Impact of drinking water supplemented 2-hydroxy-4-methylthiobutyric acid in combination with acidifier on performance, intestinal development, and microflora in broilers.

In addition to offering methionine, 2-hydroxy-4-methylthiobutyric acid (HMTBa) is also an organic acid and shows excellent bacteriostasis. Therefore, 3 experiments were conducted to determine the influence of drinking water supplemented HMTBa in combination with acidifier on performance, intestinal development, and microflora in broilers. The addition of different concentration (0.02-0.20%) of the blend of HMTBa and other acids significantly reduced the pH of water and exerted antimicrobial activity in dose-dependent manner in vitro. The outcomes from animal trial consisting of the drinking water with blended acidifier at 0.00, 0.05, 0.10, 0.15, and 0.20% indicated that the water with 0.15 or 0.20% acidifier resulted in linear and quadratic higher body weight at 42 d, gain and water consumption during 1 to 42 d (P < 0.05). In experiment 3, responding to graded blended acidifier in drinking water, birds receiving 0.10, 0.15, and 0.20% acidifier decreased the internal pH of gastrointestinal tract and muscle, and exhibited increased duodenal weight, length, villus high, and the ratio of villus high to crypt depth. Drinking water with 0.2% blended acidifier increased the abundance of probiotics (Bacteroidaceae, Ruminococcaceae, and Lachnospiraceae) and decreased the account of pathogenic bacteria such as Desulfovibrionaceae. Alternations in gut microflora were closely related to the metabolism of carbohydrate, amino acid, and vitamins. These findings, therefore, suggest that drinking water with 0.10 to 0.13% the combination HMTBa with acidifier might benefit to intestinal development and gut microbiota, and the subsequent produce a positive effect on the performance of broilers.

New Hydroxydecanoic Acid Derivatives Produced by an Dndophytic Yeast Aureobasidium pullulans AJF1 from Flowers of Aconitum carmichaeli.

Endophytes have been recognized as a source for structurally novel and biologically active secondary metabolites. Among the host plants for endophytes, some medicinal plants that produce pharmaceuticals have been reported to carry endophytes, which could also produce bioactive secondary metabolites. In this study, the medicinal plant Aconitum carmichaeli was selected as a potential source for endophytes. An endophytic microorganism, Aureobasidium pullulans AJF1, harbored in the flower of Aconitum carmichaeli, was cultured on a large scale and extracted with an organic solvent. Extensive chemical investigation of the extracts resulted in isolation of three lipid type compounds (1-3), which were identified to be (3R,5R)-3,5-dihydroxydecanoic acid (1), (3R,5R)-3-(((3R,5R)-3,5-dihydroxydecanoyl)oxy)-5-hydroxydecanoic acid (2), and (3R,5R)-3-(((3R,5R)-5-(((3R,5R)-3,5-dihydroxydecanoyl)oxy)-3-hydroxydecanoyl)oxy)-5-hydroxydecanoic acid (3) by chemical methods in combination with spectral analysis. Compounds 2 and 3 had new structures. Absolute configurations of the isolated compounds (1-3) were established using modified Mosher's method together with analysis of NMR data for their acetonide derivatives. All the isolates (1-3) were evaluated for antibiotic activities against Escherichia coli, Staphylococcus aureus, Bacillus subtilis, Pseudomonas aeruginosa, and their cytotoxicities against MCF-7 cancer cells. Unfortunately, they showed low antibiotic activities and cytotoxic activities.

Feeding broiler chicks diets containing keto- and hydroxy-acids: performance and carcass weight.

