A sensitive radioimmunoassay for adenosine in biological samples

A simple, sensitive, specific, and reproducible radioimmunoassay for the measurement of adenosine in biological materials has been developed. Adenosine antibody was obtained by immunizing rabbits with an immunogen prepared by conjugating 2′,3′-disuccinyladenosine to human serum albumin. By succinylating adenosine in samples at the 2′- and 3′-O positions with a premixed reagent consisting of succinic anhydride, triethylamine, and dioxane, the assay became sensitive enough to detect less than picomole amounts of adenosine in minute quantities of tissues. The corss-reactivity of structurally related compounds with the antibody was mostly negligible except for 2′-deoxyadenosine, whose usual concentration was very low. The use of this method made it possible to measure adenosine without any prior purification procedure. The immunoreactive materials in various biological samples disappeared during incubation of the samples with adenosine deaminase.     

A simple biological assay for relaxin measurement.

1. Relaxin (R) is considered a gestation hormone with an insulin-like molecular structure. Its physiological importance is significant in the reproduction process. 2. Different methods of biologically assaying R have been published but electrophysiology techniques on uterus and ileum of rat have never been used. 3. A protein fraction was obtained from ovarian tissue of the rat and used to measure electrophysiological activity in vivo and in vitro. 4. Protein recovered with R activity was similar to that in previous reports. 5. Reduction of 100% in contraction strength and 50% in its frequency was observed in ileum and in uterus respectively; it was only of 60%, but its frequency increased 43%. 6. Methodological considerations and some physiological aspects are discussed.     

A simple and sensitive DNA assay for plant extracts

A sensitive fluorimetric method was developed for the quantitative determination of DNA in plant (Zea mays L. and Medicago sativa L.) extracts. This method takes advantage of the specific increase in fluorescence intensity of the complex of DNA and the dye 4′,6′-diamidino-2-phenylindole (DAPI). Recovery of DNA and dissociation of histones from DNA were maximized by the addition of 2.0 molar NaCl to the homogenates. Treatment of the homogenate with chloroform to remove pigments and proteins decreased the quenching of fluorescence of the DAPI-DNA complex. The fluorescence intensity of RNA with DAPI was less than 2% of that produced by an equivalent weight of DNA. Comparisons were made between this fluorimetric DNA method and the commonly used diphenylamine assay for DNA. The diphenylamine DNA assay was more timeconsuming, less sensitive, and consistently resulted in lower estimates of DNA concentrations than did the fluorimetric DNA assay.     

A new sensitive assay for the measurement of hydroperoxides

The enzyme glutathione (GSH) peroxidase can be used to measure hydroperoxides quantitatively, easily, and specifically. A timed reaction of GSH peroxidase, coupled with the oxidation of NADPH by GSH reductase, allows a direct spectrophotometric measurement of hydroperoxide. Addition of catalase prior to the addition of GSH peroxidase permits the distinction between hydrogen peroxide and organic hydroperoxides. The solvents that can be used with the assay include methanol, ethanol, water, and aqueous solutions of detergents such as Brij 35, Triton X-100, and cetyl trimethyl ammonium bromide. The utility of the method is demonstrated by the measurement of hydrogen peroxide and organic hydroperoxides formed upon ozonolysis of an unsaturated fatty acid.     

A sensitive spectorophotometric assay for pyruvate dehydrogenase and oxoglutarate dehydrogenase complexes

The activities of pyruvate dehydrogenase and oxo-glutarate dehydrogenase can be reliably measured by coupling the production of NADH to the reduction of added cytochromec. Maximum activities required the addition of NADH-cytochromec reductase activity prepared from rat heart mitochondria. Compared to other spectrophotometric assays this method provides an eight-fold increase in sensitivity and is particularly suitable for use with small tissue samples such as needle-biopsy samples of human skeletal muscle. Measurements of activities in rat tissues showed them to be in the order skeletal muscle < liver < heart ≤brown adipose tissue. Activities in normal human skeletal muscle were similar to those of rat muscle, tn the rat tissues specific differences were seen in the relative activities of the two complexes and cytochromec oxidase suggesting tissue-specific differences in the activities of the dehydrogenases and components of the electron-transport chain.     

