FREE 3-METHOXY-4-HYDROXYPHENYLETHYLENEGLYCOL IN THE CENTRAL NERVOUS SYSTEM OF THE RAT: SEMI-AUTOMATED FLUOROMETRIC ASSAY, TURNOVER AND EFFECTS OF DRUGS

A semi-automated fluorometric assay technique for free 3-methoxy-4-hydroxyphenylethyl-eneglycol (MOPEG) in rat brain and spinal cord is described. The method is based on a simple manually-performed two-step purification procedure using column chromatography on Sephadex G 10 and DEAE-Sephadex A 25 (borate form) respectively. After isolation MOPEG is converted into a fluorophore in a continuous flow system using ethylene diamine condensation in the presence of an oxidant. The MOPEG assay is highly sensitive (detection limit 2 ng/sample) and linear, with an overall recovery of approx 75%. Specificity is demonstrated by testing a number of compounds and confirmed by gas chromatography-mass spectrometry analysis. Treatment with clozapine and haloperidol (both neuroleptics), reser-pine (impairing intraneuronal storage) or phenoxybenzamine (α-adrenoceptor blocking agent) increased the content of MOPEG both in brain and spinal cord. Cerebral levels of MOPEG were decreased after injection of a single dose of the tricyclic antidepressant desipramine and after chronic destruction of the locus coeruleus by electrolytic lesion or by the administration of the neurotoxic drug 6-hydroxy-dopamine. Animals killed by microwave irradiation did not show lower MOPEG contents in brain than decapitated animals. These results indicate that MOPEG is a measure for changes in central noradrenaline turnover and that drugs affect MOPEG in the brain and spinal cord similarly. Fractional rate constants of MOPEG in the rat brain and spinal cord were estimated with the exponential decline curves produced by treatment with pargyline. Turnover rates of 193 pmol/g/h and 167 pmol/g/h in the brain and spinal cord respectively were calculated.     

Neuropeptide Y- and somatostatin-like immunoreactivities in ganglioneuroma, ganglioneuroblastoma and neuroblastoma

Neuropeptide Y (NPY)- and somatostatin (SS)-like immunoreactivities (LI) were investigated in tumor tissues of one ganglioneuroma (GN), 3 ganglioneuroblastomas (GNB) and one neuroblastoma (NB) by radioimmunoassay. NPY-LI was detected from all 5 tumor tissues (16.4-1247 pmol/g wet tissue). Sephadex G-50 column chromatography and reverse phase high performance liquid chromatography (HPLC) revealed that most of the NPY-LI in tumor extracts was eluted in an identical position to synthetic human NPY except one GNB (case 2). In this case, most of the NPY-LI was eluted in a higher molecular weight region than synthetic human NPY in Sephadex G-50 column chromatography and in a more hydrophobic position in HPLC. SS-LI was detected from 4 tumor extracts except one GNB (case 2) (21.3-787 pmol/g wet tissue). Sephadex G-25 column chromatography and reverse phase HPLC revealed that SS-LI in tumor extracts was eluted just after the void volume and then in the same positions as SS-28 and SS-14. These results suggest that NPY, SS-14 and SS-28 exist in tumor tissues of GN, GNB and NB, and most of the NPY-LI in one GNB was a higher molecular and more hydrophobic form of NPY-LI.     

Determination of Normetanephrine, 3,4-Dihydroxyphenylethyleneglycol (Free and Total), and 3-Methoxy-4-Hydroxyphenylethyleneglycol (Free and Total) in Rat Brain by High-Performance Liquid Chromatography with Electrochemical Detection and Effects of Drugs on Regional Concentrations

