Identification of the para-isomer of tyramine in rat brain

By means of thin layer chromatography and mass spectrometry of dansylated tissue extracts tyramine has been identified in different regions of the rat CNS. The isomer present is the $\text{para}$-tyramine; no $\text{ortho}$- or $\text{meta}$-tyramine could be found.     

Evidence for 6-keto-PGF1α in adrenal cortex of the rat and effects of 6-keto-PGF1α and PGI2 on adrenal cAMP levels and steroidogenesis

Both intact cortical tissue and isolated cortical cells from the adrenal gland of the rat were analyzed for 6-keto-PGF_1α, the hydrolysis metabolite of PGI₂, using high-performance liquid chromatography and gas chromatography-mass spectrometry. 6-Keto-PGF_1α was present in both incubations of intact tissue and isolated cells of the adrenal cortex, at higher concentrations than either PGF_2α or PGE₂. Thus, the cortex does not depend upon vascular components for the synthesis of the PGI₂ metabolite. Studies $\text{in vitro}$, using isolated cortical cells exposed to 6-keto-PGF_1α (10^?6-10^?4M), show that this PG does not alter cAMP levels or steroidogenesis. Cells exposed to PGI₂ (10^?6-10^?4M), however, show a concentration-dependent increase of up to 4-fold in the levels of cAMP without altering corticosterone production. ACTH (5–200 μU/ml) increased cAMP levels up to 14-fold, and corticosterone levels up to 6-fold, in isolated cells. ACTH plus PGI₂ produced an additive increase in levels of cAMP, however, the steroidogenic response was equal to that elicited by ACTH alone. Adrenal glands of the rat perfused $\text{in situ}$ with PGI₂ showed a small decrease in corticosterone production, whereas ACTH greatly stimulated steroid release. Thus, while 6-keto-PGF_1α is present in the rat adrenal cortex, its precursor, PGI₂, is not a steroidogenic agent in this tissue although it does stimulate the accumulation of cAMP.     

Assay of liver microsomal cholesterol 7 alpha-hydroxylase using deuterated carrier and gas chromatography-mass spectrometry

The activity of cholesterol 7α-hydroxylase in rat liver microsomes was assayed by measuring the mass of 5-cholestene-3β, 7α-diol formed from endogenous cholesterol under standardized incubation conditions. After termination of incubations, a known amount of 5-[24,25,7β-²H₃]cholestene-3β,7α-diol was added. A chloroform extract of the incubation mixture was subjected to thin layer chromatography and the fraction containing 5-cholestene-3β,7α-diol was converted into trimethylsilyl ether. The trimethylsilyl ether was subjected to combined gas chromatography-mass spectrometry and the amount of unlabeled 5-cholestene-3β,7α-diol in the mixture was calculated from the ratio between the relative intensitics of the peaks at $\text{m}\text{e}$ 456 (M-90) and $\text{m}\text{e}$ 459 [M-(90 + 3)]. The precision of the method was ±2.2% (SD). The results with this method of assay of cholesterol 7α-hydroxylase were compared with those obtained with a method based on conversion of a trace amount of added [4-¹⁴C]cholesterol into 5-cholestene-3β,7α-diol.     

m-Octopamine as a substrate for monoamine oxidase.

$\text{m}$-Octopamine was characterized as substrate for monoamine oxidase (MAO) in rat brain and liver mitochondria. The $\text{K}$m and $\text{V}$max values of the brain enzyme were 735 μM and 32.5 nmoles/mg protein/30 min, and those of the liver enzyme 351 μM and 125 nmoles/mg protein/30 min, respectively. The inhibition experiments with clorgyline and deprenyl showed that $\text{m}$-octopamine was a common substrate for type A and type B MAO, though a major part of the activity was due to type A enzyme.     

Confirmation of an unexpected brain O-methylation pattern for the dopamine-derived tetrahydroisoquinoline,salsolinol

Using high resolution capillary gas chromatography, we have unequivocally separated two possible (6- and 7-)mono-O-methylated tetrahydroisoquinoline metabolites in rat brain after acute intraventricular administration of salsolinol, a cyclized dopamine/acetaldehyde derivative. 7-O-Methylsalsolinol (salsoline) constituted 94–98% of the two isomers in five brain regions examined. These results confirm the report by Bail $\text{et}$$\text{al}$. that salsolinol is largely O-methylated $\text{in}$$\text{vivo}$ (presumably by brain catechol-O-methyl transferase) on the hydroxyl situated “para” in the parent dopamine molecule. In comparison, dopamine itself, administered intraventricularly to pargyline-pretreated rats, was O-methylated exclusively on the “meta” hydroxyl group.     

