Application of liquid chromatography–mass spectrometry to measure short chain fatty acids in blood

A new liquid chromatography–mass spectrometry method is described to determine concentrations of the short chain fatty acids acetic acid, propionic acid and butyric acid (SCFAs) in human blood plasma. The method is based on reversed phase chromatography followed by post-column neutralization of the mobile phase with ammonia and a consecutive measurement of the SCFAs ammonia adducts using negative electro spray ionization. Sample preparation involved simple organic acid deproteinization, resulting in 100% recovery. SCFAs eluted baseline separated within a 25 min run cycle. A linear response was obtained in the range between 0 and 250 μmol/l (R² ranged from 0.997 to 0.9999). The limit of detection ranged from 0.05 μmol/l for propionic and butyric acid and 0.1 μmol/l for acetic acid. The method was tested by analyzing plasma of arterial blood, from portal vein and hepatic vein blood from patients undergoing a pylorus-preserving pancreaticoduodenectomy. As expected, the highest SCFA concentrations were found in portal plasma, hepatic vein levels were in between, while arterial concentrations were lowest. This newly developed method is suitable to determine SCFA concentrations in human plasma samples.     

High-Fat Diet Reduces the Formation of Butyrate,but Increases Succinate,Inflammation, Liver Fat and Cholesterol in Rats,while Dietary Fibre Counteracts These Effects

Introduction

Obesity is linked to type 2 diabetes and risk factors associated to the metabolic syndrome. Consumption of dietary fibres has been shown to have positive metabolic health effects, such as by increasing satiety, lowering blood glucose and cholesterol levels. These effects may be associated with short-chain fatty acids (SCFAs), particularly propionic and butyric acids, formed by microbial degradation of dietary fibres in colon, and by their capacity to reduce low-grade inflammation.

Objective

To investigate whether dietary fibres, giving rise to different SCFAs, would affect metabolic risk markers in low-fat and high-fat diets using a model with conventional rats for 2, 4 and 6 weeks.

Material and Methods

Conventional rats were administered low-fat or high-fat diets, for 2, 4 or 6 weeks, supplemented with fermentable dietary fibres, giving rise to different SCFA patterns (pectin – acetic acid; guar gum – propionic acid; or a mixture – butyric acid). At the end of each experimental period, liver fat, cholesterol and triglycerides, serum and caecal SCFAs, plasma cholesterol, and inflammatory cytokines were analysed. The caecal microbiota was analysed after 6 weeks.

Results and Discussion

Fermentable dietary fibre decreased weight gain, liver fat, cholesterol and triglyceride content, and changed the formation of SCFAs. The high-fat diet primarily reduced formation of SCFAs but, after a longer experimental period, the formation of propionic and acetic acids recovered. The concentration of succinic acid in the rats increased in high-fat diets with time, indicating harmful effect of high-fat consumption. The dietary fibre partly counteracted these harmful effects and reduced inflammation. Furthermore, the number of Bacteroides was higher with guar gum, while noticeably that of Akkermansia was highest with the fibre-free diet.     

Growth inhibition of Erwinia amylovora and related Erwinia species by neutralized short-chain fatty acids

Short-chain fatty acids (SCFAs) are used to preserve food and could be a tool for control of fire blight caused by Erwinia amylovora on apple, pear and related rosaceous plants. Neutralized acids were added to buffered growth media at 0.5–75 mM and tested at pHs ranging from 6.8 to 5.5. Particularly at low pH, SCFAs with a chain length exceeding that of acetic acid such as propionic acid were effective growth inhibitors of E. amylovora possibly due to uptake of free acid and its intracellular accumulation. We also observed high inhibition with monochloroacetic acid. An E. billingiae strain was as sensitive to the acids as E. amylovora or E. tasmaniensis. Fire blight symptoms on pear slices were reduced when the slices were pretreated with neutralized propionic acid. Propionic acid is well water soluble and could be applied in orchards as a control agent for fire blight.     

