High-performance liquid chromatographic determination of free and total polyamines in human serum as fluorescamine derivatives

A highly sensitive and simple fluorimetric method for the determination of free and total polyamines, spermidine, spermine, putrescine and cadaverine, in human serum by high-performance liquid chromatography is described. The polyamines, obtained after clean-up of deproteinized serum by Cellex P column chromatography, are converted to their fluorescamine derivatives in the presence of nickel ion which inhibits the reaction of interfering amines with fluorescamine, and the derivatives are separated simultaneously by reversed-phase chromatography (LiChrosorb RP-18) with a linear gradient elution. The lower limits of detection are 10 and 5 pmole for spermine and the others in 0.5 ml of serum, respectively.     

High-performance liquid chromatographic determination of indoleamines, dopamine, and norepinephrine in rat brain with fluorometric detection

A high-performance liquid chromatography-fluorescence procedure for the determination of 5-hydroxytryptamine, 5-hydroxyindoleacetic acid, tryptophan, dopamine, and norepinephrine has been developed. The method uses an ion-pairing system on an Ultrasphere ODS (5-microns) column with detector wavelength settings of excitation at 290 nm and emission at 330 nm. The procedure has been used to quantitate these indoleamines and catecholamines in rat brain tissue after homogenization in a perchloric acid solution; an aliquot of this solution is injected directly onto the HPLC column. Column sensitivities range from 6.1 pmol for tryptophan to 1.1 pmol for 5-hydroxytryptamine.     

The application of rate dialysis to the determination of free steroids in plasma

Rate dialysis is used to obtain the free steroid fraction in undiluted plasma at 37°C. The free steroid fraction is determined from the rate at which a small amount of tritiated steroid diffuses from plasma on one side of a semipermeable membrane into an identical plasma sample on the other side which lacks radioactive steroid. The method may be generally applicable to steroids since the cell permeability constant, which is a function of the volume of the dialysis cell and the area and diffusion properties of the membrane, was similar for seven steroids tested. The method requires only 0.3 ml of plasma, is simple and economical to perform, and enables up to 120 determinations to be made in one day. The free fractions of cortisol, progesterone, and estradiol-17β were measured in plasma pooled from pregnant and non-pregnant women and pregnant and lactating sows. The results were compared with those obtained for the same plasma pools by centrifugal ultrafiltration.     

Determination of protein-bound phosphate by continuous flow analysis: Automated procedure for the determination of alkali-labile phosphate

A method is described for the specific, quantitative determination of protein-bound phosphorus by a continuous flow procedure using a Technicon AutoAnalyzer. It is based on the exceptional alkali lability of serine phosphate linkages to β-elimination when the serine residues are present in a polypeptide chain. The results are reproducible within about 3, 5, or 10%, respectively, when the analytical sample contains about 100, 10, or 3 nmol of protein-bound P. The presence of less than 1 nmol protein-bound P can be detected. The method tolerates wide variations of the pH and ionic composition of the sample, making it suitable for the automatic, serial analysis of chromatographic effluent fractions. Low-molecular-weight phosphomonoesters, ribonucleic acid (phosphodiester), and nucleotide phosphates (pyrophosphate) do not react measurably. Carboxyphenyl phosphate is partially hydrolyzed (10–15%). In contrast, the release of P from various phosphoproteins is quantitative.     

A fluorometric procedure for measuring the neuraminidase activity: its application to the determination of this activity in influenza and parainfluenza viruses

A fluorometric procedure for quantitating the amount of N-acetylneuraminic acid enzymatically released by the neuraminidase activity from N-acetylneuraminyl-lactose (sialyl-lactose) has been developed. The liberated lactose is hydrolyzed with beta-galactosidase, and the released galactose is oxidized with galactose dehydrogenase and NAD+; finally, the NADH produced is measured by fluorometry (excitation at 340 nm and analysis of emitted light at 465 nm). The fluorometric assay is about 10-fold more sensitive than the spectrophotometric procedure that measures NADH at 340 nm. It readily measures amounts as little as 2 nmol of sialic acid, and does not require the use of radioactive isotopes. Interferences due to sucrose or other substances, which cause errors in some cases with the use of the periodate-thiobarbiturate method for neuraminidase activity determination, are avoided. The procedure reported here provides a sensitive, rapid, and relatively simple method (feasible with commercialized reagents) for measuring the neuraminidase activity not only in purified samples from different sources but also directly in biological materials such as viruses. The technique has been tested with some viruses recently isolated belonging to Orthomyxoviridae or Paramyxoviridae families, known to be rich in neuraminidase. Reciprocally, this method can also be employed for determining the sialic acid concentration in acylneuraminyl-lactose-containing compounds when using purified neuraminidase for hydrolysis.     