Femtomole level of analysis of biogenic amines and amino acids using functional group mass spectrometry

A general method for the determination of compounds possessing either the primary amine structure, R-CH₂-NH₂ (I), or the α-amino acid structure, RCHNH₂COOH (II), has been devised using gas chromatography and mass spectrometry. Trimethylsilyl derivatives of the biogenic amines (phenylethylamines, indoleethylamines, or Ω-amino acids) produce an intense ion at $\text{m}\text{e}$ 174 upon fragmentation; TMS derivatives of α-amino acids produce an ion at $\text{m}\text{e}$ 218. For maximum sensitivity, chromatograms were obtained with the mass spectrometer tuned to detect a single ion fragment characteristic of a group of structurally related compounds (i.e., functional group GC-MS). At $\text{m}\text{e}$ 174 up to 14 compounds of Type I, including glycine, γ-aminobutyric acid, dopamine, and 5-hydroxytryptamine, could be determined in a single analysis. Detection limits range from 10–100 femtomoles (10^?15 moles). At $\text{m}\text{e}$ 218, eight compounds of Type II, including isoleucine, phenylalanine, tyrosine, and DOPA could be determined. This technique has been applied to the assay of these compounds in extracts containing 0.1 mg mouse brain or abdominal ganglia of the marine molluse, Aplysia californica.     

Biosynthesis and excretion of meta and para tyramine in the rat

$\text{Meta}$ and $\text{para}$ tyramine, after conversion to their bis dansyl derivatives, have been identified and quantitated by mass spectrometry in rat urine. The daily $\text{para}$ to $\text{meta}$ excretion ratio of 1.69 ± 0.10 is quite constant which suggests an endogenous origin for these amines. Following intraperitoneal injection of [¹⁴C] labelled dopa and dopamine more $\text{meta}$ than $\text{para}$ tyramine is excreted; after i.p. injection of $\text{para}$ tyrosine only small amounts of $\text{para}$ tyramine could be identified. This implies that some $\text{para}$ tyramine is synthesised by a route other than dehydroxylation or decarboxylation.     

Gas chromatography and mass spectrometry of some trimethylsilyl derivatives of urinary glucuronides

Guidelines are presented to aid the recognition and interpretation of mass spectra for trimethylsilylated (TMS) glucuronides of aromatic acids and substituted phenols. Masses associated with fragmentation of the derivatized glycon include $\text{m}\text{e}$ 465, 464, 449, 392, 375, 359, 333, 331, 319, 305, 257, 243, 217, 204, 169, 147, 143, 129, 117, 103, 95, and 93, with $\text{m}\text{e}$ 375 usually being one of the most abundant masses in the spectrum. Fragmentation of the conjugate after electron impact led to the appearance of a high-abundance fragment of the aglycon having one of the following structures: (a) (TMS)_x-Ar-OTMS⁺, (b) (TMS)_x-Ar-OH⁺, or (e) (TMS)_x-AR-O⁺, where Ar indicates the aromatic ring and (TMS)_x indicates polar hydrogens (OH or NH) on the aglycon replaced by TMS groups. Nine of twelve conjugates studied are ethereally linked through a phenolic hydroxyl group; however, the remaining three are ester linked through an aromatic carboxylate group. The latter appcared to be a more labile linkage, since derivatization apparently cleaved the ester bond, forming appreciable quantities of the fully derivatized aglycon and glucopyranurono-(6→1)-lactone. Gas chromatographic retention (methylene units) and mass spectral data are presented for a number of glucuronides isolated from human urine by high-resolution liquid chromatography.     

Enzymatic formation of (20R, 22R)-20,22-dihydroxycholesterol from cholesterol and a mixture of 16O2 and 18O2: random incorporation of oxygen atoms

[4-¹⁴C]Cholesterol was incubated with an adrenocortical preparation in the presence of ¹⁶O₂ and ¹⁸O₂ devoid of significant ¹⁶O¹⁸O. Isolated (20R,22R)-20,22-dihydroxycholesterol was converted to a trimethylsilyl derivative and analyzed by gas chromatography - mass spectrometry to determine the isotope distribution of the oxygen atoms at C-20 and C-22. The ions of $\text{m}\text{e}$ 289, 291, and 293 (comprising the C₈ C-20 to C-27 side-chain and containing, respectively, ¹⁶O₂, ¹⁶O¹⁸O, and ¹⁸O₂) exhibited a binomial distribution indicating that the oxygen atoms of the vicinal glycol were drawn at random from the atomic pool of the oxygen molecules. If both side-chain hydroxyl groups had originated from the atoms of the same oxygen molecule, the ion of $\text{m}\text{e}$ 291 would have been absent.     

