Substitution of dietary oleic acid for myristic acid increases the tissue storage of α-linolenic acid and the concentration of docosahexaenoic acid in the brain, red blood cells and plasma in the rat

Various strategies have been developed to increase the cellular level of (n-3) polyunsaturated fatty acids in animals and humans. In the present study, we investigated the effect of dietary myristic acid, which represents 9% to 12% of fatty acids in milk fat, on the storage of α-linolenic acid and its conversion to highly unsaturated (n-3) fatty acid derivatives. Five isocaloric diets were designed, containing equal amounts of α-linolenic acid (1.3% of dietary fatty acids, i.e. 0.3% of dietary energy) and linoleic acid (7.0% of fatty acids, i.e. 1.5% of energy). Myristic acid was supplied from traces to high levels (0%, 5%, 10%, 20% and 30% of fatty acids, i.e. 0% to 6.6% of energy). To keep the intake of total fat and other saturated fatty acids constant, substitution was made with decreasing levels of oleic acid (76.1% to 35.5% of fatty acids, i.e. 16.7% to 7.8% of energy) that is considered to be neutral in lipid metabolism. After 8 weeks, results on physiological parameters showed that total cholesterol and low-density lipoprotein-cholesterol did not differ in the diets containing 0%, 5% and 10% myristic acid, but were significantly higher in the diet containing 30% myristic acid. In all the tissues, a significant increasing effect of the substitution of oleic acid for myristic acid was shown on the level of both α-linolenic and linoleic acids. Compared with the rats fed the diet containing no myristic acid, docosahexaenoic acid significantly increased in the brain and red blood cells of the rats fed the diet with 30% myristic acid and in the plasma of the rats fed the diet with 20% myristic acid. Arachidonic acid also increased in the brain of the rats fed the diet with 30% myristic acid. By measuring Δ6-desaturase activity, we found a significant increase in the liver of the rats fed the diet containing 10% of myristic acid but no effect at higher levels of myristic acid. These results suggest that an increase in dietary myristic acid may contribute in increasing significantly the tissue storage of α-linolenic acid and the overall bioavailability of (n-3) polyunsaturated fatty acids in the brain, red blood cells and plasma, and that mechanisms other than the single Δ6-desaturase activity are involved in this effect.     

Entropy as a factor in the binding of γ-aminobutyric acid and nipecotic acid to the γ-aminobutyric acid transport system

Nipecotic acid is one of the most potent competitive inhibitors and alternative substrates for the high-affinity -aminobutyric acid transport system in neurons, but the structural basis of this potency is unclear. Because -aminobutyrate is a highly flexible molecule in solution, it would be expected to lose rotational entropy upon binding to the transport system, a change which does not favor binding. Nipecotic acid, in contrast, is a much less flexible molecule, and one would expect the loss of conformational entropy upon binding to be smaller thus favoring the binding of nipecotic acid over -aminobutyric acid. To investigate this possibility, the thermodynamic parameters, G°, H°, and S°, were determined for the binding of -aminobutyrate and nipecotic acid to the high affinity GABA transport system in synaptosomes. In keeping with expectations, the apparent entropy change for nipecotic acid binding (112±13 J·K^–1) was more favorable than the apparent entropy change for -aminobutyric acid binding (61.3±6.6 J·K^–1). The results suggest that restricted conformation per se is an important contributory factor to the affinity of nipecotic acid for the high-affinity transport system for -aminobutyric acid.This work was conducted when both authors were at the Department of Chemistry, University of Maryland, College Park.Special issue dedicated to Dr. Elling Kvamme.     

Is the fatty acid composition of Daphnia galeata determined by the fatty acid composition of the ingested diet?

1. The fatty acid (FA) composition of Daphnia galeata and their algal food was analysed and showed many similarities, however, some significant differences were also found in the relative abundance of the FA C16 : 4ω3 and docosahexaenoic acid (DHA). Their relative abundances were much lower in daphnids than in their algal diet.
2. When daphnids were fed three distinct emulsion particles with DHA : eicosapentaenoic acid (EPA) ratios of c. 0.7, 2 and 4, the final DHA : EPA ratio in the daphnids always favoured EPA. The increase of the food DHA : EPA ratio resulted in a minor increase of DHA (to c. 2%). Feeding the animals on emulsion particles with increasing ratios of DHA : EPA, caused a minor ( c. 2%) increase of DHA level but EPA levels remained high ( c. 10%).
3. When labelled with [¹⁴C]linoleic acid and [¹⁴C]linolenic acid daphnids showed low conversion of both essential FA into C20 polyunsaturated fatty acids (PUFAs). This low conversion activity might explain the importance of C20 PUFAs as dietary compounds in the food of Daphnia.
4. The results indicate the insignificance of DHA and C16 : 4ω3 for daphnids. As EPA can be derived from C18 : 3ω3 it is not strictly essential, although it might be a significant factor in food quality for Daphnia.     

