CEREBROSPINAL FLUID HOMOVANILLIC ACID AND ISO-HOMOVANILLIC ACID: A GAS–LIQUID CHROMATOGRAPHIC METHOD

Abstract— The hexafluoroisopropyl esters of trifluoroacetylated homovanillic acid (HVA) and 3-hydroxy-4-methoxyphenylacetic acid (iso-HVA) were the derivatives chosen for assay of these phenolic acids in lumbar cerebrospinal fluid (CSF) specimens from 65 control subjects. The mean value obtained in these specimens was 40.0 ± 23.5 ng/ml. Recovery of added HVA and iso-HVA was 94 ± 2 per cent and reproducibility was ±5 per cent. CSF from Parkinson's disease patients undergoing treatment with l -DOPA showed significant increases in HVA. Iso-HVA was detected in those CSF samples obtained during l -DOPA therapy.     

Synthesis and in vitro evaluation of a novel iodinated resiniferatoxin derivative that is an agonist at the human vanilloid VR1 receptor

Using a 'directed' iodination procedure, novel iodo-resiniferatoxin congeners were synthesized from 4-acetoxy-3-methoxyphenylacetic acid and resiniferinol- 9,13,14-ortho-phenylacetate (ROPA). The 2-iodo-4-hydroxy-5-methoxyphenylacetic acid ester of resiniferinol 5 displayed high affinity binding (K(i)=0.71 nM) for the human vanilloid VR1 receptor and functioned as a partial agonist.     

IDENTIFICATION AND QUANTIFICATION OF 3-METHOXY -4-HYDROXYPHENYLLACTIC ACID (VLA) IN CEREBROSPINAL FLUID AND 3-METHOXY-4-HYDROXY PHENYLPYRUVIC ACID (VPA) IN THE URINE OF PARKINSONIAN PATIENTS TREATED WITH L-DOPA

The identification and quantification of 3-methoxy-4-hydroxyphenyllactic acid in cerebrospinal fluid, and 3-methoxy-4-hydroxyphenylpyruvic acid in the urine of Parkinsonian patients on medication with l -3,4-dihydroxyphenylalanine with or without a peripheral decarboxylase inhibitor are described. Data are also given for other catecholamine- and catecholamine precursor metabolites in urine and cerebrospinal fluid. Analyses were performed by means of gas chromatography with flame ionization or mass spectrometric detection. We have concluded that following the administration of l -3.4-dihydroxyphenylalanine + peripheral decarboxylase inhibitor. 3-methoxy-4-hydroxyphenyllactic acid must be considered as a quantitatively important metabolite of L-3.4-dihydroxyphenylalanine in cerebrospinal fluid. In addition, some comment is given on the presence of 3-methoxy-4-hydroxy-phenylpyruvic acid in the urine of these patients.     

Determination of isomeric acid dopamine metabolites in human cerebrospinal fluid by mass fragmentography

A method for the quantitative determination of the isomers 4-hydroxy-3-methoxyphenylacetic acid (HVA) and 3-hydroxy-4-methoxyphenylacetic acid (iso-HVA) in cerebrospinal fluid (CSF) by mass fragmentography has been developed. The heptafluorobutyryl methyl ester derivations of the two compounds could not be separated by gas chromatography. The relative intensity of the base peak (m/e 333) and the molecular ion (m/e 392) in the mass spectra were, however, quite different and allowed a separate determination. Contradictory to a previous report, it was found that the concentration of iso-HVA was less than 2% of the HVA level in the investigated 12 CSF samples from patients with different diseases.     

Simultaneous determination of 4-hydroxy-3-methoxy-phenylacetic (homovanillic) acid and other monoamine metabolites in human lumbar cerebrospinal fluid : An improved high-performance liquid chromatographic study with electrochemical detection

We describe a direct analysis for the simultaneous quantitative determination of 4-hydroxy-3-methoxyphenylacetic (homovanillic) acid and other monoamine metabolites in human lumbar cerebrospinal fluid, utilizing reversed-phase high-performance liquid chromatography with amperometric detection. In addition, a rapid isocratic separation was developed for homovanillic acid in the presence of other endogenous compounds.Twenty-five unselected diagnostic specimens of human lumbar cerebrospinal fluid were extracted with ethyl acetate and subsequently analyzed using the described method. Chromatographic peaks were identified on the basis of retention behavior and ratio of responses at several oxidation potentials.Although our quantitative results correlate well with the literature values, the data were not interpreted clinically since samples were obtained from routine, diagnostic testing of patients admitted to the medical or neurologic services at the Mount Sinai Hospital.     