This study evaluated reduced dietary CP and supplementing amino acid analogs to sustain growth and carcass weight in 0- to 21-day-old Cobb × Avian-48 male broiler chicks. A total of 6 diets with 3 levels of CP (22.5, 19.5, and 16.5%) and 2 sources of AA analogs, either synthetic amino acids (SA) or keto-/hydroxy-acids (KA), were assigned randomly to 36 cages (8 chicks/cage) in a 3 × 2 factorial design. For SA diets, DL-Met, DL-Met + L-Ile, and D-Met + L-Ile + L-Val were used to supplement 22.5, 19.5, and 16.5% CP diets, respectively, and for corresponding KA diets, DL-Met was replaced with methionine hydroxy analog (MHA), L-Ile was replaced with keto-Ile, and L-Val was replaced with keto-Val. Water and all isocaloric diets (3,050 kcal ME/kg) were given ad libitum. Lowering dietary CP to 16.5% reduced BW at 7, 14, and 21 D (P ≤ 0.0001) and feed intake at 8 to 14, 15 to 21, and 0 to 21 D (P ≤ 0.001). Body weight gain (BWG) was reduced and feed-to-gain ratio (FGR) was increased (P ≤ 0.003 to 0.0001) at all times for chicks fed 16.5% CP; however, chicks fed 22.5 and 19.5% CP had comparable performance. Differences in 0 to 7 D BWG (SA, 122.9 vs. KA, 113.9 g/bird; P ≤ 0.04), a 0 to 21 D FGR cumulative effect (1.45 vs. 1.51; P ≤ 0.02), and a 15 to 21 D (P ≤ 0.04) and 0 to 21 D (P ≤ 0.05) CP × AA interaction were also observed. Greater liver weight among 16.5 vs. 19.5 or 22.5% CP fed chicks was found at 14 and 21 D (P ≤ 0.0001 and P = 0.06, respectively). Lower dietary CP reduced spleen weight on day 21 birds (P ≤ 0.0005) with lighter spleens among 16.5 and 19.5% vs. the 22.5% CP fed group (0.090, 0.095, 0.119 g/100 g BW, respectively). Breast weight at 21 D was significantly less for 16.5 vs. 22.5% CP fed chicks. Fat pad weight on day 21 was heaviest among 16.5% chicks (P ≤ 0.0004). Overall, lowering dietary CP to 16.5% had a negative effect, but keto-acid supplementation supported 0 to 21 D broiler growth compared to SA; however, transamination efficiency of KA may be lower for 0 to 7D old chicks compared to older birds.

"Twin peaks": searching for 4-hydroxynonenal urinary metabolites after oral administration in rats.

4-Hydroxynonenal (HNE) is a cytotoxic and genotoxic lipid oxidation secondary product which is formed endogenously upon peroxidation of cellular n-6 fatty acids. However, it can also be formed in food or during digestion, upon peroxidation of dietary lipids. Several studies have evidenced that we are exposed through food to significant concentrations of HNE that could pose a toxicological concern. It is then of importance to known how HNE is metabolized after oral administration. Although its metabolism has been studied after intravenous administration in order to mimick endogenous formation, its in vivo fate after oral administration had never been studied. In order to identify and quantify urinary HNE metabolites after oral administration in rats, radioactive and stable isotopes of HNE were used and urine was analyzed by radio-chromatography (radio-HPLC) and chromatography coupled with High Resolution Mass Spectrometry (HPLC-HRMS). Radioactivity distribution revealed that 48% of the administered radioactivity was excreted into urine and 15% into feces after 24h, while 3% were measured in intestinal contents and 2% in major organs, mostly in the liver. Urinary radio-HPLC profiles revealed 22 major peaks accounting for 88% of the urinary radioactivity. For identification purpose, HNE and its stable isotope [1,2-(13)C]-HNE were given at equimolar dose to be able to univocally identify HNE metabolites by tracking twin peaks on HPLC-HRMS spectra. The major peak was identified as 9-hydroxy-nonenoic acid (27% of the urinary radioactivity) followed by classical HNE mercapturic acid derivatives (the mercapturic acid conjugate of di-hydroxynonane (DHN-MA), the mercapturic acid conjugate of 4-hydroxynonenoic acid (HNA-MA) in its opened and lactone form) and by metabolites that are oxidized in the terminal position. New urinary metabolites as thiomethyl and glucuronide conjugates were also evidenced. Some analyses were also performed on feces and gastro-intestinal contents, revealing the presence of tritiated water that could originate from beta-oxidation reactions.

