Effect of nicotinic acid on microviscosity in mixed liposomal system of lecithin and sphingomyelin

In rat liver plasma membrane, the molar ratio of sphingomyelin and phospholipid is approximately 1:4, whereas, the molar ratio of phospholipid and cholesterol is 3:1. Considering this ratio to be typical for a real biological membrane, we have studied the effect of anticholesterol and the vasodialatory drug nicotinic acid (NA) on the fluidity profile of a liposomal system of lipids mixed in this ratio using the fluorescence polarization probe 1,6-diphenyl-1-1,3,5-hexatriene. The study reveals that when NA is added to the aqueous dispersion of the mixed lipid system (molar ratio of lipid:NA, 1:1) it creates a more fluid environment for the probe molecule and modifies the fluidity profile of the cholesterol-incorporated liposomal system by eliminating the effect of cholesterol to some extent. The drug also affects the activation energy of diffusion of this system. These results on fluidity have been compared with those in cases of liposomes of individual lipids. The effect of NA on fluidity may be attributed to a mechanical interaction of the drug molecules with the lipid molecules.     

Molecular identification of nicotinic acid receptor

Nicotinic acid and its derivative, Acipimox, have been widely used in the treatment of hyperlipidemia. Pharmacological studies have demonstrated that they exert the beneficial effect through the activation of a Gi-protein-coupled receptor on adipocyte, which has remained elusive to date. Here we show that a novel GPCR, designated HM74b because of its high similarity to HM74, is a receptor for nicotinic acid. HM74b mRNA is found in human, murine, and rat adipose tissues. Nicotinic acid and Acipimox inhibit forskolin-stimulated intracellular cAMP accumulation in human HM74b-expressing cells and activate GTP gamma S binding in a dose-dependent manner. [3H]Nicotinic acid specifically binds to HM74b-expressing membrane and its binding is replaced by Acipimox. This finding will open a new phase of research on the physiological role of nicotinic acid and will be a clue to develop novel antihyperlipidemic drugs.     

The bacterial oxidation of nicotinic acid

N-Formylmaleamic acid, a probable intermediate in the bacterial metabolism of nicotinic acid, has been synthesized by photoisomerization of its transisomer, N-formylfumaramic acid. The compound previously reported to be N-formylmaleamic acid has been shown to be N-formylfumaramic acid.This paper is dedicated to Prof. R. Y. Stanier, who served as a model of elegant erudition to me during my graduate work with him.I thank the NIH and NSF for support     

Chromatographic and capillary electrophoretic methods for the analysis of nicotinic acid and its metabolites

Methods for the assay of nicotinic acid (NiAc) and its metabolites in biological fluids using high-performance liquid chromatography (HPLC) and capillary electrophoresis (CE) are reviewed. Most of the references cited in this review concern HPLC methods. A few CE methods that have been recently reported are also included. As these compounds are relatively polar and have a wide range of physico-chemical properties, the sample pre-treatment or clean-up process prior to analysis is included. Most HPLC methods using an isocratic elution system allow determination of a single or few metabolites, but gradient HPLC methods enable simultaneous determination of five to eight compounds. Simultaneous determination of NiAc including many metabolites in a single run can be achieved by CE. We also discuss the pharmacokinetics of NiAc and some of its metabolites.     

一株烟酸羟基化转化菌株的筛选和鉴定

从南京地区的土壤中筛选到一株高效转化烟酸为 6_羟基烟酸的菌株NA_1。形态及生理生化特征测定结果表明 ,NA_1菌株与假单胞菌属 (Pseudomonas)中的恶臭假单胞菌 (P .putida)种的特征基本一致。测定了该菌株的16SrDNA序列并根据 16SrDNA构建了系统发育树 ;在系统发育树中 ,NA_1菌株与恶臭假单胞菌形成一个类群 ,序列同源性为 99%。因此将NA_1菌株鉴定为恶臭假单胞菌     

