A sensitive radioenzymatic assay for ATP

We describe a radioenzymatic assay for ATP based on the phosphorylation of radioactively labeled sugar in the presence of the appropriate kinase and further separation from the unreacted sugar using ion-exchange resin. The sensitivity of the method as described here goes down to 10¹² to 10¹⁴ mol of ATP and could be increased using radioactive sugar of higher specific activity.     

A sensitive radioenzymatic assay for epinephrine forming enzymes

Epinephrine (E) is formed in the adrenal medulla by phenylethanolamine-N-methyltransferase (PNMT), and in other tissues. Enzymes other than PNMT may be able to synthesize E, but this has been difficult to investigate because most assays do not have E as their final product. This assay produces 3H-E from norepinephrine (NE) and 3H-S-adenosylmethionine. The 3H-E is isolated on alumina, 3H-S-adenosylmethionine is precipitated and the 3H-E is suspended in diethylhexyl phosphoric acid in toluene for scintillation counting. The assay is sensitive and linear over a wide range. E was formed by most tissues tested. Brain and adrenal contained an enzyme specific for NE, but cardiac ventricle contained an enzyme that methylated both NE and dopamine. Denervated tissues in adrenal medullectomized rats contained very little NE, but still had E and E forming enzyme present. This assay detects a non-neuronal E forming enzyme with activity in vitro and in vivo.     

A sensitive radioenzymatic assay for glycerol and acylglycerols.

A sensitive radioenzymatic assay for glycerol and acylglycerols is described. The assay depends on the quantitative phosphorylation of glycerol to glycerophosphate by glycerol kinase using [gamma-32P]ATP as a substrate. The 32P content of the formed glycerophosphate is determined and gives a measure of the original glycerol content. Acylglycerols can be determined by prior hydrolysis to glycerol. The assay is sensitive to about 0.1 nmol of glycerol and can be extended to 100 nmol. The assay can be applied to the determination of acylglycerols separated by thin-layer chromatography in amounts as low as 0.5 nmol. The assay is particularly useful in the determination of the specific activity of 14C- or 3H-labeled glycerol moeities.     

A more sensitive and specific radioenzymatic assay for catecholamines

This modification of the catechol-O-methyltransferase (COMT) based radioenzymatic assay for norepinephrine (NE) and epinephrine (E) improves sensitivity, selectivity and eliminates many inhibitors of COMT. Prior to assay, samples are extracted into heptane with diphenylborate, then into dilute acetic acid. This extraction procedure has an efficiency of 78% for NE but less than 2% for S-adenosylmethionine (SAM). The extraction procedure also excludes calcium and other COMT inhibitors present in urine, plasma and every tissue tested. This eliminates the requirement for individual standardization of tissue and urine samples. Sensitivity of the assay for NE and E is 10 and 6 pg/ml respectively in 1 ml of plasma. The intraassay coefficients of variation for NE and E are 4 and 13% and the interassay coefficients of variation for NE and E are 10 and 16% in a human plasma sample containing low catecholamine levels. The assay permits quantitation of plasma E levels that were undetectable in prior assays.     

Simultaneous single isotope radioenzymatic assay of plasma norepinephrine,epinephrine and dopamine

Modification of the original single isotope radioenzymatic assay of Passon and Peuler (1) permits the direct and simultaneous analysis of norepinephrine, epinephrine and dopamine in plasma samples of 50 μl or less. Plasma or cerebrospinal fluid without prior extraction of catecholamines or deproteinization is added directly into a mixture of 100 μl. This catechol-O-methyl-transferase-catalyzed assay is sensitive to 1 pg (20 pg/ml of plasma) for norepinephrine and epinephrine and 6 pg (120 pg/ml) for dopamine. A rapid thin layer chromatographic separation of the three ³H-methylcatecholamines contributes to the excellent specificity of the differential assay of the three catecholamines. The differential analysis of 15–20 plasma samples can be completed easily within one day. A total assay which omits the chromatographic step and, thus, measures norepinephrine plus epinephrine at the same sensitivity can be completed in 20 samples in one-half a working day.     

Catecholamine binding by adrenal medullary protein can interfere with a sensitive radioenzymatic assay for norepinephrine

Norepinephrine (NE) binds extensively to protein that copurifies with phenylethanolamine-N-methyltransferase (PNMT) prepared from bovine adrenal medulla. This binding interferes with a NE assay that employs PNMT to catalyze the transfer of a tritiated methyl group from S-adenosyl-L-methionine to the amine group of NE. It was discovered that the protein binding of endogenous NE is reversed by dialysis at pH 6.0. Preparations of PNMT intended for use in radioenzymatic assays should involve one or more purification steps at pH 6.0.     