Gas chromatographic assay of total and O-acetyl groups in bacterial lipopolysaccharides

An improved gas chromatographic method for the determination of total, i.e., amide- and ester-linked, acetyl groups in hydrolysates of bacterial lipopolysaccharides has been developed. After hydrolysis with 0.2 n HCl overnight at 100°C and adjustment of the hydrolysate to pH 3–4, 2 μl of the samples contalning 0–6 μg of acetic and 1–2 μg of propionic acid are directly injected into a gas chromatograph fitted with a 1.5-m glass column packed with Porapak QS. O-Acetyl groups can selectively be determined after hydrolysis with 0.05 n NaOH for 3–4 hr at room temperature and adjustment of the sample to pH 3–4. N-Acetyl groups can be calculated as the difference between total and O-acetyl. The above-mentioned phase allows quantitation of short-chain volatile fatty acids (S-C VFAs) up to n-valeric acid in a range of 0–6.0 μmol using an appropriate internal standard.     

Polyunsaturated fatty acids of mosquitos reared with single dietary polyunsaturates

Stock adults of Culex pipiens and tarsalis reared in crude media had a third of their phospholipid fatty acids as polyunsaturates, mainly 18C but including prominent proportions of arachidonic (20:4n6) and eicosapentaenoic (20:5n3) acids. Adults reared with synthetic media devoid of polyunsaturated fatty acids and therefore unable to fly at emergence contained no more than trace amounts of any polyunsaturate. With synthetic media containing single polyunsaturates the following findings emerged. Of four polyunsaturates known to be highly effective essential fatty acids individually 20:4n6 or 20:5n3 appeared unchanged in tissue phospholipids in proportions reflecting dietary concentrations; dietary 22:4n6 or 22:6n3 (docosahexaenoic acid) appeared also as 20:4n6 or 20:5n3, respectively, retroconverted from the administered dietary fatty acids, which were detected only in traces. Two moderately effective dietary fatty acids, 18:3n6 (γ-linolenic) and 20:3n6 (homo-γ-linolenic), which support weak flight at emergence, appeared in tissue phospholipids respectively as 18:3n6 only, or as similar proportions of 18:3n6 and 20:3n6, this latter indicating shortening to the 18C analogue as well as accumulation of the dietary 20C acid. Six other polyunsaturates [18:2n6 (linoleic), 18:3n3 (linolenic) and their 20C and 22C analogues], all considered slightly effective as essential fatty acids although unable to support proper flight, appeared in tissue phospholipid in dose-related proportions as the 18C basal n6 or n3 family analogues, with only traces of the higher analogues when these were the dietary fatty acids provided, indicating sequential chain shortening within each series, n6 or n3, no interconversion of n6 and n3 members (also shown by all other data), and efficient accumulation of the resultant 18C polyunsaturates. These findings show no capability for de novo synthesis of polyunsaturated fatty acids, afford an insight into the metabolic interrelations of diet-derived polyunsaturates and indicate a primary importance for endogenous arachidonic and eicosapentaenoic acids in mosquito essential fatty acid physiology.     

人的粪便、血浆、唾液、呼出气体中短链脂肪酸的分析

目的建立一种顶空气相色谱-串联质谱法(HS-GC/MS)快速检测人的粪便、血浆、唾液、呼出气体中短链脂肪酸(SCFAs)的方法,初步探索人的粪便、血浆、唾液、呼出气体中短链脂肪酸的相关性。方法样品无需处理直接封存于顶空进样瓶中,顶空进样;采用DB-FFAP毛细管柱(30 m×0.25 mm×0.25μm)分离;全扫描模式检测。结果人的粪便、血浆、唾液、呼出气体中均含有短链脂肪酸。在人的粪便、唾液样本中均检测到8个短链脂肪酸(乙酸、丙酸、异丁酸、丁酸、异戊酸、戊酸、异己酸、己酸);血浆、呼出气体样本中均检测到7个短链脂肪酸(未检测到异己酸)。结论初步推测人的粪便、血浆、唾液、呼出气体中的短链脂肪酸具有一定的相关性。本方法简单、快速、灵敏,可用于人的生物样品中短链脂肪酸的快速检测。     

A Colorimetric Method for the Determination of Acetic Acid in Wood Hydrolyzate Containing a Large Amount of Sulfuric Acid

A method for the determination of acetic acid in presence of a large amount of sulfuric acid has been developed. The method consists of the following procedures. The sample is neutralyzed by barium carbonate. Barium sulfate and excess of barium carbonate are filtered off. On addition of sulfuric acid, acetic acid is extracted with n-butanol from the filtrate. By the reaction of acetic and sulfuric acids in butanol layer with aniline and furfural, a red color is produced. The color produced by sulfuric acid is bleached by treating with barium carbonate powder and the absorbancy of the color produced by acetic acid is measured in a photometer. Acetic acid determination by this method is disturbed by some other acids which give soluble barium salts but the acids which give insoluble barium salts do not disturb.     