Properties of leukotriene B4 20-hydroxylase from polymorphonuclear leukocytes

Human polymorphonuclear leukocytes (PMNL) convert arachidonic acid (20:4) to a number of dihydroxy metabolites, including leukotriene B4 (LTB4) 5S,12R-dihydroxy-6,8,10,14-EEEZ-icosatetraenoic acid (isomer-1), 5S,12S-dihydroxy-6,8,10,14-EEEZ-icosatetraenoic acid, 5S,12S-dihydroxy-6,8,10,14-EZEZ-icosatetraenoic acid (5S,12S-dh-20:4), 5,6-dihydroxy-7,9,11,14-icosatetraenoic acid, and 5,15-dihydroxy-6,8,11,13-icosatetraenoic acid. LTB4 was synthesized rapidly after stimulation of PMNL with the divalent cation ionophore, A23187, but its concentration rapidly declined after about 4 min, in contrast to the other dihydroxy metabolites of 20:4 whose concentrations remained stable for at least 20 min. The amounts of polar metabolites (identified primarily as 20-hydroxy-LTB4) increased steadily with time up to 20 min. These results suggest that LTB4 may be specifically converted to its 20-hydroxy metabolite by PMNL. We prepared 3H- and 14C-labeled analogs of the dihydroxyicosatetraenoic acid metabolites described above by incubation of labeled 20:4 with PMNL. Although all of these substances were metabolized to some extent by human PMNL, LTB4 (apparent Km, 1.0 microM) was metabolized the most rapidly, followed by 5S,12S-dh-20:4 (apparent Km, 2.4 microM) and isomer-1 (apparent Km, 4.8 microM). All three substrates were shown by mass spectrometry to be converted to their 20-hydroxy metabolites. LTB4 was also metabolized to its omega-carboxy derivative. Human mononuclear leukocytes and rabbit PMNL metabolized LTB4 very slowly, whereas rat PMNL metabolized this substrate at about one-sixth the rate of human PMNL. These results demonstrate that human PMNL contain an omega-hydroxylase that specifically converts LTB4 to its 20-hydroxy metabolite. This enzyme may be important for the regulation of LTB4 levels in vivo.     

Hepatitis C virus core, NS3, NS4B and NS5A are the major immunogenic proteins in humoral immunity in chronic HCV infection

Background

Light-dependent activities against enveloped viruses in St. John's Wort (Hypericum perforatum) extracts have been extensively studied. In contrast, light-independent antiviral activity from this species has not been investigated.

Results

Here, we identify the light-independent inhibition of human immunodeficiency virus-1 (HIV-1) by highly purified fractions of chloroform extracts of H. perforatum. Both cytotoxicity and antiviral activity were evident in initial chloroform extracts, but bioassay-guided fractionation produced fractions that inhibited HIV-1 with little to no cytotoxicity. Separation of these two biological activities has not been reported for constituents responsible for the light-dependent antiviral activities. Antiviral activity was associated with more polar subfractions. GC/MS analysis of the two most active subfractions identified 3-hydroxy lauric acid as predominant in one fraction and 3-hydroxy myristic acid as predominant in the other. Synthetic 3-hydroxy lauric acid inhibited HIV infectivity without cytotoxicity, suggesting that this modified fatty acid is likely responsible for observed antiviral activity present in that fraction. As production of 3-hydroxy fatty acids by plants remains controversial, H. perforatum seedlings were grown sterilely and evaluated for presence of 3-hydroxy fatty acids by GC/MS. Small quantities of some 3-hydroxy fatty acids were detected in sterile plants, whereas different 3-hydroxy fatty acids were detected in our chloroform extracts or field-grown material.

Conclusion

Through bioguided fractionation, we have identified that 3-hydroxy lauric acid found in field grown Hypericum perforatum has anti-HIV activity. This novel anti-HIV activity can be potentially developed into inexpensive therapies, expanding the current arsenal of anti-retroviral agents.     

花生茎叶酚性成分研究

运用大孔树脂对花生茎叶提取液进行富集,不同浓度乙醇洗脱,硅胶、RP-18、Sephadex LH-20等多种材料进一步分离纯化,研究花生茎叶化学成分,并通过理化方法和光谱分析对化合物进行结构鉴定。结果表明:从花生茎叶大孔树脂10%乙醇洗脱部位中分离并鉴定了10个化合物,分别为邻苯二甲酸二异丁酯(1)、水杨酸(2)、儿茶酚(3)、对羟基苯甲酸(4)、(反)-3,4-二羟基苯丙烯酸(5)、对羟基苯酚(6)、邻苯二甲酸二丁酯(7)、3,4-二羟基苯乙醇(8)、对羟基苯乙醇(9)、3,4-二羟基苯甲酸(10)。除化合物1、2和4外,其余均为首次从该植物中分离得到。     