Castor phospholipid:diacylglycerol acyltransferase facilitates efficient metabolism of hydroxy fatty acids in transgenic Arabidopsis.

Producing unusual fatty acids (FAs) in crop plants has been a long-standing goal of green chemistry. However, expression of the enzymes that catalyze the primary synthesis of these unusual FAs in transgenic plants typically results in low levels of the desired FA. For example, seed-specific expression of castor (Ricinus communis) fatty acid hydroxylase (RcFAH) in Arabidopsis (Arabidopsis thaliana) resulted in only 17% hydroxy fatty acids (HFAs) in the seed oil. In order to increase HFA levels, we investigated castor phospholipid:diacylglycerol acyltransferase (PDAT). We cloned cDNAs encoding three putative PDAT enzymes from a castor seed cDNA library and coexpressed them with RcFAH12. One isoform, RcPDAT1A, increased HFA levels to 27%. Analysis of HFA-triacylglycerol molecular species and regiochemistry, along with analysis of the HFA content of phosphatidylcholine, indicates that RcPDAT1A functions as a PDAT in vivo. Expression of RcFAH12 alone leads to a significant decrease in FA content of seeds. Coexpression of RcPDAT1A and RcDGAT2 (for diacylglycerol acyltransferase 2) with RcFAH12 restored FA levels to nearly wild-type levels, and this was accompanied by a major increase in the mass of HFAs accumulating in the seeds. We show the usefulness of RcPDAT1A for engineering plants with high levels of HFAs and alleviating bottlenecks due to the production of unusual FAs in transgenic oilseeds.

DGAT1, DGAT2 and PDAT expression in seeds and other tissues of epoxy and hydroxy fatty acid accumulating plants.

Triacylglycerol (TAG) is the main storage lipid in plants. Acyl-CoA: diacylglycerol acyltransferase (DGAT1 and DGAT2) and phospholipid: diacylglycerol acyltransferase (PDAT) can catalyze TAG synthesis. It is unclear how these three independent genes are regulated in developing seeds, and particularly if they have specific functions in the high accumulation of unusual fatty acids in seed oil. The expression patterns of DGAT1, DGAT2 and a PDAT in relation to the accumulation of oil and epoxy and hydroxy fatty acids in developing seeds of the plant species Vernonia galamensis, Euphorbia lagascae, Stokesia laevis and castor that accumulate high levels of these fatty acids in comparison with soybean and Arabidopsis were investigated. The expression patterns of DGAT1, DGAT2 and the PDAT are consistent with all three enzymes playing a role in the high epoxy or hydroxy fatty acid accumulation in developing seeds of these plants. PDAT and DGAT2 transcript levels are present at much higher levels in developing seeds of epoxy and hydroxy fatty acid accumulating plants than in soybeans or Arabidopsis. Moreover, PDAT, DGAT1 and DGAT2 are found to be expressed in many different plant tissues, suggesting that these enzymes may have other roles in addition to seed oil accumulation. DGAT1 appears to be a major enzyme for seed oil accumulation at least in Arabidopsis and soybeans. For the epoxy and hydroxy fatty acid accumulating plants, DGAT2 and PDAT also show expression patterns consistent with a role in the selective accumulation of these unusual fatty acids in seed oil.

Lactate racemization as a rescue pathway for supplying D-lactate to the cell wall biosynthesis machinery in Lactobacillus plantarum.

