Occurrence of intercellular bridges between follicle epithelial cells in the ovary of Apis mellifica queens. Hitherto unknown intercellular bridges or fusomes between the follicle epithelial cells investing the oocytes of Apis mellifica queens have been observed both with light and electron microscopy. Usually each follicle cell has 2-3 intercellular bridges. In surfacial paraffin sections, the intercellular bridges can be seen to connect a series of follicle cells which may be branching. The intercellular bridges lie close to the egg cortex and this position is relatively constant. The width of the fusomal ring canal varies in different developmental stages. In stages 3 and 4 of oogenesis, which are the main vitellogenic stages, the intercellular bridges measure 0-5 micron, while in stages 1 and 2 they have a diameter ranging from 1-5 to 3-5 micron. In these stages the intercellular bridges are provided with numerous transverse microfilaments which disappear later. The fusomal lips are thickened and consist of electron-dense material and an additional layer of less electron-dense material both inside and outside. Ribosomes flow across the bridge. The intercellular bridges may serve to synchronize the differentiation and functional activity of the follicle epithelium during the course of oogenesis.


Studies on the de novo formation of centrioles: aster formation in the activated eggs of sea urchin. Aster formation was studied in sea-urchin eggs artificially activated by Loeb's double method. The number of asters found in an activated egg ranged from 15-40. Observation by electron microscopy revealed the presence of a centriole in a high percentage of isolated asters, strongly suggesting that these centrioles are formed de novo in the activated eggs. Using negatively stained specimens, which were isolated with 5% hexylene glycol solution containing buffer and EGTA, the whole aster could be examined by electron microscopy. A microtubular framework in the aster, extending radially from the astral centre was observed. The effect of mitotic inhibitors on aster formation was found to be similar to that on cell division.


The analysis of malignancy by cell fusion. VIII. Evidence for the intervention of an extra-chromosomal element. Diploid human fibroblasts and lymphocytes were fused with the cells of a malignant mouse melanoma and a range of hybrid clones selected for study. The ability of these clones to produce progressive tumours was assayed in nude mice. Although human chromosomes were preferentially eliminated in all the hybrid clones, the human diploid cells were as effective as mouse diploid cells in suppressing the malignancy of the mouse melanoma cells. The suppression produced by fibroblasts was again more profound than that produced by lymphocytes. Malignancy was also found to be suppressed in a hybrid clone in which a single X was the only human chromosome present; and this clone continued to give a very low take incidence even after the human X had been eliminated by back selection. Hybrids were made between the melanoma cells and diploid human fibroblasts that had been given 100 J kg-1 of gamma radiation before cell fusion. These hybrids contained no recognizable human chromosomes, but their ability to produce progressive tumours was greatly reduced compared to that of the melanoma parent cells. The take incidences given by the crosses between the melanoma cells and the irradiated human fibroblasts were, however, substantially higher than those given by the crosses between the melanoma cells and unirradiated fibroblasts. These findings suggest that the suppression of malignancy involves the activity of some extra-chromosomal element and that this element is radio-sensitive.


Substrate retention of fractured retraction fibres during detachment of trypsinized BHK21 fibroblasts. When BHK21 fibroblasts adhering to glass were incubated in trypsin they became spherical within a few minutes. They did not, however, respond simultaneously; the most trypsin-resistant cells in an unsynchronized population were of greater length, had more processes projecting from the cell body, and were more spread out than the most trypsin sensitive cells. In no instance did trypsin detach cells from their substrate, even when the incubation period was prolonged to 5 h in trypsin concentrations almost sufficient to cause cell lysis. Scanning electron-microscope observations showed that initially flat cells rounded up in trypsin to reveal persistent adhesion sites joined to the cell body by retraction fibres. Such cell could be dislodged only by agitation; when this occurred parts of the cell remained attached to the substrate in the form of small spheres and fibres; these were remnants of the retraction fibres and adhesion sites. We propose that the adhesion sites are not susceptible to proteolytic degradation, presumably because of steric hindrance, and that this causes detachment to be dependent upon mechanical dislocation. We suggest that some of the descriptions of substrate-associated 'cell exudates' in the literature may refer to these cell remnants on the substrate which consist of membrane-bound fragments of cytoplasm rather than secreted products.


Fine-structural studies of the gametes and embryo of Fucus vesiculosus L. (Phaeophyta). III. Cytokinesis and the multicellular embryo. Condensation of the chromosomes during the first cell division following fertilization of the brown alga Fucus vesiculosus L. is accompanied by the almost complete disappearance of the nuclear envelope. Golgi vesicles and other small vesicles appear within the spindle, which has paired centrioles at each end. A large amount of rough endoplasmic reticulum is in the surrounding cytoplasm during mitosis, and many vesicles at the spindle margin are encircled by stacks of endoplasmic reticulum. Annulate lamellae are observed during mitosis. The envelope which initially reforms around the chromatin in telophase has unevenly spaced nuclear pores. Cytokinesis results primarily by vesicle addition to a centripetal furrow. Mitochondria and chloroplasts concentrate around the partition site, possibly in association with microfilaments. Fibrillar material is added rapidly to the space between the daughter cells from vesicle discharge of both cells and seems to spread into the older cell wall surrounding the embryo. The rhizoid daughter cell contains numerous mitochondria and hypertrophied Golgi bodies whose vesicles increasingly pack the cell. The thallus daughter cell is packed with a variety of vesicles, and the nucleus is surrounded by many dilated cisternae of rough endoplasmic reticulum. By the four-cell stage, chloroplasts of the rhizoid cells have weakly staining lamellae, while chloroplasts of the thallus cells are actively dividing with deeply staining lamellae.


Constraints on the relative sizes of the cell populations in Hydra attenuata. The steady-state relative population sizes of the several cell populations in Hydra  attenuata were examined. In contrast to the constant average population size ratios between groups of animals, these ratios vary within limits between individual animals within a group. By maintaining animals on different feeding regimes (number of shrimp larvae ingested per day), the steady-state population size ratios were altered. The kinds of changes that occurred in these ratios suggest where controls may be operating to maintain the steady-state population sizes.


Binding of the herbicide trifluralin to Chlamydomonas flagellar tubulin. Colchicine-binding tubulin was isolated from the centre 2 axoneme microtubules from the flagella of the alga Chlamydomonas eugametos. Incubating this tubulin with 5-8 micron [14C]trifluralin resulted in significant and reproducible binding of trifluralin to tubulin. The specificity of binding was determined by demonstrating that limited or no binding occurred to flagellar membrane and matrix protein, higher plant proteins, or bovine serum albumin. It was concluded that trifluralin, at obtainable soluble concentrations, specifically inhibits microtubule-mediated processes in plants. To date no effect of trifluralin on animal microtubule systems has been reported; therefore, it is suggested that trifluralin has revealed a pharmacological difference between plant and animal tubulin.


Infrared laser damage to ciliary motion in Phragmatopoma. A glass neodymium laser was modified to make it possible to produce small lesions of 1-2 micron size with a quantitatively known amount of energy. The 1-06-micron radiation of this laser is sufficiently absorbed by water to work without the additions of dyes. Ciliary arrest in Phragmatopoma gills was produced by an amount of energy, sufficient to cause a rise in temperature of 150 degrees C in an area of 2 micron3. At these low doses the effect was fully reversible. With higher doses of laser energy the cilia stopped permanently, probably because of structural damage of the irradiated cells.


Control of cell size and cycle time in Schizosaccharomyces pombe. Steady-state and perturbed cells of Schizosaccharomyces pombe have been observed through several division cycles by time-lapse photomicrography. Perturbed cells were produced by the use of a conditional cell division cycle mutant in which nuclear division is reversibly blocked at high temperature. These experiments show that in both populations cell length at division and cell cycle duration are homeostatically controlled, probably by a primary size-control mechanism. Cycle time is indirectly controlled, as cells which have an extended cycle are on average larger at division, so that duaghters of such cells need to grow by a smaller amount and for a shorter period, before dividing again. In general, deviations from the mean are corrected within a single cycle, but in the case of very long cells the control breaks down because the cycle cannot be shortened by more than a quarter under the conditions used. These cells take more than one cycle to return to normal.


Linear increase in glycolytic activity through the cell cycle of schizosaccharomyces pombe. The glycolytic activity of 3 different synchronous system in S. pombe was studied. Synchronous cultures were produced by a selection procedure, by cyclic heat treatment, or by cloning cells from an exponentially multiplying culture. In all experiments a complex medium with 3% glucose was used. The glycolytic activity was recorded with a gasometric method, the gradient diver. A single cell in exponential growth or a small number of synchronized cells were placed in ampulla divers in which the cells progressed undistrubed through a number of cycles. An ampulla diver is in principle a narrow pipette by which a single or a few cells are removed from the mother culture. It serves next as an axenic growth chamber and at the same time as a gasometer. The divers were placed in linear saline density gradients and the gaseous exchanges taking place in the divers resulted in migration of the divers. The migration rate is a measure of the glycolytic activity of the cells. Our results show that the glycolytic activity increases in a linear fashion between sucessive divisions. The rate of increase doubles at each division. This true in all 3 synchronous systems, and we take this as an indication that the cell cycles of heat-synchronized cells do not deviate seriously from the normal.


Extensive pairing of the XY bivalent in mouse spermatocytes as visualized by whole-mount electron microscopy. Autosomes and sex chromosomes of mouse spermatocytes were examined during zygotene, pachytene, and diplotene by a whole-mount electron-microscope technique after cell dispersion in a detergent solution (Nonidet-P40). Zygotene, pachytene, and diplotene stages can be adequately identified in the preparations. Thus, asynchronous side-by-side pairing of homologous autosomes, some of them displaying attached nucleoli, defines zygotene. Pachytene is identified by complete pairing of homologues. Diplotene is characterized by disjunction of bivalents (autosomes and sex chromosomes), lack of autosomal-attached nucleoli, divergent expansions observed at lateral element endings of disassembled synaptonemal complexes, end-to-end association of the XY pair and well defined outward deformations ('bulges') along sex chromosomal axial cores. X and Y chromosomes display at pachytene an extensive side-by-side pairing segment which decreases in length as meiotic prophase advances. Each sex chromosomal axial core appears double and is formed by close apposition of 2 nearly parallel elements displayed separately along the entire length of the chromosomal core. This double structural feature suggests that each sex chromosomal axial core is presumably composed of 2 chromatid axial cores, each of which, in turn, constitutes the respective lateral elements of short synaptonemal complexes observed at the unpaired segment.