Characterization of gut microbiota and short-chain fatty acid in breastfed infants with or without breast milk jaundice

This study aims to investigate the gut microbiota and metabolites in breastfed infants with breast milk jaundice (BMJ) using gut microbiome–metabolomics. Breastfed newborns diagnosed with BMJ and those without BMJ (control group) were enrolled. Faecal samples were collected from the participants and subjected to high-throughput sequencing of the 16s rDNA V3 and V4 regions of the gut flora and metabolomics of short-chain fatty acids (SCFAs). Proteobacteria, Fimicutes and Actinobacteria were the main bacteria at the phylum level. Eshcerichia-Shigella and Enterobacteriacea were the main bacteria at the genus level. The difference between the two groups was compared. Compared to the control group, the amount of Streptococcus was significantly increased while the amount of Enterococcus was significantly decreased in the faeces from infants with BMJ. Functional prediction analysis of 16S found that biosynthesis of penicillin and cephalosporin significantly increased in the BMJ group. Gas chromatography–mass spectrometry detection of SCFAs revealed that levels of acetic acid and propionic acid were significantly lower in the BMJ group than in the control group. The reduced levels of acetic acid and propionic acid may be related to the increase in Streptococcus and decrease in Enterococcus, both of which may contribute to BMJ.     

Use of Gas-Liquid Chromatography to Determine the End Products of Growth of Lactic Acid Bacteria

A simple gas-liquid chromatographic procedure for analyzing ethanol, acetic acid, acetoin, and racemic and meso-2,3-butylene glycol in broth media is described. Overnight broth cultures were filtered or centrifuged, and the filtrate or supernatant was treated with formic acid to aid separation of volatile fatty acids. Samples were then directly analyzed by gas-liquid chromatography on a 20% Tween 80-Chromosorb W-AW column and propionic acid as an internal standard. A complete analysis took ca. 8 min. The method can be used to distinguish homofermentative from heterofermentative lactic acid bacteria based on the level of ethanol produced and citrate-utilizing from non-citrate-utilizing lactic acid bacteria based on the levels of acetic acid produced. The method also has potential in distinguishing other bacterial fermentations. Of the 13 species of lactic acid bacteria tested, Streptococcus lactis subsp. diacetylactis was the major producer of 2,3-butylene glycol (total range, 0.3 to 3.5 mM), and, except for strain DRC1, both the racemic and meso isomers were produced in approximately equal amounts.     

Alpha-linolenic acid and its conversion to longer chain n−3 fatty acids: Benefits for human health and a role in maintaining tissue n−3 fatty acid levels

There is little doubt regarding the essential nature of alpha-linolenic acid (ALA), yet the capacity of dietary ALA to maintain adequate tissue levels of long chain n−3 fatty acids remains quite controversial. This simple point remains highly debated despite evidence that removal of dietary ALA promotes n−3 fatty acid inadequacy, including that of docosahexaenoic acid (DHA), and that many experiments demonstrate that dietary inclusion of ALA raises n−3 tissue fatty acid content, including DHA. Herein we propose, based upon our previous work and that of others, that ALA is elongated and desaturated in a tissue-dependent manner. One important concept is to recognize that ALA, like many other fatty acids, rapidly undergoes β-oxidation and that the carbons are conserved and reused for synthesis of other products including cholesterol and fatty acids. This process and the differences between utilization of dietary DHA or liver-derived DHA as compared to ALA have led to the dogma that ALA is not a useful fatty acid for maintaining tissue long chain n−3 fatty acids, including DHA. Herein, we propose that indeed dietary ALA is a crucial dietary source of n−3 fatty acids and its dietary inclusion is critical for maintaining tissue long chain n−3 levels.     

Oesophagostomum radiatum: Glucose metabolism of larvae grown in vitro and adults grown in vivo

Larval stages of Oesophagostomum radiatum grown in vitro and adults grown in vivo were incubated in complex media or in a simple salt solution containing radioactive glucose. Glucose disappearance and end product accumulation of third-stage larvae in a simple salt solution indicated that they excreted CO₂ and acetic, propionic, and lactic acids. Larvae in third molt, fourth stage, and adults all excreted CO₂, acetic, propionic, and lactic acids at twice the rate of third-stage larvae plus an additional product, methylbutyric acid. Carbon dioxide arose primarily from the 3 or 4 carbons of glucose. An anaerobic atmosphere (95% N₂:5% CO₂) had no apparent effect on metabolism. When incubation was done in complex media, isobutyric and 3-methylbutyric acids were seen as major excretion products (10 and 24%, respectively). However, these acids were quantitatively minor when incubations took place in simple salts-glucose medium (1 and 0–3%, respectively).     