Lactobacillus plantarum is a lactic acid bacterium that produces d- and l-lactate using stereospecific NAD-dependent lactate dehydrogenases (LdhD and LdhL, respectively). However, reduction of glycolytic pyruvate by LdhD is not the only pathway for d-lactate production since a mutant defective in this activity still produces both lactate isomers (T. Ferain, J. N. Hobbs, Jr., J. Richardson, N. Bernard, D. Garmyn, P. Hols, N. E. Allen, and J. Delcour, J. Bacteriol. 178:5431-5437, 1996). Production of d-lactate in this species has been shown to be connected to cell wall biosynthesis through its incorporation as the last residue of the muramoyl-pentadepsipeptide peptidoglycan precursor. This particular feature leads to natural resistance to high concentrations of vancomycin. In the present study, we show that L. plantarum possesses two pathways for d-lactate production: the LdhD enzyme and a lactate racemase, whose expression requires l-lactate. We report the cloning of a six-gene operon, which is involved in lactate racemization activity and is positively regulated by l-lactate. Deletion of this operon in an L. plantarum strain that is devoid of LdhD activity leads to the exclusive production of l-lactate. As a consequence, peptidoglycan biosynthesis is affected, and growth of this mutant is d-lactate dependent. We also show that the growth defect can be partially restored by expression of the d-alanyl-d-alanine-forming Ddl ligase from Lactococcus lactis, or by supplementation with various d-2-hydroxy acids but not d-2-amino acids, leading to variable vancomycin resistance levels. This suggests that L. plantarum is unable to efficiently synthesize peptidoglycan precursors ending in d-alanine and that the cell wall biosynthesis machinery in this species is specifically dedicated to the production of peptidoglycan precursors ending in d-lactate. In this context, the lactate racemase could thus provide the bacterium with a rescue pathway for d-lactate production upon inactivation or inhibition of the LdhD enzyme.

Biosynthesis of medium chain length poly(3-hydroxyalkanoates) (mcl-PHAs) by Comamonas testosteroni during cultivation on vegetable oils.

Comamonas testosteroni has been studied for its ability to synthesize and accumulate medium chain length poly(3-hydroxyalkanoates) (mcl-PHAs) during cultivation on vegetable oils available in the local market. Castor seed oil, coconut oil, mustard oil, cotton seed oil, groundnut oil, olive oil and sesame oil were supplemented in the mineral medium as a sole source of carbon for growth and PHAs accumulation. The composition of PHAs was analysed by a coupled gas chromatography/mass spectroscopy (GC/MS). PHAs contained C6 to C14 3-hydroxy acids, with a strong presence of 3-hydroxyoctanoate when coconut oil, mustard oil, cotton seed oil and groundnut oil were supplied. 3-hydroxydecanoate was incorporated at higher concentrations when castor seed oil, olive oil and sesame oil were the substrates. Purified PHAs samples were characterized by Fourier Transform Infrared (FTIR) and 13C NMR analysis. During cultivation on various vegetable oils, C. testosteroni accumulated PHAs up to 78.5-87.5% of the cellular dry material (CDM). The efficiency of the culture to convert oil to PHAs ranged from 53.1% to 58.3% for different vegetable oils. Further more, the composition of the PHAs formed was not found to be substrate dependent as PHAs obtained from C. testosteroni during growth on variety of vegetable oils showed similar compositions; 3-hydroxyoctanoic acid and/or 3-hydroxydecanoic acid being always predominant. The polymerizing system of C. testosteroni showed higher preference for C8 and C10 monomers as longer and smaller monomers were incorporated less efficiently.

Fatty acid and hydroxy acid adaptation in three gram-negative hydrocarbon-degrading bacteria in relation to carbon source.

