A rapid enzymic procedure for the determination of picomole amounts of UDP-glucuronic acid.

A simple microassay for the determination of UDP-glucuronic acid was developed on the basis of the formation of benzo[a]pyrene 3-glucuronide catalysed by UDP-glucuronyltransferase of guinea-pig liver. As little as 1-5 pmol of UDP-glucuronic acid was detectable in extracts of heat-denatured probes of liver or cultured cells equivalent to 10-50 micrograms of cellular protein.     

A high-performance liquid chromatography method for the analysis of picomole amounts of oligosaccharides

A method for the high-performance liquid chromatography separation of tritium-reduced, acetylated oligosaccharides is described. Their highly sensitive detection in column eluant is facilitated by the use of a flow radioactivity detector. The method differentiates some structural isomers and provides resolution of high-mannose oligosaccharides comparable or superior to that of other high-performance liquid chromatography methods. The detection limit is 0.3 pmol of oligosaccharide. For the detection of radioactive oligosaccharides this method is much less laborious than scintillation counting of collected peak fractions. Generation of a continuous chromatographic trace offers a particular advantage in the detection of partially resolved peaks and the visualization of peak shape. A study of some of the factors influencing acetylation and reduction has led to the development of a robust analytical method.     

A HPLC method for determination of nicousamide in dog plasma and its application to pharmacokinetic studies

A sensitive and reproducible high performance liquid chromatography (HPLC)-UV method for determination of nicousamide, an inhibitor of rennin and transforming growth factor-beta1 (TGF-beta1) type II receptors, has been developed and validated. Following acetonitrile deproteiniation, samples were separated by isocratic reversed-phase HPLC on an Aichrom Bond-AQ C(18) column and quantified using UV detection at 320 nm. The mobile phase was acetonitrile/water (ratio 62:38 containing 0.1% H(3)PO(4)), with a flow-rate of 1.0 ml/min. A linear curve over the concentration range 5-200 ng/ml (r(2)=0.9978) was obtained. The coefficients of the variation for the intra- and inter-day precisions ranged from 1.4-10.7% and 1.8-7.1%, respectively. The percentage of relative recovery was 91.56-105.45%. The method was used to determine the plasma concentration-time profiles for nicousamide after oral doses of 30, 100 and 300 mg/kg in dogs. A nonlinear pharmacokinetics was found in dogs at doses from 30 to 300 mg/kg. Following 30 mg/kg oral dose, the C(max) and AUC in females were lower than that in male. There is a potential for accumulation in dogs following multiple doses.     

A simple HPLC method for the determination of bifendate: application to a pharmacokinetic study of bifendate liposome

A rapid, sensitive and simple high-performance liquid chromatographic (HPLC) method with ultraviolet detector (UV) has been developed for the determination of bifendate in 100 microl plasma of rats. Sample preparation was carried out by deproteinization with 100 microl of acetonitrile. A 20 microl of supernatant was directly injected into the HPLC system with methanol-double distilled water (65/35, v/v) as the mobile phase at a flow rate of 1.0 ml/min. Separation was performed with a microBondapak C(18) column at 30 degrees C. The peak was detected at 278 nm. The calibration curve was linear (r(2)=0.9989) in the concentration range of 0.028-2.80 microg/ml in plasma. The intra- and inter-day variation coefficients were not more than 6.55% and 6.07%, respectively. The limit of detection was 5 ng/ml. The mean recoveries of bifendate were ranged from 94.53% to 99.36% in plasma. The present method has been successfully applied to the pharmacokinetic study of bifendate liposome in rats.     

A fluorimetric method for continuously assaying ATPase: application to small specimens of glycerol-extracted muscle fibers.

A continuous fluorimetric method using auxiliary-coupling enzymes such as pyruvate kinase and lactate dehydrogenase for measuring ADP production to assay ATPase activity is described. This method is simpler, more rapid, and more sensitive than the previously used spectrophotometric method. The application of this method for studying the ATPase of rabbit psoas muscle fibers during Mg²⁺-ATP activation is also illustrated and discussed.     

A nonenzymatic method for determination of hydrogen peroxide and organic peroxides

Reduction of hydrogen peroxide and organic peroxides (t-butyl hydroperoxide and linoleic acid hydroperoxide) was achieved with homovanillic acid as hydrogen donor in the presence of the triethylenetetramine-Fe3+ complex. By the catalytic action of this complex, homovanillic acid is oxidized to its fluorescent dimer. Based on this reaction a fluorometric method for the measurement of the hydroperoxides mentioned above is described. The method can be extended to the determination of substrate-enzyme systems that produce hydrogen peroxide, e.g., glucose-glucose oxidase. The method allows the determination of substances such as hydrogen peroxide and t-butyl hydroperoxide with an accuracy and precision of less than 3%. Glucose can be determined with similar precision and an accuracy of 4.7%.     