(−)-Allo-pertusaric acid and (−)-dihydropertusaric acid from the lichen Pertusaria albescens

The structures of two γ-lactone carboxylic acids from the lichen Pertusaria albescens, (−)-allo-pertusaric acid and (−)dihydropertusaric acid, have been elucidated by spectroscopic and chemical methods. From P. ophthalmiza, taraxerone and a mixture of long chain aliphatic alcohols and fatty acids have been isolated.     

What makes the bioactive lipids phosphatidic acid and lysophosphatidic acid so special?

Phosphatidic acid and lysophosphatidic acid are minor but important anionic bioactive lipids involved in a number of key cellular processes, yet these molecules have a simple phosphate headgroup. To find out what is so special about these lipids, we determined the ionization behavior of phosphatidic acid (PA) and lysophosphatidic acid (LPA) in extended (flat) mixed lipid bilayers using magic angle spinning 31P NMR. Our data show two surprising results. First, despite identical phosphomonoester headgroups, LPA carries more negative charge than PA when present in a phosphatidylcholine bilayer. Dehydroxy-LPA [1-oleoyl-3-(phosphoryl)propanediol] behaves in a manner identical to that of PA, indicating that the difference in negative charge between LPA and PA is caused by the hydroxyl on the glycerol backbone of LPA and its interaction with the phosphomonoester headgroup. Second, deprotonation of phosphatidic acid and lysophosphatidic acid was found to be strongly stimulated by the inclusion of phosphatidylethanolamine in the bilayer, indicating that lipid headgroup charge depends on local lipid composition and will vary between the different subcellular locations of (L)PA. Our findings can be understood in terms of a hydrogen bond formed within the phosphomonoester headgroup of (L)PA and its destabilization by competing intra- or intermolecular hydrogen bonds. We propose that this hydrogen bonding property of (L)PA is involved in the various cellular functions of these lipids.     

Synthesis of the α,ω-dicarboxylic acid precursor of biotin by the canonical fatty acid biosynthetic pathway

Biotin synthesis requires the C7 α,ω-dicarboxylic acid, pimelic acid. Although pimelic acid was known to be primarily synthesized by a head to tail incorporation of acetate units, the synthetic mechanism was unknown. It has recently been demonstrated that in most bacteria the biotin pimelate moiety is synthesized by a modified fatty acid synthetic pathway in which the biotin synthetic intermediates are O-methyl esters disguised to resemble the canonical intermediates of the fatty acid synthetic pathway. Upon completion of the pimelate moiety, the methyl ester is cleaved. A very restricted set of bacteria have a different pathway in which the pimelate moiety is formed by cleavage of fatty acid synthetic intermediates by BioI, a member of the cytochrome P450 family.     

Biotechnological production and applications of the ω-3 polyunsaturated fatty acid docosahexaenoic acid

Docosahexaenoic acid (DHA) is a polyunsaturated fatty acid composed of 22 carbon atoms and six double bonds. Because the first double bond, as counted from the methyl terminus, is at position three, DHA belongs to the so-called -3 group. In recent years, DHA has attracted much attention because of its beneficial effect on human health. At present, fish oil is the major source of DHA, but alternatively it may be produced by use of microorganisms. Marine microorganisms may contain large quantities of DHA and are considered a potential source of this important fatty acid. Some of these organisms can be grown heterotrophically on organic substrates without light. These processes can be well controlled and DHA with constant quality can be produced all year round. This paper reviews recent advances in the biotechnological production of DHA by marine microorganisms.     

Involvement of sialic acid in the regulation of γ--aminobutyric acid uptake activity of γ-aminobutyric acid transporter 1

The γ-aminobutyric acid (GABA) transporters (GATs) have long been recognized for their key role in the uptake of neurotransmitters. The GAT1 belongs to the family of Na(+)- and Cl(-)-coupled transport proteins, which possess 12 putative transmembrane (TM) domains and three N-glycosylation sites on the extracellular loop between TM domains 3 and 4. Previously, we demonstrated that terminal trimming of N-glycans is important for the GABA uptake activity of GAT1. In this work, we examined the effect of deficiency, removal or oxidation of surface sialic acid residues on GABA uptake activity to investigate their role in the GABA uptake of GAT1. We found that the reduced concentration of sialic acid on N-glycans was paralleled by a decreased GABA uptake activity of GAT1 in Chinese hamster ovary (CHO) Lec3 cells (mutant defective in sialic acid biosynthesis) in comparison to CHO cells. Likewise, either enzymatic removal or chemical oxidation of terminal sialic acids using sialidase or sodium periodate, respectively, resulted in a strong reduction in GAT1 activity. Kinetic analysis revealed that deficiency, removal or oxidation of terminal sialic acids did not affect the K(m) GABA values. However, deficiency and removal of terminal sialic acids of GAT1 reduced the V(max) GABA values with a reduced apparent affinity for extracellular Na(+). Oxidation of cell surface sialic acids also strongly reduced V(max) without affecting both affinities of GAT1 for GABA and Na(+), respectively. These results demonstrated for the first time that the terminal sialic acid of N-linked oligosaccharides of GAT1 plays a crucial role in the GABA transport process.     

