Fast atom bombardment and collisional activation mass spectrometry of retinyl phosphate mannose synthesized by liver membranes

Fast atom bombardment (FAB) and collisional activation dissociation (CAD) mass-analysed ion kinetic energy (MIKE) spectra have confirmed the structures of retinyl phosphate (Ret-P), retinyl phosphate mannose (Ret-P-Man) and guanosine 5'-diphospho-D-mannose (GDP-Man). Ret-P-Man was made in vitro while Ret-P and GDP-Man were chemically synthesized. Positive ion FAB mass spectrometry of Ret-P showed an observable short-lived spectrum with a mass ion at m/z 367 [M + H]+, and a major fragment ion at m/z 269 [M + H - H3PO4]+. Negative ion FAB mass spectrometry of Ret-P showed a strong stable spectrum with a parent ion at m/z 365 [M - H]-, a glycerol (G) adduct ion at m/z 457 [M - H + G]- and a dimer ion at m/z 731 [2M - H]-. GDP-Man showed an intense spectrum with parent ion at m/z 604 [M - H]- and cationized species at m/z 626 [M + Na - 2H]- and 648 [M + 2Na - 3H]-. Negative ion FAB mass spectrometry of Ret-P-Man showed a parent ion at m/z 527 [M - H]- and a fragment ion at m/z 259 [C6H12PO9]-. The CAD-MIKE spectra showed structurally significant fragment ions at m/z 442 and 361 for the [M - H]- ion of GDP-Man, and at m/z 509, 406, 364 and 241 for the [M - H]- ion of Ret-P-Man. FAB and CAD-MIKE spectra have been applied successfully to confirm the structure of Ret-P-Man made in vitro from Ret-P and GDP-Man.     

Significance of renal gamma-butyrobetaine hydroxylase for carnitine biosynthesis in man

Carnitine biosynthesis was studied in man and rat. Three healthy adult men were given intravenous injections of 1 mCi of [methyl-3H]epsilon-N-trimethyl-L-lysine, a precursor of carnitine. Labeled metabolites of this compound were monitored in serum and urine at 2, 6, 12, 24, and 48 h. At least nine radioactive metabolites were detected. For each collecton period, the specific activity of urinary carnitine exceeded the average serum specific activity. In man, the amount of labeled carnitine in urine was 2 to 8 times greater than labeled gamma-butyrobetaine (the immediate precursor of carnitine). In similar experiments in rats (intravenous injection of 0.1 mCi of [methyl-3H]epsilon-N-trimethyl-L-lysine), the specific activity of carnitine in urine was always lower than the corresponding average specific activity in serum. Between 0 and 2 h after administration of labeled precursor, the animals excreted large amounts of labeled gamma-butyrobetaine but little labeled carnitine. Significant gamma-butyrobetaine, 2-oxoglutarate dioxygenase (EC 1.14.11.1) activity was found in human kidney but this activity was absent in rat kidney. The results indicate that in man and rat the kidney accumulates intravenously administered [methyl-3H]epsilon-N-trimethyl-L-lysine. This compound is metabolized predominantly to gamma-butyrobetaine in rat kidney and to carnitine in human kidney. In both species, the synthesized products are at least partially leaked (either by secretion or by passive diffusion down a concentration gradient) into the renal tubular lumen from which they are either reabsorbed into the circulation for distribution to other tissues or excreted.     

Determination of the platinum drug cis-amminedichloro(2-methylpyridine)platinum(II) in human urine by liquid chromatography-tandem mass spectrometry

