Isolation and properties of human alpha-fetoprotein from HepG2 cell cultures.

A relatively rapid 3-step fractionation method has been developed for the isolation of human alpha-fetoprotein from culture fluids of HepG2 cells applicable to large volumes. The protein exists as a complex with lipids or lipoproteins but an ethanol precipitation step is effective in separating it. Yields of 50-60% can be obtained from culture fluid containing 30-40 microg/ml. A minor fraction that appears to be a proteolytic product of the AFP is present in the final product.

Isolation and biological activity of aspidospermine and quebrachamine from an Aspidosperma tree source.

The indolealkaloids aspidospermine and quebrachamine have been isolated in crystalline form by a relatively rapid fractionation from the extract of a powdered material designated "Quebracho" derived from an Aspidosperma tree species. We present a novel isocratic LC method that provides baseline resolution of these two compounds and of the structurally related yohimbine in less than 15 min. Gas chromatography-mass spectrometry was employed to identify these compounds as well as several minor derivatives of aspidospermine during and after the purification process. Aspidospermine and quebrachamine like yohimbine have been found to possess adrenergic blocking activities for a variety of urogenital tissues.

Automated capillary electrophoresis applied to therapeutic drug monitoring.

Chromatographic separations in conjunction with physical chemical detection methods can provide extremely accurate and highly precise measurements of drugs in biological matrices. Immunochemical methods for drug analyses, on the other hand, although usually fully automated, often lack accuracy, may be subject to matrix interference effects, and frequently exhibit nonlinearities that contribute to suboptimal analytical results. Automated capillary electrophoresis (CE) promises the high accuracy and precision of physical methods with no operator intervention once the sample is loaded into the sample cup. A clean-up step is necessary for the analysis of drugs in biological matrices, but that could also be fully automated. Reagent costs per test with CE approach zero, because of the use of extremely small volumes of buffers, standards, and controls. We present data supporting CE as an alternative separation strategy for 28 commonly used therapeutic drugs. These can be resolved in less than 16 min with an optimized micelle-based buffer system.

Deficiency of copper can cause neuronal degeneration.

The aim of this article is to emphasize the important role that copper plays in the function of nerve cells. We are reporting preliminary data which suggest that the swelling of axons which we produce in rats by iminodipropionitrile, IDPN, is due to its chelating action on copper, and how conversely supplementation with copper abolishes both symptoms and lesions. The copper values we obtained by atomic absorption spectrophotometry of the spinal cord and brain from the animals fully support this contention. In comparing these results with the diseases that are known to be due to copper deficiency, namely Menkes disease in man, swayback in lambs and several neurological mutant mice, we find not only similar axonal swellings, but also amelioration of symptoms and lesions by early administration of copper. Considering the main forms in which copper is present, we discuss the cuproproteins, i.e. ceruloplasmin and metallothionein, and their role in transport and delivery of copper to various organs. Further, the many cuproenzymes i.e. superoxide dismutase, tryptophan-2,3-dioxygenase, lysine oxidase, cytochrome oxidase, monoamine oxidases, tyrosinase, dopamine-beta-hydroxylase and d-amino levulinate dehydratase are noted for their roles in the nervous system. Finally, we suggest that neuronal copper deficiency should be more fully investigated as a possible etiological factor in the more common neurodegenerative diseases, such as Alzheimer's disease and amyotrophic lateral sclerosis, ALS.

Crystallization and preliminary X-ray analysis of botulinum neurotoxin type A.

Botulinum neurotoxin serotype A was isolated from liquid culture of Clostridium botulinum. The pure Mr approximately 150,000 neurotoxin, composed of Mr approximately 50,000 light and Mr approximately 100,000 heavy chains, has been crystallized in three different crystal morphologies; all three have the same crystal form. The most suitable crystal form for X-ray analysis are bipyrimidal and crystallize in the hexagonal space group P3(1)21 (or P3(2)21) with one dimer per asymmetric unit. The unit cell dimensions are a = b = 170.5 A, c = 161.7 A. The crystals diffract to 3 A resolution.

Fatty acids are potential endogenous regulators of aldosterone secretion.