Effects of a dietary organic acid mixture and of dietary fibre levels on ileal and faecal nutrient apparent digestibility, bacterial nitrogen flow, microbial metabolite concentrations and rate of passage in the digestive tract of pigs

Six 34-kg barrows were fitted with a post-valve T-caecum cannula and assigned to six dietary treatments according to a 6 × 5 change-over design to study how a mixture of formic acid, sorbate, and benzoate (0 or 8.4 g/kg feed) influences apparent ileal and faecal digestibility coefficients, bacterial nitrogen (N) flow, microbial metabolite concentrations, and passage rate in pigs fed isoenergetic diets with medium, high, or very high fibre content (neutral-detergent fibre (NDF): 199, 224, and 248 g/kg dry matter, respectively). These barley and soya-bean meal based diets contained 0, 75, and 150 g/kg barley fibre (NDF: 577 g/kg) and 0, 8, and 16 g/kg rapeseed oil, respectively. The dietary organic acid mixture improved the apparent ileal digestibility of 14 of the 17 amino acids analysed (P < 0.05). Increasing levels of dietary fibre linearly decreased the apparent ileal digestibility of six of the 17 amino acids analysed (P < 0.05). Ileal flows of bacterial N and amino acids as assessed on the basis of purine flow were decreased by the dietary organic acid mixture (P < 0.05) but were not affected by dietary fibre level (P>0.05). As assessed on the basis of diaminopimelic acid flow, bacterial N flow was increased by both the dietary organic acid mixture and increased dietary fibre levels (P < 0.05). The dietary organic acid mixture reduced the concentration of lactic acid and increased that of acetic acid in ileal digesta (P < 0.05), while dietary fibre levels had a quadratic effect on concentrations of acetic, propionic, and butyric acid (P < 0.05). The mean retention time of Co (solute marker) and Yb (particle marker) in the large intestine decreased in a linear manner by increasing dietary fibre levels (P < 0.05) but was not affected by the dietary organic acid mixture (P>0.05). The results show that a dietary organic acid mixture has a positive effect on the apparent ileal digestibility of most amino acids irrespective of dietary fibre levels. This could be at least partly related to changes in bacterial N flow in the ileum. However, different bacterial markers showed opposite effects on bacterial N flow, which makes it questionable to use a constant bacterial marker / bacterial N ratio to estimate bacterial N flow. Increasing levels of dietary fibre had negative effects on the apparent ileal amino acid digestibilities and shortened the mean retention time of digesta in the large intestine.     

The Short-Chain Fatty Acid Uptake Fluxes by Mice on a Guar Gum Supplemented Diet Associate with Amelioration of Major Biomarkers of the Metabolic Syndrome

Studies with dietary supplementation of various types of fibers have shown beneficial effects on symptoms of the metabolic syndrome. Short-chain fatty acids (SCFAs), the main products of intestinal bacterial fermentation of dietary fiber, have been suggested to play a key role. Whether the concentration of SCFAs or their metabolism drives these beneficial effects is not yet clear. In this study we investigated the SCFA concentrations and in vivo host uptake fluxes in the absence or presence of the dietary fiber guar gum. C57Bl/6J mice were fed a high-fat diet supplemented with 0%, 5%, 7.5% or 10% of the fiber guar gum. To determine the effect on SCFA metabolism, ¹³C-labeled acetate, propionate or butyrate were infused into the cecum of mice for 6 h and the isotopic enrichment of cecal SCFAs was measured. The in vivo production, uptake and bacterial interconversion of acetate, propionate and butyrate were calculated by combining the data from the three infusion experiments in a single steady-state isotope model. Guar gum treatment decreased markers of the metabolic syndrome (body weight, adipose weight, triglycerides, glucose and insulin levels and HOMA-IR) in a dose-dependent manner. In addition, hepatic mRNA expression of genes involved in gluconeogenesis and fatty acid synthesis decreased dose-dependently by guar gum treatment. Cecal SCFA concentrations were increased compared to the control group, but no differences were observed between the different guar gum doses. Thus, no significant correlation was found between cecal SCFA concentrations and metabolic markers. In contrast, in vivo SCFA uptake fluxes by the host correlated linearly with metabolic markers. We argue that in vivo SCFA fluxes, and not concentrations, govern the protection from the metabolic syndrome by dietary fibers.     