A simple and highly sensitive radioenzymatic assay for lysophosphatidic acid quantification

The objective of the present work was to develop a simple and sensitive radioenzymatic assay to quantify lysophosphatidic acid (LPA). For that, a recombinant rat LPA acid acyltransferase (LPAAT) produced in Escherichia coli was used. In the presence of [(14)C]oleoyl-CoA, LPAAT selectively catalyzes the transformation of LPA and alkyl-LPA into [(14)C]phosphatidic acid. Acylation of LPA was complete and linear from 0 to 200 pmol with a minimal detection of 0.2 pmol. This method was used to quantify LPA in butanol-extracted lipids from bovine sera, as well as from human and mouse plasma.This radioenzymatic assay represents a new, simple, and highly sensitive method to quantify LPA in various biological fluids.     

A sensitive radioenzymatic assay for the simultaneous determination of salsolinol and dopamine

A sensitive radioenzymatic assay for the simultaneous quantitation of salsolinol and dopamine in tissues and fluids has been developed. Salsolinol and dopamine were radiolabeled by 0-methylation using the enzyme catechol-0-methyltransferase and its cosubstrate, [3H]-S-adenosylmethionine, as the methyl donor. Specificity was achieved by alumina adsorption, selective solvent extraction, thin layer chromatography, primary amine precipitation and ion pair solvent extraction. The assay was linear over a 1000 fold concentration range. Sensitivities of 2 and 3 picograms were obtained for dopamine and salsolinol, respectively. Separate assay of standard samples had a coefficient of variation of 5%. Salsolinol was formed $\text{in vitro}$ in dopamine enriched plasma and whole brain homogenates following incubation with physiologic concentrations of acetaldehyde.     

A sensitive radioenzymatic assay for (S)-5-formyltetrahydrofolate.

A highly sensitive, radioenzymatic method has been developed for the specific and quantitative estimation of (S)-5-formyltetrahydrofolate. This method is based on enzymatic cycling of the 5-formyl derivative to methylenetetrahydrofolate followed by entrapment into a stable ternary complex with thymidylate synthase and tritiated fluorodeoxyuridylate. Determination of bound radiolabeled ligand permits estimation of the original folate. The initial cycling step is catalyzed by the enzyme, methenyltetrahydrofolate synthetase, which is specific for the (S)-diastereomer of 5-formyltetrahydrofolate and generates a product which can be further cycled to tetrahydrofolate using either 10-formyltetrahydrofolate deacylase or glycinamide ribonucleotide transformylase. Tetrahydrofolate is ultimately converted to the entrapable methylene derivative in the presence of excess formaldehyde. Using this assay recovery of reference (S)-5-formyltetrahydrofolate was linear over the range 0.03-1.9 pmol with an average recovery of 83 +/- 2%. The method has been applied to estimation of plasma (S)-5-formyltetrahydrofolate from a volunteer who had been administered (R,S)-5-formyltetrahydrofolate. Where comparison was possible, estimation of plasma (S)-5-formyltetrahydrofolate by this one step ternary complex-based method yielded results that were very similar to those observed by Straw et al. (Cancer Res., 44, 3114, 1984) who used an HPLC-based method for separation of diastereomeric mixtures of reduced folates and microbiological growth dependence to determine (S)-5-formyltetrahydrofolate.     

A radioenzymatic assay for quinolinic acid

A new and rapid method for the determination of the excitotoxic tryptophan metabolite quinolinic acid is based on its enzymatic conversion to nicotinic acid mononucleotide and, in a second step utilizing [3H]ATP, further to [3H] deamido-NAD. Specificity of the assay is assured by using a highly purified preparation of the specific quinolinic acid-catabolizing enzyme, quinolinic acid phosphoribosyltransferase, in the initial step. The limit of sensitivity was found to be 2.5 pmol of quinolinic acid, sufficient to conveniently determine quinolinic acid levels in small volumes of human urine and blood plasma.     

