Simultaneous determination of cysteine sulfinic acid and cysteic acid in rat brain by high-performance liquid chromatography

A sensitive and specific assay method for cysteine sulfinic acid (CSA) and cysteic acid (CA) using high-performance liquid chromatography has been developed. The method includes post-column derivatization of various amino acids with o-phthalaldehyde in the presence of 2-mercaptoethanol. The column packed with cation-exchange resin ($\text{ISC-07}\text{S1504}$, Shimadzu Sci entific instruments, Inc., Kyoto, Japan) was used for obtaining general separation of amino acids except CSA and CA, while the separation of CSA and CA was achieved using a strong-base anion exchange ($\text{ISA-07}\text{S2504}$, Shimadzu Scientific Instruments) column. The fluorescence peak area for CSA was linear between 20 pmol and 5 nmol, whereas that for CA was 10 pmol to 5 nmol. The regional distribution of CSA, CA, and other amino acids in the rat brain was studied using this new assay method.     

Saturable binding to cell membranes of the presynanptic neurotoxin, β-Bungarotoxin

Brief exposure to the protein neurotoxin, β-bungarotoxin, is known to disrupt neuromuscular transmission irreversibly by blocking the release of transmitter from the nerve terminal. This neurotoxin also has a phospholipase A2 activity, although phospholipases in general are not very toxic. To determine if the toxicity of this molecule might result from specific binding to neural tissue, we have looked for high affinity, saturable binding using ¹²⁵I-labeled toxin. At low membrane protein concentration ¹²⁵I-labeled toxin binding was directly proportional to the amount of membrane; at fixed membrane concentration ¹²⁵I-labeled toxin showed saturable binding. It was unlikely that iodination markedly changed the toxin's properties since the iodinated toxin had a comparable binding affinity to that of native toxin as judged by competition experiments. Comparison of toxin binding to brain, liver and red blood cell membranes showed that all had high affinity binding sites with dissociation constants between one and two nanomolar. This is comparable to the concentrations previously shown to inhibit mitochondrial function. However, the density of these sites showed marked variation such that the density of sites was 13.0 pmol/mg protein for a brain membrane preparation, 2.4 pmol/mg for liver and 0.25 pmol/mg for red blood cell membranes.In earlier work we had shown that calcium uptake by brain mitochondria is inhibited at much lower toxin concentrations than is liver mitochondrial uptake. Both liver and brain mitochondria bind toxin specifically, but the density of ¹²⁵I-labeled toxin binding sites on brain mitochondrial prepartions ($\text{3.3 ± 0.3}\text{pmol/mg}$) exceeded by a factor of ten the density on liver mitochondrial preparations ($\text{0.3 ± 0.05}\text{pmol/mg}$). It is also shown that the labeled toxin does not cross synaptosomal membranes, suggesting that mitochondria may not be the site of action of the toxin in vivo. We conclude the β-bungarotoxin is an enzyme which can bind specifically with high affinity to cell membranes.     

Regional high affinity synaptosomal transport of choline in mouse brain — Influence of oxotremorine treatment

Kinetic parameters for high affinity [HA] uptake $\text{in vitro}$ in synaptosomes from different mouse brain regions were investigated. V_max was highest in the striatum [200 pmol.· mg protein^?1 · 4 min^?1], followed by the cortex [111 pmol · mg protein^?1 · 4 min^?1], hippocampus [63 pmol · mg protein^?1 · 4 min^?1], midbrain [21 pmol · mg protein^?1 · 4 min^?1] and, lowest, medulla oblongata [5 pmol · mg protein^?1 · 4 min^?1]. K_m was about the same in all brain regions [0.9–1.4 μM]. No sign of HA uptake was detected in synaptosomes from the cerebellum. A clear relationship between V_max for synaptosomal HA uptake of Ch $\text{in vitro}$ and apparent turnover of ACh $\text{in vivo}$ was found between the brain regions. Administration of oxotremorine [1 mg·kg^?1 i.p.] decreased V_max for HA uptake of Ch by 60% in the cortex and hippocampus, by 50% in the striatum and by 20% in the midbrain. This effect is in accordance with the previously observed marked decrease in turnover of ACh in these brain regions following oxotremorine treatment.     

