In vitro incorporation of elongated fatty acyl products into lipid classes in the housefly,Musca domestica L. and the American cockroach,Periplaneta americana (L.)

Thein vitro incorporation of elongated fatty acyl products into various lipid classes was studied in the American cockroach,Periplaneta americana (L.) and the houseflyMusca domestica L. Stearoyl-CoA (18∶0-CoA) and linoleoyl-CoA (18∶2-CoA) were each elongated in microsomal preparations from abdominal epidermal tissue of the adult cockroach. Incorporation of radioactive tracer into different lipid classes was determined by thin-layer chromatography (TLC). In the American cockroach, 40–45% of the total radioactive label was incorporated into the free fatty acid fraction, with smaller amounts in the triglyceride (12–31%) and phospholipid (12–19%) fractions. Of the elongated products analyzed by radio-high performance liquid chromatography (HPLC), 53–60% was found in the free fatty acid fraction. In the housefly, the substrates 18∶0-CoA and 18∶1-CoA were used to determine into which lipids the elongated products would become incorporated. The saturated fatty acyl elongated products were found mainly in the free fatty acid (41%), triglyceride (23%), and acyl-CoA (17%) fractions. The monounsaturated fatty acyl elongated products were found in the triglyceride (44%), free fatty acid (11%), acyl-CoA (35%) and phospholipid (10%) fractions in three-day-old males. In three-day-old females, the elongated products were found in the triglyceride (45%), free fatty acid (28%), acyl-CoA (11%) and phospholipid (15%) fractions. From these data, it is not possible to determine the identity of the substrate for the conversion of the elongated fatty acyl products to the corresponding hydrocarbon (Hy). In the cockroach, incubations with 18∶0-CoA and with 18∶2-CoA resulted in small incorporations into 25∶0 Hy and into 27∶2 Hy, respectively. In the housefly, incubations with 18∶1-CoA resulted in a very small production of 27∶1 Hy in mature males and 23∶1 Hy in mature female houseflies. These data support the idea that the preparation of subcellular fractions results in an uncoupling of fatty acid chain elongation from the conversion of the fatty acid to the corresponding hydrocarbon in both insects.     

Incorporation and distribution of saturated and unsaturated fatty acids into nuclear lipids of hepatic cells

Liver nuclear incorporation of stearic (18∶0), linoleic (18∶2n−6), and arachidonic (20∶4n−6) acids was studied by incubation in vitro of the [1-¹⁴C] fatty acids with nuclei, with or without the cytosol fraction at different times. The [1-¹⁴C] fatty acids were incorporated into the nuclei as free fatty acids in the following order: 18∶0>20∶4n−6≫18∶2n−6, and esterified into nuclear lipids by an acyl-CoA pathway. All [1-¹⁴C] fatty acids were esterified mainly to phospholipids and triacylglycerols and in a minor proportion to diacylglycerols. Only [1-¹⁴C] 18∶2n−6-CoA was incorporated into cholesterol esters. The incorporation was not modified by cytosol addition. The incorporation of 20∶4n−6 into nuclear phosphatidylcholine (PC) pools was also studied by incubation of liver nuclei in vitro with [1-¹⁴C]20∶4n−6-CoA, and nuclear labeled PC molecular species were determined. From the 15 PC nuclear molecular species determined, five were labeled with [1-¹⁴C]20∶4n−6-CoA: 18∶0–20∶4, 16∶0–20∶4, 18∶1–20∶4, 18∶2–20∶4, and 20∶4–20∶4. The highest specific radioactivity was found in 20∶4–20∶4 PC, which is a minor species. In conclusion, liver cell nuclei possess the necessary enzymes to incorporate exogenous saturated and unsaturated fatty acids into lipids by an acyl-CoA pathway, showing specificity for each fatty acid. Liver cell nuclei also utilize exogenous 20∶4n−6-CoA to synthesize the major molecular species of PC with 20∶4n−6 at the sn-2 position. However, the most actively synthesized is 20∶4–20∶4 PC, which is a quantitatively minor component. The labeling pattern of 20∶4–20∶4 PC would indicate that this molecular species is synthesized mainly by the de novo pathway.     