A radioimmunoassay for epinephrine and norepinephrine in tissues and plasma

An I¹²⁵ radioimmunoassay (RIA) has been developed for the measurement of plasma and tissue epinephrine (E) and norepinephrine (NE). The assay utilizes an antibody which specifically binds metanephrine. E and NE are detected by conversion to metanephrine with the enzymes catechol-0-methyltransferase and phenylethanol-amine-N-methyltransferase. The assay is very specific and will allow the measurement of E and NE in less than 500 μl of normal human plasma. E and NE concentrations were determined by both the RIA and a radioenzymatic assay in canine, human and rat biologic samples. The correlation coefficients between the two assays were .962 for E and .956 for NE. The RIA is sensitive, specific, precise and significantly less costly and time consuming than present radioenzymatic methods.     

A simplified radiometric assay for plasma norepinephrine and epinephrine

An assay for plasma norepinephrine and epinephrine levels has been developed by the modification of published procedures. The plasma norepinephrine and epinephrine assay, when compared to currently available methods, provides a substantial decrease in the assay time while providing a 10-fold increase in sensitivity which allows the analysis to be performed on 0.75 ml or less of plasma. Norepinephrine and epinephrine are converted to their O-methylated analogs in the presence of catechol-O-methyl transferase and S-adenosylmethionine-methyl-³H. Following purification of the labelled normetanephrine and metanephrine by solvent extraction and thinlayer chormatography, the amines are oxidized to vanillin, purified by solvent extraction and counted. The specificity, linearity and precision of the assay are discussed.     

A radioenzymatic assay for normetanephine in brain tissue

A simple, rapid and sensitive radioenzymatic assay for measurement of normetanephrine (NMN) in the brain has been described. The assay which is based on conversion of NMN to its N-methylated tritiated derivative metanephrine (³N-MN), by partially purified bovine adrenal phenyl-ethanolamine-N-methyl represents an extension of a previously developed procedure for measurement of urinary NMN. The sensitivity of the assay is 50 pg and results can be obtained in less than 4 hours. In rat brain, NMN concentration was 61 ± 3.4 ng/gm for hypothalamus and 82 ± 4.2 for brain stem at level of obex in male rats; 74 ±11 and 139 ± 10, respectively in female rats. Measurement of NMN in different areas of the brain may help to elucidate possible involvement of central nervous system in the pathophysiology of disease states such as hypertension.The role of central catecholamines in the pathogenesis of disease states has been investigated by a variety of techniques. These include methods which estimate relative concentrations of catecholamines in different parts of the neurones (1,2) and absolute concentrations in specific brain nuclei (3). In addition, estimates been made of catecholamine synthesis (4,%) an dturnover rates (6). Neurotransmitter actually released into the synaptic cleft cannot be measured. Moreover, part of what is released is taken up again by the neurone. The remainder is subject to catabolism and the resulting metabolic products may well reflect the amount of physiologically active transmitter. This report describes a rapid specific and sensitive assay for measurement of the O-methylated metabolite of norepinephrine, normetanephrine (NMN) in brain tissue, which is an extension of a previously developed procedure for urinary NMN (31). The metabolite is stable and readily extracted into solvents.     

A radioenzymatic assay for catecholamines and dihydroxyphenylacetic acid.

Picogram quantities of the catecholamines, dopamine, norepinephrine, and epinephrine, and the dopamine metabolite, dihydroxyphenylacetic acid, can be measured in tissue or plasma samples utilizing a rapid radioenzymatic procedure. The catechols are converted to their ³H-methylated derivatives (3-methoxytyramine, normetanephrine, metanephrine and homovanillic acid, respectively) by the enzyme catechol-O-methyltransferase with ³H-S-adenosylmethionine serving as the ³H-methyl donor. Following the enzymatic reaction, unreacted ³H-S-Adenosylmethionine is removed by precipitation and the reaction products are separated by thin layer chromatography on silica plates. The areas corresponding to the ³H-methylated derivatives are scraped into scintillation vials, eluted with aqueous buffer, extracted into nonpolar scintillation cocktail, and counted by liquid scintillation spectrometry. Using the standard assay procedure described here, over 100 tubes can be assayed in a single day with a sensitivity of 15–25 pg for all compounds measured. With the application of additional procedures, as little as 1 pg norepinephrine and epinephrine and 5–10 pg dopamine and dihydroxyphenylacetic acid can be quantified in a single sample.     

A rapid radioenzymatic assay for dopamine and N-acetyldopamine.

Dopamine (DA) was measured in various tissue extracts as [³H]methoxy-N-acetyldopamine after incubation with two partially purified enzymes, catechol-O-methyl transferase (EC 2.1.1.1) and N-acetyltransferase (EC 2.3.1.5), in the presence of [³H]adenosylmethionine and acetyl-CoA. This product can be separated quantitatively from labeled products of norepinephrine and epinephrine by solvent extraction. N-Acetyl-DA can be assayed by omitting the acetylating system from the incubation mixture. The procedure is rapid, convenient for processing large numbers of samples, and has a sensitivity of approximately 0.1 pmol. It has been used to measure DA in ganglia and in individual neurons from gastropod mollusks.     

