The effect of dietary phytosphingosine on cholesterol levels and insulin sensitivity in subjects with the metabolic syndrome.

BACKGROUND: Sphingolipids, like phytosphingosine (PS) are part of cellular membranes of yeasts, vegetables and fruits. Addition of PS to the diet decreases serum cholesterol and free fatty acid (FFA) levels in rodents and improves insulin sensitivity. OBJECTIVE: To study the effect of dietary supplementation with PS on cholesterol and glucose metabolism in humans. METHODS: Twelve men with the metabolic syndrome (MetS) (according to the International Diabetes Federation (IDF) criteria; age 51+/-2 years (mean+/-s.e.m.); body mass index (BMI) 32+/-1 kg/m(2)) were randomly assigned to 4 weeks of PS (500 mg twice daily) and 4 weeks of placebo (P) in a double-blind cross-over study, with a 4-week wash-out period between both interventions. At the end of each intervention anthropometric measures and serum lipids were measured and an intravenous glucose tolerance test (IVGTT) was performed. RESULTS: Phytosphingosine did not affect body weight and fat mass compared with P. PS decreased serum total cholesterol (5.1+/-0.3 (PS) vs 5.4+/-0.3 (P) mmol/l; P<0.05) and low-density lipoprotein (LDL)-cholesterol levels (3.1+/-0.3 (PS) vs 3.4+/-0.3 (P) mmol/l; P<0.05), whereas it did not alter serum triglyceride and high-density lipoprotein (HDL)-cholesterol levels. In addition, PS lowered fasting plasma glucose levels (6.2+/-0.3 (PS) vs 6.5+/-0.3 (P) mmol/l; P<0.05). PS increased the glucose disappearance rate (K-value) by 9.9% during the IVGTT (0.91+/-0.06 (PS) vs 0.82+/-0.05 (P) %/min; P<0.05) at similar insulin levels, compared with P, thus implying enhanced insulin sensitivity. PS induced only minor gastrointestinal side effects. CONCLUSION: Dietary supplementation of PS decreases plasma cholesterol levels and enhances insulin sensitivity in men with the MetS.

Kinetic characteristics of acidic and alkaline ceramidase in human epidermis.

It has recently become evident that at least five ceramidase (CDase) isoforms are present in human epidermis, and that specifically acidic CDase (aCDase) and alkaline CDase (alkCDase) activities increase during keratinocyte differentiation, and thus might play a pivotal role(s) in permeability barrier function. Prior to investigating their possible roles in the epidermal barrier function, it is necessary to characterize basic kinetic parameters for these enzymes, as well as to determine the effects of the established CDase inhibitors and their activities. In this study, assays for both aCDase and alkCDase activities in fully differentiated human epidermis were optimized using a radiolabeled substrate. These studies revealed that aCDase activity is substantially higher than alkCDase activity, and that both isoenzymes are inhibited by a CDase inhibitor N-oleylethanolamine. These findings were also confirmed using an in situ enzyme assay.

A novel and sensitive method for the detection of T cell stimulatory epitopes of alpha/beta- and gamma-gliadin.

BACKGROUND: It is now generally accepted that coeliac disease (CD) is caused by inflammatory T cell responses to gluten peptides bound to HLA-DQ2 or -DQ8 molecules. There is overwhelming evidence that CD patients can mount T cell responses to peptides found in both alpha-gliadin and gamma-gliadin molecules. Assays that would detect the presence or absence of such peptides in food would thus be accurate indicators of safety for consumption by CD patients. AIMS: The development of a sensitive method to detect T cell stimulatory epitopes of alpha-gliadin and gamma-gliadin molecules in food products. METHODS: Monoclonal antibodies (mAb) were raised against peptides encoding the T cell stimulatory epitopes of alpha-gliadin (amino acids (aa) 59-71) and aa gamma-gliadin (aa 142-153 and aa 147-159). These mAb competition assays were developed that quantitatively detect T cell stimulatory epitopes present on both intact proteins and peptides of sizes recognisable by CD4(+) T cells. RESULTS: With the mAb based competition assays, T cell epitopes were detected in pepsin/trypsin digests of wheat proteins and ethanol extracts of various food products, with detection levels lower than those reached with gluten specific T cells. Moreover, the presence of T cell stimulatory epitopes was also detected in preparations of barley, rye, and triticale, other cereals known to be toxic for CD patients. CONCLUSIONS: A new antibody based method has been developed, detecting the presence of T cell stimulatory gluten peptides. This can be used to further ensure the safety of food consumed by CD patients.

Is Candida albicans a trigger in the onset of coeliac disease?