The nucleolus and meiosis during microsporogenesis in Endymion non-scriptus (L.). Stages of meiosis from the bluebell Endymion non-scriptus (L.) were studied by electron microscopy. The segregated components of the nucleolus at meiotic prophase underwent fragmentation and dissolution at pachytene-diplotene. Nucleoli were absent during both meiotic divisions and reformed on the nucleolus organizer into a fibrillar mass from scattered fibrillar components at the dyad and tetrad stages. Ti is argued that the fibrillar region shows continuity through nuclear division though undergoing structural transformations in the process. Nucleolar reformation occurs on condensed nucleolus organizers. Processing of the ribosomal precursors and the resumption of RNA synthesis is discussed in relation to the dispersal of the nucleolus organizer into the fibrillar region of the reformed nucleolus.


Differentiation of the tapetum in Avena. I. The cell surface. The development of the tapetal cell surface and associated structures in Avena has been followed from cell formation to senescence. Plasmodesmata initially connect the tapetal cells to each other, the pollen mother cells, and the inner loculus wall cells. These connexions are subsequently severed, those to the sporogenous cells being broken first at the pollen mother cell surface during callose wall formation. Loss of cellulose from the tapetal walls was followed using the decline in the ability of the wall to bind the fluorescent brightener, Calcofluor White M2R New. Subplasma-membrane microtubules persist after loss of the cellulose wall. The tapetal plasma membrane facing the meiocytes then develops a series of depressions, or cups, over its surface, which are later the site of pro-orbicule formation. Sporopollenin is laid down over the pro-orbicules, to form orbicules, and over other tapetal cell surfaces. No morphological evidence was found for the intracytoplasmic formation of pro-orbicules or polymerized sporopollenin precursors. These observations on Avena are compared with those on other plants. The changes in the cell wall and associated structures, plasmodesmata and microtubules, are considered in detail, while the general significance of cell wall loss to the water relations of the tissue are assessed. Proposals that pro-orbicule formation results from non-specific accumulation of lipid at a free cell surface are rejected, instead this formation is considered to be related to the presence of a specially modified plasmamembrane surface.


Fusion and erosion of cell walls during confugation in the fussion yeast (Schizosaccharomyces pombe). Conjugation in Schizosaccharomyces pombe was studied by transmission electron microscopy. Mural and nuclear events were scored from induction, the initial event, to meiosis I, the start of sporulation. These morphogenic markers were separately identifiable as flocculation, copulation, conjugation-tube formation, cross-wall formation,  cross-wall erosion, conjugation-tube expansion, cytoplasmic fusion, de-differentiation of site of union, nuclear migration and karyogamy. The following were identified as new structural elements: sex hairs, which presumably mediate hydrogen bonding between cells during flocculation; crimp at the site of union; dark patch, which presumably serves as a leak-proof seal at the time of cross-wall erosion; suture, an electron-dense seam formed by the union of a copulant pair; and small electron-dense particles close to the site of wall erosion. No special structures on the cell wall could be identified as indicative of specific sites for potential copulatory activity. The discontinuity of the 2 cell walls at the site of union became so de-differentiated after fusion and erosion that it was no longer possible to pinpoint the site of union.


Pore-like structures in biological membranes. In freeze-fracture replicas, biological membranes appear as smooth surfaces interrupted by random globular protrusion, the intramembrane particles. Smooth areas correspond to the membrane phospholipidic domain, while intramembrane particles are the morphological counterpart of membrane proteins. In the present work, examination of membranes in a variety of cell types reveals that a number of intramembrane particles contain an electron-dense spot. The spot is thought to correspond to a minute pit in the particle, filled by the platinum used in the freeze-fracture procedure. Similar images, described previously in intramembrane particles forming the specific array of the gap junction, were interpreted as hydrophilic channels bridging the interior and the exterior of the plasma membrane. Comparison between the gap junction particles and the non-junction particles containing a dense spot suggests that these latter may too contain hydrophilic channels. The channels in random intramembrane particles would represent the morphological counterparts of the water-filled pores described in models of membrane permeability.


Invertebrate gap junctions.

Invertebrate gap junctions. Both glycerol and glutaraldehyde, the two most commonly used chemical aids in freeze-fractue studies, have been shown individually to affect the structure of certain membranes as observed in freeze-fracture replicas. The present investigation studied the effect of glycerol on the gap junctions found in a number of tissues from several invertebrate phyla. Glycerol was shown, in some of these tissues, to affect both the spatial arrangement of the particles within the membrane and their fracturing properties if the tissue had not been prefixed with glutaraldehyde. It is suggested that the effect of glycerol on invertebrate gap junctions may be of diagnostic use in enabling them to be separated into a number, or spectrum, of types. Previously B-type gap junctions have been reported only in Arthropoda. This study has extended knowledge of their occurrence into the phyla Coelenterata, Platyhelminthes and Annelida. The B-type gap junctions appear consistently to have a 12-nm particle separation, which is larger than the 10-nm separation found in vertebrate and molluscan A-type gap junctions.


Comparative study of axopodial microtubule patterns and possible mechanisms of pattern control in the centrohelidian heliozoa Acanthocystis, Raphidiophrys and Heterophrys. The axopodial microtubule pattern of 9 centrohelidians belonging to the genera Acanthocystis, Raphidiophrys and Heterophrys, as well as the fine structure of their microtubule organizing centre, the centroplast, was studied to determine the rules which govern their patterns. Microtubules capable of binding a xamimum of 4 linkers are arranged in regularly distorted hexagons and equilateral triangles. The number of microtubules present in each axoneme ranges from some 140 in Acanthocystis turfacea to as few as 6 in Heterophrys marina (Stock I). In the later species each axoneme contains a single hexagon of microtubules only. In other Heterophrys species, the central hexagon is surrounded by closely packed microtubules or by microtubules arranged in pentagons; only the central hexagon is anchored in the centroplast shell, whereas additional microtubules seem to originate from secondary nucleation sites somewhat distal to the centroplast. It is argued that the distortion of the basic unit hexagon (with alternate angles close to 134 degrees and 106 degrees) indicates that the microtubules are composed of 13 protofilaments. While in the larger Acanthocystis and Raphidiophrys species, the pattern may result from self-linkage, the arrays found in the Heterophrys species seem to favour a template-determined linkage. To explain the formation of the central hexagon in Heterophrys and balanced lateral growth in the larger microtubule arrays, a 'linker-nucleation hypothesis' is proposed. The assumption is made that graded conformational changes in the microtubule subunits not only specify the position where the next linker will bind, but that this linker, through linkage, becomes able to induce secondary microtubule nucleation, which will result in balanced lateral growth of the array. The application of this hypothesis to other microtubule systems, e.g. basal body formation, is discussed.


Response of preimplantation rat blastocysts in vitro to extracellular uterine luminal components, serum and hormones. The influence of extracellular environmental factors on preimplantation rat blastocysts was tested by determining the number of embryos which escaped from their zonae pellucidae, followed by attachment and outgrowth of trophoblast giant cells, after 72 h in culture Uterine luminal ocmponents from individual females, or hormones, were included in Dulbecco's medium which contained 4 mg/ml bovine serum albumin. In about 20% of cases, uterine fluids were embryotonic. However, uterine fluids from day-5 pregnant females, the day of implantation in the rat, were more potent in these tests than uterine fluids obtained from ovariectomized females treated with progesterone alone. The potency of a mixture of the 2 fluids was also high. Uterine fluids obtained at 14 h after an injection of oestradiol and progesterone to the ovariectomized females, were also effective in these tests. Rat serum and foetal calf serum were effective too, but steroids or insulin alone in the medium had no detectable influence on embryos. Serum or uterine luminal proteins appear to be essential for maintaining the viability of the blastocysts and for inducing the responses observed here. In the uterine fluids, some proteins released into the lumen after treatment of females with oestradiol and progesterone appear to be the biologically active components. Differences in the responses of blastocysts in vitro are compared with those in vivo.


Differentiated regions of human placental cell surface associated with exchange of materials between maternal and foetal blood: a new organelle and the binding of iron. To elucidate the method of uptake of a neutral iron compound into the placenta, isolated human chorionic villi were incubated in a medium containing the substance and were fixed at intervals and subsequently examined using transmission electron microscopy. None of the specimens examined showed evidence of vesicular transport in either micro- or macro-pinocytic vesicles which were either coated or smooth-surfaced. Iron uptake may involve attachment of particles to differentiated regions of the cell surface of the syncytiotrophoblast. These differentiated zones in the syncytial cell surface are composed of morphological distinct parts and therefore probably  merit classification as an organelle. Our interpretation of the organelle's structure is that it is flattened, with a maximum distance across of about 30 nm in the plane of the membrane. It is of otherwise uncertain shape but possesses a multilaminar structure. The external layer is apparently composed of the iron compound to be taken up. Beneath this is an electron-lucent layer possibly composed of glycoacalyx. The next innermost layer is continuous with the trilaminar unit membrane of the cell surface, however no trilaminar appearance is observed within the organelle. Beneath the membrane layer is an electron-dense plaque of amorphous material. Occasionally in favourable sections there is the suggestion of a thin electron-lucent strip interposed between the dense plaque and the membrane layer. The whole structure is composed therefore of 4 or 5 different regions. One possible function is that of initial binding in a process of active as opposed to passive transport of iron into the cell.


Differeniated regions of human placental cell surface associated with exchange of materials between maternal and foetal blood: coated vesicles. Coated vesicles may be an important component of the micropinocytic system of the human placenta. Regions of very dense reaction with glycocalyx stains are restricted to membranes within forming and fully formed coated vesicles. This is interpreted as evidence against permanently grouped specific binding sites having a role in the selective uptake of materials by micropinocytosis, and as support for theories of coated-vesicle formation which take into account the dynamic nature of membrane components. The pyroantimonate precipitation technique which was employed in an attempt to localize cations in placental tissue at term resulted in the deposition of electron-dense material in coated vesicles and basement membrane. Examination of the distribution of coated vesicles in placental tissue explants at 8--12 weeks of gestation revealed a restricted distribution of these organelles. Probably more than 89% of coated vesicles lie within the largest vesicles' diameter from the cell surface. Placental coated vesicles were isolated and examined using negative staining. A polygonally patterened structure was apparent on their surfaces. Analysis of the isolated fraction of coated vesicles using sodium dodecyl sulphate polyacrylamide gel electrophoresis shows the presence of a major protein of molecular weight 180000. This is the same molecular weight that has been given for clathrin, the major protein of the raised polygonally patterned structure on the cytoplasmic surface of coated vesicles from other sources.