A validated stable isotope dilution liquid chromatography-tandem mass spectrometry assay for the novel platinum drug cis-amminedichloro(2-methylpyridine)platinum(II) (ZD0473) in human urine has been developed. This method uses selected reaction monitoring on the transition of m/z 393 [M+NH(4)](+) to m/z 304 [M+NH(4)-NH(3)-2 x H(35)Cl](+) for ZD0473, and m/z 400 [M+NH(4)](+) to m/z 310 [M+NH(4)-NH(3)-H(35)Cl-(2)H(35)Cl](+) for the internal standard [(2)H(7)]ZD0473. Standard curves were prepared over the range from 0.15 to 50 microg/ml. The lower limit of quantitation was 0.2 microg/ml for 100 microl of urine. This simple, rapid, reliable, and sensitive method of quantitation displayed acceptable accuracy and precision over the 3 days of assay validation. A novel platinum adduct was formed during the storage of ZD0473 in human urine. The adduct did not correspond to any of the typical sulfhydryl adducts that have been identified previously for platinum drugs. Formation of the adduct was prevented by the addition of 50% (w/v) sodium chloride to the urine. The assay can be used to quantify intact ZD0473 in the urine of subjects dosed with this new platinum drug.     

用阳离子交换树脂测定尿三甲基组氨含量

因3-甲基组氨酸(3MH)从肌肉蛋白降解后,不重新参加蛋白质的合成,而从尿中排出,所以把它作为肌肉蛋白质分解的可靠指标。本文以Radha和Bess-man在1982年建立,Fitch等在1986年改进的阳离子交换树脂法。以大部国产试剂,对柱长、内径、pH等作了适当的改进,得到了满意的测定3MH的结果。从0.15到1.0μMol的三点标准曲线与Fitch等的相似,此法重复性好,回收率高,测定范围广,是较为简单,可推行的方法。用此法测定了烧伤和正常鼠尿3MH的含量,烧伤组14.5%Cv和对照组13.0%Cv,显示大鼠每天尿中排出的3MH含量比较稳定,说明烧伤和正常鼠肌肉蛋白质的分解比较恒定。烧伤组鼠尿3MH的每天排出量高于正常组35%以上,表明烧伤鼠肌肉蛋白质的分解速率高于对照组。     

Norepinephrine Metabolism in Man Using Deuterium Labelling: Origin of 4-Hydroxy-3-Methoxymandelic Acid

A double isotope labelling technique was used to simultaneously determine the in vivo turnover rates of 4-hydroxy-3-methoxyphenylglycol (HMPG) and 4-hydroxy-3-methoxymandelic acid (HMMA, VMA) and the rate of HMPG oxidation to HMMA. Six healthy men were given intravenous injections of [2H3]HMPG and [2H6]HMMA and their plasma and urine samples analysed by gas chromatography--mass spectrometry (GC/MS) for the protium and deuterium species. HMPG and HMMA production rates were calculated by isotope dilution. The rate of HMPG oxidation to HMMA was obtained from the fraction of [2H3]HMPG recovered as [2H3]HMMA. The results showed that the entire production of HMMA, 1.11 +/- 0.21 mumol/h (mean +/- SE), could be accounted for by oxidation of HMPG, 1.49 +/- 0.31 mumol/h. In another experiment designed to avoid expansion of the HMPG body pool, a tracer dose of [14C]HMPG was given to the same subjects. The levels of [14C]HMPG and [14C]HMMA were measured in urine after extraction and separation by thin layer chromatography. Urinary excretion of endogenous HMPG and HMMA was determined by GC/MS. The results showed that the endogenous HMMA fraction of the total HMPG and HMMA urinary excretion rate, 0.57 +/- 0.04, was the same as the fraction of [14C]HMPG oxidized to [14C]HMMA, 0.62 +/- 0.01. Thus, HMPG is the main intermediate in the metabolic conversion of norepinephrine and epinephrine to HMMA in man.     

Trace levels of pyrroloquinoline quinone in human and rat samples detected by gas chromatography/mass spectrometry.

A detailed procedure for the assay of free pyrroloquinoline quinone (PQQ) in human and rat samples by gas chromatography/mass spectrometry (GC/MS) has been established with stable-isotopic PQQ as internal standard. PQQ was extracted from the samples, after addition of the internal standard, with butanol under acid conditions and with Sep-Pak C18 cartridges. After derivatization of PQQ with phenyltrimethylammonium hydroxide, molecular peaks at m/z 448 and 462 were used for detection of PQQ and [U-13C]PQQ by selected ion monitoring, respectively. Trace amounts of free PQQ were detected in eight organs, plasma and urine of the human, and in three organs of the rat. The PQQ level was highest in the human spleen (5.9 +/- 3.4 ng/g tissue, followed by the pancreas and lung, and it was below detection limits for human brain and heart. Trace levels of PQQ were also found in rat small intestine, liver and testis. Our data are far below those measured by the redox cycling method of Gallop's group for human plasma, adrenal and urine.     