Adaptation of gut microbiome and host metabolic systems to lignocellulosic degradation in bamboo rats

Bamboo rats (Rhizomys pruinosus) are among the few mammals that lives on a bamboo-based diet which is mainly composed of lignocellulose. However, the mechanisms of adaptation of their gut microbiome and metabolic systems in the degradation of lignocellulose are largely unknown. Here, we conducted a multi-omics analysis on bamboo rats to investigate the interaction between their gut microbiomes and metabolic systems in the pre- and post-weaning periods, and observed significant relationships between dietary types, gut microbiome, serum metabolome and host gene expression. For comparison, published gut microbial data from the famous bamboo-eating giant panda (Ailuropoda melanoleuca) were also used for analysis. We found that the adaptation of the gut microbiome of the bamboo rat to a lignocellulose diet is related to a member switch in the order Bacteroidales from family Bacteroidaceae to family Muribaculaceae, while for the famous bamboo-eating giant panda, several aerobes and facultative anaerobes increase after weaning. The conversion of bacteria with an increased relative abundance in bamboo rats after weaning enriched diverse carbohydrate-active enzymes (CAZymes) associated with lignocellulose degradation and functionally enhanced the biosynthesis of amino acids and B vitamins. Meanwhile, the circulating concentration of short-chain fatty acids (SCFAs) derived metabolites and the metabolic capacity of linoleic acid in the host were significantly elevated. Our findings suggest that fatty acid metabolism, including linoleic acid and SCFAs, are the main energy sources for bamboo rats in response to the low-nutrient bamboo diet.Subject terms: Metagenomics, Bacterial evolution     

Maintenance of lower proportions of (n − 6) eicosanoid precursors in phospholipids of human plasma in response to added dietary (n − 3) fatty acids

Competition between the (n ? 3) and (n ? 6) types of highly unsaturated fatty acids can diminish the abundance of (n ? 6) eicosanoid precursors in a tissue, which in turn can diminish the intensity of tissue responses that are mediated by (n ? 6) eicosanoids. The mixture of 20- and 22-carbon highly unsaturated fatty acids maintained in the phospholipids of human plasma is related to the dietary intake of 18:2 (n ? 6) and 18:3 (n ?3) by empirical hyperbolic equations in a manner very similar to the relationship reported for laboratory rats (Lands, W.E.M., Morris, A. and Libelt, B. (1990) Lipids 25, 505–516). Analytical results from volunteers ingesting self-selected diets showed an inter-individual variance for the proportion of (n ? 6) eicosanoid precursors in the fatty acids of plasma phospholipids of about 5%, but the variance among multiple samples taken from the same individual throughout the day was less (about 3%), closer to the experimental variance of the analytical procedure (about 1%). The reproducibility of the results makes it likely that analysis of fatty-acid composition of plasma lipids from individuals will prove useful in estimating the diet-related tendency for severe thrombotic, arthritic of other disorders that are mediated by (n ? 6) eicosanoids. Additional constants and terms were included in the equations to account for the effects of 20- and 22-carbon highly unsaturated (n ? 3) fatty acids in the diet. A lower constant for the 20- and 22-carbon (n ? 3) fatty acids compared to that for the 18-carbon (n ? 3) fatty acid in decreasing the ability of dietary 18:2 (n ? 6) to maintain 20:4 (n ? 6) in tissue lipids confirmed the greater competitive effectiveness of the more highly unsaturated n ? 3 fatty acids in the elongation/ desaturation process. Also, a lower constant for direct incorporation of 20-carbon fatty acids of the n ? 6 vs. the n ? 3 type indicated a greater competitive effectiveness of 20:4 (n ? 6) relative to 20:5 (n ? 3) in reesterification after release from tissue lipids. The equations may be used in reverse to estimate the dietary intakes of the (n ? 3) and (n ? 6) fatty acids by using the composition of the fatty acids that had been maintained in plasma lipids.     