Coeliac disease is a T-cell-mediated autoimmune disease of the small intestine that is induced by ingestion of gluten proteins from wheat, barley, or rye. We postulate that Candida albicans is a trigger in the onset of coeliac disease. The virulence factor of C albicans-hyphal wall protein 1 (HWP1)-contains aminoacid sequences that are identical or highly homologous to known coeliac disease-related alpha-gliadin and gamma-gliadin T-cell epitopes. HWP1 is a transglutaminase substrate, and is used by C albicans to adhere to the intestinal epithelium. Furthermore, tissue transglutaminase and endomysium components could become covalently linked to the yeast. Subsequently, C albicans might function as an adjuvant that stimulates antibody formation against HWP1 and gluten, and formation of autoreactive antibodies against tissue transglutaminase and endomysium.

Structure-function relationships in the tryptophan-rich, antimicrobial peptide indolicidin.

Indolicidin is a cationic 13 amino acid peptide amide produced in the granules of bovine neutrophils with the sequence H-ILPWKWPWWPWRR-NH2. Indolicidin is both antimicrobial and, to a lesser extent, haemolytic. In order to systematically investigate structure-function relationships, the solid-phase synthesis of indolicidin and 48 distinct analogues are reported, as well as the characterization of their respective biological properties. Peptides synthesized and characterized include analogues with modified terminal functions, truncations from either terminus, an alanine scan to determine the role of each individual amino acid, specific amino acid exchanges of aromatic, charged and structural residues and several retro-, inverso- and retroinverso-analogues. Together, characterization of these analogues identifies specific residues involved in antimicrobial or haemolytic activity and suggests a core structure that may form a scaffold for the further development of peptidomimetic analogues of indolicidin.

Staphylococcus aureus resistance to human defensins and evasion of neutrophil killing via the novel virulence factor MprF is based on modification of membrane lipids with l-lysine.

Defensins, antimicrobial peptides of the innate immune system, protect human mucosal epithelia and skin against microbial infections and are produced in large amounts by neutrophils. The bacterial pathogen Staphylococcus aureus is insensitive to defensins by virtue of an unknown resistance mechanism. We describe a novel staphylococcal gene, mprF, which determines resistance to several host defense peptides such as defensins and protegrins. An mprF mutant strain was killed considerably faster by human neutrophils and exhibited attenuated virulence in mice, indicating a key role for defensin resistance in the pathogenicity of S. aureus. Analysis of membrane lipids demonstrated that the mprF mutant no longer modifies phosphatidylglycerol with l-lysine. As this unusual modification leads to a reduced negative charge of the membrane surface, MprF-mediated peptide resistance is most likely based on repulsion of the cationic peptides. Accordingly, inactivation of mprF led to increased binding of antimicrobial peptides by the bacteria. MprF has no similarity with genes of known function, but related genes were identified in the genomes of several pathogens including Mycobacterium tuberculosis, Pseudomonas aeruginosa, and Enterococcus faecalis. MprF thus constitutes a novel virulence factor, which may be of general relevance for bacterial pathogens and represents a new target for attacking multidrug resistant bacteria.

The effect of hydroxylation of linoleoyl amides on their cannabinomimetic properties.

As yet, the physiological significance of hydroxylation of anandamide and linoleoyl amides is unknown. Therefore, we investigated whether hydroxylation of ODNHEtOH and ODNH2 influences their binding abilities to the CB-1 receptor and whether it alters their reactivity towards a fatty acid amide hydrolase (FAAH) from rat brain. Neither the fatty acid amides nor their hydroxylated derivatives were able to displace the potent cannabinoid [3H]CP 55.940 from the CB-1 receptor (Ki > 1 microM). Hydroxylation of ODNHEtOH resulted in a strong reduction of the maximum rate of hydrolysis by a FAAH, but the affinity of FAAH for the substrate remained of the same order of magnitude. Hydroxylation of ODNH2 led to a decrease in the affinity of FAAH for the substrate, but its maximum rate of conversion was unaffected. Furthermore, hydroxylation of ODNHEtOH enhanced its capacity to inhibit competitively the hydrolysis of anandamide. The resulting prolonged lifetime of anandamide and other fatty acid amide derivatives may have a considerable impact on cellular signal transduction.

Dioxygenation of N-linoleoyl amides by soybean lipoxygenase-1.