Extracellular matrix synthesis in blastula and gastrula stages of normal and hybrid frog embryos. II. Autoradiographic observations on the sites of synthesis and mode of transport of galactose- and glucosamine-labelled materials. Pulse-chase labelling experiments and light- and electron-microscopic autoradiography were used to examine the sites of synthesis, mode of transport, and sites of deposition of galactose- and glucosamine-labelled materials in different developmental stages of normal developing Rana pipiens embryos and interspecific hybrid embryos formed by fertilizing the eggs of R. pipiens with the sperm of R. catesbeiana. In both normal and hybrid embryos, after 15-min pulse, grains are closely associated with juxtanuclear and cytoplasmic collections of membrane-bound vesicles which resemble the Golgi apparatus. In normal embryos following a 15-30 min pulse and a 60-min chase, grains are largely cleared from the cytoplasmic vesicles and deposited in the extracellular spaces or along cell surfaces where the extracellular spaces are relatively large. In contrast, arrested hybrid embryos given a 15-30-min pulse and a 60-min chase show a marked accumulation of grains over cytoplasmic structures such as the Golgi apparatus and vesicular elements in the cell cortex. Finally, early gastrula stage normal embryos are most active in the synthesis of galactose-labelled materials in cells above the dorsal lip of the blastopore, where cell migration is initiated.


Extracellular matrix synthesis in blastula and gastrula stages of normal and hybrid frog embryos. III. Characterization of galactose- and glucosamine-labelled materials. Normal Rana pipiens gastrulae show more incorporation of isotopically-labelled galactose and glucosamine into TCA-insoluble materials than blastulae. Interspecific hybrid embryos which undergo developmental arrest at the onset of gastrulation often synthesize reduced amounts of galactose- and glucosamine-labelled materials. These materials are high-molecular weight, but not collagen. After pronase digestion, labelled materials elute in the void volume of Sephadex G-50. Labelled materials migrate slowly on cellulose acetate, bind to several kinds of anion exchangers and elute at low ionic strength, are precipitated by cetyl pyridinium chloride (CPC) when mixed with carrier compounds, and are not degraded by Clostridium neuraminidase or Streptomyces, leech, and bovine testicular hyaluronidases.


Glycosyltrasferase activity in developing sea-urchin embryos. The developmental course of activity changes of 4 endogenous membrane glycosyltransferases: UDP-glucose : glycosyltrasferase, UDP-galactose : glycosyltransferase, GDP-mannose: glycosyltransferase, and UD-PN-acetylglucosamine : glycosyltransferase was determined in various stages of developing sea-urchin embryos from unfertilized eggs up to the 16-cell stage. The results show that there is a 2-fold increase in glycosyltransferase activity after fertilization. These results are discussed in relation to the postulated role of glycosyltransferases in mediating adhesion, recognition, and agglutination of cells and embryos.


Growth in vitro of tumour cell x fibroblast hybrids in which malignancy is suppressed. We have studied the growth in vitro of a lymphoma x fibroblast hybrid and several melanoma x fibroblast hybrids in which malignancy is suppressed. The parental cells, the hybrids, and malignant segregants derived from the hybrids were analysed for serum requirement, cloning efficiency in soft agarose, density-dependent inhibition of growth, and secretion of plasminogen-activating enzyme. One malignant segregant from the lymphoma x fibroblast cross was found by a number of criteria to have a more highly 'transformed' phenotype than the hybrid from which it was derived. However, in the case of the melanoma x fibroblast crosses, none of the parameters examined could be correlated in a direct way with malignancy.


Experimental results on mammalian cells growing in vitro in deuterated medium for neutron-scattering studies. SV-40 virus-transfromed human diploid fibroblasts (2RA) were grown in a monolayer on plastic Petri dishes in an aqueous medium deuterated to different concentrations of deuterium oxide: 10, 20, 30, up to 60%. The cells must be acclimatized to concentrations higher than 20% D2O by stepping them from a lower initial concentration during their exponential growth. The increase of cell doubling time with increasing deuterium concentrations seems to correlate, at least at 20% D20, with an initial period of suspended cell growth (lag-phase), and is qualitatively similar to that previously reported for Escherichia coli.


A substructural analysis method for structure-activity correlation of heterocyclic compounds using Wiswesser line notation. A method of substructural analysis for structure-property correlation of data sets including heterocyclic structures is described. Structural features allowing representation of occurrence and position of heteroatoms, ring fusions, and substituents are derived automatically from Wiswesser Line Notation representations. Structural feature sets of varying degrees of complexity may be derived, suitable either for sets of derivatives of a single ring system or for mixed sets containing several ring systems. The method is evaluated by correlating pKa values of 169 nitrogen heterocycles, including multisubstituted derivatives of 11 different ring systems. The technique could be carried out automatically with large machine-readable structure-property files and applied to a wide variety of properties.


Relative electron capture response of the 2-chloroethyl derivatives of some barbituric acids and anticonvulsant drugs. Data are given for the relative electron capture response of the 2-chloroethyl derivatives of ten barbituric acids and five common anticonvulsant drugs. Results indicate that these new derivatives significantly improve the detection limits of the barbituric acids but lead to no advantages for the anticonvulsant compounds. Structural features in the two classes of compounds are believed to explain the differences in the sensitivity of detection. Linear calibration plots exist for the concentration ranges 0.1-1.0 and 1.0-10.0 microgram/ml of amobarbital.


Gas chromatographic and gas chromatographic-mass spectrometric analysis of ampicillin. A method was developed for the quantitative gas chromatographic (GC) determination of ampicillin. The procedure requires silylation iwth hexamethyldisilazane, trimethylchlorosilane, trimethylsilylimidazole and N,O-bis(trimethylsilyl)acetamide in pyridine and subsequent GC on an OV-17 column, using 5 alpha-cholestane as an internal standard. The method was applied to the determination of ampicillin in some pharmaceutical products. The characteristics of the mass spectra and the derivatization GC of ampicillin are also discussed.


Separation and detection of phencyclidine in urine by gas chromatography. A rapid, sensitive and selective analytical procedure for phencyclidine and one of its metabolites in urine has bee developed. Three techniques have been studied for extraction of the drug from the biological matrix: (a) reversed-phase XAD resin, (b) charcoal absorption, and (c) solvent extraction using chloroform. Temperature-programmed gas chromatography was used to quantitate the illicit drug. Solvent extraction appears to offer the most efficient separation of the drug and its metabolite, as the recovery was 94% and the technique required only 7-8 min per sample. Reversedphase column extraction is also quite useful; although more time-consuming for an indivisual sample, it would be useful for screening purposes.


High-pressure liquid chromatographic analysis of drugs in biological fluids. IV. Determination of clofibrinic acid. A rapid, sensitive and specific high-pressure liquid chromatographic method is described for the quantitative analysis of clofibrinic acid in plasma, saliva and urine. In contrast to previously reported gas-liquid chromatographic methods, which require derivatization of clofibrinic acid before chromatography, the present method involves a simple two-step extraction procedure and chromatographic determination of the underivatized clofibrinic acid. Concentrations between 1.0 and 25.0 microgram per sample can be measured with a coefficient of variation from 1 to 6%.


The quantitative determination of metabolites of 6-mercaptopurine in biological materials. II. Advantages of a variable-wavelength HPLC spectrophotometric detector for the determination of 6-thiopurines. The advantages of a variable-wavelength spectrophotometric detector for use in high-performance liquid chromatography are demonstrated with the detection of 6-mercaptopurine metabolites in cell extracts. 6-Methylthiopurines, unsubstituted 6-thiopurines and 6-thioguanines are most sensitively detected at 291, 322, and 342 NM, respectively. Compared with detection at 254 nm, the sensitivity at these wavelengths is about one to two orders of magnitude greater. Furthermore, in the 291-355-nm range, common purines, which are normally contained in cell extracts as free bases, nucleosides and nucleotides exhibit only minute absorbances and so do not interfere in quantitative determinations of 6-thiopurine compounds.


Identification of selected antihypertensive drugs by thin-layer chromatography. A thin-layer chromatographic procedure is described for the qualitative identification of several antihypertensive drugs including certain thiazide diuretics spironolactone, triamterene, methyldopa and their metabolites. Utilization of new solvent developing systems and spray detecting reagents provides a method useful for the identification of these compounds in biologic fluids at low therapeutic concentrations. Sensitivity limits for these antihypertensive drugs are given, and alternate techniques to provide confirmatory analyses are also presented.


Determination of thiocyanate in tissues and body fluids of animals by gas chromatography with electron-capture detection. A method for the quantitative extraction of thiocyanate from biological material has been developed. Levels of 0.2-5 ppm of thiocyanate, in the presence of cyanide, were determined by gas-solid chromatography using 2-bromopropane as internal standard. Reliable results can be obtained only after careful deproteinization of the extracts with hot methanol. Cyanide can be eliminated by reaction with alkaline formaldehyde. The reproducibility of the determination of thiocyanate in biological extracts was +/-5%; the recoveries were over 90%. In vivo experiments with KS14CN-treated rats gave good agreement between the analytical results and the amount of thiocyanate determined by isotope dilution.


Detection of carbofuran and metabolites directly or as their heptafluorobutyryl derivatives using gas-liquid or high-pressure liquid chromatography with different detectors. The gas chromatography of the heptafluorobutyryl derivatives of carbofuran, 3-ketocarbofuran and 3-hydroxycarbofuran is examined with electrolytic conductivity detection (halogen mode). The reaction consists of heating the compounds with heptafluorobutyric anhydride in the presence of trimethylamine catalyst. Although as little as 200 pg of carbofuran, 400 pg of 3-ketocarbofuran and 100 pg of 3-hydroxycarbofuran can be detected by electrolytic conductivity, the minimum detectable quantities by electron capture are about 20-fold less for each derivative. Application to the analysis of carbofuran and 3-ketocarbofuran in corn, potato, turnip and wheat is carried out. This technique is compared to direct gas chromatography in the nitrogen mode and to direct high-pressure liquid chromatography with UV detection at 254 nm using extracts of field-treated turnips.