Acylated and des acyl ghrelin in human portal and systemic circulations

Octanoylation of the gastric peptide ghrelin produces an active isoform that regulates appetite and other metabolic functions. Acylated ghrelin is present in the gastrointestinal tract suggesting that octanoylation may occur in these tissues and thereby affect the acylated ghrelin in the systemic circulation. In this study blood samples were collected simultaneously from portal, arterial, peripheral venous and central venous compartments from patients undergoing laparotomy. ELISA and high sensitivity Bioplex was used to measure the concentration of acylated and des acyl ghrelin. We found median (95% confidence interval (CI)) plasma acylated ghrelin (pg/ml) was 35.8 (30.0–59.6) in the portal compartment compared to 51.5 (37.6–74.8; P < 0.05, n = 11) in the arterial, 39.3 (33.3–56.3) in the portal compartment compared to 55.0 (48.5–77.0; P < 0.001, n = 12) in the peripheral venous and 36.0 (33.1–57.4) in the portal compartment compared to 48.9 (43.3–65.6; P < 0.01, n = 15) in the central venous compartment. Median (95% CI) plasma des acyl ghrelin levels (pg/ml) was 173 (125–220) in the portal compartment compared to 136 (99.3–125; P < 0.001, n = 14)in the arterial, 186 (136–233) in the portal compartment compared to 149 (111–190; P < 0.01, n = 15) in the peripheral venous and 171 (140–208) in the portal compartment compared to 152 (119–175; P < 0.01, n = 15) the central venous compartment. We conclude that plasma acylated ghrelin concentration was significantly lower in portal compared with the systemic compartments whilst plasma des acyl ghrelin was significantly higher in portal compared with systemic compartments. These findings suggest that the liver could be involved in the regulation of circulating ghrelin.     

Beneficial effects of thyme oil on age-related changes in the phospholipid C20 and C22 polyunsaturated fatty acid composition of various rat tissues

The aim of this study was to determine any age-related changes in phospholipid polyunsaturated fatty acid composition, in particular C₂₀ and C₂₂ fatty acids in rat liver, brain, kidney and heart, and to assess and compare the effects of dietary supplementation (42.5 mg/kg body weight/day) of the natural antioxidant thyme oil and its major component thymol throughout the rat life span. The fatty acid composition in the various tissues from young (7 months) and aged (28 months) rats was determined and compared. Livers from aged control, thyme oil and thymol treated rats exhibited an increase in 22:6(n–3). In contrast, 22:6(n–3) content of brain, kidney and heart declined in aged rats in all three dietary groups. However, aged rats treated with thyme oil and thymol displayed significantly higher levels of 22:6(n–3) than the respective age-matched controls. Tissue compositions of 20:4(n–6) were found to be significantly lower in the liver and kidney from aged control rats but not those fed either thyme oil or thymol. In aged rats, the composition of 20:4(n–6) in all tissues was highest in rats fed either thyme oil or thymol. These results show that dietary supplementation with thyme oil tended to maintain higher PUFA levels in all tissues studied. The majority of protection provided by thyme oil was by virtue of its thymol component, which comprises 49% of the whole oil. Thymol administered alone did not provide significantly higher protection than the whole oil, suggesting that other components within thyme oil are also contributing antioxidant activity.     

Serum lamotrigine analysis by capillary electrophoresis

Lamotrigine, a new antiepileptic drug, is analyzed by capillary zone electrophoresis. Samples were deproteinized with acetonitrile containing an internal standard, acidified with dilute acetic acid and injected into the capillary. The drug migrated rapidly with the cationic compounds in about 3.5 min far from any interfering substances. The test was linear between 0.5–10 mg/l. The analysis time was about 5 min. The CE values correlated well with an HPLC method (r=0.97; n=35). The mean serum concentration of 121 patients on this drug was 3.7 mg/l. Incubating the serum with ß-glucuronidase for 1 h increased the peak height of lamotrigine by about 24%.     