Effect of triacylglycerol structure on absorption and metabolism of isotope-labeled palmitic and linoleic acids by humans

The effect of dietary TAG structure and fatty acid acyl TAG position on palmitic and linoleic acid metabolism was investigated in four middle-aged male subjects. The study design consisted of feeding diets containing 61 g/d of native lard (NL) or randomized lard (RL) for 28 d. Subjects then received an oral dose of either 1,3-tetradeuteriopalmitoyl-2-dideuteriolinoleoyl-rac-glycerol or a mixture of 1,3-dideuteriolinoleoyl-2-tetradeuteriopalmitoyl-rac-glycerol and 1,3-hexadeuteriopalmitoyl-2-tetradeuteriolinoleoyl-rac-glycerol. Methyl esters of plasma lipids isolated from blood samples drawn over a 2-d period were analyzed by GC-MS. Results showed that absorption of the ²H-fatty acids (²H-FA) was not influenced by TAG position. The ²H-FA at the 2-acyl TAG position were 85±4.6% retained after absorption. Substantial migration of ²H-16∶0 (31.2±8.6%) from the sn-2 TAG position to the sn-1,3 position and ²H-18∶2n−6 (52.8±6.4%) from the sn-1,3 position to the sn-2 position of chylomicron TAG occurred after initial absorption and indicates the presence of a previously unrecognized isomerization mechanism. Incorporation and turnover of the ²H-FA in chylomicron TAG, plasma TAG, and plasma cholesterol esters were not influenced by TAG acyl position. Accretion of ²H-16∶0 from the sn-2 TAG position in 1-acylphosphatidylcholine was 1.7 times higher than ²H-16∶0 from the sn-1,3 TAG positions. Acyl TAG position did not influence ²H-18∶2n−6 incorporation in PC. The concentration of ²H-18∶2n−6-derived ²H-20∶4n−6 in plasma PC from subjects fed, the RL diet was 1.5 times higher than for subjects fed the NL diet, and this result suggests that diets containing 16∶0 located at the sn-2 TAG position may inhibit 20∶4n−6 synthesis. The overall conclusion is that selective rearrangement of chylomicron TAG structures diminishes but does not totally eliminate the metabolic and physiological effects of dietary TAG structure.     

Efficiency of phospholipid remodeling via acyl-CoA:lysophospholipid acyltransferases action is modified by acyl-CoAs

This study investigated the impact of different acyl-CoAs on remodeling efficiency of PC and PE via the action of acyl-CoA:lysophospholipid acyltransferases (LPLAT). Microsomal fractions from yeast ΔALE1 mutant transformed with an empty plasmid pYES2 or plasmid carrying AtLPCAT or ALE1 encoding genes were used in the experiments. In the preliminary assays, presence of 18:1-CoA in the reaction mixture increased remodeling efficiency of [¹⁴C]PC integrated with microsomal fraction of yeast overexpressing AtLPCAT compared to assays without exogenous acyl-CoA. In further experiments, the effect of five different acyl-CoAs (16:0-CoA, 18:0-CoA, 18:1-CoA, 18:2-CoA, and 18:3-CoA) on remodeling efficiency of yeast microsomal PC and PE via the action of three LPLATs (yeast SLC1 and ALE1 and Arabidopsis LPCAT2) were tested. It has been shown that acyl-CoAs used in the experiments had different effect on the remodeling intensity of PC and PE via action of the tested acyltransferases. Moreover, each acyl-CoA used had a different effect on the tested acyltransferases. Additionally, the assays with DTNB (inhibitor of the LPLAT backward reaction) showed that remodeling of PC and PE by LPLATs can also occur through reactions other than the backward reaction carried out by these enzymes, and that acyl-CoA present in the reaction mixture affected these processes.     