Anandamide, a novel neurotransmitter, has been reported to be dioxygenated by brain lipoxygenase [1,11]. Anandamides constitute a new class of neuroregulatory fatty acid amides. However, little is known about the enzymatic dioxygenation of these lipids. Therefore, we have tested several members of the neuroactive fatty acid amide class containing a 1Z,4Z-pentadiene system whether they could be dioxygenated by soybean lipoxygenase-1, which is a model enzyme for mammalian lipoxygenases. In this study it was found that lipoxygenase-1 converts N-linoleoylethanolamide (ODNHEtOH), N-linoleoylamide (ODNH2), N-linoleoylmethylamide (ODNHMe) and N,N-linoleoyldimethylamide (ODN(Me)2 into 13-(S)-hydroperoxy-9Z,11E-octadeca-9,11-dienoyl amides derivatives. The apparent Km values for ODNHEtOH (23.6 +/- 3.7 microM), ODNH2 (8.60 +/- 0.65 microM) and linioleic acid (OD: 8.85 +/- 0.74 microM) are not significantly different. The k(cat) for ODNH2 (32.4 +/- 1.2 s(-1)) is twice as small as compared to the turnover numbers of the other substrates, viz. ODNHEtOH (61.6 +/- 5.0 s(-1)) and OD (54.4 +/- 2.0 s(-1). The results suggest that N-linoleoyl ethanolamide and N-linoleoyl amide can be readily converted by lipoxygenases in vivo.

Lipoxygenase is irreversibly inactivated by the hydroperoxides formed from the enynoic analogues of linoleic acid.

Triple bond analogues of natural fatty acids irreversibly inactivate lipoxygenase during their enzymatic conversion [Nieuwenhuizen, W. F., et al. (1995) Biochemistry 34, 10538-10545]. To gain insight into the mechanism of the irreversible inactivation of soybean lipoxygenase-1, we studied the enzymatic conversion of two linoleic acid analogues, 9(Z)-octadec-9-en-12-ynoic acid (9-ODEYA) and 12(Z)-octadec-12-en-9-ynoic acid (12-ODEYA). During the inactivation process, Fe(III)-lipoxygenase converts 9-ODEYA into three products, i.e. 11-oxooctadec-9-en-12-ynoic acid, racemic 9-hydroxy-10(E)-octadec-10-en-12-ynoic acid, and racemic 9-hydroperoxy-10(E)-octadec-10-en-12-ynoic acid. Fe(II)-lipoxygenase does not convert the inhibitor and is not inactivated by 9-ODEYA. Fe(III)-lipoxygenase converts 12-ODEYA into 13-hydroperoxy-11(Z)-octadec-11-en-9-ynoic acid (34/66 R/S), 13-hydroperoxy11(E)-octadec-11-en-9-ynoic acid (36/64 R/S), 11-hydroperoxyoctadec-12-en-9-ynoic acid (11-HP-12-ODEYA, enantiomeric composition of 33/67), and 11-oxooctadec-12-en-9-ynoic acid (11-oxo-12-ODEYA) during the inactivation process. Also, Fe(II)-lipoxygenase is inactivated by 12-ODEYA. It converts the inhibitor into the same products as Fe(III)-lipoxygenase does, but two additional products are formed, viz. 13-oxo-11(E)-octadec-11-en-9-ynoic acid and 13-oxo-11(Z)-octadec-11-en-9-ynoic acid. The purified reaction products were tested for their lipoxygenase inhibitory activities. The oxo compounds, formed in the reaction of 9-ODEYA and 12-ODEYA, do not inhibit Fe(II)- or Fe(III)-lipoxygenase. The 9- and 13-hydroperoxide products that are formed from 9-ODEYA and 12-ODEYA, respectively, oxidize Fe(II)-lipoxygenase to its Fe(III) state and are weak lipoxygenase inhibitors. 11-HP-12-ODEYA is, however, the most powerful inhibitor and is able to oxidize Fe(II)-lipoxygenase to Fe(III)-lipoxygenase. 11-HP-12-ODEYA is converted into 11-oxo-12-ODEYA by Fe(III)-lipoxygenase. We propose a mechanism for the latter reaction in which Fe(III)-lipoxygenase abstracts the bisallylic hydrogen H-11 from 11-HP-12-ODEYA, yielding a hydroperoxyl radical which is subsequently cleaved into 11-oxo-ODEYA and a hydroxyl radical which may inactivate the enzyme.

Chemical and quantum mechanical studies of the free radical C-C bond formation in the lipoxygenase-catalyzed dimerisation of octodeca-9,12-diynoic acid.

Triple bond analogues of poly-unsaturated fatty acids are well-known inactivators of lipoxygenases. In an earlier study we proposed that, since 11-oxo-octadeca-9,12-diynoic acid (11-oxo-ODYA) is the only oxygenated product formed during the irreversible inactivation of soybean lipoxygenase-1, the inactivation should proceed via a C11 centered octadeca-9,12-diynoic acid radical (ODYA radical). In the present study we investigated the lipoxygenase-catalysed formation of the ODYA radical. In the reaction of lipoxygenase with ODYA in the absence of dioxygen and in the presence of 13(S)-hydroperoxy-octadeca-9Z, 11E-dienoic acid (13-HPOD), free ODYA radicals were formed which resulted in the formation of three dimeric ODYA products in which one ODYA moiety is linked via its C9 (12%), C11 (72%) or C13 (16%) to the C11 methylene of the other ODYA moiety. With the ab initio Hartree-Fock method, using the 2,5-heptadiynyl radical as a model compound, the electron spin in the ODYA radical was calculated to be located for 12.0, 75.0 and 12.0% on carbon atoms C9, C11 and C13 of the ODYA radical, respectively. The ODYA-ODYA dimer formation could thus be explained on the basis of the electron spin distribution in the ODYA radical. The dimer formation, i. e. reaction of an ODYA radical with an ODYA molecule was compared with the reaction of the ODYA radical with dioxygen. On the basis of this comparison it is concluded that a) the ODYA dimer formation occurs at the carbon atom with the highest electron spin population; b) ODYA dimer formation is predominantly a kinetically determined process; c) the electron spin distribution in the ODYA radical can be used to predict the composition of the dimer mixture; and d) the regiospecific oxygen addition in the formation of 11-oxo-ODYA is enzymatically controlled.