Immunoassay for honey bee cytochrome c in single animals with cytochrome c-coated bacteriophages: A sensitive tool for the study of caste formation in the honey bee, Apis Mellifera

The development of a sensitive viroimmunoassay for honey bee cytochrome $\text{c}$ and its usage for early detection of caste differentiation is described. Pure honey bee cytochrome $\text{c}$ was isolated from workers and used to produce antibodies in rabbits. Bacteriophage T4 was chemically modified by covalent attachment of honey bee cytochrome $\text{c}$ using tolylene-2,4-diisocyanate as a cross-linking agent. The immunospecific inactivation of this bacteriophage-cytochrome c conjugate by anti-cytochrome $\text{c}$ antibodies can be inhibited by free cytochrome $\text{c}$. In quantitative determinations, 50% inhibition is reproducibly achieved at a concentration of 6 ng/ml (5 pmol/ml) and as little as 0.3 ng/ml (0.25 pmol/ml) could be detected by this system. Cytochrome $\text{c}$ concentrations were measured in individual animals and substantial differences between corresponding larval stages of worker and queen bees are reported.     

Receptor-like stereospecific binding of a pyrethroid insecticide to mouse brain membranes

A heterogeneous particulate fraction of mouse brain homogenates binds NRDC 157 (3-phenoxybenzyl [1$\text{R}$,$\text{cis}$]-3-(2,2-dibromovinyl)-2,2- dimethylcyclopropanecarboxylate), a potent pyrethroid insecticide, stereospecifically and with high affinity. Stereospecific binding is a minor component of total binding (2.8%); the remainder of observed binding is predominantly nonspecific and unsaturable. Stereospecific binding is half-saturated at 4×10^?8$\text{M}$ and fully saturated at concentrations in excess of 1×10^?7$\text{M}$. The stereospecific binding capacity of this preparation was 200–250 pmoles of NRDC 157 per gram equivalent of brain tissue (2.3–2.8 pmol/mg protein). This binding site may represent the neural receptor involved in the stereospecific toxic action of pyrethroids.     

Characterization and application of a radioimmunoassay for beta-endorphin using an antiserum with negligible cross-reactivity against beta-lipotropin

High avidity antisera against β-endorphin $\text{(β}{}_{\mathrm{<mtext>h</mtext>}}\text{-}\text{EP}\text{)}$ were obtained in two of five rabbits immunized with unconjugated synthetic human $\text{β}{}_{\mathrm{<mtext>h</mtext>}}\text{-}\text{EP}$. One of these antisera (K-7762) cross-reacted 1.5% on a molar basis with β-lipotropin $\text{(β}{}_{\mathrm{<mtext>h</mtext>}}\text{-}\text{LPH}\text{)}$ and did not recognize leucine-enkephalin in a concentration as high as 0.2 mmol/l. The cross-reaction with methionine-enkephalin $\text{(β}{}_{\mathrm{<mtext>h</mtext>}}\text{-}\text{LPH}\text{61–65)}$ was 9%, while that with α-endorphin ($\text{β}{}_{\mathrm{<mtext>h</mtext>}}\text{-}\text{LPH}$ 61–76) was 69%. This implied that the specific recognition site was in the amino-terminal region of $\text{β}{}_{\mathrm{<mtext>h</mtext>}}\text{-}\text{EP}$. Although this sequence is present in $\text{β}{}_{\mathrm{<mtext>h</mtext>}}\text{-}\text{LPH}$ it was poorly recognized by the antiserum, suggesting that the free amino-terminal is essential. This interpretation was supported by the finding that $\text{α-N-}\text{acetyl}\text{-β}{}_{\mathrm{<mtext>h</mtext>}}\text{-}\text{EP}$ was equally poorly recognized by the antiserum. The sensitivity of the radioimmunoassay was 1.9 pmol/l. $\text{β}{}_{\mathrm{<mtext>h</mtext>}}\text{-}\text{EP}$ was not detectable (< 3 pmol/l) in 26 of 27 extracted plasma samples in healthy blood donors, in one it was 5 pmol/l. In five of six patients with an enlarged sella turcica, but without clinical and laboratory evidence of pituitary dysfunction, $\text{β}{}_{\mathrm{<mtext>h</mtext>}}\text{-}\text{EP}$ was detectable ($\text{5 ± 3}\text{pmol/l}$; mean ± S.D.) after metyrapone. $\text{β}{}_{\mathrm{<mtext>h</mtext>}}\text{-}\text{EP}$ was elevated in Addison's disease (23, 54 and 76 pmol/l), Nelson's syndrome (37, 39 and 109 pmol/l), ectopic ACTH production (27, 59 and 76 pmol/l), but only detectable in one of three samples from patients with Cushing's disease (7 pmol/l). Gel chromatography of extracts of porcine pituitary revealed only one immunoreactive peak co-eluting with synthetic human $\text{β}{}_{\mathrm{<mtext>h</mtext>}}\text{-}\text{EP}$. The specificity of the antiserum K-7762 was such that the $\text{β}{}_{\mathrm{<mtext>h</mtext>}}\text{-}\text{EP}$ concentration in plasma extracts could be reliably estimated by radioimmunoassay without prior chromatography.     