Simultaneous determination of nicotinic acid and its metabolites in rat urine by micellar electrokinetic chromatography with photodiode array detection

Nicotinic acid, nicotinamide and their possible metabolites were successfully separated within 17 min by micellar electrokinetic chromatography using 50 mM borate buffer (pH 9.0) containing 150 mM sodium dodecyl sulfate as the running buffer. Calibration curves for all compounds showed good linearity in a range of 5 μg/ml and 250 μg/ml with good correlation. The present method did not require any clean-up procedures and made it possible to determine all metabolites without interference on a photodiode array detector. Urine samples collected from Wistar male rats were analyzed after high-dose oral or intravenous administration of nicotinic acid or nicotinamide. Metabolic pathways of nicotinic acid in male Wistar rats are also discussed.     

大肠杆菌来源的喹啉酸磷酸核糖转移酶和烟酸磷酸核糖转移酶的表达纯化及酶活性的初步检测

【目的】在原核表达体系中实现大肠杆菌来源的喹啉酸磷酸核糖转移酶(Quinolinic acid phosphoribosyl transferase,QPRT)和烟酸磷酸核糖转移酶(Nicotinic acidphosphoribosyl transferase,NaPPT)的表达与纯化,并利用酶的生物催化作用实现2,3-二羧酸喹啉的2位选择性脱羧得到烟酸【。方法】通过PCR扩增分别得到编码QPRT和NaPPT的基因片段,构建成原核表达质粒pET28a-NadC和pRSETB-PncB,在Escherichia coli(E.coli)中对其进行表达,在体外对目标蛋白进行纯化并利用高效液相色谱法(HPLC)检测酶催化反应的发生。【结果】成功表达纯化得到QPRT和NaPPT,检测结果表明在这两个酶的生物催化作用下可实现喹啉酸的2位选择性脱羧。     

Reversed-phase high-performance liquid chromatography of nicotinic acid mononucleotide for measurement of quinolinate phosphoribosyltransferase

A system has been developed for the determination of quinolinate phosphoribosyltransferase (QPRT) activity in liver and kidney homogenates using HPLC. A product, nicotinic acid mononucleotide (NaMN), is separated by reversed-phase chromatography (a Tosoh ODS 80TS was used as an analytical column) using a mixture of 10 mM KH₂PO₄–K₂HPO₄ buffer (pH 7.0) containing 1.48 g/l tetra-n-butylammonium bromide–acetonitrile (9:1, v/v) as a mobile phase. The flow-rate was 1.0 ml/min, the detection wavelength was 265 nm. The column temperature was maintained at 40°C. Under these conditions, NaMN was eluted at about 8.1 min. Sample preparation was very straightforward. The reaction mixture of QPRT assay was stopped by immersing the tube into a boiling water bath, the resulting supernatant was filtered, and the filtrate was directly injected into a HPLC system. The total HPLC analysis time was approximately 20 min.     

Biotransformation of 3-cyanopyridine to nicotinic acid by free and immobilized cells of recombinant Escherichia coli

An efficient biocatalytic process for the production of nicotinic acid (niacin) from 3-cyanopyridine was developed using cells of recombinant Escherichia coli JM109 harboring the nitrilase gene from Alcaligenes faecalis MTCC 126. The freely suspended cells of the biocatalyst were found to withstand higher concentrations of the substrate and the product without any signs of substrate inhibition. Immobilization of the cells further enhanced their substrate tolerance, stability and reusability in repetitive cycles of nicotinic acid production. Under optimized conditions (37 °C, 100 mM Tris buffer, pH 7.5) for the immobilized cells, the recombinant biocatalyst achieved a 100% conversion of 1 M 3-cyanopyridine to nicotinic acid within 5 h at a cell mass concentration (fresh weight) of 500 mg/mL. The high substrate/product tolerance and stability of the immobilized whole cell biocatalyst confers its potential industrial use.     