A new, fast and sensitive assay for normetanephrine (NM), free and total 3,4-dihydroxyphenylethyleneglycol (DOPEG), and free and total 3-methoxy-4-hydroxyphenylethyleneglycol (MOPEG) in brain tissue is described. The method is based on high-performance liquid chromatography with electrochemical detection. Small Sephadex G 10 columns were used for prepurification. This permitted the additional isolation and quantification of tyrosine, 3,4-dihydroxyphenylalanine, noradrenaline, dopamine, 3-methoxytyramine, 3,4-dihydroxyphenylacetic acid, homovanillic acid, and 5-hydroxyindoleacetic acid. The compounds were determined in six brain areas (striatum, cortex, hippocampus, hypothalamus, brainstem, and cerebellum). Most DOPEG and MOPEG in rat brain was present in the conjugated form, except for the cerebellum, where about 80% of MOPEG was nonconjugated. No postmortem effects on MOPEG levels were observed; a slight increase in DOPEG in certain brain areas was found in microwave-killed rats. The effects of clonidine, yohimbine, N,N-dipropyl-5,6-ADTN, and chlorpromazine on the concentrations of the five noradrenaline (NA) metabolites were determined. Free and total DOPEG and MOPEG provide similar information on NA metabolism, whereas NM (after monoamine oxidase inhibition) reflects a different type of interaction of drugs with NA metabolism. The similarity in the pattern of drug-induced changes in NA metabolism in the various brain areas suggests that adrenoreceptors mediating NA metabolism are homogeneously distributed throughout the brain.     

Simultaneous microdetermination of homovanillic acid and 5-hydroxyindoleacetic acid in brain tissue using Sephadex G-10 and QAE-Sephadex A-25

Homovanillic acid (HVA) and 5-hydroxyindoleacetic acid (5-HIAA) in animal brains were simultaneously purified by two steps of column chromatography on Sephadex G-10 and QAE-Sephadex A-25. Perchloric acid extracts of brain tissue were directly passed through a column of Sephadex G-10. The gel retained both HVA and 5-HIAA, thereby separating them from Cl0^?₄ which interferes with subsequent purification process and from endogenous substances which give blank fluorescence. HVA was loosely adsorbed on the gel and was easily desorbed with dilute acetic acid. This effluent was successively passed onto a column of QAE-Sephadex A-25 placed beneath the G-10 column and the adsorbed HVA was eluted with 0.1 M Na₂HPO₄. The 5-HIAA remaining on the Sephadex G-10 without being desorbed by acetic acid was eluted with dilute ammonia. The recovery of both acid metabolites by this column procedure was more than 90%. Thus, it is possible to determine the levels of HVA and 5-HIAA in single brains of small rodents.     

Radioimmunoassays for prostaglandins. II. Measurement of prostaglandin E2 and the 13,14-dihydro-15-keto metabolites of the E and F series. Description of a reliable technique with a universal applicability

Human plasma samples of 1 ml were processed according to three different procedures prior to Radioimmunoassay (RIA) of Prostaglandin F_2α (PGF_2α). Serial dilutions of ethyl acetate extracts as such, or combined with either silicic acid of Sephadex G-25 chromatography were assessed for linearity, homogeneity and parallelism with the corresponding standard dose response line. For plasma extracts used as such, non-parallelism is observed. Subsequent chromatography on silicic acid of such extracts gave only a limited linear and parallel portion upon serial dilution. However, purification of the extracts by gelfiltration on Sephadex G-25 results in linear and parallel lines over the full range of the standard dose response line (B/B_o 0.9-0.2).Upon comparison of separation by polyethylene glycol (PEG) and Dextran coated charcoal (DCC) in these systems, PEG proved to give the best results. It was found that in the Sephadex G-25 procedure, separation by PEG is essential. The method of gelfiltration on Sephadex G-25 is simple and reliable. Intra- and inter-assay coefficients of variation are 6% and 12%, respectively. Accuracy, as measured by recovery of added known amounts of PGF_2α is 97.6%.     

Monoamines and Their Precursors and Metabolites in the Chicken Brain, Pineal, and Retina: Regional Distribution and Day/Night Variations

