Microchemistry of microdissected hypothalamic nuclear areas.
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Animals↗
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Pancreatic islets subjected to different concentrations of glucose in vitro. A study with special regard to mitochondrial changes.
Isolated pancreatic islets of mice and gerbils were cultured for 6 days at low (2mM) or high (20mM) concentrations of glucose after which they were studied using qualitative and quantitative electron microscopy, histo- and microchemistry, and X-ray microanalysis. Compared with the islets cultured at high glucose, those subjected to low glucose exhibited enhanced succinate dehydrogenase activity, a decreased content of adenosine triphosphate, and an increased volume of B-cell mitochondria which often were rounded or oval.
Adenosine Triphosphate↗
Enzymic preparation of neurosecretosome- and pituicyte-enriched fractions from the rat neurohypophysis.
A microcellular dispersion procedure for the rat neurohypophysis was developed, comprising tissue softening and dissociation using a special sieving sytringe. In preparatory studies the influence of mesh width, and treatment with trypsin, pronase or collagenase-hyaluronidase was investigated using light and electron microscopy, as well as with microchemistry by means of protein and lactate dehydrogenase activity determinations. Trypsinization gave the best results. In the final adopted procedure, 3 incubated neurohypophyses were sequentially sieved through a 200- and a 50-mum mesh. The resulting 50-mul dispersion was found to contain numerous ultrastructurally well-preserved pinched-off axonal endings (neurosecretosomes), and pituicytes often revealing processes. On the basis of DNA and oxytocin assays 11% of the pituicytes and 28% of the axonal cytoplasm were recovered. Oxytocin immunofluorescence microscopy showed hormone within the neurosecretosomes, but often also in the cytoplasm of pituicytes. Microdensity gradient centrifugation was performed on neurohypophyseal disperions, in order to obtain fractions enriched for neurosecretosomes and pituicytes. Fractions were characterized by means of phase contrast, oxytocin immunofluorescence and electron microscopy, as well as by oxytocin and DNA assays as respective markers. With a 10:14:22% (w/v) Ficoll gradient, fractions were obtained for which the relative purification was by a factor of 4 on the basis of DNA/oxytocin ratios.
Animals↗
Acidosis and growth in nonuremic renal disease.
Our data demonstrate that correction of acidosis is sustained in children with type 1 RTA when alkali therapy is given in doses of 5 to 14 mEq/kg/day. The large doses are required as a result of renal bicarbonate-wasting. Children with type 1 RTA and acidosis who have significant growth impairment experience catch-up growth and attain normal stature for their age when correction of acidosis is sustained. Whether chronic acidosis impairs growth in any clinical condition except type 1 RTA is not settled. Whether sustained correction of acidosis with alkali therapy will allow attainment of normal stature in children with nonuremic diffuse renal disease is not yet determined. With the increasing availability of microchemistry and microgasometry and the new standards for growth based on mean-parent height [40], it can be anticipated that answers to these clinically important questions will be forthcoming.
Acidosis↗
Perspectives on the use of microspectrophotometry in the diagnosis of pretumorous processes and malignant tumors.
Comparative microspectrophotometric study of the nuclear Feulgen DNA content in pretumorous processes and cancers (larynx, endometrium, large intestine, and stomach) has made it possible to formulate an exponential law of DNA accumulation in cell populations in proliferating and malignant tissue. The dynamics of changes in the "index of DNA accumulation" can be used as an objective quantitative diagnostic test. A scale of malignant transformation was proposed with which the degree of probability of malignancy in pretumorous processes could be estimated. The data obtained allowed the author to propose a hypothesis of DNA superproduction as the basic cause of progressive neoplastic growth.
Chromosomes↗
Application of a microchemical technique to the elucidation of enzyme activity profiles within single human mammary tumors.