Large and small forms of cholecystokinin in human plasma: measurement using high pressure liquid chromatography and radioimmunoassay

Defining the role of cholecystokinin (CCK) in gut physiology and disease has proved difficult because of problems with development of radioimmunoassays and because CCK exists in several different molecular forms which have different biological actions. In order to measure small (8 amino acid residues, CCK 8) and large (33 and 39 residues) forms of CCK in plasma we have developed high pressure liquid chromatographic (HPLC) fractionation of plasma prior to radioimmunoassay. Fasting plasma CCK 8 and CCK $\text{33}\text{39}$ levels were usually undetectable (< 3 and < 6 pmol/1, respectively). After a liquid fat meal both CCK 8 and CCK $\text{33}\text{39}$ levels were significantly elevated at 5 min (11.3±3.3 and 11.6±2.6 pmol/1, respectively). Peak CCK 8 levels occurred at 30 min (15.0±4.4 pmol/1) while peak CCK $\text{33}\text{39}$ levels occurred at 120 min (16.7±4.9 pmol/1. Total CCK levels showed a biphasic response to the meal. These CCK 8 and CCK $\text{33}\text{39}$ responses to oral fat are consistent with a role for these hormones in the regulation of gallbladder emptying and pancreatic secretion.     

A rapid isotope dilution method for analysis of indole-3-acetic acid and indoleacetyl aspartic acid from small amounts of plant tissue

A method is described for analysis of indole-3-acetic acid and indoleacetyl aspartic acid in plant tissue. Methanolic extracts are purified on two small columns prior to HPLC using an electrochemical detector. $\text{In}$,$\text{vivo}$ amounts are calculated by isotope-dilution analysis. The method requires only 1 g of tissue, results in relative high recoveries, and is sensitive to the 20 pmol range or below. Typical results using light-grown $\text{Pisum}$,$\text{sativum}$ cv. Little Marvel tissue are given. This report is the first determination of the natural level of the aspartate derivative in growing plant tissues.     

Metabolism and uptake of L-pipecolic acid by brain and heart

Metabolism and uptake of $\text{L}$-[1-¹⁴C]pipecolate was studied in the rat through tail vein injection at low (30 μg/kg) and high (30 mg/kg) doses. No radioactive compound other than $\text{L}$-pipecolate was detected in the brain or heart samples 0.5 to 60 min after injection. The contents of $\text{L}$-pipecolate in the brain dropped rapidly to reach a plateau value 2 min after injection both in the low and high dose experiments (from 0.06 to 0.05 and from 86 to 55 nmole/g brain, respectively). Similar results were observed for the heart except that the heart had $\text{L}$-pipecolate contents 2–3 fold higher than the brain at every time interval. The influx of $\text{L}$-pipecolate to the brain, as measured by the plasma/brain ratio of its contents, was 3 fold lower than the heart at each sampling point throughout the course of measurement for both dosages. The influx of $\text{L}$-pipecolate from the plasma to the heart reached an equilibrium, i.e., plasma/heart = 1, 60 min after injection for both dosages; the plasma to brain ratio was 3 at 60 min. These results indicate that there is a blood-brain transport barrier for $\text{L}$-pipecolate in the rat, which may account for the differences in neuronal effects of $\text{L}$-pipecolate when administered intracerebrally or intraperitoneally.     