Levels of norepinephrine, epinephrine, dopamine, and serotonin (5-HT) and their precursors [tyrosine, L-3,4-dihydroxyphenylalanine, tryptophan, and 5-hydroxytryptophan (5-HTP)] and metabolites [3,4-dihydroxyphenylacetic acid (DOPAC), 3-methoxytyramine (3-MT), homovanillic acid, 3-methoxy-4-hydroxyphenylglycol, and 5-hydroxyindoleacetic acid (5-HIAA)] were determined concurrently in samples of chick retina, pineal gland, and nine selected areas of the brain (optic lobes, thalamus, hypothalamus, optic chiasm, pons/medulla, cerebellum, neostriatum/ectostriatum, hyperstriatum, and basal forebrain) using HPLC coupled with a coulometric electrode array detection system. The norepinephrine level was highest in the pineal gland, but it was also widely distributed throughout the chick brain, with the thalamus and hypothalamus showing substantial levels. The dopamine level was highest in the basal forebrain. The epinephrine level was highest in the hypothalamus. The thalamus and hypothalamus showed the highest levels of 5-HT. Daytime levels (1100 h) of these compounds were compared with levels in chicks killed in the middle of the dark phase (2300 h). In the brain areas examined, no day/night variations in levels of norepinephrine, epinephrine, dopamine, or 5-HT were seen, although significant nocturnal changes in levels of their metabolites were observed in some areas. Pineal levels of 5-HIAA decreased significantly at night. The retina showed significant nocturnal increases in 5-HTP, 5-HT, and 5-HIAA levels. Retinal levels of 3-MT and DOPAC were significantly decreased at night.     

The gel-filtration behaviour of proteins related to their molecular weights over a wide range

1. Correlation between elution volume, V_e, and molecular weight was investigated for gel filtration of proteins of molecular weights ranging from 3500 (glucagon) to 820000 (α-crystallin) on Sephadex G-200 columns at pH7·5. 2. Allowing for uncertainties in the molecular weights, the results for most of the carbohydrate-free globular proteins fitted a smooth V_e–log(mol.wt.) curve. In the lower part of the molecular-weight range the results were similar to those obtained with Sephadex G-75 and G-100 gels. 3. V_e–log(mol.wt.) curves based on results with the three gels are taken to represent the behaviour of `typical' globular proteins, and are proposed as standard data for the uniform interpretation of gel-filtration experiments. 4. Some glycoproteins, including γ-globulins and fibrinogen, do not conform to the standard relationship. The effect of shape and carbohydrate content on the gel-filtration behaviour of proteins is discussed. 5. As predicted by the theoretical studies of other authors, correlation exists between the gel-filtration behaviour and diffusion coefficients of proteins. 6. The lower molecular-weight limit for complete exclusion of typical globular proteins from Sephadex G-200 varies with the swelling of the gel, but is usually >10⁶. 7. The concentration-dependent dissociation of glutamate dehydrogenase was observed in experiments with Sephadex G-200, and the sub-unit molecular weight estimated as 250000. The free sub-units readily lose enzymic activity. 8. Recognition of the atypical gel-filtration behaviour of γ-globulins necessitates an alteration to several molecular weights previously estimated with Sephadex G-100 (Andrews, 1964). New values are: yeast glucose 6-phosphate dehydrogenase, 128000; bovine intestinal alkaline phosphatase, 130000; Aerobacter aerogenes glycerol dehydrogenase, 140000; milk alkaline phosphatase, 180000.     

Effect of l-Tryptophan on Mouse Brain 5-Hydroxytryptamine: Comparison of Values Obtained Using a Fluorimetric Assay and a Liquid Chromatographic Assay with Electrochemical Detection

Abstract: 5-Hydroxytryptamine (5-HT) in mouse brain and spinal cord was assayed in the same samples using a fluorimetric assay and a high pressure liquid chromatographic (HPLC) assay with electrochemical detection. The HPLC assay was able to detect levels of 5-hydroxytryptamine as low as 0.2-0.5 pmol. With the column (Vydac cation exchange), solvent system (acetate/citrate buffer, 0.1 or 0.2 M, pH 4.8-5.2) extraction procedure and electrode potential (+0.55 V) used, the HPLC assay was specific for 5-HT. When the electrode potential was increased to +0.9 V tryptamine could also be detected, with a longer retention time than 5-hydroxytryptamine. The percentage increase in mouse brain 5-hydroxytryptamine after pargyline (75 mg/kg) and pargyline + l -tryptophan (100 mg/kg) was very similar whether measured by fluorimetry or HPLC, although the fluorimetric assay gave consistently higher absolute values (24–32%) in both control and drug-treated animals. l -Tryptophan (25, 50 and 100 mg/kg) also increased brain 5-hydroxytryptamine with similar percentage increases with either assay method. There was a significant correlation ( P < 0.001) between the values obtained with the two assay methods. The results confirm the use of HPLC with electrochemical detection as a sensitive and specific assay method for 5-hydroxytryptamine and indicate its potential use for the assay of tryptamine, and the importance of determining the electroactivity and retention characteristics of any drugs used.     

