mM in the incubation medium significantly reduced the incorporation of [¹⁴C] acetate into free fatty acids (p<0.01) and total phospholipids (p<0.001) but not triglycerides. Chlorpromazine also altered the pattern of arterial phospholipids synthesized from [¹⁴C] acetate by significantly increasing the relative proportion of phosphatidylinositol plus phosphatidylserine (p<0.02) and reducing the relative proportion of sphingomyelin (p<0.001). [¹⁴C] Acetate incorporation into the combined fractions of steryl esters plus hydrocarbons and sterols plus diglycerides was also significantly reduced (p<0.001) by 0.25 mM chlorpromazine. Studies with [¹⁴C] mevalonate showed that chlorpromazine is also an inhibitor of sterol biosynthesis in arterial tissues as evidenced by 35–40% reductions (p<0.05) in the formation of¹⁴C-labeled squalene and C₂₇ sterols.     

cis-9,trans-11 and trans-10,cis-12 CLA affect lipid metabolism differently in primary white and brown adipocytes of Djungarian hamsters

We explored whether CLA isomers and other C₁₈ FA affect (i) lipid content and FA concentrations in total adipocyte lipids, (ii) FA synthesis from glucose in TAG and phospholipids of primary brown (BAT) and white adipocytes (WAT), and (iii) mRNA expression of uncoupling protein 1 (UCP1) in primary brown adipocytes of Djungarian hamsters (Phodopus sungorus). c9,t11-CLA, oleic, linoleic, and α-linolenic acid increased whereas t10,c12-CLA decreased lipid accumulation in both adipocyte types. t10,c12-CLA treatment affected FA composition mainly in BAT cells. CLA incorporation into lipids, in particular c9,t11-CLA, was higher in BAT. In both cell types, t10,c12-CLA treatment reduced the incorporation of glucose ¹³C carbon into FA of TAG and phospholipids, whereas c9,t11-CLA, linoleic, and α-linolenic acid either did not influence or dose-dependently increased glucose carbon incorporation into FA. UCP1 mRNA expression was inhibited by t10,c12-CLA but increased by c9,t11-CLA, linoleic, and α-linolenic acid. It is concluded that c9,t11-CLA and t10,c12-CLA have distinctly different effects on lipid metabolism in primary adipocytes. The effects of c9,t11-CLA are similar to those of other unsaturated C₁₈ FA. The opposite effects of c9,t11-CLA and t10,c12-CLA are evident in both WAT and BAT cultures; however, brown adipocytes seem to be more susceptible to CLA treatment.