Molybdenum-dependent degradation of nicotinic acid by Bacillus sp. DSM 2923

Abstract Growth of Bacillus sp. DSM 2923 on nicotinic acid in mineral medium was dependent on the concentration of sodium molybdate added. Addition of increasing amounts of tungstate to the medium resulted in an inhibition of growth on nicotinic acid or 6-hydroxynicotinic acid as sole source of carbon and energy. Chlorate-resistant mutants were isolated which were not able to degrade nicotinic acid and 6-hydroxynicotinic acid nor to reduce nitrate. Additionally, enzyme activities of nicotinic acid dehydrogenase and 6-hydroxynicotinic acid dehydrogenase increased with increasing concentrations of molybdate (10⁻⁸ to 10⁻⁶ M) added to the medium, and decreased with increasing amounts of tungstate (10⁻⁶ to 10⁻⁵ M) in the medium.     

恶臭假单胞菌NA-1菌株烟酸羟基化酶活性的诱导和转化条件的研究

恶臭假单胞菌NA-1菌株的培养和产酶特性与已报道的产酶菌株粘质沙雷氏菌(Serratiamarcescens)IFO12648和荧光假单胞菌(Psudomonasfluorescens)TN5有所不同,主要反映在最适碳源及浓度、最适诱导剂浓度和最适培养温度等方面。最适的转化条件是温度为30℃,pH为7.0,烟酸的浓度为3%。采用初步优化后的条件和流加底物的方式进行4L上罐生产,恶臭假单胞菌NA-1菌株的6-羟基烟酸产率可达到108.39gL。     

Structure-activity relationships of trans-substituted-propenoic acid derivatives on the nicotinic acid receptor HCA2 (GPR109A)

Nicotinic acid (niacin) has been used for decades as an antidyslipidemic drug in man. Its main target is the hydroxy-carboxylic acid receptor HCA2 (GPR109A), a G protein-coupled receptor. Other acids and esters such as methyl fumarate also interact with the receptor, which constituted the basis for the current study. We synthesized a novel series of substituted propenoic acids, such as fumaric acid esters, fumaric acid amides and cinnamic acid derivatives, and determined their affinities for the HCA2 receptor. We observed a rather restricted binding pocket on the receptor with trans-cinnamic acid being the largest planar ligand in our series with appreciable affinity for the receptor. Molecular modeling and analysis of the structure-activity relationships in the series suggest a planar trans-propenoic acid pharmacophore with a maximum length of 8 Å and out-of-plane orientation of the larger substituents.     

Synthesis and biological evaluation of novel hydrogen sulfide releasing nicotinic acid derivatives

Twelve novel hybrids of slowly releasing hydrogen sulfide donor ADT-OH combined with nicotinic acid were synthesized. All of their structures had been confirmed by ¹H NMR, ¹³C NMR and MS spectra. The target compounds were evaluated for their neuroprotective effects on hippocampal neuron HT22 cells against glutamate-induced injury at the concentrations of 1–100 μM with MTT assay, and their toxicity on HT22 cells untreated by glutamine at the concentration of 100 μM. The active compound was further investigated for its effect on ischemic infarct volume by intraperitoneal injection at 3 h after ischemia in mice models of permanent middle cerebral artery occlusion (pMCAO). The results showed that all the compounds significantly protected HT22 cells from glutamate-induced damage at most of the experimental concentrations, and had no or little neurotoxicity on normal HT22 cells at the high concentration. More importantly, compound A6 significantly reduced infarct volume in the pMCAO model. These results suggested that compound A6 may be promising for further evaluation for the intervention of cerebral ischemic injury.     