Radioimmunoassays for prostaglandins. I. Technical validation of prostaglandin F2α measurements in human plasma using Sephadex G-25 gelfiltration

Human plasma samples of 1 ml were processed according to three different procedures prior to Radioimmunoassay (RIA) of Prostaglandin F_2α (PGF_2α). Serial dilutions of ethyl acetate extracts as such, or combined with either silicic acid of Sephadex G-25 chromatography were assessed for linearity, homogeneity and parallelism with the corresponding standard dose response line. For plasma extracts used as such, non-parallelism is observed. Subsequent chromatography on silicic acid of such extracts gave only a limited linear and parallel portion upon serial dilution. However, purification of the extracts by gelfiltration on Sephadex G-25 results in linear and parallel lines over the full range of the standard dose response line (B/B_o 0.9-0.2).Upon comparison of separation by polyethylene glycol (PEG) and Dextran coated charcoal (DCC) in these systems, PEG proved to give the best results. It was found that in the Sephadex G-25 procedure, separation by PEG is essential. The method of gelfiltration on Sephadex G-25 is simple and reliable. Intra- and inter-assay coefficients of variation are 6% and 12%, respectively. Accuracy, as measured by recovery of added known amounts of PGF_2α is 97.6%.     

Precursors in the biosynthesis of vasopressin and oxytocin in the rat. Characteristics of all the components in high-performance liquid chromatography.

A reverse-phase high performance liquid-chromatography (h.p.l.c.) protocol has been developed, whereby all the major known posterior-pituitary components that are derived from the processing of pro-oxytocin and pro-vasopressin can be separated one from another. Thus, in a single chromatographic step, it has been possible to separate vasopressin (VP), oxytocin (OT), oxytocin-neurophysin (rOT-Np), vasopressin-neurophysin (rVP-Np) and vasopressin-glycopeptide (rVP-GP) from acid extracts of the neurointermediate lobes of rat pituitary glands. All these peptides except rVP-GP were labelled in the neural lobe by 24h after a hypothalamic injection of [35S]cysteine, whereas all except VP were labelled by 24h after a similar injection of [3H]leucine. Three major labelled proteins were isolated from 20 min [35S]cysteine-injected rats when extracts of the supraoptic nucleus were subjected to Sephadex G-75 chromatography, h.p.l.c. and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Immunoprecipitation with antisera raised against rat neurophysins, VP and OT revealed 21000- and 19000-mol.wt. common precursors to VP and rVP-Np and a 15000-mol.wt. common precursor to OT and rOT-Np. Some immunoreactive rVP-Np could occasionally be detected in the Vo of Sephadex G-75 chromatograms of Wistar rat supraoptic-nucleus extracts, but no evidence of [35S]neurophysin in this fraction was obtained from h.p.l.c. fingerprinting of its S-carboxymethylated tryptic digests. Radioimmunoassay for rVP-Np and rOT-Np revealed that about 70-80% of the total recovered immunoreactive neurophysin (IR-Np) in the supraoptic nucleus eluted from Sephadex G-75 and h.p.l.c. in the positions of rVP-Np and rOT-Np. Evidence is presented for an approx. 20000-mol.wt. rOT-Np in both Wistar and Brattleboro rats and for an approx. 20000-mol.wt. component in the Brattleboro rat that is recognized by vasopressin-neurophysin antisera.     