Phospholipid synthesis by chick retinal microsomes: Fatty acid preference and effect of fatty acid binding protein

The acylation of 1-palmitoyl-sn-glycerophosphocholine (1-16∶0-GPC) or 1-palmitoyl-sn-glycerophosphoethanolamine (1-16∶0-GPE) was measured using the microsomal fraction prepared from retinas of 14–15-day-old chick embryos. Rates of incorporation of exogenously supplied fatty acids into diacyl-GPC were generally 5–7 times greater than into diacyl-GPE. Substrate preferences for incorporation into diacyl-GPC and diacyl-GPE were, respectively, 18∶2>18∶3=20∶5>20∶4>18∶1>22∶6=18∶0 and 18∶2>22∶6≽18∶3=18∶0≽20∶4=18∶1>20∶5. The apparent selectivities were not consistent with the reported fatty acid compositions of these lipid classes. The addition of partially purified fatty acid binding protein (FABP) to the reaction had no effect either on overall rates of incorporation or on the substrate preference. When fatty acyl-CoA substrates were used, rates of incorporation of the 18∶0 derivative were much higher than with the fatty acid, while rates with other fatty acyl-CoA were similar to those with the respective fatty acid. Substrate preferences for CoA derivatives incorporated into diacyl-GPC were: 18∶0>20∶4>18∶2≽22∶6, and into diacyl-GPE: 20∶4=22∶6>18∶0>18∶2. Polyunsaturated fatty acyl CoA (PUFA-CoA) were thus favored for incorporation into diacyl-GPE, and to a lesser extent into diacyl-GPC, a result that is consistent with composition data. When purified FABP was added to the reactions, there was an increase in the incorporation of 18∶0-CoA and a decrease or no change in the incorporation of PUFA-CoA. The deacylation/reacylation cycle thus appears to play a role in the modification of phospholipid composition. The data are not consistent, however, with a role for FABP in directing PUFA toward membrane lipid synthesis.     

Distribution of the major fatty acids of human milk betweensn-2 andsn-1,3 positions of triacylglycerols

Human milk triacylgycerols (TAG) were analyzed by tandem mass spectrometry. The SIMPLEX method and a simple linear model were used to interpret the distribution of fatty acids between thesn-2 andsn-1,3 positions in 24 major molecular weight groups of TAG. The number of regio-isomeric pairs of TAG varied between 3 and 18 in each of these groups. Hexadecanoic (16∶0), tetradecanoic (14∶0) and dodecanoic acids (12∶0) typically occupied thesn-2 position in TAG containing less than 54 acyl carbons, whereas long-chain C18 and C20 acids were predominantly located at the primary positions. The positions of the three fatty acids within a TAG molecule were shown to depend on the fatty acid combination. The maximum of 12∶0 in thesn-2 position appeared at acyl carbon number (ACN) 48, the maxima of 14∶0 were at ACN 44 and ACN 50, and for 16∶0 at ACN 46 and 52.     

Triacylglycerols of human milk: Rapid analysis by ammonia negative ion tandem mass spectrometry

Human milk traicylglycerols (TAG) were analyzed by ammonia negative ion chemical ionization tandem mass spectrometry. The deprotonated molecular ions of triacylglycerols were fractionated at the first mass spectrometry (MS) stage. Twenty-nine of the deprotonated TAG ions were further analyzed based on their collisionally activated (CA) spectra. The tandem MS analysis covered eleven major acyl carbon number fractions, two of which contained odd carbon number fatty acids. Fatty acids of 28 different molecular weights were recorded from the daughter spectra. Hexadecanoic acid was present in all CA spectra, octadecenoic acid in the CA spectra of all mono- and higher unsaturated TAG, and octadecadienoic acid in the CA spectra of all di- and higher unsaturated TAG. The major fatty acid combinations in triacylglycerols were: with 0 double bonds (DB), 12∶0/12∶0/16∶0; with 1 DB, 12∶0/16∶0/18∶1; with 2 DB, 16∶0/18∶1/18∶1; with 3 DB, 16∶0/18∶2/18∶1; with 4 DB, 18∶2/18∶1/18∶1; and with 5 DB, 18∶2/18∶2/18∶1; hexadecanoic acid typically occupied thesn-2 position. The most abundant TAG was shown to besn-18∶1–16∶0–18∶1, comprising about 10% of all triacylglycerols.     