Bioelectrochemical transformation of nicotinic acid into 6-hydroxynicotinic acid on Pseudomonas fluorescens TN5-immobilized column electrolytic flow system

Pseudomonas fluorescens TN5 catalyzes the hydroxylation of nicotinic acid (NA) into 6-hydroxynicotinic acid (6HNA), an important compound as a starting material for the synthesis of a new type of pesticides. Under aerobic conditions, however, 6HNA is metabolized in the P. fluorescens cells. The use of Fe(CN)₆³⁻ as an extracellular electron acceptor enhances the biotransformation of NA into 6HNA and completely suppresses the subsequent oxidation of 6HNA. The function of the P. fluorescens cell was combined with the electrode process by immobilizing the P. fluorescens cells on the carbon fiber electrode surface in the column, where Fe(CN)₆³⁻ was used as an electron transfer mediator. Continuous-flow electrolysis of NA in the presence of Fe(CN)₆³⁻ at the P. fluorescens-immobilized column electrode realized the accelerated and complete transformation of NA into 6HNA without any by-product.     

Effect of polyunsaturated fatty acid supplementation on biophysical parameters and chilling sensitivity of ewe oocytes.

Fat supplementation in the diet influences reproductive performance of lactating ruminants. Changes in the fat supply alter fatty acid composition and this can affect physical properties of cell membranes. This study examined the effect of rumen bypass polyunsaturated fatty acid (PUFA) supplementation on oocyte quality, chilling sensitivity, and lipid phase transition in oocytes of the sheep. Ewes were fed a diet supplemented with calcium soaps of fish oil for 13 weeks. More follicles and oocytes were found in the ovaries of ewes supplemented with PUFA than in control ewes. The number of high-quality oocytes was higher in ewes fed PUFA than in control ewes (74.3 and 57.0%, P < 0.05, respectively). Evaluation of phospholipid fatty acid composition indicated that PUFA were present in small proportions in oocytes, and eicosapentaenoic acid and docosahexaenoic acid were absent. Supplementation with PUFA increased the proportion of long chain unsaturated fatty acid in the plasma and cumulus cells phospholipids by 7.4 and 12.7%, respectively (P < 0.05). Integrity of oocyte membranes following chilling (16 degrees C, 15 min) was improved by PUFA supplementation increasing from 62.5 to 90.0% (P < 0.05). Due to changes in the oocyte's fatty acid profile, physical properties of the membrane were changed and the midpoint temperature of lipid transition reduced by 11 degrees C. These results suggest that supplementation of rumen bypass PUFA to ruminant diets can change fatty acid composition of follicle components and influence parameters such as number and quality of oocytes and their chilling resistance.     

Inhibition of human P450 enzymes by nicotinic acid and nicotinamide

Nicotinic acid has been used as a cholesterol-lowering agent for a few decades already, whereas the cytoprotective and antiviral properties of nicotinamide are slowly gaining attention. In both cases however, very high doses are needed to achieve a therapeutic effect, resulting in blood concentrations sometimes as high as 15 mM. Based on their common pyridine functionality, we hypothesized that these two molecules could inhibit human P450 enzymes. In vitro inhibition studies demonstrate that, at their therapeutic concentrations, both nicotinic acid and nicotinamide inhibit CYP2D6 (Ki = 3.8 +/- 0.3 and 19 +/- 4 mM, respectively). Nicotinamide also inhibits CYP3A4 (Ki = 13 +/- 3 mM) and CYP2E1 (Ki = 13 +/- 8 mM). As expected for nitrogen-containing heteroaromatic molecules, spectrophotometric analysis indicates that the inhibition occurs via coordination of the pyridine nitrogen atom to the heme iron.     