Mechanism of lipoxygenase inactivation by the linoleic acid analogue octadeca-9,12-diynoic acid.

During the irreversible inactivation of soybean Fe(III)-lipoxygenase [Fe(III)-LOX] by octadeca-9,12-diynoic acid (ODYA), significant quantities of 11-oxooctadeca-9,12 diynoic acid (11-oxo-ODYA) are formed [Nieuwenhuizen, W. F., et al. (1995) Biochemistry 34, 10538-10545]. To elucidate the inactivation mechanism, a quantitative study into the relationship between the inactivation and 11-oxo-ODYA formation was carried out. The following observations were made (1) LOX (0.84 microM) was completely inactivated by 10 to 80 microM ODYA. However, at ODYA concentrations greater than 100 microM, LOX was only partially inactivated, and there was no inactivation at all at ODYA concentrations above 750 microM. The average number of turnovers in which 11-oxo-ODYA was formed increased from 1.2 to 12 when the ODYA concentration increased from 1 to 50 microM and then decreased again to 1.2 at 1000 microM ODYA. (2) The enzyme that was not irreversibly inactivated by ODYA was in the Fe(III) form at ODYA concentrations below 10 microM but in the Fe(II) form at ODYA concentrations greater than 100 microM. (3) In the presence of 750 microM ODYA and 25 microM 13(S)-hydroperoxy-9Z,11E-octadecadienoic acid, all of the enzyme was inactivated. On the basis of these results, it is proposed that the dioxygenation product of ODYA is 11-hydroperoxyoctadeca-9,12-diynoic acid (11-HP-ODYA), which can convert Fe(II)-LOX into its Fe(III) form. However 11-HP-ODYA is converted into 11-oxo-ODYA, which cannot perform the oxidation. It is proposed that the inactivating agent is either 11-HP-ODYA or the 11-peroxy-octadeca-9,12-diynoic acid radical (11-peroxy-ODYA radical), formed from the ODYA radical and O2. The oxidation of Fe(II)-LOX into its Fe(III) form as well as the inactivation of Fe(III)-LOX is competitively inhibited by ODYA

Fe(III)-lipoxygenase converts its suicide-type inhibitor octadeca-9,12-diynoic acid into 11-oxooctadeca-9,12-diynoic acid.

Triple bond analogues of polyunsaturated fatty acids irreversibly inactivate lipoxygenases. During the inactivation the inhibitors are converted enzymatically [Kühn, H., et al. (1984) Eur. J. Biochem. 139, 577-583]. Since the converted inhibitor molecules may hold important information about the inactivation mechanism, we have determined the structure of the product that is formed during the irreversible inactivation of soybean lipoxygenase-1 by octadeca-9,12-diynoic acid (ODYA), the triple bond analogue of linoleic acid. This product is formed only in the presence of Fe(III)-lipoxygenase-1 and O2. It was purified by C18 solid phase extraction and reversed phase HPLC and was identified with UV, IR, and NMR spectroscopic and mass spectrometric techniques as the novel lipoxygenase product, 11-oxooctadeca-9,12-diynoic acid (11-oxo-ODYA). It is estimated that each lipoxygenase molecule produces 8-10 11-oxo-ODYA molecules before it is inactivated. Furthermore, we have shown that in a secondary reaction 3-4 molecules of 11-oxo-ODYA are covalently attached per lipoxygenase molecule, most likely, to solvent-exposed amino groups. This leads to the formation of a N-penten-4-yn-3-one chromophore, RC(NHX)=CHC(O)C=CR1, in which X stands for the protein and R or R1 for CH3(CH2)4- or -(CH2)7COOH, respectively. Fe(II)- and Fe(III)-lipoxygenase remain active upon reaction with purified 11-oxo-ODYA. It is concluded that (a) several enzymatic turnovers are required for the complete inactivation of lipoxygenase by ODYA and (b) covalent attachment of 11-oxo-ODYA occurs outside the active site and is not the cause of the inactivation.