The lipids of three gram-negative bacteria, Acinetobacter calcoaceticus, Marinobacter aquaeolei, and Pseudomonas oleovorans grown on mineral media supplemented with ammonium acetate or hydrocarbons, were isolated, purified, and their structures determined. Three pools of lipids were isolated according to a sequential procedure: unbound lipids extracted with organic solvents, comprising metabolic lipids and the main part of membrane lipids, OH--labile lipids (mainly ester-bound in the lipopolysaccharides, LPS) and H+-labile lipids (mainly amide-bound in the LPS). Unsaturated FA composition gave evidence for an aerobic desaturation pathway for the synthesis of these acids in A. calcoaceticus and M. aquaeolei, a nonclassic route in gram-negative bacteria. Surprisingly, both aerobic and anaerobic pathways are operating in the studied strain of P. oleovorans. The increase of the proportion of saturated FA observed for the strain of P. oleovorans grown on light hydrocarbons would increase the temperature transition of the lipids for maintaining the inner membrane fluidity. An opposite phenomenon occurs in A. calcoaceticus and M. aquaeolei grown on solid or highly viscous C19 hydrocarbons. The increases of FA < C18 when the bacteria were grown on n-nonadecane, or of iso-FA in cultures on isononadecane would decrease the transition temperature of the lipids, to maintain the fluidity of the inner membranes. Moreover, P. oleovorans grown on hydrocarbons greatly decreases the proportion of P-hydroxy acids of LPS, thus likely maintaining the physical properties of the outer membrane. By contrast, no dramatic change in hydroxy acid composition occurred in the other two bacteria.

Characterization of metabolic pathway of linoleic acid 9-hydroperoxide in cytosolic fraction of potato tubers and identification of reaction products.

Potato tubers are shown to contain a unique lipoxygenase pathway to form 9-hydroperoxy-10,12-octadecadienoic acid (9-HPODE) from linoleic acid. Here, we report the metabolic pathway of 9-HPODE in the cytosolic fraction and the characterization of enzymes involved in the conversion of metabolites. The analysis of enzymatic reaction products at pH 5.5 revealed the formation of 9-keto-10,12-octadecadienoic acid, 9-hydroxy-10,12-octadecadienoic acid, 9,10-epoxy-11-hydroxy-12-octadecenoic acid, 9,10,13-trihydroxy-11-octadecenoic acid, and 9,12,13-trihydroxy-10-octadecenoic acid. The cytosolic enzymes were separated by anion-exchange chromatography into two fractions E1 and E2, having molecular masses of 66 and 54 kDa, respectively. The enzyme fraction E1 only produced 9-keto-10,12-octadecadienoic acid, whereas E2 formed other products. The enzyme E1 showed higher reactivity with 13- and 9-hydroperoxide of alpha-linolenic acid than 9-HPODE, but no reaction with hydroxy fatty acids. In contrast, the enzyme E2 showed the highest reactivity with 9-HPODE, followed by hydroperoxides of alpha-linolenic acid and arachidonic acid. We also evaluated the antibacterial activity of hydroxy fatty acids against Erwinia carotovora T-29, a bacterium infecting potato tubers. Growth of the bacteria was suppressed more potently with 9- or 13-hydroxy fatty acids than dihydroxy or trihydroxy fatty acids, suggesting a role for the metabolites in the resistance of bacterial infection.

Incorporation of 1-chlorooctadecane into FA and beta-hydroxy acids of Marinobacter hydrocarbonoclasticus.

The lipids of the gram-negative marine bacterium Marinobacter hydrocarbonoclasticus, cultivated in synthetic seawater supplemented with 1-chlorooctadecane as sole source of carbon, were isolated, purified, and their structures determined. Three pools of lipids were isolated according to the sequential procedure used: unbound lipids extracted by organic solvents, ester-bound lipids released under alkaline conditions, and amide-bound lipids released by acid hydrolysis. FA isolated from the unbound lipids included omega-chlorinated (21%, w/w, of this fraction; C16 predominant) and nonchlorinated compounds (22%, w/w; C18 predominant). These acids were accompanied by a high proportion of omega-chloro-C18 alcohols (43%, w/w) and a lower amount of omega-chloro-beta-hydroxy-C18, -C16, and -C14 acids (5%, w/w). These data, together with the isolation from the culture medium of gamma-butyrolactone, suggested a metabolism of 1-chlorooctadecane through oxidation into omega-chloro acid and then the classic beta-oxidation pathway. The analysis of the ester-bound and amide-bound lipids revealed that significant amounts of omega-chloro-beta-hydroxy C10-C12 acids were incorporated into the lipopolysaccharides of the bacterium. Incorporation of these omega-chloro-beta-hydroxy acids into the lipopolysaccharides represents a novel route for chloroalkane assimilation in hydrocarbonoclastic gram-negative bacteria. The formation of chlorinated hydroxy acids, like the omega-chloro FA in the cellular lipids, could account for an incomplete mineralization of chloroparaffins in the environment.