Purification of the acyl-CoA elongase complex from developing rapeseed and characterization of the 3-ketoacyl-CoA synthase and the 3-hydroxyacyl-CoA dehydratase

Oleoyl-CoA elongase catalyzes four successive reactions: condensation of malonyl-CoA to oleoyl-CoA, reduction, dehydration, and another reduction. Evidence supporting this mechanism and the multienzymatic nature of the elongation complex are reported. A particulate membrane fraction from rapeseed is able to elongate intermediates (R,S) 3-hydroxy-20∶0-CoA and (E) 2,3–20∶1-CoA to very long chain fatty acids in the presence of malonyl-CoA. Studies of the 3-ketoacyl-CoA synthase activities showed that maximal activity could be measured by using 15 to 30 μM 18∶1-CoA and 30 μM malonyl-CoA, and that 18∶0-CoA and 18∶1-CoA were the best substrates. Comparison of the condensation and the overall elongation activities indicated that condensation is the rate-limiting step of the elongation process. The 3-hydroxyacyl-CoA dehydratase activity was maximal in the presence of 75 μM Triton X-100 and 25 μg of proteins. Finally, the acyl-CoA elongase complex was solubilized and purified. During the purification process, the 3-hydroxyacyl-CoA dehydratase copurified with the elongase complex, strongly suggesting that this enzyme belongs to the elongase complex. The apparent molecular mass of 700 kDa determined for the elongase complex, and the fact that four different polypeptide bands were detected after sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis of the purified fraction, further suggest that the acyl-CoA elongase is a multienzymatic complex.     

Storage lipid accumulation and acyltransferase action in developing flaxseed

Investigations of storage lipid synthesis in developing flaxseed (Linum usitatissimum) provide useful information for designing strategies to enhance the oil content and nutritional value of this crop. Lipid content and changes in the FA composition during seed development were examined in two cultivars of flax (AC Emerson and Vimy). The oil content on a dry weight basis increased steadily until about 20 d after flowering (DAF). The proportion of α-linolenic acid (α-18∶3, 18∶3cisΔ^{9, 12, 15}) in TAG increased during seed development in both cultivars while the proportions of linoleic acid (18∶2cisΔ^{9, 12}) and saturated FA decreased. The developmental and substrate specificity characteristics of microsomal DAG acyltransferase (DGAT, Ec 2.3.1.20) and lysophosphatidic acid acyltransferase (LPAAT, EC 2.3.1.51) were examined using cultivar AC Emerson. The maximal acyltransferase specific activities occurred in the range of 8–14 DAF, during rapid lipid accumulation on a per seed basis. Acyl-CoA of EPA (20∶5cisΔ^5,8,11,14,17) or DHA (22∶6cis ^{4,7,10,13,16,19}) were included in the specificity studies. DGAT displayed enhanced specificity for α-18∶3-CoA, whereas the preferred substrate of LPAAT was 18∶2-CoA. Both enzymes could use EPA- or DHA-CoA to varying extents. Developing flax embryos were able to take up and incorporate these nutritional FA into TAG and other intermediates in the TAG-formation pathway. This study suggests that if the appropriate acyl-CoA-dependent desaturation/elongation pathways are introduced and efficiently expressed in flax, this may lead to the conversion of α-18∶3-CoA into EPA-CoA, thereby providing an activated substrate for TAG formation.     

Effect of Δ9-tetrahydrocannabinol and merthiolate on acyltransferase activities in guinea pig liver microsomesand merthiolate on acyltransferase activities in guinea pig liver microsomes

Δ⁹-tetrahydrocannabinol (THC) and merthiolate have been utilized as lysophospholipid acyltransferase inhibitors in metabolic studies. However, their effects on acyltransferases other than lysophosphatidylcholine:acyl-CoA acyltransferase (LPCAT) are not known. We have therefore investigated the effectiveness of THC and merthiolate in inhibiting the acylation of lysophosphatidylcholine, lysophosphatidylethanolamine, lysophosphatidylserine, lysophosphatidylinositol (LPI) and lysophosphatidic acid (LPA) in guinea pig liver microsomes using oleoyl-CoA and arachidonoyl-CoA as acyl donors. THC inhibited LPCAT and lysophosphatidylethanolamine: acyl-CoA acyltransferase (LPEAT) by 40–50%, but had no effect or only slightly increased the activities of the other acyltransferases when assayed with oleoyl-CoA as the acyl donor. The results obtained with arachidonoyl-CoA were similar to those with oleoyl-CoA, with the exception of a 40% inhibition of lysophosphatidylserine:acyl-CoA acyltransferase (LPSAT) at concentrations of 50 μM or higher. At similar concentrations, merthiolate was more effective than THC in inhibiting the acyltransferases examined. Selective effects on the acyltransferases were observed at low concentrations of merthiolate (20 μM or less). Thus, LPCAT was most susceptible, followed by LPI acyltransferases, LPSAT, LPEAT and lysophosphatidic acid:acyl-CoA acyltransferases (LPAAT). The presence of LPA did not affect the inhibition of LPCAT by merthiolate. Thus the resilience of LPAAT to merthiolate inhibition was not due to chelation of the compound by the acidic lysolipid. Thiol reagents includingN-ethylmaleiamide, 5,5′-dithio-bis-nitrobenzoic acid, iodoacetate, β-mercaptoethanol and dithiothreitol had little or no effect on the acyltransferases relative to equimolar concentrations of merthiolate. The above results indicate that merthiolate is a much more effective inhibitor of lysophospholipid:acyl-CoA acyltransferases than is THC, and that the selectivity exhibited by merthiolate may be due to direct and specific interaction with the acyltransferases.     