Use of chemical ionization for GC–MS metabolite profiling

Metabolite profiling is commonly performed by GC–MS of methoximated trimethylsilyl derivatives. The popularity of this technique owes much to the robust, library searchable spectra produced by electron ionization (EI). However, due to extensive fragmentation, EI spectra of trimethylsilyl derivatives are commonly dominated by trimethylsilyl fragments (e.g. m/z 73 and 147) and higher m/z fragment ions with structural information are at low abundance. Consequently different metabolites can have similar EI spectra, and this presents problems for identification of “unknowns” and the detection and deconvolution of overlapping peaks. The aim of this work is to explore use of positive chemical ionization (CI) as an adjunct to EI for GC–MS metabolite profiling. Two reagent gases differing in proton affinity (CH₄ and NH₃) were used to analyse 111 metabolite standards and extracts from plant samples. NH₃-CI mass spectra were simple and generally dominated by [MH]⁺ and/or the adduct [M+NH₄]⁺. For the 111 metabolite standards, m/z 73 and 147 were less than 3% of basepeak in NH₃-CI and less than 30% of basepeak in CH₄-CI. With CH₄-CI, [MH]⁺ was generally present but at lower relative abundance than for NH₃-CI. CH₄-CI spectra were commonly dominated by losses of CH₄ [M+1-16]⁺, 1–3 TMSOH [M+1-nx90]⁺, and combinations of CH₄ and TMSOH losses [M+1-nx90-16]⁺. CH₄-CI and NH₃-CI mass spectra are presented for 111 common metabolites, and CI is used with real samples to help identify overlapping peaks and aid identification via determination of the pseudomolecular ion with NH₃-CI and structural information with CH₄-CI.     

Identification of a new kinin in human urine

The types of kinins excreted in fresh urine of dogs, rats, and humans were compared. Urinary kinins were separated by reverse-phase (C18) high performance liquid chromatography and quantitated by radioimmunoassay using an antibody directed against the COOH-terminal region of the peptide. Kinins were found in the following proportions: 53 +/- 3% bradykinin, 23 +/- 4% Lys-bradykinin, and 13 +/- 7% des-Arg1-bradykinin in dog urine; 67 +/- 6% bradykinin, 6 +/- 3% Lys-bradykinin, and 10 +/- 3% des-Arg1-bradykinin in rat urine; and 12 +/- 4% bradykinin, 30 +/- 3% Lys-bradykinin, 2 +/- 1% des-Arg1-bradykinin, and 41 +/- 3% unknown kinin in human urine. The unknown kinin was purified from a pool of human urine. Amino acid sequencing revealed a structure similar to Lys-bradykinin except that proline in position 4 was replaced by alanine ([Ala3]Lys-bradykinin). Synthetic and endogenous [Ala3]Lys-bradykinins had similar high performance liquid chromotography elution volumes and both had vasodilator activity and contracted the rat uterus. Human urinary kallikrein incubated with semipurified human low molecular weight kininogen released 76% of the total kinins as Lys-bradykinin, 7% as bradykinin, and 17% as [Ala3]Lys-bradykinin. In contrast, rat urinary kallikrein released 86% bradykinin, 18% Lys-bradykinin, and negligible amounts of [Ala3]Lys-bradykinin. The study revealed the presence of a new kinin, [Ala3]Lys-bradykinin, in human urine and it also proves that the types of kinins generated intrarenally are species-dependent.     