Determination of 3-Methoxytyramine in Rat Brain by HPLC with Electrochemical Detection and Its Correlation with Dopamine Function After Administration of a Monoamine Oxidase Inhibitor and L-3,4-Dihydroxyphenylalanine

3-Methoxytyramine (3-MT), normally a minor metabolite of 3,4-dihydroxyphenylethylamine (dopamine) in brain, becomes the sole product of metabolism following the administration of a monoamine oxidase (MAO) inhibitor. A simplified reverse-phase HPLC method for 3-MT employing electrochemical detection is fully described. This method has a detection limit of 0.1 microgram/g brain wet weight and is sensitive enough to detect 3-MT in individual brain regions after rats have been pretreated with an MAO inhibitor. Administration of tranylcypromine (TCP, 10 mg/kg) and L-3,4-dihydroxyphenylalanine (L-DOPA) (10-50 mg/kg) produced a dose-dependent linear increase in 3-MT concentrations in the dopaminergic brain regions n. caudatus (r = 0.95; p less than 0.01) and n. accumbens (r = 0.96; p less than 0.01). This treatment also produced a dose-dependent increase in behavioural activity in rats (r = 0.88; p less than 0.01). Furthermore, a good correlation was found between the activity responses of individual rats and the accumulation of 3-MT after TCP/L-DOPA in both n. caudatus (r = 0.76; p less than 0.01) and n. accumbens (r = 0.84; p less than 0.01). These data describe a simple and sensitive HPLC analysis technique for 3-MT and demonstrate that following administration of an MAO inhibitor this metabolite may provide a useful monitor of central dopamine function.     

A method for the assay of conjugated 3,4-dihydroxyphenylglycol,a major noradrenaline metabolite in the rat brain

We present the first published procedure for the measurement of endogenous conjugated 3,4-dihydroxyphenylglycol (DOPEG) in the rat brain. Conjugated DOPEG is estimated from brain extracts after enzymic hydrolysis, isolation of hydrolysed DOPEG on alumina, methylation of DOPEG to 3-methoxy-4-hydroxyphenylglycol (MOPEG) and gas chromatographic quantification of MOPEG. The level of conjugated DOPEG in the CNS of rats (65.7 +/- 0.7 ng/g whole brain tissue corrected for recovery) almost equals the level of conjugated MOPEG. The sensitivity of the method is about 6 ng/g brain tissue. After inhibition of monoamine oxidase with clorgyline (30 mg/kg) conjugated DOPEG and MOPEG both disappeared from the brain with a half-life of about 1 h. Turnover calculations indicate that conjugated DOPEG and MOPEG are the two major noradrenaline end-metabolites in the rat brain. The method of estimating conjugated DOPEG also allows the measurement of noradrenaline, dopamine and total MOPEG in an extract from one half of a rat brain.     

Possible existence of high molecular precursor proteins for substance P biosynthesis in rabbit spinal ganglia

The presence of precursor protein for substance P (SP) was examined. Sephadex G-75 chromatography of extracts from rabbit spinal ganglia incubated with [³⁵S]methionine gave two radioactive peaks. In the lower molecular weight peak SP was identified by radioimmunoassay, Sephadex G-15 and TLC. When higher molecular weight proteins were incubated with spinal ganglia microsomal preparation and applied to Sephadex G-75, G-15, TLC and HPLC, ³⁵S-labeled SP was identified and characterized as authentic by immunoprecipitation followed by Sephadex G-15. The amount of ³⁵S-labeled SP was reduced by prior heating of ganglia homogenates, addition of N-ethylmaleimide or p-chloromercuribenzoic acid but not by cycloheximide. Characterization of higher molecular weight proteins by Sephadex G-200, gel-permeation chromatography and chromatofocusing revealed that the proteins were of approx. 100,000 and 7000 dalton with isoelectric points of 9.0, 8.4 and 7.8. These results suggest that the processing from a precursor protein to SP may involve several steps and our high molecular weight protein of 7000 dalton may be one of these intermediate precursor peptides for SP.     

Comparison of the substrate specificities of protein phosphatases involved in the regulation of glycogen metabolism in rabbit skeletal muscle.