Properties of diacylglycerol acyltransferase from spinach leaves

Diacylglycerol-acyltransferase (EC 2.3.1.20) was partially purified and characterized from spinach leaves. The enzyme had a pH optimum of 8.0 and activity was stimulated 2-fold by the addition of 20 mM Mn²⁺ or Mg²⁺. Diolein and dipalmitin were examined assn-1,2-diacylglycerol substrates. Only diolein gave detectable triacylglycerol synthesis. In addition, the saturation kinetics of the enzyme with 16∶0-CoA, 18∶0-CoA and 18∶1-CoA were examined. The highest apparent, Km was observed with 18∶1-CoA, the lowest with 16∶0-CoA. Endogenous spinach leaf glycerolipids were extracted and analyzed. The leaves contained 70 nmol triacylglycerol (g fresh weight)⁻.     

Plackett-burman design for determining the preference of Rhizomucor miehei lipase for FA in acidolysis reactions with coconut oil

The preference of lipase (EC 3.1.1.3) from Rhizomucor miehei in the incorporation of 11 FA, ranging from C10∶0 to C22∶6, into coconut oil TAG during acidolysis was studied by applying the Plackett-Burman experimental design. Enzymatic acidolysis reactions were carried out in hexane at 37°C for 48 h with coconut oil (0.1 M) and a mixture of 11 FA at a TAG to FA molar ratio of 1∶1. Lipase was used at the 5 wt% level. The incorporation of FA into coconut oil TAG was determined by GC. The lipase showed preference for long-chain saturated FA for incorporation into coconut oil TAG. The FA with 18 carbon atoms showed a high incorporation rate (18∶1>18∶1>18∶3). The lipase showed the least preference for the incorporation of 12∶0, which occurs in maximal concentration (46%), whereas the most preferred FA, 18∶0, occurs at a very low concentration (<2%) in coconut oil. The overall preference of lipase for the incorporation of different FA into coconut oil TAG was 18∶0>18∶2, 22∶0>18∶1, 18∶3, 14∶0, 20∶4, 22∶6>16∶0>12∶0≫10∶0.     

Generation and remodeling of highly polyunsaturated molecular species of rat hepatocyte phospholipids

Freshly isolated rat hepatocytes were incubated for 20 min with [U-¹⁴C]glycerol in the presence or absence of unlabeled linoleic (18∶2n-6), arachidonic (20∶4n-6), or docosahexaenoic (22∶6n-3) acid, added as albumin complex in 10% ethanol. Most of the radioactivity (≈95%) recovered in hepatocyte lipids was present in phosphatidylcholine (PC), phosphatidylethanolamine (PF), and triacylglycerol (TAG). The presence of exogenous fatty acids resulted in (i) higher incorporation of [U-¹⁴C]glycerol, (ii) higher percentage of label in TAG, and (iii) enhanced formation of PC and PE molecular species bearing the exogenous fatty acid at both the sn-1 and sn-2 positions of glycerol. In each case, these molecular species contained 60 to 70% of the label in that lipid class. Further incubation of the cells for 40 and 80 min in the absence of labeled substrate and exogenous fatty acids resulted in a redistribution of label among PC and PE molecular species due to deacylation-reacylation at the sn-1 position of glycerol.     