PHOTOOXIDATIVE CONSUMPTION AND PHOTOREDUCTIVE FORMATION OF ASCORBIC ACID IN GREEN LEAVES

1. From leaves of barley and spinach, cellular components wereisolated and brought together under various conditions to investigatethe fate of ascorbic acid as affected by the components in thelight and dark. 2. A new colorimetric method for assaying ascorbic acid andsome other reducing substances was devised, measuring the colorof molybdenum-blue developed by the substances in the presenceof excess amounts of phosphomolybdate and inorganic acid. 3. The photooxidation of ascorbic acid by green and yellow filtrates,prepared from green and etiolated leaves of barley, was studiedby the ordinary as well as the new colorimetric method. In thepresence of oxygen, the oxidation of ascorbic acid was foundto be accelerated by light in the green filtrate, but not inthe yellow filtrate. 4. The oxidation of the endogenous reducing substance containedin the supernatant fraction of spinach leaf extracts was studiedin the presence of washed chloroplasts (spinach). In the presenceof oxygen, the rate of oxidation in the light was markedly higherthan in the dark. From the changes in absorption spectrum accompanyingthe reaction, the endogenous reducing substance in questionwas identified as ascorbic acid. 5. The occurrence of an endogenous precursor of ascorbic acidin spinach leaf extracts was disclosed. The photoreduction ofthis precursor into ascorbic acid was studied in the precenceof spinach chloroplasts. A specific inhibition of this reactionby phosphoglycerate and glycerophosphate was discovered. 6. The experimental results obtained were discussed in connectionwith the role of ascorbic acid in photosynthesis. (Received September 13, 1960; )     

Ds-erythro-2-amino-4-ethoxy-3-hydroxybutanoic acid from the fruiting bodies of the edible mushroom,Lyophyllum ulmarium

A new β-hydroxy amino acid isolated from the fruiting bodies of Lyophyllum ulmarium was identified as D_s,-erythro-2-amino-4-ethoxy-3-hydroxybutanoic acid by chemical degradation and spectroscopic analyses.     

INTRACRANIAL CONVERSION OF LINOLEIC ACID TO ARACHIDONIC ACID: EVIDENCE FOR LACK OF Δ8 DESATURASE IN THE BRAIN

Eleven-day old rats were given intracranial injection of [1-¹⁴C]linoleic acid (all cis 9,12 octadecadienoic acid) and sacrificed after 8 h. Analysis of brain fatty acids showed that 16:0, 18:2, 20:2,20:3 and 20:4 were labeled. Separation by AgN03:Si02 TLC plates followed by reductive ozonolysis characterized thc polyunsaturated fatty acids as 18:2 (Δ9,12), 20:2 (Δ11,14), 20:3 (Δ8,11,14) and 20:4 (Δ5,8,11,14). A smaller amount of 18:3 (Δ6,9,12) was also identified. This initially suggested 20:2 (A1 1,14) as an intermediate in the optional pathway of biosynthesis of arachidonate. However, when [l-14C]eicosadienoic acid (Δ1 1,141 itself was injected in the brain it was converted to 20:3 (Δ5,11,14) (a non-methylene interrupted double bond system) rather than the expected 20:3 (Δ8,11,14). Only a small amount of arachidonate was formed from 20:2 (Δ11,14). Thus it was concluded that 20:2 (Δ11,14) was not an intermediate in the pathways of arachidonate biosynthesis due to lack of Δ⁵ desaturase in thc brain which agrees with the findings of SPKECRER & LEE (1975) in rat liver.     

OCCURRENCE OF N-ACETYL-l-GLUTAMIC ACID IN THE HUMAN BRAIN

A substance apparently identical with N-acetyl-l -glutamic acid was isolated from an aqueous extract from human brain by a combination of paper and ion exchange chromatography. The isolated substance does not react with ninhydrin reagent but yields glutamic acid upon acid hydrolysis. Acetyl hydrazide was identified by paper chromatography of hydrazinolysates of the isolated substance and N-acetyl-l -glutamic acid. The configuration was determined with l -specific hog kidney acylase.     