Determination of 5-methyltetrahydrofolate (13C-labeled and unlabeled) in human plasma and urine by combined liquid chromatography mass spectrometry

The association of folates with the prevention of neural tube defects and reduced risk of other chronic diseases has stimulated interest in the development of techniques for the study of their bioavailability in humans. Stable isotope protocols differentiate between oral and/or intravenous test doses of folate and natural levels of folate already present in the body. An liquid chromatography/mass spectrometry (LC/MS) procedure is described that has been validated for the determination of [13C]5-methyltetrahydropteroyl monoglutamic acid ([13C]5-CH3H4PteGlu) in plasma and urine, following oral dosing of volunteers with different labeled folates. Folate binding protein affinity columns were used for sample purification prior to LC/MS determination. Chromatographic separation was achieved using a Superspher 100RP18 (4 microm) column and mobile phase of 0.1 mol/L acetic acid (pH 3.3):acetonitrile (90:10; 250 microL/min). Selected ion monitoring was conducted on the [M-H](-) ion: m/z 458 and 459 for analyzing 5-CH3H4PteGlu; m/z 464 [M+6-H](-) to determine 5-CH3H4PteGlu derived from the label dose; m/z 444 for analysis of 2H4PteGlu internal standard, and m/z 446 and 478 to confirm that there was no direct absorption of unmetabolized compounds. Calibration was linear over the range 0-9 x 10(-9) mol/L; the limits of detection and quantification were 0.2 x 10(-9) and 0.55 x 10(-9) mol/L, respectively. The mean coefficient of variation of the ratios (m/z 463/458) was 7.4%. The method has potential applications for other key folates involved in one-carbon metabolism.     

Glutamic acid residues of bacteriorhodopsin at the extracellular surface as determinants for conformation and dynamics as revealed by site-directed solid-state 13C NMR

We recorded (13)C NMR spectra of [3-(13)C]Ala- and [1-(13)C]Val-labeled bacteriorhodopsin (bR) and a variety of its mutants, E9Q, E74Q, E194Q/E204Q (2Glu), E9Q/E194Q/E204Q (3Glu), and E9Q/E74Q/E194Q/E204Q (4Glu), to clarify contributions of the extracellular (EC) Glu residues to the conformation and dynamics of bR. Replacement of Glu-9 or Glu-74 and Glu-194/204 at the EC surface by glutamine(s) induced significant conformational changes in the cytoplasmic (CP) surface structure. These changes occurred in the C-terminal alpha-helix and loops, and also those of the EC surface, as viewed from (13)C NMR spectra of [3-(13)C]Ala- and [1-(13)C]Val-labeled proteins. Additional conformational changes in the transmembrane alpha-helices were induced as modified retinal-protein interactions for multiple mutants involving the E194Q/E204Q pair. Significant dynamic changes were induced for the triple or quadruple mutants, as shown by broadened (13)C NMR peaks of [1-(13)C]Val-labeled proteins. These changes were due to acquired global fluctuation motions of the order of 10(-4)-10(-5) s as a result of disorganized trimeric form. In such mutants (13)C NMR signals from Val residues of [1-(13)C]Val-labeled triple and quadruple mutants near the CP and EC surfaces (including 8.7-A depth from the surface) were substantially suppressed, as shown by comparative (13)C NMR studies with and without 40 micro M Mn(2+) ion. We conclude that these Glu residues at the EC surface play an important role in maintaining the native secondary structure of bR in the purple membrane.     

Trifluoroacetic anhydride-catalyzed nitration of toluene as an approach to the specific analysis of nitrate by gas chromatography-mass spectrometry.