Diatoms and Plants Acyl-CoA:lysophosphatidylcholine Acyltransferases (LPCATs) Exhibit Diverse Substrate Specificity and Biochemical Properties

The search of the Phaeodactylum tricornutum genome database revealed the existence of six genes potentially encoding lysophospholipid acyltransferases. One of these genes, Phatr3_J20460, after introduction to yeast ale1 mutant disrupted in the LPCAT gene, produced a very active acyl-CoA:lysophosphatidylcholine (LPCAT) enzyme. Using in vitro assays applying different radioactive and non-radioactive substrates and microsomal fractions from such yeast, we have characterized the biochemical properties and substrate specificities of this PtLPCAT1. We have found that the substrate specificity of this enzyme indicates that it can completely supply phosphatidylcholine (PC) with all fatty acids connected with a biosynthetic pathway of very long-chain polyunsaturated fatty acids (VLC-PUFAs) used further for the desaturation process. Additionally, we have shown that biochemical properties of the PtLPCAT1 in comparison to plant LPCATs are in some cases similar (such as the dependency of its activity on pH value), differ moderately (such as in response to temperature changes), or express completely different properties (such as in reaction to calcium and magnesium ions or toward some acyl-CoA with 20C polyunsaturated fatty acids). Moreover, the obtained results suggest that cloned “Phatr3_J20460” gene can be useful in oilseeds plant engineering toward efficient production of VLC-PUFA as LPCAT it encodes can (contrary to plant LPCATs) introduce 20:4-CoA (n-3) to PC for further desaturation to 20:5 (EPA, eicosapentaenoic acid).     

Pathways for fatty acid elongation and desaturation in Neurospora crassa

Neurospora crassa incorporated exogenous deuterated palmitate (16∶0) and ¹⁴C-labeled oleate (18∶1^Δ9) into cell lipids. Of the exogenous 18∶1^Δ9 incorporated, 59% was desaturated to 18∶2^Δ9,12 and 18∶3^Δ9,12,15. Of the exogenous 16∶0 incorporated, 20% was elongated to 18∶0, while 37% was elongated and desaturated into 18∶1^Δ9, 18∶2^Δ9,12, and 18∶3^Δ9,12,15. The mass of unsaturated fatty acids in phospholipid and triacylglycerol is 12 times greater than the mass of 18∶0. Deuterium label incorporation in unsaturated fatty acids is only twofold greater than in 18∶0, indicating a sixfold preferential use of 16∶0 for saturated fatty acid synthesis. These results indicate that the release of 16∶0 from fatty acid synthase is a key control point that influences fatty acid composition in Neurospora.     

Analysis of positional distribution of fatty acids in palm oil by13C NMR spectroscopy

The¹³C NMR spectrum of the carbonyl carbons of the acyl groups of triacylglycerols of palm oil has been shown to give the composition of saturated, oleic and linoleic acyl groups at the 1,3-positions and at the 2-position of the glycerol moiety. Except for the lack of differentiation of the saturated fatty acids, the¹³C NMR technique provides the same information as the tedious enzymatic hydrolysis cum fatty acid analysis. The carbonyl carbon of the linolenic acyl group (18∶3,[cis, cis, cis]-9, 12, 15) has a chemical shift which is only 0.005 ppm to low frequency of that of the linoleic acyl group (18∶2,[cis, cis]-9, 12), so that the two resonances may not be distinguishable (or resolved) even at a high magnetic field.     

Membrane lipid profile of an edible basidiomycete <Emphasis Type="Italic">Lentinula edodes</Emphasis> during growth and cell differentiation

The basidiomycetous mushroom Lentinula edodes (Shiitake) exhibits a unique process of cell differentiation termed “fruiting-body formation”. To clarify the relationship between membrane lipids and fruiting-body formation in this fungus, we investigated variations in levels of phospholipids, cerebrosides, fatty acyl residues in the major phospholipids, and fatty acyl and sphingoid base residues in cerebrosides during vegetative growth and fruiting-body formation. PC, PE, and PS were the primary phospholipids in the cells of L. edodes. After a shift in growth temperature of L. edodes mycelia has been shifted from 25 to 18°C, the proportion of unsaturated FA (UFA), such as linoleic acid (18∶2) and oleic acid (18∶1), increased. In contrast, during fruiting-body formation induced by the temperature downshift to 18°C, 18∶2 of PC in the primordia and fruiting bodies decreased, and the UFA of PF and 18∶1 of PC increased compared with the proportions in mycelia growing at 18°C. These results showed that the proportions of fatty acyl residues in PC and PE differed during fruiting-body formation in L. edodes. Moreover, the amount of cerebrosides in primordia increased compared with those in mycelia and fruiting bodies and, in these differentiating tissues, the proportion of 2-hydroxypentadecanoic acid increased whereas that of 2-hydroxyoctadecanoic acid decreased compared with that in the mycelia. However, the proportion of sphingoid base residues in cerebrosides did not change during fruiting-body formation in L. edodes.     