Muscle extracts were subjected to fractionation with ethanol, chromatography on DEAE-cellulose, precipitation with (NH4)2SO4 and gel filtration on Sephadex G-200. These fractions were assayed for protein phosphatase activities by using the following seven phosphoprotein substrates: phosphorylase a, glycogen synthase b1, glycogen synthase b2, phosphorylase kinase (phosphorylated in either the alpha-subunit or the beta-subunit), histone H1 and histone H2B. Three protein phosphatases with distinctive specificities were resolved by the final gel-filtration step and were termed I, II and III. Protein phosphatase-I, apparent mol.wt. 300000, was an active histone phosphatase, but it accounted for only 10-15% of the glycogen synthase phosphatase-1 and glycogen synthase phosphatase-2 activities and 2-3% of the phosphorylase kinase phosphatase and phosphorylase phosphatase activity recovered from the Sephadex G-200 column. Protein phosphatase-II, apparent mol.wt. 170000, possessed histone phosphatase activity similar to that of protein phosphatase-I. It possessed more than 95% of the activity towards the alpha-subunit of phosphorylase kinase that was recovered from Sephadex G-200. It accounted for 10-15% of the glycogen synthase phosphatase-1 and glycogen synthase phosphatase-2 activity, but less than 5% of the activity against the beta-subunit of phosphorylase kinase and 1-2% of the phosphorylase phosphatase activity recovered from Sephadex G-200. Protein phosphatase-III was the most active histone phosphatase. It possessed 95% of the phosphorylase phosphatase and beta-phosphorylase kinase phosphatase activities, and 75% of the glycogen synthase phosphatase-1 and glycogen synthase phosphatase-2 activities recovered from Sephadex G-200. It accounted for less than 5% of the alpha-phosphorylase kinase phosphatase activity. Protein phosphatase-III was sometimes eluted from Sephadex-G-200 as a species of apparent mol.wt. 75000(termed IIIA), sometimes as a species of mol.wt. 46000(termed IIIB) and sometimes as a mixture of both components. The substrate specificities of protein phosphatases-IIA and -IIB were identical. These findings, taken with the observation that phosphorylase phosphatase, beta-phosphorylase kinase phosphatase, glycogen synthase phosphatase-1 and glycogen synthase phosphatase-2 activities co-purified up to the Sephadex G-200 step, suggest that a single protein phosphatase (protein phosphatase-III) catalyses each of the dephosphorylation reactions that inhibit glycogenolysis or stimulate glycogen synthesis. This contention is further supported by results presented in the following paper [Cohen, P., Nimmo, G.A. & Antoniw, J.F. (1977) Biochem. J. 1628 435-444] which describes a heat-stable protein that is a specific inhibitor of protein phosphatase-III.     

Purification and Some Properties of Acidocin 8912, a Novel Bacteriocin Produced by Lactobacillus acidophilus TK8912

Acidocin 8912, a bacteriocin produced by Lactobacillus acidophilus TK8912, was purified by ammonium sulfate fractionation and successive chromatographies on CM-cellulose, Sephadex G-50, Sephadex G-25, and reversed-phase HPLC on Aquapore RP-300. The purified acidocin 8912 migrated as a single band on SDS–PAGE. The molecular weight was estimated to be 5200 by SDS–PAGE, and 5400 by HPLC gel filtration on TSKgel G3000PW_XL. Both the amino acid composition and the N-terminal amino acid sequence analysis indicated that acidocin 8912 was a peptide composed of presumably 50 amino acids containing a Lys residue at the N-terminus. The purified acidocin 8912 showed a bactericidal effect on sensitive cells but not a bacteriolytic effect.     

Isolation and Properties of a New Kallikrein Inhibitor from Tityus serrulatus Venom

The kallikrein inhibitor-peptide content of Tityus serrulatus scorpion crude venom was purified by Sephadex G-50 and Sephadex G-25 fine gel filtration chromatographies, followed by two steps of reverse-phase column on HPLC. The isolated inhibitor peptide was homogeneous in its N-terminal and partial amino acid sequence, showing a molecular weight of 4.489 Da by mass spectrometry and amino acid analysis. The peptide was tested with rat plasma and urine kallikrein, which resulting in an inhibition with similar afinity to both enzymes, showing an IC₅₀ of 14.3 μM after 13 and 8 min, respectively, using kininogen as substrate on the isolated guinea-pig ileum bioassay. The porcine pancreatic kallikrein showed after 10 min an IC₅₀ value of 12.6 μM with H-D-Val-Leu-Arg-pNA HCl as substrate. In addition, the isolated peptide significantly inhibited porcine pancreatic kallikrein with values in the range of apparent or absolute calculated peptide K _i = 2.5 μM. The inhibitor was heat resistant and stable at pH values less than 5.     