The nitration of aromatic compounds by electrophilic substitution is often utilized in analyses of nitrate concentrations in physiological samples by gas chromatographic methods. Problems associated with the use of concentrated sulfuric acid, which is normally used to catalyze this reaction, led us to investigate an alternative method. We describe here a facile GC/MS assay for nitrate in plasma or urine samples which takes advantage of the ability of trifluoroacetic anhydride (TFAA) to catalyze the nitration of aromatics. Toluene, utilized as both reaction solvent and electrophile, was shown to react with nitrate in the presence of TFAA to quantitatively produce the three nitrotoluene isomers (ratio o-:m:p-, approx 57:3:40). Following the incorporation of 15N-labeled nitrate as internal standard, nitrotoluene was quantified using GC/MS by analysis of the selected the ion pairs m/z 120 and 121 (M+ -OH) for the o-isomer or m/z 137 and 138 (molecular ion, M+) for the p-isomer. The limit of detection for nitrate after TFAA-catalyzed conversion to nitrotoluene was less than 100 fmol on column (s/n; 40:1). The TFAA-based GC/MS assay was compared with that utilizing the usual catalyst, concentrated sulfuric acid. With the exception of samples containing nitroarginine analogues, good correlation was found for urine or plasma samples analyzed using either a standard sulfuric acid-catalyzed method or the TFAA-catalyzed procedure. Nitroarginine analogues, which can be present in samples following their use as nitric oxide synthase inhibitors, did not decompose under the conditions of the TFAA-catalyzed assay and, hence, do not give rise to significant interference with nitrate analysis in this procedure. In contrast, catalytic sulfuric acid caused nitroarginine analogues to decompose (essentially quantitatively) and cause spuriously high nitrate levels in samples. The use of TFAA as a catalyst for the nitration of toluene enables a facile and sensitive GC/MS analysis for nitrate which offers improved safety and sample integrity.     

Biosynthesis of methanopterin

The biosynthetic pathway for the generation of the methylated pterin in methanopterins was determined for the methanogenic bacteria Methanococcus volta and Methanobacterium formicicum. Extracts of M. volta were found to readily cleave L-7,8-dihydroneopterin to 7,8-dihydro-6-(hydroxymethyl)pterin, which was confirmed to be a precursor of the pterin portion of the methanopterin. [methylene-2H]-6-(Hydroxymethyl)pterin was incorporated into methanopterin by growing cells of M. volta to an extent of 30%. Both the C-11 and C-12 methyl groups of methanopterin originate from [methyl-2H3]methionine, as confirmed by the incorporation of two C2H3 groups into 6-ethyl-7-methylpterin, a pterin-containing fragment derived from methanopterin. Cells grown in the presence of [methylene-2H]-6-(hydroxymethyl)pterin, [ethyl-2H4]-6-[1 (RS)-hydroxyethyl]pterin, [methyl-2H3]-6- (hydroxymethyl)-7-methylpterin, [ethyl-2H4, methyl-2H3]-6-[1 (RS)-hydroxyethyl]-7-methylpterin, and [1-ethyl-3H]-6-[1 (RS)-hydroxyethyl]-7-methylpterin showed that only the non-7-methylated pterins were incorporated into methanopterin. Cells extracts of M. formicicum readily condensed synthetic [methylene-3H]-7,8-H2-6-(hydroxymethyl)pterin-PP with methaniline to generate demethylated methanopterin, which is then methylated to methanopterin by the cell extract in the presence of S-adenosylmethionine. These observations indicate that the pterin portion of methanopterin is biosynthetically derived from 7,8-H2-6-(hydroxymethyl)pterin, which is coupled to methaniline by a pathway analogous to the biosynthesis of folic acid. This pathway for the biosynthesis of methanopterin represents the first example of the modification of the specificity of a coenzyme through a methylation reaction.     

Development of a method for determination of the malondialdehyde-deoxyguanosine adduct in urine using liquid chromatography-tandem mass spectrometry

A procedure is described for the quantification of the major malondialdehyde deoxyguanosine adduct, pyrimido[1,2-alpha]purin-10(3H)-one-deoxyribose (M(1)GdR) in urine. M(1)GdR is isolated from urine by a combination of C(18) solid-phase extraction and immunoaffinity chromatography. Sodium borohydride treatment reduces M(1)GdR to the 5,6-dihydro derivative, which is quantified by liquid chromatography-mass spectrometry. Authentic [7,9-15N,8-13C]M(1)GdR is added to urine as an internal standard. A detection limit of 50 fmol M(1)GdR/ml urine is achieved starting with 5 ml of urine. Analysis of urine samples from control rats or rats treated with CCl(4) indicates that the levels of M(1)GdR are below the detection limit of the assay. This method is easily adaptable to the analysis of M(1)GdR in DNA samples or biological fluids.     