Molecular weight distribution and regioisomeric structure of triacylglycerols in some common human milk substitutes

Triacylglycerol (TAG) molecular weight distribution and regioisomeric structure of selected molecular weight species in human milk and in 32 human milk substitutes was determined. Negative ion chemical ionization mass spectrometry was used to determine the molecular weight distribution and collisionally induced dissociation tandem mass spectrometry applied to identify the sn-2 and sn-1/3 positions of fatty acids in TAG. The main molecular weight species of human milk TAG in decreasing order of abundance were 52∶2, 52∶3, 52∶1, 54∶3, 50∶2, 50∶1, 54∶4, 48∶1, 54∶2, 48∶2, 46∶1, 52∶4, and 50∶3 (acyl carbon number/number of double bonds), constituting 83 mol% of total TAG molecular species. In human milk substitutes, the proportion of the corresponding molecular weight species varied from 33 to 87 mol%. The main TAG regioisomers within the molecular weight species 52∶2, 52∶3, and 50∶1 in human milk were 18∶1-16∶0-18∶1 (83 mol%), 18∶1-16∶0-18∶2 (83 mol%), and 18∶1-16∶0-16∶0 (80 mol%), respectively. In human milk substitutes, the corresponding proportions varied in a wide range of 0–82 mol%, 0–100 mol%, and 0–73 mol%, respectively. Although TAG structures in some human milk substitutes closely resembled those in human milk, the great variation among samples leads to the conclusion that it is still possible to improve the TAG composition in human milk substitutes by applying novel methods to synthesize structured TAG.     

Formation of a thiamine disulfide complex with fatty acid: Mechanism of formation of the complex

The formation of complexes between thiamine disulfide (TDS) orO-acetyl thiamine disulfide (O-acetyl TDS) and fatty acid or fatty acid methyl ester in methanol has been studied by fluorescence quenching and¹³C NMR relaxation (T₁) measurements. The association constants (K-values) of TDS andO-acetyl TDS with fatty acids (from 11∶0 to 18∶0, and 18∶1, 18∶2, 18∶3 and 20∶4) and fatty acid methyl esters have been determined. These values do not depend on either the number of carbon atoms or the degree of unsaturation of the fatty acid. The K-values of TDS andO-acetyl TDS with fatty acid were 7.8 M⁻¹ and 5.1 M⁻¹, respectively. The K-values of TDS andO-acetyl TDS with fatty acid methyl ester were very small. These results show that the-OH moiety in TDS and the-COOH moiety in the fatty acid are necessary for formation of the complex     

A simple HPLC method for the simultaneous analysis of phosphatidylcholine and its partial hydrolysis products 1- and 2-acyl lysophosphatidylcholine

A simple HPLC method for the simultaneous analysis of phosphatidylcholine (PC), 1-acyl lysophosphatidylcholine (1-acyl LPC), and 2-acyl lysophosphatidylcholine (2-acyl LPC) with refractive index detection is described. The separation of these three compounds was achieved on a Waters (Milford, MA) Spherisorb amino phase column using a mixture of ethanol and an aqueous oxalic acid solution as eluent. The optimal mixture of ethanol to oxalic acid solution was 92∶8 (vol/vol). PC and the two regioisomers of LPC eluted within 15 min. The calibration curves were linear in a concentration range from 0.05 to 2.5 mM. Natural PC or LPC eluted in a single peak, despite the diversity in the fatty acid composition. There was no rearrangement between 1-acyl LPC and 2-acyl LPC during analysis or storage in ethanol within 23 h. This method is thus especially suitable for studying reactions on PC and acyl migration in LPC.