Biosynthesis of 3-hydroxy fatty acids, the pheromone components of female mallard ducks, by cell-free preparations from the uropygial gland

Diesters of 3-hydroxy C8, C10, and C12 acids, the female mallard duck pheromones, were found as the major products of the uropygial glands only during the breeding season. The 3-hydroxy acids were identified by mass spectrometry of the trimethylsilyl ethers of the methyl esters and of the diols derived from LiAlH4 reduction of the hydroxy acids. A cell-free extract from the gland catalyzed conversion of dodecanoic acid to 3-hydroxydodecanoic acid which was identified by radio thin-layer and radio gas chromatographic analysis of the enzymic products as methyl-3-acetoxydodecanoate and as diacetate of the diol generated by LiAlH4 reduction of the enzymic product. The enzymic introduction of the hydroxyl group at C-3 was catalyzed mainly by a 50,000g pellet prepared from a 1000g supernatant obtained from the cell-free extract. This reaction required ATP, CoA, and O2, and the CoA ester of the acid was more efficiently converted than the free acid to the 3-hydroxy acid. KCN at 1 mM and 50% CO did not inhibit the reaction. 3H from 3H2O was incorporated into 3-hydroxydodecanoic acid during the enzymic synthesis of this acid from dodecanoic acid. Mass spectrometry of the 3-hydroxy acid generated by the particulate fraction in the presence of H2 18O showed that 18O was incorporated as expected from hydration of a delta 2 double bond. From the above results it is tentatively concluded that peroxisomal acyl-CoA oxidase converts the acyl-CoA to the 2-enoyl-CoA which is hydrated to generate the 3-hydroxy acid.     

Novel biotransformations of 4-chlorobiphenyl by a Pseudomonas sp

A bacterium, tentatively identified as a representative of the genus Pseudomonas (strain MB86), was isolated from soil contaminated by wood-preservation chemicals by using 4-chlorobenzoate as an enrichment substrate. The pseudomonad was able to grow on 4-chlorobenzoic acid and 4-chlorobiphenyl as sole carbon and energy sources. Spent culture medium from 4-chlorobiphenyl-grown cells contained 4-chlorobenzoic acid, 4'-chloroacetophenone, 2-hydroxy,2-[4'-chlorophenyl] ethane, and 2-oxo,2-[4'-chlorophenyl] ethanol as metabolites. 4'-Chloroacetophenone was produced in large amounts, possibly as a dead-end metabolite.     

Metabolism of 18:2(n - 6), 18:3(n - 3), 20:4(n - 6) and 20:5(n - 3) by the fungus Gaeumannomyces graminis: identification of metabolites formed by 8-hydroxylation and by w2 and w3 oxygenation.

The present study was aimed at developing a cell-free preparation of Gaeumannomyces graminis to biosynthesize w2-hydroxy, w3-hydroxy and related metabolites of essential fatty acids. 14C-labelled linoleic acid (18:2(n - 6)), linolenic acid (18:3(n - 3)), arachidonic acid (20:4(n - 6)) and eicosapentaenoic acid (20:5(n - 3)) were incubated with the cytosolic and microsomal fractions and NADPH. Significant metabolism was only found in the cytosol. The main products were purified by high-performance liquid chromatography and identified by gas chromatography-mass spectrometry (GC-MS). 18:2(n - 6) was metabolized mainly to 8-hydroxy-9,12-octadecadienoic acid (8-HODE), while the w2 and the w3 alcohols were formed in relatively small amounts. The absolute configuration of the 8-hydroxyl was found to be R by ozonolysis of the diastereoisomeric (-)-menthoxycarbonyl derivative of 8-HODE and GC-MS analysis. In analogy, 18:3(n - 3) was converted to 8-hydroxy-9,12,15-octadecatrienoic acid and to smaller amounts of the 15,16-diol (15,16-DiHODE). In contrast, 8-hydroxy metabolites of 20:4(n - 6) or 20:5(n - 3) could not be detected. 20:4(n - 6) was efficiently converted to 18(R)-hydroxyeicosatetraenoic acid (18(R)-HETE) and 19(R)-HETE and to traces of 17-HETE, while 20:5(n - 3) was mainly metabolized to the 17,18-diol (17,18-DiHETE) and to smaller amounts of the w2 alcohol. In conclusion, the cytosol of G. graminis can be used for stereoselective biosynthesis of some hydroxy metabolites of essential fatty acids.     