Met-enkephalin immunoreactivity in blood platelets

Picogram amounts (50–150 pg/mg protein) of immunoreactive met-enkephalin material (met-enkephalin in IR) were detected by radioimmunoassay in human, rat and rabbit platelets. Characterization of this material by thin-layer chromatography, gel filtration chromatography and high-pressure liquid chromatography indicated that it behaves identically with synthetic met-enkephalin. No high molecular weight met-enkephalin IR could be detected in the platelet extracts, even after trypsin hydrolysis, using two antisera which are able to recognize some of the putative met-enkephalin precursors present in the adrenal gland or striatum. $\text{In vitro}$, thrombin released platelet met-enkephalin in IR concomitantly with 5-hydroxytryptamine (5-HT), suggesting a common subcellular localization, i.e. the 5-HT storing organelles, for met-enkephalin IR and the amine. $\text{In vivo}$, platelet met-enkephalin IR in the Sprague-Dawley rat was affected neither by adrenalectomy nor by hypophysectomy. Thirteen- and 18-week-old spontaneous hypertensive rats (SHR) had lower platelet concentrations of met-enkephalin in IR than age matched normotensive Wistar-Kyoto rats.     

Synthesis and relative stereochemistry of the four mercapturic acids derived from styrene oxide and N-acetylcysteine

The chemical reaction between (±)-styrene oxide and N-acetylcysteine produces both positional isomers ($\text{1}$ and $\text{2}$) as a mixture of diastereoisomers with a preference for the benzylic thioether isomer $\text{1}$ (2 : 1). Synthesis of the mercapturic acid conjugates from either (+)- or (?)-styrene oxide produces only two of the four possible stereoisomers. The single diastereoisomers of $\text{1}$ and $\text{2}$ were separated by high pressure liquid chromatography (HPLC) and identified by ¹H- and ¹³C-nuclear magnetic resonance (NMR). The relative stereochemistry at the benzylic carbon center of the mercapturic acid conjugates was assigned on the basis of the established chemical correlation between optically pure styrene oxide and its precursor mandelic acid, and considerations on the mechanism of ring opening of epoxides by sulfur nucleophiles. The stereochemical definition of the isomers $\text{3}$–$\text{6}$ should prove useful in investigations of the biotransformation of the glutathione (GSH) conjugates of styrene oxide.     

Analysis of microquantities of choline and its esters utilizing gas chromatography-chemical ionization mass spectrometry.

Gas chromatography-chemical ionization mass spectrometry has been applied successfully in the analysis of choline and its esters. This approach serves to extend further the potential of existing gas chromatographic procedures which are capable of the microestimation of choline esters following their N-demethylation by either chemical or physical means. Typical fragmentation patterns with ions at $\text{m}\text{e}\text{= 72}$ and $\text{m}\text{e}\text{= (M + 1)}$ were obtained for each choline ester derivative. When methane was used as the reactant gas, the above fragments were approximately of equal abundance for each ester. Use of isobutane as reactant gas yielded almost 80% of the (M + 1) fragment, and only approximately 5% of the fragment ion at $\text{m}\text{e}\text{= 72}$. Recovery of all fragments was linear for nondeuterated as well as deuterated analogs of choline ester derivatives. Recovery, as evident from the analysis of records of relative ratios of injected isotopic variants of these esters, indicated that this analysis of choline esters using chemical ionization mass spectrometry coupled with gas chromatography is quantitative and highly reproducible.     