Reversed-phase liquid chromatography with amperometric detection of lipophilic dopamine analogues and determination of brain and serum concentrations after sample clean-up on small sephadex G-10 columns

The liquid chromatographic determination of N-alkylated analogues of dopamine is described. The retention and separation of these compounds, ranging from dopamine to N,N-dibutyl-dopamine, was studied on four bonded-phase columns, of which Nucleosil 5 C₁, was chosen for routine use. The compounds were detected by a rotating disc amperometric detector. Samples of rat brain and serum were taken through a clean-up step on small Sephadex G-10 columns from which the dopamine analogues eluted in the same fraction as dopamine. The overall recovery was 70–90% from brain tissue and 60–70% from serum or plasma. The limit of detection for the catechol-containing compounds in tissue was 40–100 pg, for O-methylated ones 100–200 pg. The method is applied to the determination of dopamine analogues in rat brain after peripheral administration.     

DT-n-PROPYLACETATE AND GABA DEGRADATION. PREFERENTIAL INHIBITION OF SUCCINIC SEMIALDEHYDE DEHYDROGENASE AND INDIRECT INHIBITION OF GABA-TRANSAMINASE

The kinetic constants for 4-aminobutyrate: 2-oxoglutarate aminotransferase (GABA-trans-aminase) and succinate-semialdehyde: NAD⁺ oxidoreductase (SSA-DH) have been determined using rat brain homogenate. The Michaelis constants for GABA-T at saturated substrate concentrations were calculated to be K_gaba= 1.5 mM, K_2-OG= 0.25 mM, K_GLU= 620 μM, and K_SSA= 87 μm. The V_max for the reaction using GABA and 2-oxoglutarate (2-OG) as substrates (forward reaction) was found to be 35.2 μmol/ These results indicate that MOPEG is a measure for changes in central noradrenaline turnover and that drugs affect MOPEG in the brain and spinal cord similarly. Fractional rate constants of MOPEG in the rat brain and spinal cord were estimated with the exponential decline curves produced by treatment with pargyline. Turnover rates of 193 pmol/gh and 167 pmol/g These results indicate that MOPEG is a measure for changes in central noradrenaline turnover and that drugs affect MOPEG in the brain and spinal cord similarly. Fractional rate constants of MOPEG in the rat brain and spinal cord were estimated with the exponential decline curves produced by treatment with pargyline. Turnover rates of 193 pmol/g/h and 167 pmol/g/h in the brain and spinal cord respectively were calculated. The kinetics of GABA-T have been shown to be consistent with a Ping Pong Bi Bi mechanism. Substrate inhibition of the forward reaction, through formation of a dead-end complex, was found to occur with 2-OG (K_i 3.3 mM), whereas GABA was found to be a product inhibitor of the reverse reaction (K_i= 0.6 mM). Using the appropriate Haldane relationship, a K_eq of 0.04 for GGBA-T was found, indicating that the reaction was strongly biased towards GABA. For SSA-DH, the K_m of SSA was determined (9.1 μM) and the V_max was 27.5 μmol/ These results indicate that MOPEG is a measure for changes in central noradrenaline turnover and that drugs affect MOPEG in the brain and spinal cord similarly. Fractional rate constants of MOPEG in the rat brain and spinal cord were estimated with the exponential decline curves produced by treatment with pargyline. Turnover rates of 193 pmol/g/h and 167 pmol/g These results indicate that MOPEG is a measure for changes in central noradrenaline turnover and that drugs affect MOPEG in the brain and spinal cord similarly. Fractional rate constants of MOPEG in the rat brain and spinal cord were estimated with the exponential decline curves produced by treatment with pargyline. Turnover rates of 193 pmol/g/h and 167 pmol/g/h in the brain and spinal cord respectively were calculated. h. The effect of di-n-propylacetate (DPA) on both GABA-T and SSA-DH was measured. DPA inhibited SSA-DH competitively with respect to SSA, giving a K_i of 0.5 mM. GABA-T was only slightly inhibited. The K_i of DPA for the forward reaction was 23.2 mM with respect to GABA, which was 40-50 times higher than that for SSA-DH. For the reverse reaction the K_i of DPA was found to be nearly the same (15.2 mM with respect to Glu and 22.9 mM with respect to SSA). These results suggest that GABA accumulation in the brain, after administration of DPA in vivo, is caused by SSA-DH inhibition. Two mechanisms are indicated by the data. (1) The higher level of SSA, which results from inhibition of SSA-DH, initiates the reverse reaction of GABA-T, thus increasing the level of GABA via conversion of SSA. (2) The degradation of GABA is inhibited by SSA, since SSA has a strong inhibitory effect on the forward reaction, as calculated from the present data.     