Poly-beta-hydroxyalkanoates consumption during degradation of 2,4,6-trichlorophenol by Sphingopyxis chilensis S37.

AIMS: To analyse the possible effect of poly-beta-hydroxyalkanoate (PHA) consumption on 2,4,6-trichlorophenol (2,4,6-TCP) degradation during starvation by Sphingopyxis chilensis S37 strain, which stores PHAs and degrades 2,4,6-TCP. METHODS AND RESULTS: The strain was inoculated in saline solution supplemented with 2,4,6-TCP (25-400 microm). Chlorophenol degradation was followed both spectrophotometrically and by chlorine released; viable bacterial counts were also determined. Cells starved for 24, 48 or 72 h were incubated with 25 microm of 2,4,6-TCP and PHA in cells investigated by spectrofluorimetric and flow cytometry. Results demonstrated that starvation decreased the ability to degrade 2,4,6-TCP. After 72 h of starvation, degradation of 2,4,6-TCP decreased to less than 10% and the relative PHA content diminished to ca 50% during the first 24 h. CONCLUSION: Utilization of PHA may be an important factor for the degradation of toxic compounds, such as 2,4,6-TCP, in bacterial strains unable to use this toxic compound as carbon and energy source. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study describing a relationship between intracellular PHA consumption and 2,4,6-TCP degradation. Therefore, PHAs provides an endogenous carbon and energy source under starvation and can play a significant role in the degradation of toxic compounds.

Production of polyesters consisting of medium chain length 3-hydroxyalkanoic acids by Pseudomonas mendocina 0806 from various carbon sources.

Pseudomonas mendocina strain 0806 was isolated from oil-contaminated soil and found to produce polyesters consisting of medium chain length 3-hydroxyalkanoates (mclPHAs). The monomers of mclPHAs contained even numbers of carbon atoms, such as 3-hydroxyhexanoate (HHx or C6), 3-hydroxyoctanoate (HO or C8), and/or 3-hydroxydecanoate (HD or C10) as major components when grown on many carbon sources unrelated to their monomeric structures, such as glucose, citric acid, and carbon sources related to their monomeric structures, such as myristic acid, octanoate, or oleic acid. On the other hand, PHA containing both even and odd numbers of hydroxyalkanoates (HA) monomers was synthesized when the strain was grown on tridecanoic acid. The molar ratio of carbon to nitrogen (C/N) had a significant effect on PHA composition: the strain produced PHAs containing 97-99% of HD monomer when grown in a glucose ammonium sulfate medium of C/N<20, and 20% HO, and 80% of the HD monomer when growth was conducted in media containing C/N>40. It was demonstrated that the HO/HD ratio in the polymers remained constant in media with a constant C/N ratio, regardless of the glucose concentration. Up to 3.6 g/L cell dry weight containing 45% of PHAs was produced when the strain was grown for 48 h in a medium containing 20 g/L glucose with a C/N ratio of 40.

Efficient production of polyhydroxyalkanoates from plant oils by Alcaligenes eutrophus and its recombinant strain.

The ability of Alcaligenes eutrophus to grow and produce polyhydroxyalkanoates (PHA) on plant oils was evaluated. When olive oil, corn oil, or palm oil was fed as a sole carbon source, the wild-type strain of A. eutrophus grew well and accumulated poly(3-hydroxybutyrate) homopolymer up to approximately 80% (w/w) of the cell dry weight during its stationary growth phase. In addition, a recombinant strain of A. eutrophus PHB-4 (a PHA-negative mutant), harboring a PHA synthase gene from Aeromonas caviae, was revealed to produce a random copolyester of 3-hydroxybutyrate and 3-hydroxyhexanoate from these plant oils with a high cellular content (approximately 80% w/w). The mole fraction of 3-hydroxyhexanoate units was 4-5 mol% whatever the structure of the triglycerides fed. The polyesters produced by the A. eutrophus strains from olive oil were 200-400 kDa (the number-average molecular mass). The results demonstrate that renewable and inexpensive plant oils are excellent carbon sources for efficient production of PHA using A. eutrophus strains.