A one-step enzymatic assay for the measurement of 17 beta-hydroxy- and 17-oxo-steroid profiles in biological samples

We describe a simple enzymatic method for the sensitive and specific detection and quantitation of families of hydroxy- and oxo-steroids in biological mixtures. Analysis of the profiles of individual steroids may be achieved following their chromatographic separation. The objectives of this analytical system are, therefore, different from conventional methods which are designed to measure single steroids with a high degree of specificity. The method employs highly purified and active bacterial hydroxysteroid dehydrogenases (HSD) which promote stereospecific, nicotinamide nucleotide-dependent oxidations and reductions at specified positions of steroids. In the presence of catalytic quantities of steroids these enzymes promote the transfer of hydrogen (transhydrogenation) between NADH and NAD analogues. A recently purified 17 beta-HSD from an Alcaligenes species (D. W. Payne and P. Talalay, J. biol. Chem., 260, 13468-13655, 1985) shows almost complete specificity for the 17 beta-hydroxy- and 17-oxo-groups of both C18 and C19 steroids. This enzyme catalyzes steroid-dependent transhydrogenation between NADH and the thionicotinamide analogue of NAD (S-NAD). When these components are incubated at pH 8.5 in the presence of minute quantities of steroid substrates, S-NADH (measured at 398 nm where NADH does not absorb) accumulates at a constant rate which is proportional to the concentrations of steroid and enzyme. The linear increase in absorbance with time is a measure of the total concentration of 17 beta-hydroxy- and 17-oxo-steroids, and can be used to detect subpicomol quantities of steroids. The method is illustrated by the detection and identification of free and conjugated androgens in human serum following their separation by high pressure liquid chromatography. The specificity of the transhydrogenase assay is completely dependent on the specificity of the enzyme and is thus applicable to the detection of other hydroxy- and oxo-steroids by making use of HSDs with appropriate specificities (e.g. 3 alpha-HSD for the measurement of 3 alpha-hydroxy- and 3-oxo-steroids). The simple one-step reaction lends itself to automation, needs no auxiliary detection systems, and requires only an inexpensive colorimeter.     

Structure of dihydroxyacetone phosphate dimethyl acetal, a stable dihydroxyacetone phosphate precursor, in the crystalline state

Crystal and molecular structures of four different salts of a dihydroxyacetone phosphate (DHAP) precursor, its dimethyl acetal [2,2-dimethoxy-1,3-propanediol phosphate, C(5)H(13)O(7)P, (MeO)(2)DHAP]: (cha)(2)[(MeO)(2)DHAP].H(2)O (6a), (cha)[(MeO)(2)DHAP] (6b), Na(2)[(MeO)(2)DHAP].5.75H(2)O (6c) and K(2)[(MeO)(2)DHAP].H(2)O (6d), along with the cyclohexylammonium (cha) salt of its phenyl ester (cha)[(MeO)(2)DHAP(Ph)] (6e) are described. In the (MeO)(2)DHAP mono- and dianions, slightly different orientation of the phosphate group in relation to the acetal carbon atom is observed, with a delicate tendency of phosphate group to be located antiperiplanar in the monoanions and anticlinal in the dianions. The 2,2-dimethoxy-1,3-propandiol moiety, (MeO)(2)DHA, seems to be very rigid and its conformation is independent of phosphorylation, the ionization state of the inserted phosphate group and its additional substitution. The overall structures of the cyclohexylammonium (6a,b) and potassium salts (6d) have a double-layered architecture, while the sodium cation network in 6c forms the system of channels, which are filled up with the [(MeO)(2)DHAP](2-) ions. The different architectures of 6c and 6d crystals result from the different ways in which the relevant dianions coordinate to sodium and potassium ions and affect also the hydrogen bonding system observed in 6c and 6d crystals.     

A sensitive enzymatic assay for determination of cholesterol in lipid extracts

A procedure for the determination of free and total cholesterol in lipid extracts is described. The method for free cholesterol employs cholesterol oxidase to generate H2O2 and peroxidase to catalyze the reaction of H2O2 with o-dianisidine to yield a colored product. For the determination of total cholesterol, cholesterol ester hydrolase is included.     

A sensitive assay for the determination of propionyl-CoA in biological material

A method has been developed for the determination of low amounts of propionyl-CoA in biological material. The method involves ¹⁴CO₂ fixation by propionyl-CoA in the presence of purified propionyl-CoA carboxylase. Values for propionyl-CoA in rat liver in vivo and in isolated rat livers perfused in the presence of propionate are reported.     