Is the concentration of γ-aminobutyric acid in the nerve terminal regulated via product inhibition of glutamic acid decarboxylase?

Fractions of synaptosomes were used to study the regulation of -aminobutyric acid (GABA) synthesis. The isolated synaptosomes were superfused in media of various compositions. [³H]GABA and GABA released into the medium or remaining in the synaptosomes were analyzed by liquid scintillation and HPLC techniques. Different conditions, designed to increase the GABA efflux rate were used: the rate of superfusion was varied and the concentrations of K⁺ and Ca²⁺ were altered. Stimulation of GABA efflux was paralleled with an increased synthesis of GABA, since, in spite of the increased GABA efflux, a relatively constant intraterminal level was found. The findings suggest that the intraterminal concentration of GABA and thus also its synthesis is regulated via product inhibition. In addition, [³H]GABA, exogenous, and GABA, endogenous, responded to external stimulae (Ca²⁺, veretradine, various GABA concentrations and the glutaminase inhibitor diazo-nor-leucine) in a way which was compatible with them being localized in and/or released from different compartments.     

Modelling the reaction course of N-acetylneuraminic acid synthesis from N-acetyl-d-glucosamine—new strategies for the optimisation of neuraminic acid synthesis

In this work, a model describing the complete enzyme catalysed synthesis of N-acetylneuraminic acid (Neu5Ac) from N-acetyl-d-glucosamine (GlcNAc) is presented. It includes the combined reaction steps of epimerisation from GlcNAc to N-acetyl-d-mannosamine (ManNAc) and the aldol condensation of ManNAc with sodium pyruvate yielding Neu5Ac. The model is expedient to predict the reaction course for various initial and feed concentrations and therefore to calculate reaction times and yields. The equilibrium constants calculated from the kinetic constants via the Haldane relationship correspond with experimental values very well (0.26 calculated and 0.24 experimental value for the epimerisation, 27.4 l mol⁻¹ calculated and 28.7 l mol⁻¹ experimental for the aldol condensation). The actual relevance of the model is shown by a scale-up. Using the model, an optimisation of reaction conditions in consideration of different targets is possible. Exemplarily, it is presented how the optimal ratio of the two enzymes in the reaction can be determined and how the composition of the reaction solution in a fed-batch reactor can be designed to meet downstream processing needs.     

The effect of genetic complexity on the time-course of ribonucleic acid–deoxyribonucleic acid hybridization

1. The rate of RNA-DNA hybridization was studied under conditions of RNA excess, with RNA synthesized in vitro. The initial rate of the reaction was proportional to the initial RNA concentration. Throughout the observed course of the reaction there was a linear relationship between the reciprocal of the amount of RNA hybridized/mug. of DNA and the reciprocal of time. The slope of the reciprocal plot was inversely proportional to the initial RNA concentration. 2. A comparison was made of the hybridization of DNA from Escherichia coli and from bacteriophages T4 and lambda with homologous RNA. The initial rate of hybridization was inversely proportional to the genetic complexity of the hybridizing system. The slope of the reciprocal-time plot was directly proportional to genetic complexity. These results are interpreted to indicate that the rate of hybridization reflects the mean concentration of the various unique RNA species in a preparation.     

Probing the fatty acid binding site of β-lactoglobulins

The interactions of fatty acids with porcine and bovine β-lactoglobulins were measured using tryptophan fluorescence enhancement. In the case of bovine β-lactoglobulin, the apparent binding constants for most of the saturated and unsaturated fatty acids were in the range of 10^?7 M at neutralpH. Bovine β-lactoglobulin displays only one high affinity binding site for palmitate with an apparent dissociation constant of 1·10^?7 M. The strength of the binding was decreasing in the following way: palmitate > stearate > myristate > arachidate > laurate. Caprylic and capric acids are not bound at all. The affinity of β-lactoglobulin for palmitate decreased as thepH of the incubation medium was lowered and BLG/palmitate complex was not observed atpH's lower than 4.5. Surprisingly, chemically modified bovine β-lactoglobulin and porcine β-lactoglobulin did not bind fatty acids in the applied conditions.     