Insulin-like growth factor I (IGF-I) production and the presence of IGF-I receptors in rat medullary thyroid carcinoma cell line 6-23 (clone 6)

To clarify whether insulin-like growth factor I (IGF-I) is an autocrine growth factor of rat medullary thyroid carcinoma (MTC) cell line, 6-23 (clone 6), IGF-I binding to MTC cell membranes, IGF-I levels in the conditioned culture medium of MTC cells and the effects of IGF-I on methyl-[3H]thymidine incorporation to MTC cells were examined. Scatchard analysis of saturation binding studies revealed the association constant and the maximal binding capacity were 1.0 x 10(9) M-1 and 199 fmol/mg of membrane protein, respectively. The binding of [125I]IGF-I to MTC cell membranes was inhibited by unlabeled IGF-I, IGF-II and insulin; the relative potencies were IGF-I greater than IGF-II much greater than insulin, suggesting the presence of type I IGF receptors in MTC cells. IGF-I levels in the conditioned culture medium of MTC cells were 120 +/- 3 pM (mean + SE). IGF-I (10(-10) to 10(-8) M) dose-dependently stimulated methyl-[3H]thymidine incorporation to MTC cells. These findings suggest a possible role of IGF-I as an autocrine growth factor for MTC cells.     

Metabolism of 1-methyladenosine

1-[methyl-8-14C] Adenosine was synthesized and its metabolic fate was determined in intact rat. It was found that approximately 57% of 1-[methyl-8-14C] adenosine administered iv was excreted unchanged in the urine and 33% of the excreted radioactivity in the urine was associated with the major metabolite 1-methyl-hypoxanthine and about 4.5% was associated with 1-methylinosine. Very little adenosine or N6-methyladenosine was formed. It is concluded that 1-methyladenosine is initially deaminated by adenosine deaminase to 1-methylinosine which is then cleaved by nucleoside phosphorylase to 1-methylhypoxanthine.     

Determination of fexofenadine in human plasma and urine by liquid chromatography-mass spectrometry

A sensitive method was developed to determine fexofenadine in human plasma and urine by HPLC-electrospray mass spectrometry with MDL 026042 as internal standard. Extraction was carried out on C18 solid-phase extraction cartridges. The mobile phases used for HPLC were: (A) 12 mM ammonium acetate in water and (B) acetonitrile. Chromatographic separation was achieved on a LUNA CN column (10 cm x 2.0 mm I.D., particle size 3 microm) using a linear gradient from 40% B to 60% B in 10 min. The mass spectrometer was operated in the selected ion monitoring mode using the respective MH+ ions, m/z 502.3 for fexofenadine and m/z 530.3 for the internal standard. The limit of quantification achieved with this method was 0.5 ng/ml in plasma and 1.0 ng in 50 microl of urine. The method described was successfully applied to the determination of fexofenadine in human plasma and urine in pharmacokinetic studies.     