Determination of hydrazine metabolites of isoniazid in human urine by gas chromatography. A method is described for the determination of isoniazid, acetylisoniazid, acetylhydrazine, diacetylhydrazine and hydrazine in urine. Isoniazid, acetylhydrazine and hydrazine are reacted in aqueous solution  with p-chlorobenzaldehyde to form hydrazones. Following the addition of appropriate internal standards, these hydrazones are then extracted into an organic solvent and determined by gas chromatography using a nitrogen-sensitive detector. Acetylisoniazid and diacetylhydrazine are determined similarly after hydrolysis to isoniazid and acetylhydrazine, respectively.


High-pressure liquid chromatographic analysis of drugs in biological fluids. V. Analysis of acebutolol and its major metabolite. A high-pressure liquid chromatographic analysis for acebutolol and its major metabolite in blood, plasma and urine is reported. The analysis, in which the above mentioned compounds are chromatographed as ion pairs with dodecyl sulfonic acid, uses a simple and rapid method of sample preparation. The technique is more sensitive and rapid than those previously reported and it has equivalent or better reproducibility. The method is applied to the measurement in blood of acebutolol and its acetyl metabolite after a single oral dose.


Simultaneous analysis of bases, nucleosides and nucleoside mono- and polyphosphates by high-performance liquid chromatography. A single-column high-performance liquid chromatographic system for the simultaneous separation of bases, nucleosides and nucleoside mono- and polyphosphates has been developed, in which a strong porous anion-exchange resin (Aminex A-14) is used. The chromatographic run, carried out at 55 degrees and at alkaline pH by using a linear gradient both of ionic strength and pH, takes less than 225 min. The quantitative application of the described procedure to the analysis of cell nucleotide pools is reported.


Separation of alkyl derivatives of uracil by solvophobic adsorption chromatography on spheron. Derivatives of such related substances as cytosine, uracil, thymine, 6-methyl-uracil, 5-ethyluracil, 5-propyluracil, 5-isopropyluracil, 5-cyclopropyluracil, 5-allyluracil, 5,6-trimethyleneuracil, 6-cyclopropyluracil, 5-cyclobutyluracil and 5-tert-butyluracil have been separated on a column of Spheron P-300. Retention on the column was found to depend on the size of the non-polar part of the molecule. The chromatographic behaviour is analyzed according to the theory of solvophobic chromatography.


System for the identification of novel prostaglandins. A system for structural analysis based on thin-layer and gas chromatographic and mass spectrometric data is presented. It permits the rapid and unequivocal identification of structurally closely related prostaglandins and their products containing microbiologically introduced hydroxyl groups at positions C-16, -17, -18, -19, and -20. Novel criteria are described by which distinctions can be made between stereoisomers.


Mechanism of the reaction gas chromatography of phenylmercury(II) compounds. By combining radio-gas chromatography with liquid scintillation counting and with thin-layer chromatography-autoradiography, the conversion of symmetrical phenylmercury(II) compounds into phenylmercury(II) chloride and mercury(II) chloride in gas chromatography in the nanogram range has been demonstrated. The chlorine atoms necessary for this reaction are supplied by the Si-Cl-groups of cholorosilanized materials. The composition of the eluates depends on the concentration of the Si-Cl-groups.


Simple sensitive and specific gas chromatographic method for the quantification of diltiazem human body fluids. A gas cromatographic method for the determination of the benzothiazepine diltiazem together with its major metabolite desacetydilitiazem, is described. Silylation of the desacetyl derivative separates the metabolite from the parent drug on a 1% OV-17 column and cyclopam is used as an internal reference standard. The compounds are analysed by means of a nitrogen detector which allows the determination of 10 ng/ml of both compounds in plasma. The method has been used to determine both diltiazem and its desacetyl derivative in plasma obtained from healthy volunteers after oral doses of 60-210 mg of diltiazem.


Solvent suppression of the decomposition of phenobarbital during on-column methylation with trimethylanilinium hydroxide. Gas-liquid chromatographic (GLC) analysis of phenobarbital by on-column methylation with trimethylanilinium hydroxide gives rise to a major decomposition product, N-methyl-2-phenylbutyramide, in addition to the methylated barbiturate, N,N'-dimethylphenobarbital. This reaction occurs nearly exclusively in the solution phase in the injection port of the gas chromatography. A mechanism for the decomposition reaction consistent with the available information is presented. This decomposition is shown to be inhibited by certain solvents and this effect forms the basis of a new analytical technique for the simultaneous GLC analysis of phenobarbital, primidone, and diphenylhydantoin.


Determination of paraoxon by combined bovine liver esterase inhibition and gas-liquid chromatography. A combined gas-liquid chromatography and enzyme procedure is described for the determination of organophosphorus compounds that show anticarboxylesterase activity in vitro. The method is based on the enzyme hydrolysis of 1-naphthyl acetate and on the determination of both the release 1-naphthol and the unhydrolyzed 1-naphthyl acetate by gas-liquid chromatography. With bovine liver as the source of enzyme the detection limit for paraoxon was 13 ppb.


Column liquid chromatography of tricyclic antidepressants. A column liquid chromatographic system for the analysis of tricyclic antidepressants in serum is described. A high separation efficiency can be obtained with a mixture of ethyl acetate, n-hexane and methylamine as eluent on a silica gel column. The retention is easily regulated by varying the concentration of n-hexane, the modifier methylamine and the water content of the ethyl acetate. Examples are given of separation of test mixtures of tricyclic antidepressants and of some of these drugs in serum. UV detection permits determinations down to the 10-ng level in serum.


Rapid, sensitive gas-liquid chromatographic method for determination of amitryptyline and nortriptyline in plasma using a nitrogen-sensitive detector. A simple gas chromatographic method for determination of amitriptyline and nortriptyline in plasma using a nitrogen-sensitive detector is described. Concentrations of both drugs as low as 10 ng per ml plasma were measured. The precision and accuracy of the method are within acceptable limits.


Specific and sensitive method for the determination of gamma-aminobutyric acid using gas chromatography with electron-capture or mass fragmentographic detection. A gas chromatographic method for the determination of gamma-aminobutyric acid (GABA) in brain tissue is described. After microwave fixation, the brains were dissected and homogenized in 0.1 N formic acid; delta-amino-n-valeric acid (AVA), a homologue of GABA, was then added as an internal standard. After centrifugation, aliquots of the supernatant were treated with cation-exchange paper to adsorb the amino acids. The eluates of this paper were dried and the residues subjected to reaction with trifluoroacetic anhydride and hexafluoroisopropanol. After removal of the derivatization reagents by evaporation, the residues were dissolved in ethyl acetate and an aliquot was analysed by gas chromatography with electron-capture or mass fragmentographic detection. Quantitation can be carried out by either peak-height or peak-area measurements. The specificity of this method has been demonstrated with brain tissue by simultaneous mass fragmentographic analysis. The sensitivity is comparable to that of mass fragmentographic methods and is in the femtomole range. The method is simple and readily automated.


High-performance liquid chromatographic analysis of partial glycerides and other technical lipid mixtures. A high-performance liquid chromatographic (HPLC) technique for the analysis of partial glyceride mixtures is described and some technical details are discussed. Gradient elution HPLC with a "moving-wire" detector linked to a computer is used for quantitative analysis. The method described has been applied to the analysis of mono-, di- and triglyceride mixtures, food emulsifiers, certain seed fats, and technical products such as polymer additives.


[Contribution to the standardization of results in paper and thin-layer chromatography (author's transl)]. Chromatographic indexes that facilitate a better reproducibility of paper chromatographic results have been proposed, using the homologous series of coloured derivatives of secondary amines as standard substances. When using dimethylformamide as the stationary phase the value of the indexes proved to be independent of the concentration of the impregnating solution of the stationary phase, of the temperature changes during the chromatographic process, of the ways of impregnation and of the skills of workers. It depends only on the type of the stationary phase and on the composition of the mobile phase. The obtained indexes can contribute to a better reproducibility of RF values and to an easier and more objective identification of substances.


Gas chromatographic method for the determination of free and total solanesol in tabacco. Solanesol, a trisesquiterpenoid alcohol in tobacco leaf, has been shown to be an important precursor of the tumorigenic polynuclear aromatic hydrocarbons of smoke. Thus, a rapid, reproducible method for the determination of leaf solanesol levels is desirable. We developed procedures based on high-temperature gas chromatography for the analyses of free and total solanesol. The alcohol, as its trimethylsilyl derivative, was separated and quantified on a short Dexsil 300 GC column, with 1,3-dimyristin as an internal standard. The free alcohol was determined by direct derivatization of ground tobacco and its hexane extract with N,O-bis(trimethylsily)-acetamide-dimethylformamide reagents. For total solanesol, the ground tobacco or its hexane extract was saponified with ethanolic potassium hydroxide. Acidification and hexane extraction yielded samples suitable for silylation and gas chromatographic analysis. Evaluation of the various methods indicated that free solanesol was best determined in the hexane extract of tobacco, and total solanesol after saponification of ground tobacco. Total solanesol levels in commercially important tobacco types were determined.


Quantitative determination of the herbicide paraquat in human plasma by gas chromatographic and mass spectrometric methods. The gas chromatographic (GC) determination of the herbicide paraquat, the 1,1'-dimethyl-4,4'-dipyridyl cation in human plasma is described. In poisoning cases, plasma concentrations provide a necessary index of the severity of intoxication and a means of monitoring subsequent therapy. The methods may be extended to the specific trace analysis of paraquat in body fluids of post-mortem tissue. Reduction of fully ionised paraquat salts with sodium borohydride yields a hexahydro derivative, a diene, amenable to solvent extraction and GC. Employing 1,1'-diethyl-4,4'-dipyridyl dichloride as the internal standard, plasma concentrations of 0.1 microgram/ml (+/- 6% S.D.) may be determined with flame ionisation detection and 0.025 microgram/ml with nitrogen-selective flame ionisation. Further enhancement of specificity is achieved using selected ion monitoring mass spectrometry and the value of this technique in forensic analysis is illustrated.


Determination of the rodenticide difenacoum in biological materials by high-pressure liquid chromatography with confirmation of identity by mass spectrometry. A method for determining difenacoum in liver, plasma, urine and feedingstuffs by high-pressure liquid chromatography is described. Samples are cleaned up by molecular exclusion chromatography on porous glass. In some cases this also serves for determination; if not, the separated difenacoum is determined on an adsorption column. Identity is confirmed by chemical ionisation mass spectrometry. Recoveries at levels of 0.025-5 ppm from plasma were 101-113% by exclusion chromatography alone and 93-101% after adsorption chromatography. Recoveries from liver after both chromatographic steps were 62-86%. Reasons for the lower recoveries from liver are suggested.