Adrenal glomerulosa cells washed with delipidated albumin produced increased amounts of aldosterone in response to angiotensin-II (AII) or (Bu)2cAMP. Albumin treatment also increased binding of 125I-labeled AII to high affinity binding sites on adrenal cells. Lipid extracts of albumin solutions that were used to wash cells inhibited AII binding and aldosterone responses by washed glomerulosa cells. Chromatographic fractionation and mass spectroscopic analysis indicated that the inhibitors removed from cells by albumin were long chain fatty acids. Exogenous fatty acids not only inhibited AII binding, but they inhibited basal aldosterone production and increments in aldosterone caused by AII or dbcAMP, suggesting an effect on postreceptor steps in aldosteronogenesis. The most potent and most abundant fatty acids removed from adrenal cells were oleic, linoleic, and arachidonic. These fatty acids inhibited at micromolar concentrations in the absence of albumin and at somewhat higher concentrations in its presence. Cells that had been washed, then inhibited by exogenous oleic acid in vitro, were restored to their enhanced responsiveness by a second albumin wash, making it unlikely that cell damage is the mechanism of inhibition by fatty acids. Responses of fasciculata cells were not potentiated by albumin washes, and cortisol production was less sensitive than aldosterone production to exogenous fatty acids. Binding of ANP to glomerulosa cells was not affected by albumin or fatty acids. These results combined with clinical correlations make it plausible that unesterified fatty acids are naturally occurring regulators of the adrenal glomerulosa. Insulin's ability to lower plasma levels of fatty acids may be one way that it causes sodium retention.

A SepPak HPLC method for tricyclic antidepressant drugs in human vitreous humor.

Death from tricyclic antidepressant overdose has become an all-too-common occurrence. Several factors, including postmortem concentration changes, can render blood and tissue samples useless for the determination of antidepressant drug concentrations. We present here an efficient method of solid-phase extraction for these drugs from vitreous humor and a reversed-phase, isocratic high-performance liquid chromatographic method for the simultaneous quantitation of amitriptyline, doxepin, and imipramine and their desmethylated metabolites.

Estimation of human dose of staphylococcal enterotoxin A from a large outbreak of staphylococcal food poisoning involving chocolate milk.

An outbreak of gastroenteritis in a school district in the United States was determined to be staphylococcal food poisoning due to 2% chocolate milk containing staphylococcal enterotoxin A (SEA). Twelve one-half pint (approx 0.28 l) cartons of the 2% chocolate milk from this outbreak were analyzed for the quantity of SEA present in the milk. The amount of SEA in the cartons varied from 94 to 184 ng with the average being 144 ng (mean = 139 +/- 45). The attack rate for vomiting among those who consumed more than one carton was greater (38.3%) than among those who consumed only one carton (31.5%) with the highest attack rate among those who consumed three or more cartons (44.4%).

Structural analysis of staphylococcal enterotoxins B and C1 using circular dichroism and fluorescence spectroscopy.

Secondary and tertiary structural parameters of two functionally and serologically related proteins, staphylococcal enterotoxins B and C1, have been determined by using circular dichroism and fluorescence spectroscopy. The secondary structures derived from the respective far-UV circular dichroic spectra were 9.5% alpha-helix, 55.0% beta-pleated sheets, 16.5% beta-turns, and 19.0% random coils for enterotoxin B and 15.0% alpha-helix, 38.0% beta-pleated sheets, 25.5% beta-turns, and 21.5% random coils for staphylococcal enterotoxin C1. The values matched well with the secondary structures derived from the amino acid sequences (Chou and Fasman method). Seven antigenic sites have been predicted for both staphylococcal enterotoxins B and C1 by using the hydrophilicity and the secondary structure information. Three of these antigenic sites appear similar. Fluorescence quantum yield of the single tryptophan residue (Trp-197) of both the enterotoxins showed the tryptophan residue in staphylococcal enterotoxin B to be approximately 46% more fluorescent than in staphylococcal enterotoxin C1. Tryptophan fluorescence quenching by the surface quencher I- and the neutral quencher acrylamide revealed that the single tryptophan residue in each of the enterotoxins is buried in the protein matrix and is not accessible to the surface quencher I-. The tryptophan residue in staphylococcal enterotoxin C1 is 14% less accessible to acrylamide than in staphylococcal enterotoxin B. The data, in general, reflect several similarities and significant differences between the two related enterotoxins.