Lactic acid bacteria as probiotics for the nose?

Several studies have recently pointed towards an increased occurrence and prevalence of several taxa of the lactic acid bacteria (LAB) in the microbiota of the upper respiratory tract (URT) under healthy conditions versus disease. These include several species of the Lactobacillales such as Lacticaseibacillus casei, Lactococcus lactis and Dolosigranulum pigrum. In addition to physiological studies on their potential beneficial functions and their long history of safe use as probiotics in other human body sites, LAB are thus increasingly to be explored as alternative or complementary treatment for URT diseases. This review highlights the importance of lactic acid bacteria in the respiratory tract and their potential as topical probiotics for this body site. We focus on the potential probiotic properties and adaptation factors that are needed for a bacterial strain to optimally exert its beneficial activity in the respiratory tract. Furthermore, we discuss a range of in silico, in vitro and in vivo models needed to obtain better insights into the efficacy and adaptation factors specifically for URT probiotics. Such knowledge will facilitate optimal strain selection in order to conduct rigorous clinical studies with the most suitable probiotic strains. Despite convincing evidence from microbiome association and in vitro studies, the clinical evidence for oral or topical probiotics for common URT diseases such as chronic rhinosinusitis (CRS) needs further substantiation.     

Further purification and characterization of the acid α-glucosidase

1. Centrifugation of rat liver acid glucosidase, which had been purified by adsorption on dextran gel, on a density gradient of sucrose showed the enzyme to be impure. 2. Preliminary purification of the enzyme before the gel filtration improved the final degree of purity of this preparation. Disc gel electrophoresis of this preparation showed a single band of protein. 3. The sedimentation co-efficient and the molecular weight determined on a sucrose gradient were 4.9-5.1s and 76000-83000 respectively for the rat liver enzyme, and 5.6s and 97000 for the acid alpha-glucosidase purified by means of the same procedure from the human kidney. 4. The Michaelis constants of rat liver and human kidney enzyme were 4.7x10(-3)m and 13.6x10(-3)m respectively with maltose as substrate. 5. The enzyme from both tissues was inhibited by tris and by erythritol. The inhibition of the rat liver acid glucosidase by erythritol was competitive.     

Effects of γ-rays on Poly-L-glutamic acid and Poly-D,L-glutamic acid in the solid state

Summary Poly-L-glutamic acid and poly-D,L-glutamic acid, as models of proteins, were irradiated with⁶⁰Co--radiation in air and under vacuo to examine whether or not the changes caused by the exposure to ionizing radiation depend on the conformations of polypeptides.It was found that theG- values (yield of main-chain scissions per 100 eV of energy absorbed) of both polypeptides are approximately equal for the irradiation in air, while under vacuo theG- value of poly-D,L-glutamic acid is larger than that of poly-L-glutamic acid. This observation for irradiation under vacuo was ascribed to the stabilizing effect of intramolecular hydrogen bond bridges in poly-L-glutamic acid. It was also found that the-helical structure of poly-L-glutamic acid is destroyed by the exposure to ionizing radiation.     

Insufficient folic acid intake in the Netherlands: what about the future?

BACKGROUND: In 1993 all women of childbearing age in the Netherlands were advised to take a daily 0.5 mg folic acid pill to reduce the risk for neural tube defects. This study describes both recent and past awareness and use of folic acid supplements in relation to socio-economic status in the Northern Netherlands. The consequences of a recent report of the Dutch Health Council report will be discussed as well. METHODS: In the most recent cross-sectional study (November 2000), pregnant women filled out a questionnaire. Out of 473 women, 461 were willing to cooperate. The highest fulfilled level of education was taken as an indicator for socio-economic status. RESULTS: Seventy-seven percent (n = 357) of the respondents had heard about folic acid before being pregnant. Sixty-three percent (n = 289) knew about the protective effect for NTDs and 33% (n = 151) knew the entire advised period. Sixty-one percent (n = 265) of the respondents used folic acid in some part of the advised period and 36% (n = 164) used it in the entire advised period. Higher educated women knew more about folic acid and used it significantly more often in the periconceptional period than lower educated women. CONCLUSIONS: Because compliance to proper use of folic acid was poor, food fortification in the Netherlands must be seriously considered. The Dutch Health Council wants to limit the fortification of food products to those products that are especially aimed for women who wish to become pregnant. The fortification of specific products instead of staple foods is a missed chance to reduce NTDs and possibly other birth defects and cardiovascular defects as well.