Chromatographic behaviour of nucleic acid constituents and of phenols on chitosan thin layers. The chromatographic characteristics of several nucleic acid constituents and of 36 phenols have been investigated on mixed layers of powdered chitosan and microcrystalline cellulose, with water, water-methanol mixtures and aqueous salt solutions at different pH values at mobile phases. The behaviour of the phenols was strongly correlated with the form in which these compounds were present in solution and therefore with the pH of the eluent. Chitosan was more effective than PEI- and DEAE-cellulose as adsorbent in separating phenols. Analytical applications of chitosan layers are reported.


Dansyl hydrazine as a fluorimetric reagent for thin-layer chromatographic analysis of reducing sugars. Reducing sugars, particularly aldoses, react readily with dansyl hydrazine. The fluorescent hydrazones produced can be separated by thin-layer chromatography and determined quantitatively by spectrofluorimetry after elution from the chromatograms.


[Gas chromatographic determination of the volatile fatty acids (C1-C5) including lactic acid after conversion into their benzyl esters by phenyldiazomethane. Determination of the acids in silages (author's transl)]. A gas chromatographic method for the determination of the volatile fatty acids (C1-C5) including lactic acid is described. The acids are converted into their benzyl esters by means of phenyldiazomethane without previous purification of the reagent by distillation. The benzyl esters are well separated from the solvent as well as from one another. Silicone SE-30 is used as the stationary phase. An application of the method on silage acids is demonstrated.


Gas chromatographic determination of methylguanidine, guanidine and agmatine as their hexafluoroacetyletonates. Seven derivatives of each of methylguanidine, guanidine and agmatine have been prepared, and the specificity and volatility of their gas chromatographic detection have been studied. The hexafluoroacetylacetonates have been found to be the most specific for the three guanidines, and are highly sensitive to alkali flame ionization and electron-capture detections. These derivatives are also fairly resistant to hydrolysis occurring in the derivatization process.


Gas chromatographic determination of apomorphine in plasma. A method is described for the analysis of 1 to 10 microgram/ml concentrations of apomorphine in plasma. The procedure is based on ethyl acetate extraction, a back extraction cleanup-step, derivatization with heptafluorobutyric anhydride, and gas chromatography on a 3% OV-17 column using flame ionization detection. N-n-Propylnorapomorphine is employed as an internal standard and quantitative relative recoveries of drug are realized with relative standard deviations of 4.6%. The method permits analysis of apomorphine in the presence of its two monomethyl ether metabolites, apocodeine and isoapocodeine. The latter compounds are also chromatographically resolved as their heptofluorobutyrate derivatives.


Quantitative determination of thebaine in Papaver bracteatum by high-pressure liquid chromatography. A method is described for the quantitative analysis of thebaine from Papaver bracteatum, using a single high-pressure liquid chromatographic column. The procedure gives base-line separation of thebaine without the need for gradient elution equipment, and can be completed within 12 min. Thebaine isolated by this technique was shown to be pure, regardless of the age of plant or plant part from which it was obtained.


Sensitive thin-layer chromatographic method for urine screening of barbiturates. A simple and sensitive thin-layer chromatographic (TLC) method is described for the detection of barbiturates and other sedatives at therapeutic levels in the urine. The method consists of an extraction followed by TLC on a single plate in a solvent system which separates the barbiturates from most of the other drugs. The upper portion of the plate is sprayed with a new visualization reagent, N,2,6-trichloro-p-benzoquinone imine, followed by heating. The barbiturates appear as blue spots. This procedure is capable of detecting barbiturates at levels of 0.1 mg/dl in urine.


[Separation and fluorimetric determination of adrenaline and noradrenaline. Combination of a high-pressure liquid chromatograph with an automatic analysis system]. The possibilities for a high-pressure liquid chromatographic analysis combined with automatic fluorimetric detection of the catecholamines adrenaline and noradrenaline are described. The optimal conditions are given for a fast separation by ion exchange and reversed-phase chromatography, and for the sensitive fluorimetric determination of adrenaline by the trihydroxyindole technique when a high excess of noradrenaline is present.


Methods for the rapid separation and estimation of the major lipids of arteries and other tissues by thin-layer chromatography on small plates followed by microchemical assays. Methods are described for the rapid separation of the major individual phospholipids and neutral lipids of tissues by thin-layer chromatography on small glass plates (75 X 75 mm), and for the specific microchemical estimation of separated lipids and for determination of fatty acid composition and radioactivity. The overall method, involving tissues extraction, thin-layer chromatographic separation and assay has been evaluated using pure standards and biological samples and gives good reproducibility and almost complete recovery of lipids.


Determination of amitriptyline and some of its metabolites in blood by high-pressure liquid chromatography. Conditions for the determination of amitriptyline and some of its metabolites in serum on a reversed-phase material (C-8) by high-pressure liquid chromatography with UV detection at 254 nm were systematically investigated. The separation of tricyclic antidepressants is best carried out on a phase system consisting of C-8 bonded-phase material as the stationary phase and water--methanol--dichloromethane--propylamine as the mobile phase. The precision and detection limit of the method and the extraction efficiency were established. A chromatogram of a serum extract from a patient treated with amitriptyline is shown. Serum levels of amitriptyline and its four main metabolites (nortriptyline, desmethylnortriptyline, trans-10-hydroxy-amitriptyline and trans-10-hydroxy-nortriptyline) in a patient receiving 150 mg of amitriptyline daily, are reported.


Characterization of human expired air: a promising investigative and diagnostic technique. Expired air samples have been collected from a carefully selected population of normal healthy human subjects under controlled experimental conditions. The samples were concentrated and analyzed by quantitative, reproducible and sensitive techniques which resulted in well-defined composite compositional and occurrence profiles of the organic constituents present in normal expired air. The composite profiles provide valuable baseline information upon which suitable correlation studies may be advanced toward the use of human expired air for diagnostic purposes.


A systematic approach to thermal conductivity detector design I. Use of computer-aided data acquisition for the evaluation of cell contributions to peak shape. The design of thermal conductivity detectors for open tubular gas chromatography was systematically investigated with the aid of modern digital data acquisition techniques. Two important factors in such a design are sensitivity and peak broadening. The latter factor was the principle thrust of this work and contributions to peak shape from both cell design and volume were analyzed by the method of statistical moments. Peak variance (the mean square displacement along the x-axis of a chromatographic peak) was shown to correlate directly with cell volume in cells of the same design but both variance and asymmetry were found to be design dependent.


Erucic acid in edible fats and oils: a collaborative study on determination by open-tubular (capillary) gas-liquid chromatography. An open-tubular (capillary) column gas-liquid chromatographic method for the determination of the specific isomer of docosenoic acid known as erucic acid (cis-docos-13-enoic) in the presence of other docosenoic acid isomers present in partially hydrogenated marine oils has been evaluated collaboratively. With wall-coated columns and the liquid phase SILAR-5CP, nine laboratories successfully analysed mixtures of partially hydrogenated marine oils, corn oil and rapeseed oil with a nominal content of 10% erucic acid, compatible with the regulations of the European Economic Community.


Sampling and extra-column effects in high-performance liquid chromatography; influence of peak skew on plate count calculations. Chromatographic resolution and analysis accuracy can be seriously affected by broadening of very sharp bands associated with both high-performance size-exclusion chromatography and other liquid chromatography methods using less than 5 micron particles. The effect on column plate count by extra-column band broadening from injection systems, detectors, connectors and "guard" columns is discussed and exemplified with experimental data. The special cases of tailing and doublet peaks for a single species are investigated. These effects are eliminated by a special sample injection technique. Computer simulation studies demonstrated that serious errors can result from calculating column plate count with non-Gaussian peaks. The maximum allowable peak tailing is suggested and a new method for quantitating peak skew is proposed.


GC-MS measurement of deuterium in succinic anhydride and TMS-succinate. An isotope memory effect with succinic anhydride. Direct GC-MS of succinic and glutaric anhydrides has been investigated, and shown to be feasible. In the GC-MS determination of deuterium in succinic anhydride, an isotope memory effect was observed, shown to be due to strong adsorption of solute onto the column. This phenomenon was investigated, together with possible methods for its suppression. Succinate TMS ester also was found to be a suitable derivative for direct GC-MS of deutero-succinic acid.


Analysis of some drug substances by high-performance liquid chromatography. A high-performance liquid chromatographic system using a silica gel column and methylene chloride:methanol:ammonium hydroxide as a solvent for the analysis of some drug substances is described. The procedure described is suitable for the analysis of pharmaceutical dosage forms.


Electron capture gas chromatographic analysis of the amine metabolites of pesticides: derivatization of anilines. A number of amines have been shown to result from metabolism of various pesticides. From an epidemiological standpoint, it may be possible to monitor human exposure to these pesticides through the excretion of their corresponding amines in urine. An investigation has been initiated to develop and apply methods of analysis of amines in human urine. The results of a survey of derivatization techniques involving several substituted anilines are presented. These include conditions for derivatization, utilizing a number of halo- and nitro- substituted reagents; electron capture and gas chromatographic properties of the derivatives; and stability of the derivatives to extraction and column chromatography for purposes of separation and cleanup. The recoveries of anilines from spiked water and urine samples at the 1.0 ppm and 0.1 ppm levels were between 85 and 90%. The advantages and disadvantages of the various derivatives and techniques are discussed and a rationale is presented for the preliminary selection of a particular derivative for application of the analysis of aniline metabolites in urine.


Bactericidal effect of anaerobic broth exposed to atmospheric oxygen tested on Peptostreptococcus anaerobius. Peptostreptococcus anaerobius strain VPI 4330-1 was used as the test organism in an evaluation of the bactericidal effect of anaerobic broth exposed to air. The test organism, grown under anaerobic conditions in Trypticase soy broth, was diluted in buffered salt solution, and about 2 x 10(4) cells were suspended in 10 ml of an aerated broth. Ninety percent of the cells were killed within 15 min in actinomyces broth and within 50 min in Trypticase soy broth. All cells survived for 2 h in fluid thioglycolate medium. Addition of DABCO [1,4-diazabicyclo (2.2.2) octane] or mannitol to Trypticase soy broth did not influence the death rate of the organism, whereas superoxide dismutase decreased the death rate. Addition of catalase or manganese dioxide to the broth kept all the cells viable for 2 h. Of the three broth media tested, actinomyces broth reduced oxygen at the highest rate and Trypticase soy broth reduced it at the slowest rate. Hydrogen peroxide could be demonstrated in actinomyces broth and in Trypticase soy broth but not in fluid thioglycolate medium. In addition to catalase, manganese dioxide also removed all hydrogen peroxide from Trypticase soy broth, and superoxide dismutase significantly decreased the concentration of hydrogen peroxide in the broth. The results suggest that hydrogen peroxide mediated the toxic effect of atmospheric oxygen in these broth media.