Alteration of the fatty acid profile of Streptomyces coelicolor by replacement of the initiation enzyme 3-ketoacyl acyl carrier protein synthase III (FabH)

The first elongation step of fatty acid biosynthesis by a type II dissociated fatty acid synthases is catalyzed by 3-ketoacyl-acyl carrier protein (ACP) synthase III (KASIII, FabH). This enzyme, encoded by the fabH gene, catalyzes a decarboxylative condensation between an acyl coenzyme A (CoA) primer and malonyl-ACP. In organisms such as Escherichia coli, which generate only straight-chain fatty acids (SCFAs), FabH has a substrate preference for acetyl-CoA. In streptomycetes and other organisms which produce a mixture of both SCFAs and branched-chain fatty acids (BCFAs), FabH has been shown to utilize straight- and branched-chain acyl-CoA substrates. We report herein the generation of a Streptomyces coelicolor mutant (YL/ecFabH) in which the chromosomal copy of the fabH gene has been replaced and the essential process of fatty acid biosynthesis is initiated by plasmid-based expression of the E. coli FabH (bearing only 35% amino acid identity to the Streptomyces enzyme). The YL/ecFabH mutant produces predominantly SCFAs (86%). In contrast, BCFAs predominate (~70%) in both the S. coelicolor parental strain and S. coelicolor YL/sgFabH (a ΔfabH mutant carrying a plasmid expressing the Streptomyces glaucescens FabH). These results provide the first unequivocal evidence that the substrate specificity of FabH observed in vitro is a determinant of the fatty acid made in an organism. The YL/ecFabH strain grows significantly slower on both solid and liquid media. The levels of FabH activity in cell extracts of YL/ecFabH were also significantly lower than those in cell extracts of YL/sgFabH, suggesting that a decreased rate of fatty acid synthesis may account for the observed decreased growth rate. The production of low levels of BCFAs in YL/ecFabH suggests either that the E. coli FabH is more tolerant of different acyl-CoAs substrates than previously thought or that there is an additional pathway for initiation of BCFA biosynthesis in Streptomyces coelicolor.     

The effect of dietary n-3 polyunsaturated fatty acids supplementation of rams on semen quality and subsequent quality of liquid stored semen

The objective of this study was to examine the effect of dietary n-3 polyunsaturated fatty acid (PUFA) supplementation of rams on semen quality and subsequent sperm function of liquid stored semen. Mature rams of proven fertility were individually housed and were blocked according to breed, body weight, and body condition score and randomly allocated within block to one of two dietary treatments (N = 7 per treatment). Rams were offered a base diet of hay and concentrate, with the concentrate enriched with either: (1) saturated palmitic acid (CON) or (2) high n-3 PUFA fish oil (FO) supplements. Both lipid supplements were added at 2% (wt/wt) of the total diet as fed and both were partially rumen-protected. The animals were fed their respective diets for a total of 9 weeks and blood samples were collected on weeks 0 (pre-experimental), 4, and 9, relative to initial allocation of diet (week 0), for measurement of plasma concentration of fatty acids, metabolites, insulin like growth factor 1 (IGF-1) and insulin. Semen was collected from each ram (on 1 day in each week) in weeks 4, 5, 7, 8, and 9, and each ejaculate was assessed for volume, wave motion, and concentration of sperm, after which it was diluted in a skim milk-based extender and stored at 4 °C. A second ejaculate was collected on weeks 4, 7, and 9, centrifuged, and the sperm frozen for subsequent lipid analysis. A sample of semen from each ram was assessed at 24, 48, and 72 hours after collection for sperm progressive linear motion, ability to penetrate artificial mucus, and the ability to resist lipid peroxidation (at 24 and 48 hours only) using the thiobarbituric acid reactive substances assay. There was no effect of diet on plasma insulin concentrations or on any of the metabolites measured, however, there was a diet by week interaction for plasma IGF-1 concentration (P < 0.05). This was manifested as the FO supplemented rams having higher IGF-1 concentrations on week 9 compared with the control treatment (P < 0.05), but not at the earlier sampling dates. Compared with the pre-experimental values, supplementation with FO increased plasma concentrations of total n-3 PUFAs by 3.1-fold and decreased n-6 PUFA concentrations by 1.84-fold. Consequently, the ratio of n-6 to n-3 PUFA was decreased in the FO-supplemented rams (P < 0.001). Dietary supplementation with FO increased the concentration of eicosapentaenoic acid in sperm from week 4 to 9 by 2.7-fold (P < 0.05) leading to a 1.5-fold increase in total n-3 PUFA in the same period. Ejaculates collected from rams supplemented with FO yielded a higher semen concentration (P < 0.05), however, there was no difference between diets on any of the other semen quality parameters including semen volume, wave motion, progressive linear motion, ability to penetrate artificial mucus, or ability to resist lipid peroxidation. In conclusion, dietary supplementation of rams with n-3 PUFA successfully increased the n-3 PUFA content of plasma and sperm but has limited effects on the quality of liquid stored semen.