Synthesis of 2-hydroxy acid from 2-amino acid by Clostridium butyricum.

Cultures of Clostridium butyricum type strain in synthetic medium supplemented with various L-2-amino acids revealed the presence of the corresponding 2-hydroxy acid. This metabolite is able to produce the polyester poly(2-hydroxyalkanoic acid). The bioconversion is not stereoselective since D-2-amino acids were also converted. Chiral GC analysis demonstrated that only D-enantiomer is formed from L-leucine.

Formation of novel poly(hydroxyalkanoates) from long-chain fatty acids.

Poly(hydroxyalkanoates) (PHAs) were isolated from Pseudomonas aeruginosa 44T1 cultivated on euphorbia oil and castor oil. With the aid of 2-D proton NMR spectra and proton-detected multiple bond coherence NMR spectra the structures of the PHAs were determined. In addition to the usual PHA constituents (C6-C14 3-hydroxy fatty acids), PHAs formed from euphorbia oil contained delta 8,9-epoxy-3-hydroxy-5c-tetradecenoate, and probably delta 6,7-epoxy-3-hydroxydodecanoate and delta 4,5-epoxy-3-hydroxydecanoate. These novel constituents account for approximately 15% of the total amount of monomers and are clearly generated via beta-oxidation of vernolic acid (delta 12,13-epoxy-9c-octadecenoic acid), the main component of euphorbia oil. In PHAs formed from castor oil, 7% of the monomers found were derived from ricinoleic acid (12-hydroxy-9c-octadecenoic acid). The presence of 3,8-dihydroxy-5c-tetradecenoate was clearly demonstrated. Furthermore, NMR analysis strongly suggested the presence of 3,6-dihydroxydodecanoate, 6-hydroxy-3c-dodecenoate, and 4-hydroxydecanoate.

Dietary supplementation with ethyl ester concentrates of fish oil (n-3) and borage oil (n-6) polyunsaturated fatty acids induces epidermal generation of local putative anti-inflammatory metabolites.

Clinical reports have attributed the amelioration of chronic inflammatory skin disorders to the presence of certain polyunsaturated fatty acids (PUFA) in dietary oils. To test the hypothesis of a local modulatory effect of these PUFA in the epidermis, the basal diet of normal guinea pigs was supplemented with ethyl esters of either fish oil [rich in eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA)] or borage oil [rich in gamma-linolenic acid (GLA)]. Our data demonstrated that dietary oils influence the distribution of PUFA in epidermal phospholipids and the epidermal levels of PUFA-derived hydroxy fatty acids. Specifically, animals supplemented with ethyl esters of fish oil markedly incorporated EPA and DHA into epidermal phospholipids, which paralleled the epidermal accumulation of 15-hydroxyeicosapentaenoic acid (15-HEPE) and 17-hydroxydocosahexaenoic acid (17-HDoHE). Similarly, animals supplemented with esters of borage oil preferentially incorporated dihomogammalinolenic acid (DGLA), the epidermal elongase product of GLA, into the epidermal phospholipids, which also was accompanied by epidermal accumulation of 15-hydroxyeicosatrienoic acid (15-HETrE). By factoring the epidermal levels of the 15-lipoxygenase products and their relative inhibitory potencies, we evolved a measure of the overall potential of dietary oils to exert local anti-inflammatory effect. For example, the leukotriene inhibition potentials (LIP) of both fish oil and borage oil were greatly enhanced when compared to controls. Thus, the altered profiles of epidermal 15-lipoxygenase products generated from particular dietary oils may be responsible, at least in part, for reported ameliorative effects of oils on chronic inflammatory skin disorders.