八种脑-肠肽侧脑室内注射对大鼠基础胃酸分泌的影响

用乌拉坦麻醉大鼠作急性实验,采用连续灌流胃并收集流出液的方法,观察向侧脑室内注射微量脑-肠肽对大鼠基础胃酸分泌的影响。实验结果如下:(1)雨蛙肽、八肽胆囊收缩素、促甲状腺素释放激素及四肽胃泌素均使总酸排出量增加;(2)生长抑素、胰多肽、P 物质、胰高血糖素则使总酸排出量减少;(3)上述肽类用侧脑室注射的剂量作肌肉注射,除四肽胃泌素也产生明显的刺激胃酸分泌作用外,对胃酸分泌均无明显影响。以上结果提示,脑内的一些肽类可能以神经递质或调制物的方式,参与中枢对胃酸分泌的调节。     

RELATIONSHIP BETWEEN ORAL AND NONORAL EVALUATION OF TEXTURE IN ACID MILK GELS

The objectives of this study were to compare oral and nonoral sensory evaluation for discrimination of texture of acid skim milk gels and to establish whether nonoral attributes could be correlated to the oral perception of texture. Trained panelists (n = 13) identified 11 nonoral (visual and in-hand) and 4 oral attributes during a preliminary profiling session that could be used to discriminate textures (P < 0.001) in a range of acid gels prepared with different solid contents and heat treatment of the milks. Both methods of sensory appraisal were found to discriminate between gels. Correlation analysis showed high interrelationship between individual oral and nonoral attributes (P < 0.01). Principal component analysis revealed that all 4 oral attributes could be combined into one single attribute (PC1), with equal relative importance of the individual attributes in explaining the variance in the oral sensory data set. Canonical correlation analysis revealed good correlation between the oral and nonoral set of attributes (R² > 87.5%).     

下丘脑外侧区注射TRH对大鼠胃酸分泌的影响

本文采用连续收集胃腔灌流法,观察下丘脑外侧区(LHA)注射促甲状腺激素释放激素(TRH)对大鼠胃酸分泌的影响,并分析TRH在LHA促进胃酸分泌的作用机制。结果表明:(1)LHA注射TRH(1μg)明显地刺激胃酸分泌;(2)预先向LHA注射酚妥拉明(10μg)、美多心安(5μg)及胃泌素抗体1μl(1:640)并不影响TRH的泌酸作用,如预先向LHA注射阿托品(5μg)则可消除TRH的泌酸效应;(3)垂体摘除及肾上腺切除均不影响TRH的泌酸作用;(4)隔下迷走神经切断后,LHA注入TRH的泌酸效应仍然出现,但持续时间显著缩短;腹腔交感神经节摘除后,TRH仍能促进胃酸分泌,但分泌量少而平稳。以上结果提示:LHA是TRH中枢泌酸效应的有关结构之一,其中枢机制是通过胆碱能M受体中介的,腹腔交感神经节和膈下迷走神经是TRH泌酸效应的传出途径。前者引起的泌酸反应出现较早且引起泌酸高峰,但持续时间短;后者则引起低平的持续分泌。     

EFFECTS OF GAMMA-HYDROXYBUTYRIC ACID AND OTHER HYPNOTICS ON GLUCOSE UPTAKE IN VIVO AND IN VITRO

Abstract— (1) The effects of gamma-hydroxybutyrate, imidazole-4-acetic acid and pento-barbitone on mouse brain glucose, glycogen and lactate levels have been studied. All the drugs significantly increased the brain glucose content, but did not significantly alter brain glycogen levels. The increase in brain glucose following imidazole-4-acetic acid or hypnotic doses of pentobarbitone was matched by corresponding decreases in the lactate level; this was not the case with gamma-hydroxybutyrate where the total glucose equivalents in the brain, expressed as the tissue level of (glucose) + (lactate/2), were significantly increased.
(2) All drugs except imidazole-4-acetic acid significantly decreased the rate of appearance of [¹⁴C]glucose into the bloodstream in vivo but had no effect on the uptake of glucose into rat diaphragm in vitro when present at 2·5 mM concentration.
(3) Only imidazole-4-acetic acid significantly inhibited glucose uptake into the brain in vivo but at 2·5 mM had no significant effect on glucose uptake into rat cerebral cortical slices in vitro.
(4) It is concluded that the very large increase in brain glucose level observed following the injection of hypnotic doses of gamma-hydroxybutyrate cannot be explained in terms of an increased net uptake of glucose into the brain.     

Novel biotransformations of 4-chlorobiphenyl by a Pseudomonas sp.

A bacterium, tentatively identified as a representative of the genus Pseudomonas (strain MB86), was isolated from soil contaminated by wood-preservation chemicals by using 4-chlorobenzoate as an enrichment substrate. The pseudomonad was able to grow on 4-chlorobenzoic acid and 4-chlorobiphenyl as sole carbon and energy sources. Spent culture medium from 4-chlorobiphenyl-grown cells contained 4-chlorobenzoic acid, 4'-chloroacetophenone, 2-hydroxy,2-[4'-chlorophenyl] ethane, and 2-oxo,2-[4'-chlorophenyl] ethanol as metabolites. 4'-Chloroacetophenone was produced in large amounts, possibly as a dead-end metabolite.