A method for determination of pyridine nucleotides using a single extract

Intracellular redox levels play an important role in physiology and pathophysiology. The principal intracellular reductant is NADPH, which is required for both the proper activity of the entire antioxidant system and important prooxidant enzymes such as nitric oxide synthase and NADPH oxidase. Thus an easy and accurate measurement of NADPH is very desirable. The method described in this paper is based on the fact that NADH and NADPH (not NAD(+) and NADP(+)) affect absorbance at 340 nm. A single cell extract is separated into three aliquots (A(1), A(2), and A(3)). A(1) is untreated and the absorbance at 340 nm is measured. A(2) is treated with an enzyme that converts all of the NADP(+) to NADPH and then the absorbance at 340 nm is measured. A(3) is treated with an enzyme that converts all of the NADPH to NADP(+) and then the absorbance at 340 nm is measured. A(1) - A(3) is the NADPH content and A(2) - A(1) is the NADP(+) content of the extract. Using this method, we have obtained full recovery of all added nucleotides from cell extracts, thus making the method suitable for the quick determination of NADP(+) and NADPH in living cells. We conclude that this method for the measurement of NADP(+) and NADPH is rapid, simple, accurate, and reliable.     

A radiometric method for the determination of NADH in subpicomole amounts

A radiometric method has been devised for the determination of small quantities of NADH formed in preceding dehydrogenase reactions. In a coupled enzymatic reaction, phosphoglycerate kinase (PGK) catalyzes the transfer of [32P]orthophosphate from [gamma-32P]ATP to 3-phosphoglycerate; the intermediate, 1,3-[1-32P]diphosphoglycerate, is dephosphorylated by glyceraldehyde-3-phosphate dehydrogenase (GAP-DH). [32P]Orthophosphate is released proportionally to NADH and can be measured after adsorption of [gamma-32P]ATP to activated charcoal. With this method, 0.2 pmol of NADH are detectable in the presence of a 10(4)-fold excess of NAD over NADH.     

A new method for the determination of the permittivity of small samples in the microwave range and its application to hemoglobin single crystals

A cavity perturbation method for the absolute determination of the complex permittivity of small samples in the microwave range is developed and tested. Samples with volumes less than 0.4 mm3, for example protein powder or single crystals of macromolecules, may be investigated in a temperature range between 180 and 300 K, using this method. As an application the complex permittivity of hemoglobin single crystals is determined at three frequencies, v = 3.06 GHz, v = 8.76 GHz and v = 17.0 GHz in a temperature range between 180 and 300 K.     

A specific method for the determination of millimicrogram amounts of urinary oestrone

1. A new combined radioactivity-fluorescence method is described for urinary oestrone, which involves acid hydrolysis, extraction and purification of the phenolic fraction, saponification, a Girard T separation and alumina chromatography of acetylated oestrone. 2. Sulphuric acid fluorescence is used for quantitation and specificity is achieved by the addition of tritiated oestrone to the urine hydrolysate. This radioactive tracer functions both as an internal corrector for purification losses and enables the demonstration of constant specific activity through the oestrone peaks to act as an index of specificity in each determination. 3. By using one-fifth of a 24hr. urine sample, 1mug. of urinary oestrone/24hr. can be determined with an accuracy of +/-4%. Fluorescence emission spectra from processed urine samples are identical with that of authentic oestrone acetate. 4. The advantages of the method are its high sensitivity and specificity, which is achieved with relative convenience.     

A novel method to study contraction characteristics of a single cardiac myocyte using carbon fibers

To facilitate cardiac muscle research, we developed a novel method by which the force and length of a single ventricular myocyte can be recorded with a pair of carbon graphite fibers attached firmly to both ends. One fiber was stiff, whereas the other fiber was compliant to allow the recording of force and shortening during twitch contractions. The image of the compliant carbon fiber was projected onto a pair of photodiodes, and their output was fed to a piezoelectric transducer after variable amplifications to alter the effective compliance of the carbon fiber. Thus contraction of the myocyte was induced under virtually isometric conditions as well as under auxotonic conditions. We obtained a bell-shaped relation between the compliance under an auxotonic load and the work output of the myocyte, which was directly related to myocyte performance in the heart. Because it is easy to attach myocytes to the experimental apparatus, the present method would allow us to study cardiac muscle mechanics at the cellular and molecular levels.     