Hydroperoxide-dependent epoxidation of unsaturated fatty acids in the broad bean (Vicia faba L.).

Incubation of linoleic acid with the 105,000g particle fraction of the homogenate of the broad bean (Vicia faba L.) led to the formation of the following products: 13(S)-hydroxy-9(Z),11(E)-octadecadienoic acid, 9,10-epoxy-12(Z)-octadecenoic acid (9(R),10(S)/9(S)/10(R), 80/20), 12,13-epoxy-9(Z)-octadecenoic acid (12(S),13(R)/12(R)/13(S), 64/36), and 9,10-epoxy-13(S)-hydroxy-11(E)-octadecenoic acid (9(S),10(R)/9(R),10(S), 91/9). Oleic acid incubated with the enzyme preparation in the presence of 13(S)-hydroperoxy-9(Z),11(E)-octadecadienoic acid or cumene hydroperoxide was converted into 9,10-epoxyoctadecanoic acid (9(R),10(S)/9(S),10(R), 79/21). Two enzyme activities were involved in the formation of the products, an omega 6-lipoxygenase and a hydroperoxide-dependent epoxygenase. The lipoxygenase, but not the epoxygenase, was inhibited by low concentrations of 5,8,11,14-eicosatetraynoic acid and nordihydroguaiaretic acid. In contrast, the epoxygenase, but not the lipoxygenase, was readily inactivated in the presence of 13(S)-hydroperoxy-9(Z),11(E)-octadecadienoic acid. Studies with 18O2-labeled 13(S)-hydroperoxy-9(Z),11(E)-octadecadienoic acid showed that the epoxide oxygens of 9,10-epoxyoctadecanoic acid and of 9,10-epoxy-13(S)-hydroxy-11(E)-octadecenoic acid were derived from hydroperoxide and not from molecular oxygen.

Organic acids and branched-chain amino acids in body fluids before and after multiple exchange transfusions in maple syrup urine disease.

We successfully treated a critically ill infant with the classical type of maple syrup urine disease by multiple exchange transfusions via a peripheral artery and vein and with positive calorie supplementation in the early stage of therapy. Clinical improvement was definite after the plasma leucine level fell below 1 mmol/l. There was a close linear correlation between plasma concentrations of branched-chain amino acids and their corresponding branched-chain alpha-keto acids and branched-chain alpha-hydroxy acids. alpha-Hydroxy acids were more easily excreted in the urine than alpha-keto acids and amino acids. Our studies on urinary organic acids supported the existence of minor metabolic pathways of branched-chain alpha-keto acids, although they were not thought to be important in eliminating accumulated alpha-keto acids. Urinary excretion of succinic acid and alpha-ketoglutaric acid, which are components of the citric acid cycle, increased transiently during the patient's convalescence. The cerebrospinal fluid/plasma ratios for branched-chain amino acids, alpha-keto acids, and alpha-hydroxy acids were very high before the transfusions and decreased after improvement. The cerebrospinal fluid/plasma ratios for 5-carbon acids, alpha-ketoisovaleric acid and alpha-hydroxyisovaleric acid were much higher than for other branched-chain acids not only in the patient but also in normal controls. Cerebrospinal fluid levels of alpha-ketoisocaproic acid and alpha-hydroxyisovaleric acid were as high as 1 mmol/l in our patient.

L-2-Hydroxyglutaric aciduria: an inborn error of metabolism?

A 5-year-old boy, excreting large amounts of 2-hydroxyglutaric acid in the urine (3.3-7.6 mmol/l), is described. The patient presented with psychomotor retardation and dystrophy. His skeletal age was delayed. The EEG was not well differentiated; it resembled that observed in 2-year-old children. There was a severe anaemia, which reacted well to iron supplements. The 2-hydroxyglutaric acid was found to have the L-configuration, as analysed by capillary gas chromatography of the O-acetylated di-(-)-2-butyl ester derivative. The relation of L-2-hydroxyglutarate excretion to known metabolic pathways is discussed.