N-acetylation of dopamine and tyramine by mosquito pupae (Aedes togoi)

The $\text{N-}\text{acetyltransferase}$ (NAT) activity of mosquito pupae was measured by a radioenzymatic assay, using [¹⁴C]-, [³H]dopamine, [¹⁴C]tyramine or [¹⁴C]acetyl-CoA. The pupal extract could also generate acetyl-CoA from ATP, acetate and CoA for this acetylation reaction. Both the dopamine- and tyramine-NAT reactions proceeded linearly up to 20 min at an optimum pH of 8.4. It is possible that the same enzyme is involved in the acetylation of both biogenic amines as shown by the competitive inhibition kinetics obtained, and the similarities of the NAT reaction with both amines, in the presence of metal chelators, metal ions, SH reagents and MAO inhibitors. Mn²⁺ stimulated and Zn²⁺ inhibited the reaction. The specific activity of NAT in individual pupae measured soon after pupation showed no significant difference between the male and female pupae: the values obtained were, respectively, $\text{893 ± 57}$ and $\text{861 ± 30}$ pmol [¹⁴C]NAcT formed/min per mg protein and $\text{21.9 ± 1.2}$ and $\text{22.0 ± 1.4}$ pmol [³H]NADA formed/min per mg protein.     

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.     

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.     

Accurate and efficient method for quantification of urinary histamine by gas chromatography negative ion chemical ionization mass spectrometry

Because of the recognized inaccuracy and unreliability of currently available methods for the quantification of histamine in biological fluids, a method for quantification of urinary histamine by stable isotope dilution assay with negative ion chemical ionization mass spectrometry has been developed. Following the addition of [²H₄]histamine to 1 ml of urine, histamine is extracted into butanol, back-extracted into HCl, derivatized to the pentafluorobenzyl derivative (CH₂C₆F₅)₃-histamine, extracted into methylene chloride, and then quantified with negative ion chemical ionization mass spectrometry by selected ion monitoring of the ratio of ions $\text{m}\text{z}$$\text{430}\text{434}$. Twenty samples can be assayed in 2 days. Precision of the assay is ±2.7% and the accuracy is 97.6%. Lower limits of sensitivity are approximately 100–500 fg injected on-column. This assay provides a very sensitive, accurate, and efficient method for the quantification of histamine in human urine.     

Gamma amino butyric acid (GABA) levels in hypophyseal stalk plasma of rats

To determine the possible physiologic contribution of GABA to the tonic hypothalamic inhibition of adenohypophyseal prolactin secretion, we compared GABA levels in hypophyseal stalk plasma with those found in the peripheral circulation. Hypophyseal stalk blood was collected $\text{via}$ a parapharyngeal approach from 8 urethane anesthetized diestrus rats. Peripheral blood was collected simultaneously from the external jugular vein of the same rat at a rate similar to hypophyseal stalk blood flow. Blood samples resulting from a single 4 hr collection per animal were centrifuged, and the plasma stored frozen before ethanol extraction and assay using a radioreceptor method. GABA levels in hypophyseal stalk plasma ($\text{909±171}\text{pmol/ml}\text{;}\text{X}\text{±}\text{S.E.M.}$) were not significantly higher than levels in peripheral plasma (845±182; p>0.05), indicating little or no secretion of GABA by the median eminence.     

Quantitation of guanosine 5',3'-bisdiphosphate in extracts from bacterial cells by ion-pair reverse-phase high-performance liquid chromatography

A new method to determine the guanosine 5′,3′-bisdiphosphate (ppGpp) concentration in bacterial cultures has been developed. Cells in which the nucleotide pool was stabilized by treatment with formaldehyde were concentrated from samples of cultures and lysed by alkali. The ppGpp was separated from other nucleotides on a reverse phase C₁₈ column by ion-pair high performance liquid chromatography using an isocratic elution system. The area of the peak corresponding to ppGpp in elution profiles obtained by monitoring the absorbance of the eluant at 254 nm was used to quantitate ppGpp. The method allows detection of as little as 1 pmol of ppGpp per A₄₆₀ of culture, with $\text{2.5}\text{pmol}\text{A}{}_{460}$ being quantitated with greater than 90% accuracy.     