The role of tryptophan residues in heparin-antithrombin interactions

A single tryptophan residue on antithrombin has been modified with dimethyl-(2-hydroxy-5-nitrobenzyl)sulfonium bromide. This alteration led to a 500-fold reduction in the heparin-dependent acceleration of thrombin-modified antithrombin interactions, as well as a 10-fold decrease in the avidity of the modified protease inhibitor for mucopolysaccharide. Preincubation of antithrombin with the octasaccharide binding domain of heparin prior to treatment with dimethyl-(2-hydroxy-5-nitrobenzyl)sulfonium bromide was able to suppress modification of the critical tryptophan and preserve the functional capacities of the protease inhibitor. Fluorescence quenching experiments indicated that the modifiable tryptophan groups of antithrombin were exposed to the solvent environment. Based upon these data, it was proposed that the loss of “heparin cofactor” activity of antithrombin must be predominantly due to an inability of the modified protease inhibitor to undergo a conformational transition required for mucopolysaccharide-dependent “activation” of the macromolecule.     

A sensitive method for determination of 5-fluorouracil and 5-fluoro-2'-deoxyuridine in human plasma by high-pressure liquid chromatography

Polymerization-competent extracts of suspension-cultured HeLa cells and porcine brain tissue were assayed for tubulin content. Five different methods were used to assay identically prepared extracts: two types of sodium dodecyl sulfate-containing acrylamide gels, a DEAE retention assay, a colchicine-binding assay, and a radioimmunoassay. The colchicine-binding and radioimmunoassay results were in close agreement and are therefore considered reliable assays for tubulin content in cell extracts. The DEAE retention assay gave slight overestimates, but the gel methods seriously overestimated tubulin content. Based on data from colchicine binding and radioimmunoassay, the proportion of soluble cell protein which is tubulin is 4.3% for HeLa cell extracts and 12.1% for brain tissue extracts.     

Gas chromatographic determination of low concentrations of benzoic acid in human plasma and urine

A method for the determination of benzoic acid down to concentrations of 10 ng/ml in plasma or urine is described. After addition of an internal standard, benzoic acid is extracted at acid pH into diethyl ether. Both compounds are derivatized with pentafluorobenzyl bromide. The derivatives are determined by gas chromatography using a ⁴³Ni electron-capture detector. Hippuric acid is hydrolysed in plasma and urine and total benzoic acid is determined by the same technique.     

Reversed-phase liquid chromatographic determination of plasma levels of adriamycin and adriamycinol

A method is given for the determination of adriamycin and its main metabolite, adriamycinol in plasma from cancer patients after administration of adriamycin as the free drug or as a complex with DNA.Adriamycin and adriamycinol are extracted in a column from 1 ml of plasma (pH 8.6) using a mixture of chloroform—1-heptanol (8:2). After re-extraction into phosphate buffer pH 2.2, the separation is performed as reversed-phase liquid chromatography on a LiChrosorb RP-2 (5 μm) column with a mobile phase of acetonitrile—water, acidified with phosphoric acid.The precision by quantitation with photometric detection was better than 5% within the range 50–300 ng/ml. Plasma levels of adriamycin and adriamycinol in a cancer patient are presented in this paper.     

A direct fluorometric assay of benzo[a]pyrene hydroxylase.

A technique to measure the activity of pyruvate carboxylase spectrophotometrically in crude liver homogenates is described. The assay is based on the transformation of oxaloacetate, which is formed during the carboxylation reaction, into citrate in the presence of excess acetyl CoA and citrate synthase. After removal of pyruvate with KBH₄ and of protein with HClO₄, citrate is cleaved with citrate lyase into oxaloacetate and acetate, and oxaloacetate then is measured spectrophotometrically. Optimal concentrations of pyruvate, Mg²⁺, ATP, and KHCO₃ for the carboxylation reaction and the V_max were in good correlation with the data found by others using [¹⁴C]pyruvate.     

New electron-capture gas-liquid chromatographic method for the determination of mexiletine plasma levels in man

A method for the determination of mexiletine in human plasma by gas—liquid chromatography with electron-capture detection is described. Plasma samples are extracted at pH 12 with dichloromethane after addition of the internal standard, the 2,4-methyl analogue of mexiletine. A derivative is obtained using heptafluorobutyric anhydride; according to gas chromatography—mass spectrometry it is a monoheptafluorobutyryl compound. The minimum detectable amount of mexiletine is 5 pg. Accurate determinations of human plasma levels were performed after oral or intravenous treatment.