Δ1-11-oxa-11-deoxycortisol: A new antiglucocorticoid with activity in vivo

A new steroid-like compound, Δ¹-11-oxa-11-deoxycortisol, was tested in a one-week growth suppression, thymus suppression and adrenal weight suppression bioassay for possible glucocorticoid antagonist activity in $\text{vivo}$. We hypothesized that this compound would have antiglucocorticoid activity based on previous studies of 11-deoxycortisol and Δ^1,9(11)-11-deoxycortisol, which were optimal glucocorticoid antagonists $\text{in vivo}$ in adrenalectomized rats, but which lost antiglucocorticoid activity in intact animals, apparently due to adrenal 11β-hydroxylation. Thus, Δ¹-11-oxa-11-deoxycortisol, a compound which cannot undergo llβ-hydroxylation, was synthesized and tested as an antiglucocorticoid. This analog had an affinity for the rat thymus glucocorticoid receptor similar to that of its parent compounds (Ki 0.9-3.1×10^?7M). A dose of 1 $\text{mg}\text{rat}$ antagonized the effect of 15μg of dexamethasone in the growth suppression assay (p<0.05) and in the thymus suppression assay (p<0.06), but did not antagonize dexamethasone-induced adrenal weight suppression. Δ¹-11-oxa-11-deoxycortisol did not exhibit glucocorticoid activity in any of the three assays. These data suggest that Δ¹-11-oxa-11-deoxycortisol may be a pure competitive antagonist of dexamethasone.     

Changes in adrenal dopamine concentration after metyrapone or ACTH administration: implications for the in vivo regulation of dopamine beta-hydroxylase by glucocorticoids.

$\text{In vitro}$ studies have demonstrated that rat adrenal dopamine beta-hydroxylase activity is controlled by neural input and by glucocorticoid production. However, beta-hydroxylation of dopamine $\text{in vivo}$ is a first-order reaction and may be considerably slower than the maximal rate determined by $\text{in vitro}$ methods. To estimate the $\text{in vivo}$ reaction rate the concentrations of dopamine (substrate) and of beta-hydroxylated catecholamine (product) were measured as a function of endogenous glucocorticoid production. Beta-hydroxylated catecholamine changed little but dopamine was increased 2-fold or more 17.5 h after the inhibition of steroidogenesis with metyrapone. Dopamine was also increased by metyrapone in animals with pre-existing adrenal denervation. ACTH 17.5 h before sacrifice caused only slight changes in normal rats but reduced the increase in dopamine caused by stress. The results indicate that adrenal dopamine concentration is inversely related to glucocorticoid production at a given level of neural input and provide $\text{in vivo}$ evidence that glucocorticoids maintain dopamine beta-hydroxylase activity in the adrenal gland.     

Behavioral comparisons of the stereoisomers of tetrahydrocannabinols

The potencies of (?)-$\text{trans}$-Δ⁹-THC, (+)-$\text{trans}$-Δ⁹-THC, (+)-$\text{cis}$-Δ⁹-THC, (?)-$\text{trans}$-Δ⁸-THC and (+)-$\text{trans}$-Δ⁸-THC were compared in several different species. (?)-$\text{trans}$-Δ⁹-THC was 100 times more potent than (+)-$\text{trans}$-Δ⁹-THC in depressing schedule-controlled responding in monkeys. The (+)-$\text{trans}$ isomers were less effective than their corresponding (?)-$\text{trans}$ isomers in the dog static-ataxia test, but potency ratios could not be determined due to a lack of dose-responsiveness of the (+)-$\text{trans}$ isomers. However, it appeared that their potency differed by at least ten fold. The potency of (+)-$\text{cis}$-Δ⁹-THC in the dog static-ataxia test was comparable to that of (+)-$\text{trans}$-Δ⁹-THC. The hypothermia in mice produced by the (?) isomers of $\text{trans}$-Δ⁹-THC and $\text{trans}$-Δ⁸-THC were 9.1 and 30.4 times greater than that produced by their respective (+)-isomers. Also, the potency ratio of the (+)- and (?)-$\text{trans}$-Δ⁹-THC was 5.6 as measured by depression of spontaneous activity in mice. The magnitude of the potency ratios of the THC stereo-isomers is dependent upon the species and the pharmacological test used.     

Design of a stable isotope dilution gas chromatography/mass spectrometric assay for cAMP: Comparison with standard protein-binding and radioimmunoassay methods

A stable isotope dilution assay is presented in which picomole quantities of cAMP can be determined with high precision and selectivity using gas chromatography and electron impact mass spectrometry with multiple ion detection techniques. Using synthetic [2,8-²H₂,6-¹⁵N]-cAMP as the internal standard, suitable specificity was obtained by monitoring the (MCH₃)⁺ fragment ions of the trimethylsilyl derivatives of cAMP and the internal standard at $\text{m}\text{z}$ 530 and $\text{m}\text{z}$ 533, respectively. The sensitivity of the assay as judged from the lower limit of detection of the mass spectrometer was 3.0 pmol. Rat liver and human urine cAMP levels were assayed using gas chromatography/mass spectrometry and were compared with levels determined by protein-binding assays and radioimmunoassays for the same samples. The intraassay coefficients of variation of the gas chromatography/mass spectrometry assay were 5.3% for the rat liver sample (cAMP level 832 pmol/g) and 6.0% for the urine sample (cAMP level 2.50 μmol/liter). Comparison of the levels of cAMP determined by the three assay methods showed correlation to within 10% variation.     

