A sensitive analytical method for the detection and quantitation of adenosine in biological samples

A new method for the measurement of adenosine in biological materials has been developed. The method is based on the combined principles of isotope dilution and enzymatic catalysis using a highly specific adenosine kinase isolated from rat heart. By differential centrifugation and gel filtration, this adenosine kinase was obtained free of adenosine deaminase and other enzymes which would have been a source of error in the use of this enzyme in the adenosine assay. The cardiac adenosine kinase was shown to be highly specific and to exhibit an apparent K_m for adenosine of 0.35 μM. Using this enzyme, unknown quantities of adenosine could be detected by measuring the effect of their addition on the conversion of radioisotopic adenosine to 5′-AMP in the kinase reaction. In this procedure, as little as 20 pmoles of adenosine could be detected. To test the applicability of the assay, measurements of the tissue content of this nucleoside were made in samples of dog and rat hearts frozen in situ under control, hypoxic, or ischemic conditions. The assay has several advantageous features when compared to other existing methods used to measure adenosine: a minimum of sample preparation is required before the actual assay procedure; many samples can be processed per day by a single operator; single determinations can be done on as little as 5 μl of sample, and the specificity of the assay can be readily checked by treatment of samples with adenosine deaminase.     

A rapid and sensitive radioimmunoassay for norethisterone

A direct radioimmunoassay for the measurement of norethisterone (NET) in unextracted serum samples was developed. The combined use of a highly specific unpurified antiserum and heat treatment of diluted serum samples obviated both extraction and chromatographic procedures. The direct NET assay fulfilled all the quality control parameters. When this assay was compared with other methods involving either solvent extraction and/or chromatographic purification procedures, no significant differences were observed. The overall results were interpreted as demonstrating that this simple, rapid and reliable NET assay can be used as a helpful tool in metabolic and pharmacokinetic studies of this contraceptive progestogen.     

A sensitive method for quantitative analysis of hydrogen peroxide and oxidase substrates in biological samples]

The sensitive method for peroxidative assays of hydrogen peroxide (up to 5 nmole in 1 ml) and oxidases' substrates (glucose, ethanol) has been developed by use of a non-cancerogenic chromogen--3,3', 5,5'-tetramethylbenzidine (TMB), horseradish peroxidase and corresponding oxidases. The proposed modification of the known method consists in using decreased concentration of TMB (0.05-0.1 mM) and stopping the reaction by adding of acid to pH 1.4-2.1. This approach allowed to enhance the sensitivity of assays of corresponding analytes (7-10 times) and decrease the costs of analysis.     

A practically sensitive radioimmunoassay for cyclic CMP by 2'-O-acetylation

An improved method for the determination of subnanogram quantities of zinc has been devised using a tungsten filament for vaporization in a low-pressure microwave-induced helium plasma emission spectrometer. Desolvation and ashing in an air atmosphere of zinc containing samples in the presence of 1,10-phenanthroline (8 mm) and potassium chloride (3 mm) prevent fractional vaporization and yields a single, sharp emission signal. Analysis of nanogram quantities of zinc metalloenzymes contained in sample volumes of 5 μl illustrates the use of this method. The coefficient of variation for 0.14 ng of zinc in 75 ng of carboxypeptidase A is 3.5%, with a detection limit of 3 pg.     

A sensitive mass spectrometry method for simultaneous quantification of DNA methylation and hydroxymethylation levels in biological samples

The recent discovery of 5-hydroxymethyl-cytosine (5hmC) in embryonic stem cells and postmitotic neurons has triggered the need for quantitative measurements of both 5-methyl-cytosine (5mC) and 5hmC in the same sample. We have developed a method using liquid chromatography electrospray ionization tandem mass spectrometry with multiple reaction monitoring (LC–ESI–MS/MS–MRM) to simultaneously measure levels of 5mC and 5hmC in digested genomic DNA. This method is fast, robust, and accurate, and it is more sensitive than the current 5hmC quantitation methods such as end labeling with thin layer chromatography and radiolabeling by glycosylation. Only 50 ng of digested genomic DNA is required to measure the presence of 0.1% 5hmC in DNA from mouse embryonic stem cells. Using this procedure, we show that human induced pluripotent stem cells exhibit a dramatic increase in 5mC and 5hmC levels compared with parental fibroblast cells, suggesting a dynamic regulation of DNA methylation and hydroxymethylation during cellular reprogramming.     

An efficient and sensitive method for preparing cDNA libraries from scarce biological samples

The preparation and high-throughput sequencing of cDNA libraries from samples of small RNA is a powerful tool to quantify known small RNAs (such as microRNAs) and to discover novel RNA species. Interest in identifying the small RNA repertoire present in tissues and in biofluids has grown substantially with the findings that small RNAs can serve as indicators of biological conditions and disease states. Here we describe a novel and straightforward method to clone cDNA libraries from small quantities of input RNA. This method permits the generation of cDNA libraries from sub-picogram quantities of RNA robustly, efficiently and reproducibly. We demonstrate that the method provides a significant improvement in sensitivity compared to previous cloning methods while maintaining reproducible identification of diverse small RNA species. This method should have widespread applications in a variety of contexts, including biomarker discovery from scarce samples of human tissue or body fluids.     