200 Kd neurofilament protein binds Al, Cu and Zn.

200 Kd human and bovine neurofilament proteins were isolated from spinal cord and purified to homogeneity. The purified proteins were shown to be metal-binding proteins which bind at least one mole of Al, one mole of Cu and four moles of Zn. Neither dephosphorylation of the protein nor equilibrium dialysis against 25 mM EDTA, pH 7.0, removed these metals. This is the first report of a human protein which stoichiometrically binds Al.

Measurement of copper in biological samples by flame or electrothermal atomic absorption spectrometry.

Guidelines presented here allow for copper analysis of biological materials by methods that are very sensitive, that require little sample preparation, that have few chemical or spectral interferences, that are inexpensive, and that require only usual care in contamination control. The commercial instruments for FAAS and ETAAS from Perkin-Elmer, from Varian, and from Instrumentation Laboratories Inc. (Allied Analytical Systems) all work well in either the flame or the flameless mode. Background correction techniques are not essential for copper analysis if care is taken with the sample preparation to minimize the background signals. Different types of burners will work adequately if one makes certain that the viscosity of the sample and the control products are similar to the calibration standards. Further, dilution of samples is preferred over increasing the viscosity of the calibration standards by the addition of a protein containing solution or a substance such as glycerol. A 1:10 dilution of blood plasma or serum with dilute nitric acid or water is all that is necessary for copper analysis by the FFAS methods. Cation and anion effects should be tested by bracketing the concentrations of the ions found in the sample with known amounts of ions in the sample solutions. Increasing the concentrations of the ions thought to interfere while keeping the copper concentration constant is another way to test for ion interferences.(ABSTRACT TRUNCATED AT 250 WORDS)

Detection of staphylococcal enterotoxins by enzyme-linked immunosorbent assays and radioimmunoassays: comparison of monoclonal and polyclonal antibody systems.

Murine monoclonal antibodies reactive with at least one of the serological types of staphylococcal enterotoxin were examined for use in assay systems for the detection of enterotoxin at the level of 1.0 ng of enterotoxin per ml. An antibody sandwich enzyme-linked immunosorbent assay was devised for each toxin type by identifying an effective antibody pair. One antibody (the coating antibody) was coated onto a polystyrene plate and removed the enterotoxin from the test solution; the second antibody (the probing antibody) was conjugated to horseradish peroxidase and detected the captured toxin. Enterotoxins A and E could be detected in the same system by the use of cross-reacting monoclonal antibodies. All subtypes of enterotoxin C could be detected in one assay system. Two effective systems were described for each of types B and D. Each of these systems, when compared with the homologous enterotoxin-specific polyclonal rabbit antibody systems, was found to compare favorably. The monoclonal enzyme-linked immunosorbent assay systems for the detection of enterotoxins A and C2 were examined for a variety of food extracts; no abnormal interference could be detected from these extracts. The monoclonal antibody systems were also compared with the homologous enterotoxin-specific polyclonal serum for the detection of enterotoxin by the competitive radioimmunoassay (RIA). Single monoclonal antibodies generally did not perform as well in the RIA as did the homologous toxin-specific polyclonal serum. However, pools of monoclonal antibodies were prepared that approached the sensitivity and precision of the polyclonal system for the detection of each toxin by the RIA.

Regulation of aminotransferase-glutamate dehydrogenase interactions by carbamyl phosphate synthase-I, Mg2+ plus leucine versus citrate and malate.

Citrate, malate, and high levels of ATP dissociate the mitochondrial aspartate aminotransferase-glutamate dehydrogenase complex and have an inhibitory effect on the latter enzyme. These effects are opposed by Mg2+, leucine, Mg2+ plus ATP, and carbamyl phosphate synthase-I. In addition, Mg2+ directly facilitates formation of a complex between glutamate dehydrogenase and the aminotransferase and displaces the aminotransferase from the inner mitochondrial membrane which could enable it to interact with glutamate dehydrogenase in the matrix. Zn2+ also favors an aminotransferase-glutamate dehydrogenase complex. It, however, is a potent inhibitor of and has a high affinity for glutamate dehydrogenase. Leucine, however, enhances binding of Mg2+ and decreases binding of and the effect of Zn2+ on the enzyme. Thus, since both metal ions enhance enzyme-enzyme interaction and Zn2+ is a more potent inhibitor, the addition of leucine in the presence of both metal ions results in activation of glutamate dehydrogenase without disruption of the enzyme-enzyme complex. Furthermore, the combination of leucine plus Mg2+ produces slightly more activation than leucine alone. These results indicate that leucine, carbamyl phosphate synthase-I, and its substrate and cofactor, ATP and Mg2+, operate synergistically to facilitate glutamate dehydrogenase activity and interaction between this enzyme and the aminotransferase. Alternatively, Krebs cycle intermediates, such as citrate and malate, have opposing effects.