An ultramicrochemical technique has been adapted to the evolution of enzyme profiles within individual human mammary tumors. Tandem observation of adjacent stained and lyophilized sections permitted dissection of microgram quantities of freeze-dried material within confirmed regions of malignancy. Enzymes frequently monitored to examine glycolytic, respiratory, and metastatic capacity were microanalyzed successfully: lactic dehydrogenase (LDH), phosphoglucose isomerase (PGI), malate dehydrogenase (MDH), acid phosphatase (AP), aldolase (ALD), glucose-6-phosphate dehydrogenase (G6PDH), pyruvate kinase (PK), alpha-glycerophosphate dehydrogenase (alpha-GOPDH), hexokinase (HK), and phosphofructokinase (PRK). All enzyme activities were higher in infiltrating ductal carcinomas than in fibroadenomas. Extracts of tumor cells mixed in varying proportions with brain or muscle extracts of rat evidenced no modification of expected activity. The technical adaptation described provided a sensitive methodology to resolve problems of relication, profile analysis, sample quantity, and selectivity within heterogeneous tissues.
Acid Phosphatase↗
Recent advances in bioluminescence and chemiluminescence assay.
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Adenosine Triphosphate↗
Preclinical toxicological study of phosphonoacetic acid: determination in blood by selected ion monitoring.
Confirmed observations of the inhibition of oncogenic viruses by phosphonoacetic acid led to preclinical trials in animal model systems. Circulating phosphonoacetic acid was detected in the blood of mouse, rabbit and monkey after oral or subcutaneous administration of the drug. Phosphonoacetic acid is quantified in blood, after removing proteins and lipids, as the trimethylsilylated derivative, by monitoring the intensity of the protonated molecular ion and also that of phosphonopropionic acid (internal standard) using chemical ionization mass spectrometry combined with gas chromatography. The detection limit is 20 ng ml-1 when 0.2 ml serum is analyzed. A dosage of 230 mg kg-1 day-1 by continuous infusion is proposed for therapeutic trials in monkeys. This dose is well tolerated and results in slowly increasing blood levels of phosphonoacetic acid which reach a maximum of approximately 50 microgram ml-1 in a four day infusion, and decrease to 2 microgram ml-1 24 h after termination.
Animals↗
Determination of lithium in microlitre amounts of human body fluids at therapeutic and normal levels by stable isotope dilution and field desorption mass spectrometry.
The use of field desorption mass spectrometry for the determination of lithium in body fluids at therapeutic levels--ppm region--as well as at the normal level--ppb region--has been developed. The use of a stable isotope enriched internal standard, together with the outstanding sensitivity of field desorption for alkali cations and the high specificity of mass spectrometry, allows a quantitative determination of lithium in microlitre amounts of body fluids, such as plasma, saliva and urine. The assay allows a determination of lithium even at ultratrace concentrations where routine spectroscopic procedures cannot be applied. Analysis of plasma required a simple protein precipitation, whereas saliva and urine could be analysed without treatment. The precision of the data obtained ranged from 2--10%. The time consumption for one analysis in routine work is about 20--30 min.
Humans↗
Determination of uracil and thymine and their nucleosides and nucleotides in picomole amounts by gas chromatography mass spectrometry selected ion monitoring.
A stable isotope dilution method is presented by which uracil (Ura) and thymine (Thy) can be determined with high precision and sensitivity at the picomole level utilizing stable isotope dilution and gas chromatography electron impact mass spectrometry in the selected ion monitoring mode. [15N2]Ura and [2H3]Thy served as internal standards. The molecular ions as well as the [M-CH3]+ ion fragments of silylated Ura and Thy (Ura-TMS and Thy-TMS) were suitable for the assay which provides evidence of specificity, if identical results are obtained at both ions. Nucleosides and nucleotides of Ura and Thy were determined following quantitative hydrolysis in 6 N HCl at 180 degrees C for two hours. Other hydrolysis procedures did not give satisfactory results. Levels of free Ura and Thy were measured in human and rat plasma after solvent extraction with a sensitivity of 20-40 pm ml-1 demonstrating ready applicability of the assay method to biological samples. The potential physiological role of circulating Ura and Thy is discussed.
Animals↗
[Hygienic significance of sterigmatocystin in vegetable foods. 1. Analytical detection of sterigmatocystin].
The present paper gives a survey of the chemical, physical and biological properties of the mycotoxin sterigmatocystin. It must be regarded as toxic to warm-blooded animals and as cancerogenic. It is likely to occur in foods; therefore, its analytical detection is necessary. With reference to known confirmatory reactions, a method is described for the semi-quantitative determination of sterigmatocystin in fruit and vegetables. This method permits to detect 20 microgram/kg of food by means of thin-layer chromatography, after column-chromatographic purification on silica gel. The identity is confirmed by derivatization to the semi-acetal by treatment with trifluoro-acetic anhydride. The method is suited especially for the routine control of foods.