A sensitive automated method for adenosine triphosphatase kinetics.

An automated method for measuring adenosine triphosphatase (ATPase) activity is described. A modified version of a Technicon Autoanalyzer utilizing a sensitive colourimetric technique for determining inorganic phosphate concentrations (1 nmol/ml) allows investigations on enzymes of low specific activities. Dialysis may be used for measuring tissue homogenate activities or bypassed by examining purified enzyme preparations. When combined with a gradient apparatus, the proposed technique is particularly well suited for the study of enzyme kinetics in relation to cation or anion concentrations.     

A sensitive radioimmunoassay for detecting products translated from cloned DNA fragments.

A simple and sensitive radioimmunoassay using E. coli β-galactosidase as a model protein has been developed for the detection of specific translation products of foreign gene fragments cloned into plasmid or phage vectors. This immunoassay is based upon the coupling to an insoluble matrix of F(ab)′₂ fragments derived from the specific antiserum by pepsin digestion. The in situ analysis of phage plaques or of bacterial colonies is performed by overlaying the phage plaques or lysed bacterial colonies with a cellulose filter to which F(ab)′₂ fragments have been chemically coupled. The antigen bound to the filter is detected by subsequent incubations with undigested antiserum and with ¹²⁵I-labeled Staphylococcus aureus protein A followed by autoradiography. By coupling the F(ab)′₂ fragments to the wells of a plastic microtiter plate, liquid cultures can be analyzed quantitatively for the presence of antigen, making possible the analysis of heterogeneous cultures by sib selection. The detection threshold of the microtiter plate assay for liquid culture is shown to be <2 × 10⁸ molecules, or about 1 molecule of β-galactosidase per cell. The in situ immunoassay for bacterial colonies, which permits examination of about 1000 clones per plate, can easily detect microcolonies producing about 10 molecules of β-galactosidase per cell, while the in situ phage plaque assay, also capable of screening about 1000 plaques per plate, is even more sensitive, detecting <1 × 10⁷ molecules per bacteriophage plaque.     

A simple and sensitive solid-phase radioimmunoassay for measuring the transferrin content of human biological fluids: its application to seminal plasma

A highly sensitive and reproducible radioimmunoassay was established to detect transferrin in human fluids. By this technique, applied to seminal fluid, transferrin levels (micrograms/ml) were found in normozoospermic individuals (64.49 +/- 25.41) at level higher than in oligozoospermic (38.93 +/- 21.35), azoospermic (19.49 +/- 10.23), or vasectomized (19.61 +/- 8.95) subjects. A relationship between transferrin and spermatozoid concentration in sperm was shown. These results reinforce previous findings that seminal transferrin can be used as a reliable clinical marker of Sertoli Cell function.     

Highly sensitive determination of saquinavir in biological samples using liquid chromatography-tandem mass spectrometry

A selective and sensitive method for the determination of the HIV protease inhibitor saquinavir in human plasma, saliva, and urine using liquid-liquid extraction and LC-MS-MS has been developed, validated, and applied to samples of a healthy individual. After extraction with ethyl acetate, sample extracts were chromatographed isocratically within 5 min on Kromasil RP-18. The drug was detected with tandem mass spectrometry in the selected reaction monitoring mode using an electrospray ion source and 2H(5)-saquinavir as internal standard. The limit of quantification was 0.05 ng/mL. The accuracy of the method varied between -1 and +10% (SD within-batch) and the precision ranged from +4 to +10% (SD batch-to-batch). The method is linear at least within 0.05 and 87.6 ng/mL. After a regular oral dose (600 mg) saquinavir concentrations were detectable for 48 h in plasma and were well correlated with saliva concentrations (r(2)=0.9348, mean saliva/plasma ratio 1:15.1). The method is well suited for low saquinavir concentrations in different matrices.     

A sensitive atomic fluorescence system for metals in biological systems

An atomic fluorescence spectrometric system for trace elemental determinations in biological samples is described. A heated graphite atomization furnace is used, with continuous sample introduction. Carbonic anhydrase and DNA dependent RNA polymerase enzymes are employed as application models, and accurate Zn determinations at the 10^?7m level are made on enzyme samples of 0.1 mg and less with a precision of 1–2%. The instrumentation is relatively simple, the system is versatile and has excellent stability.     

A micro-technique for preparing homogenates of biological samples

A method is described for preparing homogenates of insects, mites, nematodes, or fungal spores for electrophoretic analysis which overcomes the disadvantages of existing methods. It is also applicable to the homogenization of microsamples of animal tissue. The method involves grinding the samples in glass tubes, closed at one end, prepared from melting-point capillaries. A range of plungers is fabricated from the stainless-steel plungers from broken microsyringes, or from stainless-steel wire. The plunger is operated by hand to homogenize a sample in 2-20 microliter of buffer in a tube. The techniques for handling the samples before and after homogenization and for labeling and centrifuging them are described. Compared with existing methods, the procedure minimizes sample loss and risk of cross-contamination and eliminates the possibility of overheating the sample during homogenization.