A sensitive assay method for the measurement of S-adenosylmethionine in tissue

An assay method has been developed for the measurement of tissue levels of S-adenosylmethionine based upon the ability of this compound to activate tripolyphosphatase associated with S-adenosylmethionine synthetase beta prepared from rat liver. The method has been used to measure S-adenosylmethionine levels in rat liver after feeding rats on various concentrations of methionine in the diet. The results obtained by this method agree well with those measured by the spectrophotometric method. The limit of sensitivity of the assay was about 0.1 nmol of S-adenosylmethionine in an incubation volume of 0.1 ml (10(-6) M).     

A sensitive radioisotopic assay of pyruvate dehydrogenase complex in human muscle tissue.

A radioactive assay for the determination of pyruvate dehydrogenase complex activity in muscle tissue has been developed. The assay measures the rate of acetyl-CoA formation from pyruvate in a reaction mixture containing NAD+ and CoASH. The acetyl-CoA is determined as [14C]citrate after condensation with [14C]-oxaloacetate by citrate synthase. The method is specific and sensitive to the picomole range of acetyl-CoA formed. In eleven normal subjects, the active form of pyruvate dehydrogenase (PDCa) in resting human skeletal muscle samples obtained using the needle biopsy technique was 0.44 +/- 0.16 (SD) mumol acetyl-CoA.min-1.g-1 wet wt. Total pyruvate dehydrogenase complex (PDCt) activity was determined after activation by pretreating the muscle homogenate with Ca2+, Mg2+, dichloroacetate, glucose, and hexokinase. The mean value for PDCt was 1.69 +/- 0.32 mumol acetyl-CoA.min-1.g-1 wet wt, n = 11. The precision of the method was determined by analyzing 4-5 samples of the same muscle piece. The coefficient of variation for PDCa was 8% and for PDCt 5%.     

A rapid sensitive method for the measurement of guanine ribonucleotides in bacterial and environmental extracts.

A method was devised for the quantitative determination of guanine ribonucleotides (GTP, GDP, and GMP) in extracts of biological materlals. The technique is based upon selective enzymatic hydrolysis of UTP and ATP contained within the cell extracts, followed by a quantitative determination of GTP. GTP is measured using a nucleoside diphosphate kinase-firefly luciferase coupled bioluminescent reaction, during which the GTP is enzymatically coupled to ATP production, resulting in ATP-dependent light emission. The methods are simple and reproducible and extremely sensitive (≤ 10^?9m GTP), and require no preparatory chromatographic separation procedures. Methods are also presented for the enzymatic conversions of GDP and GMP to GTP in addition to the determination of GTP.     

Enzymatic measurement of ethanol or NAD in acid extracts of biological samples

An enzymatic method for the measurement of ethanol has been developed to permit analyses with unneutralized acid extracts of blood, liver, cell suspensions, or other biological materials. Components of the assay mixture include NAD, yeast alcohol dehydrogenase, tris(hydroxymethyl)aminomethane (Tris), and lysine. Tris is a trapping agent for the reaction product, acetaldehyde. Lysine is used to maintain the pH at 9.7 where oxidation of ethanol is quantitative and most rapid, even when as much as 0.2 ml of 0.5 n HClO₄ is added. Lysine also causes the reaction to be 2 to 4 times faster than it is when either glycine or 2-amino-2-methyl-1-propanol is used as the buffer. The assay is linear up to an ethanol concentration of 0.125 mm in the reaction mixture and is complete by 4 min. By substituting ethanol for NAD in the reagents, the assay performs equally well in measuring NAD.     

A rapid and sensitive assay for determination of cholesterol in membrane lipid extracts

A commercially available enzymatic assay (Boehringer Monotest) was modified to allow a rapid and sensitive determination of cholesterol in membrane lipid extracts. This was achieved by adding 0.5% Triton X-100 to the reagent solution. The detergent did not interfere with the assay. The relationship between the amount of cholesterol per assay and the absorbance at 500 nm was linear up to 100 μg. The recovery in the assay was better than 95%. The assay was applied to the determination of cholesterol in erythrocyte membrane lipid extracts.