Cellular fatty acid composition of isolates from Legionnaires disease. The cellular fatty acids of four isolates from Legionnaires disease and two antigenically related isolates were identified by gas chromatography, mass spectrometry, and associated techniques. The six isolates had essentially the same fatty acid composition, which was characterized by large amounts (greater than 80%) of branched-chain acids.


Statistical determination of endotoxin content in influenza virus vaccine by the limulus amoebocyte lysate test. To determine laboratory-to-laboratory variability of the limulus amoebocyte lysate (LAL) test for evaluating endotoxin content in influenza virus vaccine, a collaborative study was designed. Participants were six influenza virus vaccine manufacturers and the Bureau of Biologics (BoB). Lysate lot 116, Reference Influenza Virus Vaccine for Endotoxin Assay E-1, and four test vaccines having different ratios of LAL activity relative to reference E-1 were supplied by the BoB. Each laboratory used its normal test procedure. All vaccines were coded. One pair (reference and test) of vaccines per day was tested for 4 days a week over a 4-week period. All data were analyzed at the BoB. The degree of variability experienced by testing laboratories was estimated by the study. This estimate did not conflict with experience gained from previous routine testing in any of the participating laboratories. A statistical approach to the evaluation of LAL data from testing influenza virus vaccine for endotoxin content was developed based upon the overall variation obtained from the collaborative study.


Aeromonas hydrophila in acute diarrheal disease: detection of enterotoxin and biotyping of strains. Eleven isolates of Aeromonas species from human stool cultures were found to produce enterotoxin activity as determined by assay of culture filtrates in rabbit intestinal loops and rabbit skin and on adrenal Y1 cells. Hemolysin(s) and a cytotoxic protein were found to interfere in all three assay systems but could be inactivated upon heating at 56 degrees C or by specific antihemolysin. Biotyping of each isolate was performed with a conventional test system and with API 50E and APIZYM kit systems (Analytab, Inc.). No single test of the more than 70 biochemical reactions investigated was found to correlate with enterotoxigenicity in the strains of Aeromonas hydrophila examined. All strains were found to belong to ideal phenotypes of A. hydrophila, but each strain possessed its own biochemical profile.


Degradation of parathyroid hormone and fragment production by the isolated perfused dog kidney. The effect of glomerular filtration rate and perfusate CA++ concentrations. The renal degradation of intact bovine parathyroid hormone (b-PTH 1-84) was studied with the isolated perfused dog kidney. Disappearance of b-PTH 1-84 from the perfusate occurred concomitantly with the appearance of smaller molecular weight forms of immunoreactive parathyroid hormone (PTH). These smaller molecular weight PTH fragments included both carboxyl and amino terminal regions of the PTH peptide. Perfusate from kidneys with lower glomerular filtration rates (GFR) contained b-PTH 1-84 for longer periods of time than kidneys with higher GFRs, and perfusate from kidneys with lower GFRs demonstrated greater accumulation of carboxyl terminal PTH fragments. Perfusate containing high Ca++ concentrations retarded, and perfusate with low Ca++ concentrations accelerated the rate of degradation of b-PTH 1-84 by the kidney. These studies, therefore document the production of PTH fragments during the course of intact hormone degradation by the kidney. They also demonstrate renal clearance of the PTH fragments produced and define the effects of glomerular filtration rate and calcium concentrations on degradation of intact hormone and the clearance of PTH fragments.


Pharmacologic and hemodynamic influences on the rate of isovolumic left ventricular relaxation in the normal conscious dog. We studied the effects of acute pharmacologic and hemodynamic interventions on isovolumic left ventricular relaxation in 19 conscious dogs using micromanometer tip catheters. Isoproterenol (11 studies) augmented peak rate of rise of left ventricular pressure [(+) dP/dt] by 1,275+/-227 (SE) mm Hg/s (P < 0.001) and dP/dt at an isopressure point of 35 mm Hg during isovolumic relaxation [(-) dP/dt(35)] by 435+/-80 mm Hg/s (P < 0.001). Peak (-) dP/dt decreased by 467+/-89 mm Hg/s (P < 0.002). The time constant, T, derived from the logarithmic fall of pressure during isovolumic relaxation, shortened from 20+/-2.8 to 14.9+/-1.8 ms (P < 0.003). Calcium (11 studies) increased peak (+) dP/dt and (-) dP/dt(35) (both P < 0.0001); peak (-) dP/dt was unchanged. T shortened from 20.4+/-1.8 to 17.3+/-1.5 ms (P < 0.002). Volume (13 studies) did not affect either dP/dt or T. Phenylephrine (13 studies) augmented peak (-) dP/dt, but reduced (-) dP/dt(35) (both P < 0.01); T lengthened from 22.1+/-1.5 to 32.5+/-1.5 ms (P < 0.01). In 15 studies, rapid atrial pacing increased peak (+) dP/dt and (-) dP/dt(35) (both P < 0.01). In the first post-pacing beat, peak (-) dP/dt and (-) dP/dt(35) decreased (both P < 0.01), although peak (+) dP/dt increased further. T paralleled values of (-) dP/dt(35). In five dogs, beta adrenergic blockade had no significant effect on any variable after calcium, volume, or phenylephrine infusion or during or after atrial pacing when the pre-and post-propranolol states were compared. We conclude that positive inotropic interventions augment both left ventricular contraction and relaxation. The changes in isovolumic relaxation are independent of alterations in sympathetic tone produced by beta-adrenergic blockade. Peak (-) dP/dt may not be a valid measure of left ventricular relaxation rate during acute alterations in inotropic state or afterload.


Prekallikrein deficiency in a kindred with kininogen deficiency and Fitzgerald trait clotting defect. Evidence that high molecular weight kininogen and prekallikrein exist as a complex in normal human plasma. Plasma from an individual with a hereditary deficiency of kininogens is deficient in kininogen antigens; heterozygous relatives are partially deficient in plasma kininogen antigens. In addition, plasma from the proband is partially deficient in functional and antigenic properties of a plasma prekallikrein, and the relatives heterozygous for kininogen deficiency are also partially deficient in the plasma prekallikrein. It is possible that the defects are both inherited and that the inheritance of a deficiency of prekallikrein is genetically linked to the inheritance of a deficiency of kininogen. Alternatively, it is possible that the deficiency of prekallikrein may be due to its hypercatabolism which could be a consequence of a deficiency of high molecular weight kininogen that may stabilize the prekallikrein in plasma. Evidence to support this possibility is presented by the fact that prekallikrein and high molecular weight kininogen apparently exist as a complex in normal plasma, because monospecific antiserum to kininogen removed both high molecular weight kininogen and prekallikrein from plasma, and vice versa. Moreover, prekallikrein was not adsorbed from kininogen-deficient plasma by antiserum to kininogen unless high molecular weight kininogen was first added to the plasma. Low molecular weight kininogen did not participate in these reactions.


Purification of high molecular weight kininogen and the role of this agent in blood coagulation. Recent studies of individuals with high molecular weight (HMW) kininogen deficiency established the importance of this plasma protein for in vitro initiation of blood coagulation. In the present study, HMW-kininogen was highly purified from human plasma by monitoring its clot-promoting activity, using Fitzgerald trait plasma as a substrate. This preparation of HMW-kininogen revealed a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (mol wt: 120,000) and released 1% of its weight as bradykinin upon incubation with plasma kallikrein. HMW-kininogen specifically repaired impaired surface-mediated plasma reactions of Fitzgerald trait plasma, but did not affect those of Hageman trait and Fletcher trait plasma. Kinin release from HMW-kininogen by trypsin, but not by plasma kallikrein, resulted in total loss of clot-promoting activity. No inhibitors of coagulation were found when all kinin activity was removed from HMW-kininogen by trypsin. The roles of HMW-kininogen, Hageman factor (HF, Factor XII), plasma prekallikrein (Fletcher factor), and plasma thromboplastin antecedent (PTA, Factor XI) in blood coagulation were studied in a purified system. HMW-kininogen was absolutely required for activation of PTA by HF and ellagic acid. The yield of activated PTA was proportional to the amount of HF, HMW-kininogen, and PTA in the mixtures, suggesting that, to activate PTA, these three proteins might form a complex in the presence of ellagic acid. No fragmentation of HF was found under these conditions. In contrast to HF, HF-fragments (mol wt: 30,000) activated PTA in the absence of HMW-kininogen and ellagic acid. Thus, it appears that in the present study PTA was activated in two distinct ways. Which pathway is the major one in whole plasma remains to be determined.


Small airways in idiopathic pulmonary fibrosis. Comparison of morphologic and physiologic observations. 18 patients with idiopathic pulmonary fibrosis were studied to determine if they had morphologic evidence of small airways disease and if physiologic testing could predict morphologic findings. In the presence of normal airway function by standard physiologic studies (forced expiratory volume in 1 s/forced vital capacity and airway resistance by plethysmography), dynamic compliance, maximum expiratory flow-volume curves, and maximum flowstatic recoil curves were measured to detect physiologic alterations consistent with small airways abnormalities. These physiologic data were then compared with estimates of small airways diameter made in lung biopsy specimens.94% (17 of 18) of the patients had peribronchiolar fibrosis or peribronchiolar inflammation or bronchiolitis. 67% (12 of 18) had an overall estimate of small airways diameter of "narrowed," whereas 33% (6 of 18) had airways that overall were "not narrowed." 59% (10 of 17) had frequency-dependent dynamic compliance, 50% (9 of 18) had abnormal maximum expiratory flow-volume curves, and 39% (7 of 18) had abnormal maximum flow-static recoil curves. Comparisons between morphologic and physiologic data revealed a significant correlation between the results of dynamic compliance and the overall estimate of small airways diameter (P = 0.001), and the results of maximum flow-volume curves and the overall estimate of small airways diameter (P = 0.009); there was no significant correlation between the results of maximum flow-static recoil curves and the overall estimate of small airways diameter (P = 0.1). THE RESULTS OF THIS STUDY SUGGEST THAT: (a) idiopathic pulmonary fibrosis is a disease of small airways as well as alveoli; (b) dynamic compliance and the maximum expiratory flow-volume curve can predict the overall status of small airways diameter in idiopathic pulmonary fibrosis; and (c) whereas the maximum flowstatic recoil curve predicts the overall estimate of small airways diameter in most patients with this disease, it is the least sensitive of these three monitors of small airways.