A sensitive fluorometric assay for measuring xanthine dehydrogenase and oxidase in tissues

The conversion of xanthine dehydrogenase to a free radical producing oxidase is an important component of oxygen-mediated tissue injury. Current assays for these enzymes are of limited sensitivity, making it difficult to analyze activities in organ biopsies or cultured cells. The xanthine oxidase-catalyzed conversion of pterin (2-amino-4-hydroxypteridine) to isoxanthopterin provides the basis for a fluorometric assay which is 100-500 times more sensitive than the traditional spectrophotometric assay of urate formation from xanthine. Enzyme activity as low as 0.1 pmol min-1 ml-1 can be measured with the fluorometric pterin assay. Xanthine oxidase is assayed in the presence of pterin only, while combined xanthine dehydrogenase plus oxidase activity is determined with methylene blue which replaces NAD+ as an electron acceptor. The relative proportions and specific activities of xanthine oxidase and dehydrogenase determined by the fluorometric pterin assay are comparable with the spectrophotometric measurement of activities present in rat liver, intestine, kidney, and plasma. The assay has been successfully applied to brain, human kidney, and cultured mammalian cells, where xanthine dehydrogenase and oxidase activities are too low to detect spectrophotometrically.     

A rapid and sensitive assay for plasma testosterone in adults and children

A simplified method based on competitive protein binding is described for the determination of plasma testosterone concentration. The method involves extraction of a small volume of plasma and purification by a single thin-layer chromatogram. Sample testosterone displaces testosterone-³H from testosterone-binding globulin in dilute pregnancy plasma. Protein-bound testosterone is precipitated with ammonium sulfate and the testosterone-H³ in the supernatant determined by liquid scintillation counting. Similar treatment of standards provides an intrinsic correction for recovery and blank. Normal values for children are 0–50 ng/100 ml, for women 10–80 ng/100 ml, and for adult males 300–1200 ng/100 ml. Typical data from patients with endocrine disorders are included.     

A sensitive competitive protein binding assay for vitamin D in plasma

A sensitive protein binding assay for vitamin D is described. The vitamin D3 was extracted from plasma with diethyl ether and methylene chloride. The lipid extract was purified in Sephadex LH-20 followed by Lipidex 5000 and finally by high pressure liquid chromatography on a Zorbax Sil column (0.79 x 25 cm) developed in 0.25:99.75 isopropanol: methylene chloride. The vitamin D fraction was collected and quantitated by competitive protein binding assay with a 1/50,000 dilution of sheep plasma in 0.05 M potassium phosphate buffer (pH 7.5) containing 0.01% gelatin. [H3]-25-Hydroxyvitamin D3 was used as a radioactive tracer in the assay. We found that under these conditions, sheep plasma had equal affinity for vitamin D2 and vitamin D3 and could detect as little as 0.1 ng of vitamin D. When rat, cow, or human plasma was substituted for the sheep plasma, the decline in sensitivity to vitamin D2 was fivefold to tenfold. With this assay, we found excellent agreement (r = 0.98) between the results obtained by competitive protein binding analysis and direct U.V. absorbance analysis by high pressure liquid chromatography.     

A new radioenzymatic assay for histamine using purified histamine N-methyltransferase

Radioenzymatic assays for histamine (Hm) have found wide application. However, these procedures may lack the sensitivity necessary to quantify Hm in certain biological samples, such as human plasma. Purification of histamine N-methyltransferase (HNMT) has permitted the development of a new and highly sensitive radioenzymatic assay for Hm. HNMT was purified by sequential ion exchange, hydrophobic and molecular exclusion chromatography. The use of purified HNMT in the Hm assay has allowed the inclusion of high specific activity tritiated S-adenosyl-L-methionine ([3H]SAME) and the development of a simplified solvent extraction product isolation procedure. This assay has a sensitivity of approximately 2 picograms and is specific for Hm. Hm was easily quantified in human plasma and was found to be 303 +/- 81 pg/ml (mean +/- SD) in 8 male subjects. Substantial blank reduction and increased product conversion occur when purified HNMT is utilized in the Hm radioenzymatic assay, thus, increasing the sensitivity and possibly improving the specificity of this procedure.