Validated HPLC method for the determination of gabapentin in human plasma using pre-column derivatization with 1-fluoro-2,4-dinitrobenzene and its application to a pharmacokinetic study

A rapid, sensitive and accurate high-performance liquid chromatographic method with UV detection was developed and validated for the quantification of gabapentin in human plasma. Gabapentin was quantified using pre-column derivatization with 1-fluoro-2,4-dinitrobenzene following protein precipitation of plasma with acetonitrile. Amlodipine was used as internal standard. The chromatographic separation was carried out on a Nova-Pak C(18) column using a mixture of 50 mM NaH(2)PO(4) (pH=2.5)-acetonitrile (30:70, v/v) as mobile phase with UV detection at 360 nm. The flow rate was set at 1.5 ml/min. The method was linear over the range of 0.05-5 microg/ml of gabapentin in plasma (r(2)>0.999). The within-day and between-day precision values were in the range of 2-5%. The limit of quantification of the method was 0.05 microg/ml. The method was successfully used to study the pharmacokinetics of gabapentin in healthy volunteers.     

Development and validation of an HPLC method for the determination of dibenzoylmethane in rat plasma and its application to the pharmacokinetic study

A highly sensitive and simple high-performance liquid chromatographic (HPLC) assay has been developed and validated for the quantification of dibenzoylmethane (DBM) in rat plasma. DBM and internal standard (I.S.) 1-(5-chloro-2-hydroxy-4-methylphenyl)-3-phenyl-1,3-propanedione (CHMPP) were extracted from rat plasma by ethyl acetate/methanol (95:5, v/v) and analyzed using reverse-phase gradient elution with a Phenomenex Gemini C18 5-mum column. A gradient of mobile phase (mobile phase A: water/methanol (80:20, v/v) with 0.1% TFA and mobile phase B: acetonitrile with 0.1% TFA) at a flow rate of 0.2 mL/min, and ultraviolet (UV) detection at 335 nm were utilized. The lower limit of quantification (LLOQ) using 50 microL rat plasma was 0.05 microg/mL. The calibration curve was linear over a concentration range of 0.05-20 microg/mL. The mean recoveries were 80.6+/-5.7, 83.4+/-1.6 and 77.1+/-3.4% with quality control (QC) level of 0.05, 1 and 20 microg/mL, respectively. Intra- and inter-day assay accuracy and precision fulfilled US FDA guidance for industry bioanalytical method validation. Stability studies showed that DBM was stable in rat plasma after 4h incubation at room temperature, one month storage at -80 degrees C and three freeze/thaw cycles, as well as in reconstitute buffer for 48 h at 4 degrees C. The utility of the assay was confirmed by the successful analysis of plasma samples from DBM pharmacokinetics studies in the rats after oral and intravenous administrations.     

A double-labeling procedure for sequence analysis of picomole amounts of nonradioactive RNA fragments.

A double-labeling procedure for sequence analysis of nonradioactive polyribonucleotides is detailed, which is based on controlled endonucleolytic degradation of 3'-terminally (3H)-labeled oligonucleotide-(3') dialcohols and 5"-terminal analysis of the partial (3H)-labeled fragments following their separation according to chain length by polyethyleneimine- (PEI-)cellulose TLC and detection by fluorography. Undesired nonradioactive partial digestion products are eliminated by periodate oxidation. The 5'-termini are assayed by enzymic incorporation of (32p)-label into the isolated fragments, enzymic release of (32p)-labeled nucleoside-(5') monophosphates, two-dimensional PEI-cellulose chromatography, and autoradiography. Using this procedure, as little as 0.1 - 0.3 A260 unit of tRNA is needed to sequence all fragments in complete ribonuclease T1 and A digests, whereas radioactive derivative methods previously described by us1-4 required 4 - 6 A260 units.     

A colorimetric method for the determination of indole, and its application to assay of tryptophanase.

Indole reacts with sodium nitrite and glycine-HCl buffer, pH 2.6, to form a red color that is stable for more than 1 week. The reaction is reproducible and is linear over a wide range of indole concentrations (0.05–1.00 μmol). Twelve indole derivatives, including tryptophan, and 17 protein amine acids do not interfere. Indole-3-acetic acid, indole-3-acrylic acid, indole-3-pyruvic acid, 5-indole carboxylic acid, and 5-hydroxyindole-3-acetic acid interfere to varying extents (16–27%). Free indole was determined in biological material containing tryptophan by the present method. The method is also applicable to the assay of tryptophanase activity without prior indole extraction.