Animals↗
[Determination and the hygienic-toxicologic significance of patulin in fruit and fruit products].
The combination of extracting, chromatographic and fluorescence densitometric steps permits to determine patulin in fruits and fruit products with great precision and sensitivity. The limit of detection is 10 microgram/kg; the recovery rates range from 86 to 92% with a variation coefficient lying between 5.6 and 13.6%. Interferences due to patulin-simulating substances are widely excluded. Patulin concentrations ranging from 0.3 to 42 mg/kg were found in the brown-rotten portions of apples. Juices domestically prepared from healthy fruits and fruits rid of rotten portions, respectively, contained no patulin. On the contrary, patulin concentrations ranging from 0.02 to 0.3 mg/l were observed in commercial apple juices. As to products with subsequent mould infection, patulin values up to 50 mg/l were found in apple juices, and up to 0.4 mg/kg in peach preserves. In a further 24 commercial fruit and vegetable juices, patulin was not detectable even not in cider, in cereals which had gone mouldy spontaneously, and in bread samples. The fruits and fruit products were analysed not only for patulin, but also for aflatoxin and ochratoxin; the latter two, however, were found in none of the samples examined. The possibilities of manufacturing patulin-free products are discussed.
Chromatography, Thin Layer↗
[Determination of aluminum in foods].
In connection with studies on the changes of functional properties of food constituents (e. g. solubility of proteins) by means of aluminium compounds, the elaboration of an appropriate method for the quantitative determination of aluminium has been necessary. The organic samples are mineralized with 80% perchloric acid; in case of fat-containing foods, after fat removal. After reaction with aluminon (ammonium salt of aurin tricarboxylic acid), aluminium is determined photometrically at 530 nm. The limit of detection lies between 1 and 2 ppm; the recovery rate is 103%; the standard deviation is +/- 10%. The determination (without mineralization) requires about 1 hours. The authors analyzed, inter alia, milk (1.2--1.7 ppm A1), fat cheese (9 ppm), micora (4 ppm), aluminium-stabilized protein texturates (300--1500 ppm).
Aluminum↗
[Determination of residues of the fungicide benzimidazole carbamic acid methyl ester and its metabolites 2-aminobenzimidazole and benzimidazole].
The authors describe a method for extracting and determining benzimidazole carbamic acid methyl ester (MBC) in the presence of its metabolites 2-aminobenzimidazole and benzimidazole. The determination is performed thin-layer and paper chromatographically using visual spot comparison. A sensitive biological method is employed for the detection of MBC. The metabolites are assayed by chemical procedures. The smallest amounts of residues that the authors could detect were: 0.01 p.p.m. of MBC and approximately 0.2 p.p.m. of the metabolites, the recovery rate ranging from 70--85%.
Benzimidazoles↗
The use of microchemical techniques for the identification of new transmitter molecules in neurons.
The author reviews available microtechniques and results on cellular analysis of single neurons emphasizing the identification of putative neurotransmitter molecules. Highly sensitive microquantitative methods for identification and assay of known and putative neurotransmitters have been developed within the last 15 years. Several of the methods reported in the article fulfill the requirements of both specificity and sensitivity for exploring and measuring the level of transmitter molecules in single neurons. Some of the available techniques, mostly those which associate GC or TLC with MS, have been able to unambiguously demonstrate the presence of putative neurotransmitter molecules, or at least of substances which are strongly suspected of fulfilling a functional role in the nervous system.
Animals↗
Submicrogram assay for scopolamine in plasma and urine.
A GLC-mass spectrometric method for scopolamine, sensitive to 50 pg/ml for a 4-ml plasma or urine sample, was developed. The method used a deuterated internal standard to minimize variability in absolute recovery in the extraction procedure. Scopoline and deuterated scopoline were formed from the base-catalyzed hydrolysis of scopolamine and the internal standard and were analyzed as the heptafluorobutyrates, using a GLC-mass spectrometric system by monitoring the m/e 138 and 141 fragments, respectively.
Chromatography, Gas↗