Chromatographic and biologic analysis of ME and OVLT LHRH

The luteinizing hormone (LH)-releasing activities a pooled rat organum vasculosum lamina terminalis (OVLT) and median eminence (ME) tissues were evaluated for chromatographic and biologic similarity and compared to those of synthetic decapeptide LH-releasing hormone (LHRH). The LHRH detected in these extracts appeared similar chromatographically (Sephadex G-25) to synthetic LHRH. These extracts, as well as synthetic LHRH, were also capable of stimulating dose dependent gonadotropin release form cultures rat gonadotrophs. These findings suggest a physiological role of the LHRH present in the rat OVLT in the control of gonadotropin secretion.     

Tryptophan over-producing cell suspensions of Catharanthus roseus (L) G. Don and their up-scaling in stirred tank bioreactor: detection of a phenolic compound with antioxidant potential

Five cell suspension lines of Catharanthus roseus resistant to 5-methyl tryptophan (5-MT; an analogue of tryptophan) were selected and characterized for growth, free tryptophan content and terpenoid indole alkaloid accumulation. These lines showed differential tolerance to analogue-induced growth inhibition by 30 to 70 mg/l 5-MT supplementation (LD₅₀?=?7–15 mg/l). Lines P40, D40, N30, D50 and P70 recorded growth indices (i.e. percent increment over the initial inoculum weight) of 840.9, 765.0, 643.9, 585.7 and 356.5 in the absence and, 656.7, 573.9, 705.8, 489.0 and 236.0 in the presence of 5-MT after 40 days of culture, respectively. A corresponding increment in the free tryptophan level ranging from 46.7 to 160.0 μg/g dry weight in the absence and 168.0 to 468.0 μg/g dry weight in the presence was noted in the variant lines. Higher tryptophan accumulation of 368.0 and 468.0 g/g dry weight in lines N30 and P40 in 5-MT presence also resulted in higher alkaloid accumulation (0.65 to 0.90 % dry weight) in them. High-performance liquid chromatography (HPLC) analysis of the crude alkaloid extracts of the selected lines did not show the presence of any pharmaceutically important monomeric or dimeric alkaloids except catharanthine in traces in the N30 line that was also unique in terms of a chlorophyllous green phenotype. The N30 line under optimized up-scaling conditions in a 7-l stirred tank bioreactor using Murashige and Skoog medium containing 2 mg/l α-naphthalene acetic acid and 0.2 mg/l kinetin attained 18-folds biomass accumulation within 8 weeks. Interestingly, the cell biomass yield was enhanced to 30-folds if 30 mg/l 5-MT was added in the bioreactor vessel one week prior to harvest. Crude alkaloid extract of the cells grown in shake flask and this bioreactor batch also showed the formation of yellow-coloured crystals which upon ¹HNMR and ESI-MS analysis indicated a phenolic identity. This crude alkaloid extract of bioreactor-harvested cells containing this compound at 50 μg/ml concentration registered 65.21, 17.75, 97.0, 100 % more total antioxidant capacity, reducing power, total phenolic content, and ferric-reducing antioxidant power, respectively, when compared with that of extracts of cells grown in shake flask cultures. The latter, however, showed 57.47 % better radical scavenging activity (DPPH) than the bioreactor-harvested cells.