Stabilized analysis of antidepressant drugs by solvent-recycled liquid chromatography: procedure and proposed resolution mechanisms for chromatography.

In this efficient extraction and isocratic liquid-chromatographic procedure for measuring eight antidepressant drugs in serum (amoxapine, 8-hydroxyamoxapine, doxepin, desmethyldoxepin, imipramine, desipramine, amitriptyline, and nortriptyline), they are extracted from serum as a group by use of disposable solid-phase cyanopropyl columns; the eluate is injected directly onto a Zorbax cyanopropyl analytical column. This sample preparation circumvents potential problems of drug instability associated with the usual evaporating/concentrating techniques. Recycling the acetic acid/acetonitrile/n-butylamine mobile phase not only maintains a stable, cost-effective system, it also prolongs column life. Standard curves for all eight drugs are linear to 1000 micrograms/L; the detection limit is 8-10 micrograms/L. For all drugs and metabolites, within-run CVs were 0.9 to 2.2%, between-run CVs 2.1 to 3.8%. Analytical recovery of the solid-phase extraction step was 85 to 100% (mean = 94.5%) for all analytes. Standards and controls, stored frozen in drug-free serum, are stable for at least six months. We also report a study of separation mechanisms.

Effect of combined nitroglycerin and dobutamine infusion in left ventricular dysfunction.

The immediate therapy of severe left ventricular (LV) failure after acute myocardial infarction (AMI) frequently requires simultaneous preload reduction, pump output augmentation, and maintenance of systemic blood pressure. Therefore the effects of intravenous nitroglycerin (NG) and dobutamine (DB) were evaluated in 12 patients with severe LV failure following AMI. Nitroglycerin achieved salutary lowering of abnormally elevated LV filling pressure (23 to 14 mm Hg, p less than 0.001) while DB markedly augmented LV pump function (cardiac index rose from 1.7 to 2.5 L/min/m2, p less than 0.005). Notably, the combined infusion of NG + DB simultaneously decreased preload (LV filling pressure 23 to 14 mm Hg, p less than 0.001) and markedly enhanced LV pump performance (cardiac index increased from 1.7 to 2.4 L/min/m2, p less than 0.001). Minor decline in mean systemic blood pressure with NG (72 to 66 mm Hg, p less than 0.05) was rapidly reversed by DB addition (69 mm Hg, p greater than 0.05). Both agents were well tolerated without clinical or ECG evidence of myocardial ischemia or dysrhythmias. Thus the principally venodilator effects of NG minimize systemic hypotension while salutary augmentation of cardiac function in AMI with LV failure is achieved by NG + DB.

Effects of contaminants in blood-collection devices on measurements of therapeutic drugs.

Substances in evacuated blood-collection devices produce gas-chromatographic peaks with retention times similar to those of drugs. All tubes tested except the serum separator tubes contained tris(2-butoxyethyl) phosphate, as determined by mass spectrometry. In addition, the partition coefficient during the solvent-extraction step increased for some drugs by as much as 40%, while for other drugs it decreased by as much as 30%, depending on the drug and the collection tube. The Becton Dickinson serum separator tube also contains several other compounds identified by mass spectrometry to be dibasic esters used in the preparation of the polyester gel. The Corning serum separator tube and the Venoject serum tube contain still other impurities. The Becton Dickinson royal-blue-stoppered tube contained the least amount of impurities of the tubes tested. CVs of 25 commonly measured drugs in plasma pools increased from 5% for samples collected in glass tubes to greater than 20% for samples collected in evacuated blood-collection tubes. Clearly, accuracy and precision of measurements of therapeutic drugs can be seriously compromised if an inappropriate blood-collection device is used.