On the mechanism of polyuria in potassium depletion. The role of polydipsia. The association of potassium (K) depletion with polyuria and a concentrating defect is established, but the extent to which these defects could be secondary to an effect of low K on water intake has not been systematically investigated. To determine whether hypokalemia has a primary effect to increase thirst and whether any resultant polyuria and polydipsia contribute to the concentrating defect, we studied three groups of rats kept in metabolic cages for 15 days. The groups were set up as follows: group 1, normal diets and ad lib. fluids (n = 12); group 2, K-deficient diet on ad lib. fluids (n = 12); and group 3, K-deficient diet and fluid intake matched to group 1 (n = 14). Daily urine flow and urinary osmolality of groups 1 and 3 were not significantly different throughout the study. In contrast, as of day 6, group 2 rats consistently had a higher fluid intake (P < 0.0025), higher urine flow (P < 0.001), and lower urinary osmolality (P < 0.001) than the other two groups. These alterations in fluid intake and urine flow preceded a defect in maximal concentrating ability. On day 7, maximal urinary osmolality was 2,599+/-138 msmol/kg in rats on K-deficient intake and 2,567+/-142 msmol/kg in controls. To determine whether this primary polydipsia is itself responsible for the development of the concentrating defect, the three groups of rats were dehydrated on day 15. Despite different levels of fluid intake, maximal urinary osmolality was impaired equally in groups 2 and 3 (1,703 and 1,511 msmol/kg, respectively), as compared to rats in group 1 (2,414 msmol/kg), P < 0.001. We therefore conclude that K depletion stimulates thirst, and the resultant increase in water intake is largely responsible for the observed polyuria. After 15 days of a K-deficient diet, the impaired maximal urinary concentration in hypokalemia, however, was not related to increased water intake, since fluid restriction did not abolish the renal concentrating defect.


Role of 1,25-dihydroxyvitamin D3 on intestinal phosphate absorption in rats with a normal vitamin D supply. In vitamin D-deficient rats, impaired intestinal phosphorus (P) absorption can be corrected by 1,25-dihydroxyvitamin D(3)[1,25-(OH)(2)D(3)]. In the present study, it was investigated whether changes in 1,25-(OH)(2)D(3) production can influence intestinal P transport also in animals with a normal supply of vitamin D. The intestinal P absorption was evaluated in rats using both the in situ duodenal loop technique and the determination of the overall gastrointestinal absorption under three conditions known to influence the production of 1,25-(OH)(2)D(3): (a) variation in dietary P, (b) thyroparathyroidectomy (TPTX) with or without administration of parathyroid hormone (PTH), and (c) treatment with disodium ethane-1-hydroxy-1,1-diphosphonate (EHDP). In all circumstances changes in duodenal absorption paralleled the changes in the overall fractional absorption. (a) Lowering dietary P stimulated P absorption. (b) TPTX decreased P absorption. This effect was corrected either by the administration of PTH or by the administration of 1,25-(OH)(2)D(3). (c) EHDP, when given at a dose known to inhibit 1,25-(OH)(2)D(3) formation, decreased the duodenal P absorption in both intact and TPTX animals. This effect was corrected by 1,25-(OH)(2)D(3). In the TPTX-EHDP-treated animals, the administration of PTH did not rectify the low duodenal P absorption. These results support the thesis that, in rats with normal vitamin D supply, variations in the endogenous production of 1,25-(OH)(2)D(3) change the rate of P absorption. However, these changes are in such magnitude that they are of relatively small importance when compared to the effect of variation in the dietary intake of P. These results also strongly suggest that the action of PTH on duodenal P transport is mediated by its effect on 1,25-(OH)(2)D(3) production, inasmuch as the effect of the hormone is abolished after blocking the renal 1-hydroxylation with EHDP.


Estimation of somatomedin-C levels in normals and patients with pituitary disease by radioimmunoassay. The development of a radioimmunoassay for somatomedin-C has for the first time made it possible to discriminate between serum concentrations of a single peptide or closely related group of peptides and the net somatomedin activity measured by less specific bioassay and radioreceptor techniques. Antibodies to human somatomedin-C were raised in rabbits using a somatomedin-C ovalbumin complex as the antigen. A variety of peptide hormones at concentrations up to 1 muM are not recognized by the antibody. Insulin at concentrations >0.1 muM cross reacts in a non-parallel fashion; purified somatomedin-A is only 3% as active as somatomedin-C; and radiolabeled cloned rat liver multiplication stimulating activity does not bind to the antibody. Immunoreactive somatomedin-C can also be quantitated in the sera of a variety of subhuman species. Unusual assay kinetics, which are manifest when reactants are incubated under classic "equilibrium" assay conditions, appear to result from the failure of (125)I-somatomedin-C to readily equilibrate with the somatomedin-C serum binding protein complex. It is, therefore, necessary to use nonequilibrium assay conditions to quantitate somatomedin-C in serum. With this assay it is possible to detect somatomedin-C in normal subjects using as little as 0.25 mul of unextracted serum. Serum somatomedin-C concentrations in normal subjects were lowest in cord blood and rose rapidly during the first 4 yr of life to near adult levels. In 23 normal adult volunteers, the mean serum somatomedin-C concentration was 1.50+/-0.10 U/ml (SEM) when compared to a pooled adult serum standard. 19 children with hypopituitary dwarfism had concentrations below 0.20 U/ml. 17 of these were below 0.1 U/ml, the lower limit of sensitivity of the assay. The mean concentration in 14 adults with active acromegaly was 6.28+/-0.37 U/ml (SEM), five times greater than the normal volunteers. Significant increases in serum somatomedin-C concentrations were observed in 8 of 10 hypopituitary children within 72 h after the parenteral administration of human growth hormone. Three patients with Cushing's disease had elevated serum somatomedin-C concentrations (2.61+/-0.14 U/ml [SEM]). Three patients with hyperprolactinemia had normal concentrations (1.74+/-0.11 U/ml [SEM]).The important new discovery brought to light by quantitation of immunoassayable somatomedin in patient sera is that all previously used assays detect, in addition to somatomedin-C, serum substances that are not under as stringent growth hormone control.


A critical assessment of the roles of circulating hydrogen ion and lactate in the production of exercise-induced asthma. To evaluate the roles of circulating hydrogen ion and lactate in the production of exercise-induced asthma, two experiments were performed. In the first, we exercised six asthmatic subjects to exhaustion on a bicycle ergometer while recording arterial pH at periodic intervals. Multiple aspects of pulmonary mechanics were measured before and after the work load. After recovery, the identical procedures were repeated, but sufficient quantities of sodium bicarbonate were infused to keep the pH at the pre-exercise level. In both experiments, statistically identical attacks of asthma were induced. To study the effect of lactate, five subjects were exercised on several occasions in order to determine the lowest level of work, and hence arterial lactate, that was reproducibly associated with an acute asthma attack. When this was known, sufficient quantities of sodium lactate were infused into the resting subjects so as to equal or exceed the amount produced with exercise. Pulmonary mechanics were not altered with this intervention. These findings demonstrate that lactic acidemia is not the cause of exercise-induced asthma.


Intestinal assimilation of a tetrapeptide in the rat. Obligate function of brush border aminopeptidase. The small intestine is capable of taking up peptide nutrients of two or three amino acid residues, but the mechanism of intestinal assimilation of larger oligopeptides has not been established. The amino-oligopeptidase of the intestinal brush border possesses high specificity for oligopeptides having bulky side chains and is a candidate for a crucial role in the overall assimilation of dietary protein. Rat jejunum was used for in vitro gut sac and in vivo perfusion experiments with Gly-l-Leu-Gly-Gly (2 mM) as the test substrate with analysis of parent peptide and products by automatic ion-exchange chromatography. In these experiments, the tetrapeptide disappeared rapidly from the test solution (20 mumol/s per cm(2) in vitro; 17 mumol/s per cm(2) in vivo) by sequential removal of amino acid residues from the N-terminus to yield amino acids and the C-terminal dipeptide. In gut sac experiments, 61-100% of these products of hydrolysis appeared in the incubation medium and the remainder in the tissue. In contrast, only small amounts of hydrolytic products were found within intestinal lumen in vivo.Gly-l-Pro (10 mM), a peptide known to be transported intact but not to be hydrolyzed by the brush border aminopeptidase, failed to inhibit Gly-l-Leu-Gly-Gly disappearance suggesting that the tetrapeptide does not utilize the known intact transport mechanism. Hypoxic conditions (N(2) atmosphere) in vitro markedly inhibited transport of glucose, leucine, and Gly-Gly but failed to impair Gly-l-Leu-Gly-Gly disappearance suggesting that the first step in assimilation of the tetrapeptide does not involve a transport process. Disappearance of the tetrapeptide was completely blocked by l-leucyl-beta-naphthylamide (10 mM), a specific substrate for brush border aminopeptidase and by the phthalimido derivative of l-leucine bromomethyl ketone, a potent peptidase inhibitor. Hence, the amino-oligopeptidase at the intestinal surface appears to be essential for the initial stages of assimilation of this model tetrapeptide.


Eosinophilopoietin. A circulating low molecular weight peptide-like substance which stimulates the production of eosinophils in mice. In earlier studies, methods were developed to raise specific antibodies in rabbits against purified suspensions of mouse or human eosinophils. On administration of antieosinophil serum (AES) to mice, the mature eosinophils in tissues, peripheral blood, and bone marrow were depleted, while the immature eosinophil pool in the bone marrow was observed to proliferate. The current investigations explore the generation of eosinophilopoietic factors during AES-induced eosinophilopenia. Mice received three injections of AES, one every other day. As the peripheral eosinophil counts started to recover after the last AES injection, the serum was collected and transferred to normal animals. Within 2 days the recipients showed an increase in peripheral blood as well as in bone marrow eosinophils. The rise in bone marrow eosinophils was due to newly formed cells as evidenced by increased uptake of [(3)H]thymidine. The generation of eosinophilopoietic activity was specifically related to depletion of eosinophils but not neutrophils. The eosinophilopoietic activity was: (a) dependent on the volume of serum transferred, (b) lost on dialysis, and (c) largely heat labile. The activity eluted as a low molecular weight substance on G-25 Sephadex and was digested by pronase but not by trypsin. Active fractions collected from G-25 columns were not chemotactic for the eosinophils in vitro. Thus, specific depletion of mature eosinophils generates a low molecular weight peptide which stimulates eosinophilopoiesis in vivo. It is suggested that this substance be named eosinophilopoietin.