Hydroxylation of beta-phenylethylamine in the rat

$\text{ortho}$, $\text{meta}$ and $\text{para}$ Tyramine, as their dansyl derivatives, have been identified and quantitated by mass spectrometry in rat urine. After intraperitoneal injection of phenylethylamine labelled either with deuterium or tritium in the presence of pargyline, the amount of urinary tyramines excreted was 0.8% ($\text{para}$, 0.74%; $\text{meta}$, 0.12%; $\text{ortho}$, 0.008%) of the injected dose. The advantages of involving stable isotopes and mass spectrometry in metabolic studies of pharmacologically active compounds is discussed.     

Determination of the kinetic constants of fructose transport and phosphorylation in the perfused rat liver

The kinetics of fructose uptake was determined in perfused rat liver during steady-state fructose elimination. On the basis of the corresponding values of fructose concentration in the affluent and in the effluent medium, and the fructose and ATP concentration in biopsies, the kinetics of membrane transport and intracellular phosphorylation in the intact organ was calculated according to a model system. Carrier-mediated fructose transport has a high $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ (67 mM) and $\text{V}$ (30 μmoles · min^?1 ·g^?1). The calculated kinetic constants of the intracellular phosphorylation were compared with values obtained with an acid-treated rat liver high speed supernatant (values given in parentheses). $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ with fructose 1.0 mM (0.7 mM), $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ with ATP 0.54 mM (0.37 mM), $\text{V}$ 10.3 μmoles · min^?1 · g^?1 (10.1 μmoles · min^?1 · g^?1, calculated on the basis of the highest measured rate of fructose uptake correcting the ATP concentration to saturating values). The kinetics of fructose uptake reveals that at Physiological fructose concentrations the membrane transport limits the rate of fructose uptake, thus protecting the liver from severe depletion of adenine nucleotides.     

Partial characterization of N-acetyltransferase activity from cerebral ganglia and malpighian tubules of Periplaneta americana

Octopamine-N-acetyltransferase (NAT) activity from Malpighian tubules and cerebral ganglia of Periplaneta americana was studied using high performance liquid chromatography with coulometric electrochemical detection. The enzyme from these tissues is highly soluble and temperature-resistant with maximal activity demonstrated at 50°C. The pH profiles of enzyme activity from Malpighian tubules and cerebral ganglia differ markedly although, in both tissues, strong activity is evident under alkaline conditions. Kinetic analyses indicate that the enzyme present in Malpighian tubules has a K_m for octopamine of 0.40 mM with a maximum velocity of 37.9 nmol N-acetyl-p-octopamine (N-acOA) produced/mg protein/min; the equivalent values for acetyl CoA were determined as 0.32 mM and 36.9 nmol N-acOA/mg protein/min. NAT from cerebral ganglia showed K_ms for octopamine and acetyl CoA of 0.33 and 0.19 mM, respectively, and the maximum velocity with octopamine was 6.8 nmol N-acOA/mg protein/min. Analysis of NAT activity from a variety of cockroach tissues indicates that the enzyme is widely distributed, with the gut and associated Malpighian tubules, conglobate gland and salivary glands showing the greatest activity.     

Metabolic activation of procarbazine

Procarbazine chemical degradation and rat $\text{in vitro}$ and $\text{in vivo}$ metabolism have been investigated. Procarbazine was rapidly oxidized to the azo derivative in aqueous solution in the presence of oxygen. $\text{In vitro}$ rat liver supernatany and microsomal preparations oxidize the azo function to azoxy isomers and further hydroxylate these metabolites in a manner that may by analogous to 1,2-dimethylhydrazine metabolism. The hydroxylated metabolites are activated species that chemically react to give methylating and alkylating agents. An additional metabolic pathway was observed $\text{in vivo}$. This suggests that procarbazine may be converted to free radical intermediates that decompose to give methane and N-isopropyltoluamide. Procarbazine metabolites have been separated and identified using high performance liquid chromatography and direct probe chemical ionization mass spectrometry.