Insulin receptors in rat brain cortex. Kinetic evidence for a receptor subtype in the central nervous system

Binding kinetics of porcine ¹²⁵I-insulin were studied in synaptosomal and microsomal fractions of rat brain cortex. Receptor binding was temperature- and pH-dependent with optimum at 4°C and pH 8.0–8.3. At 15°C, steady state binding was heterogenous, and Scatchard analysis revealed two classes of receptors with K_d of 2 nmol/l and 40 nmol/l in amounts of 50 pmol/g and 200 pmol/g of membrane protein. Dissociation kinetics were biexponential with $\text{T}\text{1}\text{2}$ of about 5 min and 180 min, and in contrast to other cell-types, not influenced by negative cooperativity. No receptor-mediated insulin degradation was detectable at 37°C in the presence of bacitracin. Insulin analogues inhibited ¹²⁵I-insulin binding with potencies relative to porcine insulin (%): human insulin 100, rat insulin (I+II) 71, coypu insulin 47, rat multiplication stimulating activity 8, porcine proinsulin 5, among which the three last values were significantly higher than in rat liver and fat cells. No competition was observed with porcine relaxin and mouse nerve growth factor up to about 1 μmol/l. Receptors were present in all regions of central nervous system with highest concentrations in the cerebral cortex, cerebellum and olfactory bulb, and lowest in the pons, medulla oblongata and spinal cord. In conclusion, insulin receptors in rat brain cortex are functionally different from other tissues regarding the insulin specificity and the absence of negative cooperativity. It is suggested that an insulin receptor subtype in rat brain mediates the growth activity of insulin on nerve cells.     

The effects of quipazine on serotonin metabolism in rat brain.

Quipazine, 2-(1-piperazinyl)-quinoline, is a drug that has been reported to stimulate serotonin receptors in brain. We therefore studied the effect of quipazine on several parameters of serotonin metabolism in rat brain. Quipazine caused a slight, dose-related elevation of serotonin levels and decrease in 5-hydroxyindoleacetic acid levels for 2–4 hrs after it was administered. The decrease in 5-hydroxyindoleacetic acid levels was probably due primarily to a depression of 5-hydroxyindole synthesis, since quipazine also decreased the rate of 5-hydroxytryptophan accumulation after NSD 1015, the rate of serotonin decline after α-propyldopacetamide, and the rate of 5-hydroxyindoleacetic acid accumulation after probenecid. The elevation of serotonin was probably due to weak inhibition of monoamine oxidase. Quipazine reversibly inhibited the oxidation of serotonin by rat brain monoamine oxidase $\text{in}$$\text{vitro}$ and protected against the irreversible inactivation of the enzyme $\text{in}$$\text{vivo}$. Quipazine also was a potent inhibitor of serotonin uptake into brain synaptosomes $\text{in}$$\text{vitro}$ and attained concentrations in brain higher than the $\text{in}$$\text{vitro}$ IC₅₀. However, quipazine did not prevent the depletion of brain serotonin by p-chloroamphetamine $\text{in}$$\text{vivo}$. In addition to stimulating serotonin receptors in brain, quipazine may inhibit monoamine oxidase and serotonin reuptake $\text{in}$$\text{vivo}$.     

Peptide purification by gel filtration in aqueous pyridine--ammonia.

A method is described for the purification of peptides by gel filtration on Sephadex. The success of the method is due mainly to the use of 70% ($\text{v}\text{v}$) pyridine-30% ($\text{v}\text{v}$) 1 m aqueous ammonia, an excellent volatile solvent for peptides which does not degrade Sephadex. The method has been used to purify all of the major peptides of cowpea chlorotic mottle virus coat protein, after an initial fractionation by ion-exchange chromatography, and selected separations are used to illustrate the degree of fractionation which can be achieved.     

Sulfation of gastrin in Zollinger-Ellison sera: evidence for association between sulfation and proteolytic processing

Sulfated gastrins were quantitated in sera from 15 patients with the Zollinger-Ellison syndrome (ZES) by specific radioimmunoassays. The total concentration of gastrin varied from 174 to 285000 pmol/1. Sulfated gastrins constituted $\text{44.8±5.5\%}$ (mean ± S.E.M.) of the gastrins in ZES sera compared with $\text{37.7±1.9\%}$ in sera from 100 control subjects ($\text{P>0.1}$). There was no correlation between gastrin concentration and sulfation ($\text{r=0.40}$). Gel and ion-exchange chromatography showed that up to 90% of the gastrins could be in the sulfated form. The highest degree of sulfation was found in sera where the small gastrin components dominated. Thus, the percentage of small gastrins (G-17 and G-14) correlated with the degree of sulfation ($\text{N = 15}$, $\text{r=0.75}$, $\text{P<0.01}$). We suggest therefore that proteolytic processing of the gastrin precursor and sulfation of tyrosyl are associated.     

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.