Bile acid excretion: the alternate pathway in the hamster. The quantitative significance of renal excretion of bile acid ester sulfates as an alternate excretory pathway was evaluated in hamsters. After bile duct ligation, total serum bile acid fell from a mean level of 454 microgram/ml at 24 h to 64 microgram/ml by 96 h. During this period the bulk of the bile acid pool could be accounted for as esterified bile acids in urine. Renal pedicle ligation of animals with bile duct obstruction led to retention of the bile acid ester sulfates in serum. Thioacetamide hepatotoxicity diminished ester sulfation of bile acids causing diminished renal secretion with relatively greater retention of nonesterified bile acids in serum. We conclude that secretion of esterified bile acids by the kidney is an efficient alternate pathway for maintaining bile acid excretion in obstructive biliary tract disease. Coexistent hepatocellular disease diminishes ester sulfation and the effectiveness of the alternate pathway in maintaining bile acid excretion.


Regulation of mammary and adipose tissue lipoprotein lipase and blood triacylglycerol in rats during late pregnancy. Effect of prostaglandins. The effects of several prostaglandins on lipoprotein lipase activity of mammary gland and adipose tissue and serum triacylglycerol were studied during late pregnancy in rats. Prostaglandins were injected twice daily for 2 days before and once on the day of analysis. In rats pregnant 20 days, prostaglandin F(2alpha) (PGF(2alpha)) increased the activity of lipoprotein lipase in mammary gland fourfold, reduced the activity in adipose tissue about 60%, and decreased serum concentration of triacylglycerol 50%. PGF(2alpha) also reduced serum concentration of progesterone 90% and increased that of prolactin fivefold, but had no effect on serum concentrations of either immuno-reactive insulin or 17beta-estradiol. Injections of 13,14-dihydro-15-keto PGF(2alpha), a metabolite of PGF(2alpha), had similar effects in rats pregnant 20 days, whereas prostaglandins E(1) and E(2) did not. In rats pregnant 16 days, PGF(2alpha) did not affect lipoprotein lipase activity in the tissues or the concentration of triacylglycerol and prolactin in serum, although it decreased serum progesterone 80%.2-Br-alpha-ergocryptine prevented the increase in serum prolactin in response to PGF(2alpha), but did not alter the effect of PGF(2alpha) on lipoprotein lipase activity or serum triacylglycerol. Progesterone completely blocked the effects of PGF(2alpha) on lipoprotein lipase activity and serum triacylglycerol and prolactin concentrations. These findings indicate that the changes in lipoprotein lipase activity and serum triacylglycerol in PGF(2alpha)-treated rats are probably related to the inhibitory action of PGF(2alpha) on progesterone secretion. They also suggest that endogenous F prostaglandins may play a role in the regulation of lipoprotein lipase activity in mammary gland and adipose tissue near parturition.


Absence of the eighth component of complement in association with systemic lupus erythematosus-like disease. A 56-yr-old black woman with absence of the eighth component of complement and a disease compatible with systemic lupus erythematosus is described. Her disease is characterized by the presence of photosensitive "malar" rash, alopecia, polyarthritis, and nephrotic syndrome. Hemolytic and immunochemical assays of the serum complement components were normal, except for C8 which was undetectable. Hemolytic activity could be restored to normal by the addition of functionally pure C8. In vitro tests to investigate the functional integrity of the classical and alternative pathways indicated that the functions mediated by activation of C3 and C5 were intact whereas heatlabile bactericidal activity was totally absent. Addition of C8 restored this function to normal levels. One of two brothers of the proband was shown to have serum C8 levels approaching 50% of normal indicating the hereditary nature of the defect. HLA typing studies showed that the normal brother had identical haplotypes to the proband while the proposed heterozygous brother only shared one haplotype with the patient, suggesting that the gene controlling the C8 defect was not closely linked to the major histocompatibility complex. If the association of a connective tissue disease and absence of a terminal component of complement is not coincidental, these results suggest that C8 deficiency may be associated with a subtle defect in the defense mechanisms to viral infection leading to viral persistance and perhaps to diseases such as systemic lupus erythematosus where chronic viral infections have been implicated.


Deficiency of carnitine in cachectic cirrhotic patients. Carnitine is synthesized from lysine and methionine. In the rat, inadequate intake of either of these essential amino acids causes carnitine depletion. Inasmuch as protein deficiency is common in the hospital population, we have investigated the possible occurrence of nosocomial carnitine deficiency. Fasting serum carnitine concentration was measured in 16 normal and 247 patients in 16 disease groups. Normal range of carnitine was 55-103 muM. Only the cirrhotic group showed significant (P < 0.05) hypocarnitinemia. 14 of 36 hospitalized cirrhotics had subnormal values for serum carnitine. The creatinine/height index, midarm muscle circumference, and triceps skin-fold thickness indicated protein-calorie starvation in the 14 hypocarnitinemic liver patients. In six of the hypocarnitinemic cirrhotics (average serum level 50% of normal), spontaneous dietary intakes of carnitine, lysine, and methionine were measured and found to be only 5-15% as great as in six normocarnitinemic, healthy controls. When these six cirrhotic and six normal subjects were given the same lysine-rich, methionine-rich, and carnitine-free nutritional intake, the normals maintained normal serum carnitine levels and excreted 100 mumol/day, whereas the cirrhotics' serum level fell to 25% of normal, and urinary excretion declined to 15 mumol/day. Seven hypocarnitinemic cirrhotics died. Postmortem concentrations of carnitine in liver, muscle, heart, kidney, and brain averaged only one-fourth to one-third those in corresponding tissues of eight normally nourished nonhepatic patients who died after an acute illness of a 1-3-day duration. THESE DATA SHOW THAT CARNITINE DEPLETION IS COMMON IN PATIENTS HOSPITALIZED FOR ADVANCED CIRRHOSIS, AND THAT IT RESULTS FROM THREE FACTORS: substandard intake of dietary carnitine; substandard intake of lysine and methionine, the precursors for endogenous carnitine synthesis; and loss of capacity to synthesize carnitine from lysine and methionine.


Sites and mechanisms of localization of technetium-99m phosphorus radiopharmaceuticals in acute myocardial infarcts and other tissues. This study was performed to elucidate the localization at the cellular level of technetium-99m phosphorus ((99m)Tc-P) radiopharmaceuticals in acute myocardial infarcts and the mechanisms responsible for (99m)Tc-P uptake in acute myocardial infarcts and other tissues. In 20 dogs with proximal left anterior descending coronary arterial ligation for 1-3 days, elevated calcium levels were measured at all sites of increased (99m)Tc-P uptake (acute myocardial infarcts, necrotic thoracotomy muscle, lactating breast, and normal bone); however, a consistent linear relationship between (99m)Tc-P and calcium levels was not observed. A strong correlation (r = 0.95 and 0.99, n = 2 dogs) was demonstrated between levels of (3)H-diphosphonate and (99m)Tc-P in infarcted myocardium. Autoradiographic studies with (3)H-diphosphonate revealed extensive labeling in the infarct periphery which contained necrotic muscle cells with features of severe calcium overloading, including widespread hypercontraction as well as more selective formation of mitochondrial calcific deposits. Autoradiography also demonstrated labeling of a small population of damaged border zone muscle cells which exhibited prominent accumulation of lipid droplets and focal, early mitochondrial calcification. Cell fractionation studies revealed major localization of both (99m)Tc-P and calcium in the soluble supernate and membrane-debris fractions of infarcted myocardium and less than 2% of total (99m)Tc-P and calcium in the mitochondrial fractions; however, electron microscopic examination showed that mitochondria with calcific deposits were not preserved in the mitochondrial fractions. In vitro studies evaluating the role of serum protein binding on tissue uptake of (99m)Tc-P agents demonstrated that, in spite of significant complexing with serum proteins, serum (99m)Tc-P activity retained the ability to adsorp to calcium hydroxyapatite and amorphous calcium phosphate. In vivo studies showed that concentration of human serum albumin (labeled with iodine-131) in infarcted myocardium reached a maximum of only 3.8 times normal after a circulation time of 96 h, whereas (99m)Tc-P uptake was at least 10 times normal after a circulation time as short as 1 h. It is concluded that: (a) (99m)Tc-P uptake in acutely infarcted myocardium, and possibly other types of soft tissue damage, is limited to necrotic and severely injured cells; (b) concentration of (99m)Tc-P results from selective adsorption of (99m)Tc-P with various forms of tissue calcium stores, including amorphous calcium phosphate, crystalline hydroxyapatite, and calcium complexed with myofibrils and other macromolecules, possibly supplemented by calcium-independent complexing with organic macromolecules; and (c) lack of a linear relationship between (99m)Tc-P and tissue calcium levels mainly results from local differences in composition and physicochemical properties of tissue calcium stores and from local variations in levels of blood flow for delivery of (99m)Tc-P agents.


Autoantibodies to the insulin receptor. Effect on the insulin-receptor interaction in IM-9 lymphocytes. The serum of some patients with insulin-resistant "diabetes" contains antibodies that bind to and block the cell membrane receptors for insulin. In this report, we have characterized the effects of the antireceptor antibodies on the interaction of (125)I-insulin with its receptor on the human lymphoblastoid cell line IM-9. Up to 95% of specific insulin binding can be inhibited by pretreatment of the cells with these immunoglobulins. The onset of the inhibitory effect is time- and temperature-dependent, and the effect is reversed extremely slowly if the cells are suspended in a large excess of antibody-free buffer. These features of antibody binding can be easily distinguished from those for insulin binding to its receptor. The inhibitory effect of the antibodies can be reversed by exposure of the cells to conditions known to elute surface immunoglobulins. The three antireceptor sera studied appear to alter the insulin-receptor interaction in different ways. Two antisera markedly reduce receptor affinity through combined effects on the insulin association and dissociation rates, and, additionally, have smaller effects on available receptor number. A third antiserum primarily affects available receptor number and has little effect on receptor affinity. All three antisera inhibit the capacity of insulin to promote negatively cooperative site-site interactions among insulin receptors. The data suggest that these autoantibodies to the insulin receptor bind to different determinants on the receptor and may therefore be useful as unique probes of insulin receptor structure and function.