Zinc potentiation of androgen receptor binding to nuclei in vitro

Zn2+ potentiates binding of the 4.5S [3H]dihydrotestosterone-receptor complex to isolated rat prostate Dunning tumor nuclei in vitro when assayed in the presence of 300 microM ZnCl2, 3 mM MgCl2, 0.25 M sucrose, 5 mM mercaptoethanol, 0.15 M KCl, and 50 mM tris(hydroxymethyl)aminomethane, pH 7.5. In the presence of 5 mM mercaptoethanol, the concentration of 50 microM total Zn2+ required to promote half-maximal receptor binding to nuclei corresponds to a free Zn2+ concentration of 50 nM. The receptor-nuclear interaction appears to be selective for Zn2+; other divalent cations when added at a concentration of 1 mM to a buffer containing 5 mM mercaptoethanol are less effective (Ni2+) or have essentially no effect (Ca2+, Mg2+, Mn2+, Co2+, Cu2+, and Cd2+). Zn2+ does not alter the sedimentation rate of the 4.5S [3H]dihydrotestosterone receptor in the presence of mercaptoethanol; however, in the absence of mercaptoethanol, Zn2+ causes the receptor to aggregate. Zn2+-dependent nuclear binding of the 4.5S [3H]dihydrotestosterone receptor is saturable at 1.4 X 10(-13) mol of receptor sites/mg of DNA, corresponding to approximately 1150 sites/nucleus. In the presence of excess nuclei, up to 60% of added receptor is nuclear bound. An apparent binding constant for the receptor-nuclear interaction of 10(13) M-1 was approximated. Pyridoxal 5'-phosphate (less than or equal to 10 mM), but not 0.4 M KCl, inhibits Zn2+-dependent nuclear binding of the [3H]dihydrotestosterone receptor. Up to 66% of nuclear-bound receptor can be extracted in buffer containing 3 mM ethylenediaminetetraacetic acid plus either 0.4 M KCl or 10 mM pyridoxal 5'-phosphate.(ABSTRACT TRUNCATED AT 250 WORDS)     

Involvement of Protein Kinase C in γ-Aminobutyric Acid Release from Xenopus Oocytes Injected with Rat Brain mRNA

Abstract: Involvement of protein kinase C (PKC) in the release of γ-aminobutyric acid (GABA) was examined in Xenopus laevis oocytes injected with mRNA from rat cerebellum, as compared with findings in slices of rat cerebellum. The mRNA-injected oocytes preloaded with [³H]GABA showed spontaneous release of [³H]GABA, ∼0.5% of GABA content per 1 min. Stimulation with either Ca²⁺ ionophore (A23187) or a high K⁺ concentration increased the release of [³H]GABA from slices of rat deep cerebellar nucleus and mRNA-injected oocytes but not from noninjected and water-injected oocytes. 12- O -Tetradecanoylphorbol 13-acetate (10–300 n M ) but not 4α-phorbol 12,13-didecanoate (300 n M ) potentiated the A23187-stimulated release of [³H]GABA from slices and from mRNA-injected oocytes, in a concentration-dependent manner. Thus, machinery associated with release processes of GABA can be expressed in oocytes by injecting rat cerebellar mRNA, and PKC participates in GABA release from the functionally expressed GABAergic nerve terminals.     

Use of chemical ionization for GC–MS metabolite profiling

Metabolite profiling is commonly performed by GC–MS of methoximated trimethylsilyl derivatives. The popularity of this technique owes much to the robust, library searchable spectra produced by electron ionization (EI). However, due to extensive fragmentation, EI spectra of trimethylsilyl derivatives are commonly dominated by trimethylsilyl fragments (e.g. m/z 73 and 147) and higher m/z fragment ions with structural information are at low abundance. Consequently different metabolites can have similar EI spectra, and this presents problems for identification of “unknowns” and the detection and deconvolution of overlapping peaks. The aim of this work is to explore use of positive chemical ionization (CI) as an adjunct to EI for GC–MS metabolite profiling. Two reagent gases differing in proton affinity (CH₄ and NH₃) were used to analyse 111 metabolite standards and extracts from plant samples. NH₃-CI mass spectra were simple and generally dominated by [MH]⁺ and/or the adduct [M+NH₄]⁺. For the 111 metabolite standards, m/z 73 and 147 were less than 3% of basepeak in NH₃-CI and less than 30% of basepeak in CH₄-CI. With CH₄-CI, [MH]⁺ was generally present but at lower relative abundance than for NH₃-CI. CH₄-CI spectra were commonly dominated by losses of CH₄ [M+1-16]⁺, 1–3 TMSOH [M+1-nx90]⁺, and combinations of CH₄ and TMSOH losses [M+1-nx90-16]⁺. CH₄-CI and NH₃-CI mass spectra are presented for 111 common metabolites, and CI is used with real samples to help identify overlapping peaks and aid identification via determination of the pseudomolecular ion with NH₃-CI and structural information with CH₄-CI.