Enhancement of arachidonic acid signaling pathway by nicotinic acid receptor HM74A

HM74A is a G protein-coupled receptor for nicotinic acid (niacin), which has been used clinically to treat dyslipidemia for decades. The molecular mechanisms whereby niacin exerts its pleiotropic effects on lipid metabolism remain largely unknown. In addition, the most common side effect in niacin therapy is skin flushing that is caused by prostaglandin release, suggesting that the phospholipase A(2) (PLA(2))/arachidonic acid (AA) pathway is involved. Various eicosanoids have been shown to activate peroxisome-proliferator activated receptors (PPAR) that play a diverse array of roles in lipid metabolism. To further elucidate the potential roles of HM74A in mediating the therapeutic effects and/or side effects of niacin, we sought to explore the signaling events upon HM74A activation. Here we demonstrated that HM74A synergistically enhanced UTP- and bradykinin-mediated AA release in a pertussis toxin-sensitive manner in A431 cells. Activation of HM74A also led to Ca(2+)-mobilization and enhanced bradykinin-promoted Ca(2+)-mobilization through Gi protein. While HM74A increased ERK1/2 activation by the bradykinin receptor, it had no effects on UTP-promoted ERK1/2 activation.Furthermore, UTP- and bradykinin-mediated AA release was significantly decreased in the presence of both MAPK kinase inhibitor PD 098059 and PKC inhibitor GF 109203X. However, the synergistic effects of HM74A were not dramatically affected by co-treatment with both inhibitors, indicating the cross-talk occurred at the receptor level. Finally, stimulation of A431 cells transiently transfected with PPRE-luciferase with AA significantly induced luciferase activity, mimicking the effects of PPARgamma agonist rosiglitazone, suggesting that alteration of AA signaling pathway can regulate gene expression via endogenous PPARs.     

Miscibility of phosphatidylcholine binary mixtures in unilamellar vesicles: Phase equilibria

Summary Miscibility among phospholipids with different lipid chain-lengths or with different head groups has attracted a number of research efforts because of its significance in biological membrane structure and function. The general consensus about the miscibility of phosphatidylcholines with varying lipid chainlengths appears to be that binary mixtures of phospholipids with a difference of two carbon atoms in the lipid chain mix well at the main phase transition. Miscibility between phosphatidylcholines with differences of four carbon atoms appears to be inconclusive. Previous reports on the phase transition of binary phospholipid mixtures are concerned mainly with multilamellar vesicles and are mostly limited to the main transition. In the present study, unilamellar vesicles were used and miscibility in binary systems between dimyristoyl-, dipalmitoyl- and distearoyl-phosphatidylcholines at pretransition, as well as main transition temperatures was evaluated by constructing phase diagrams. Two methods were used to monitor the phase transitions: differential scanning microcalorimetry and optical absorbance methods. The optical method has the advantage that unilamellar vesicles of dilute phospholipid concentrations can be used. The liquidus and solidus phase boundaries were determined by the onset temperature of heating and cooling scans, respectively, because the completion temperature of a phase transition has no meaning in binary solutions. Dimyristoyl- and distearoyl-phosphatidylcholines. where the difference in the, lipid chain-length is four carbon atoms, mixed well even at pretransition temperature.     

Isolation of new 6-methylnicotinic-acid-degrading bacteria, one of which catalyses the regioselective hydroxylation of nicotinic acid at position C2

2-Hydroxynicotinic acid is an important building block for herbicides and pharmaceuticals. Enrichment strategies to increase the chances of finding microorganisms capable of hydroxylating at the C2 position and to avoid the degradation of nicotinic acid via the usual intermediate, 6-hydroxynicotinic acid, were used. Three bacterial strains (Mena 23/3–3c, Mena 25/4–1, and Mena 25/ 4–3) were isolated from enrichment cultures with 6-methylnicotinic acid as the sole source of carbon and energy. Partial characterization of these strains indicated that they represent new bacterial species. All three strains completely degraded 6-methylnicotinic acid, and evidence is presented that the first step in the degradation pathway of strain Mena 23/3–3c is hydroxylation at the C2 position. Resting cells of this strain grown on 6-methylnicotinic acid also hydroxylated nicotinic acid at the C2 position, but did not further degrade the product. Strain Mena 23/ 3–3c showed the highest degree of 16S rRNA sequence similarity to members of the genera Ralstonia and Burkholderia. Received: 4 April 1997 / Accepted: 10 June 1997