Structure of interphase nuclei in relation to the cell cycle. Chromatin organization in mouse L cells temperature-sensitive for DNA replication.

Mutant lines of mouse L cells, TS A1S9, and TS C1, show temperature-sensitive (TS) DNA synthesis and cell division when shifted from 34 degrees to 38.5 degrees C. With TS A1S9 the decline in DNA synthesis begins after 6-8 h at 38.5 degrees C and is most marked at about 24 h. Most cells in S, G2, or M at temperature upshift complete one mitosis and accumulate in the subsequent interphase at G1 or early S as a result of expression of a primary defect, failure of elongation of newly made small DNA fragments. Heat inactivation of TS C1 cells is more rapid; they fail to complete the interphase in progress at temperature upshift and accumulate at late S or G2. Inhibition of both cell types is reversible on return to 34 degrees C. Cell and nuclear growth continues during inhibition of replication. Expression of both TS mutations leads to a marked change in gross organization of chromatin as revealed by electron microscopy. Nuclei of wild-type cells at 34 degrees and 38.5 degrees C and mutant cells at 34 degrees C show a range of aggregation of condensed chromatin from small dispersed bodies to large discrete clumps, with the majority in an intermediate state. In TS cells at 38.5 degrees C, condensed chromatin bodies in the central nuclear region become disaggregated into small clumps dispersed through the nucleus. Morphometric estimation of volume of condensed chromatin indicates that this process is not due to complete decondensation of chromatin fibrils, but rather involves dispersal of large condensed chromatin bodies into finer aggregates and loosening of fibrils within the aggregates. The dispersed condition is reversed in nuclei which resume DNA synthesis when TS cells are downshifted from 38.5 degrees to 34 degrees C. The morphological observations are consistent with the hypothesis that condensed chromatin normally undergoes an ordered cycle of transient, localized disaggregation and reaggregation associated with replication. In temperature-inactivated mutants, normal progressive disaggregation presumably occurs, but subsequent lack of chromatin replication prevents reaggregation.


Unusual thick and thin filament packing in a crustacean muscle.

The proximal accessory flexor (PAF) of the myochordotonal organ (MCO) in the meropodite of crayfish walking legs contains two populations of muscle fibers which are distinguishable by their diameters. The large accessory (LA) fibers are 40-80 micrometer in diam and are similar in ultrastructure to other slow crustacean fibers. The small accessory (SA) fibers are 1-12 micrometer in diam and have a unique myofilament distribution at normal body lengths. There is extensive double overlap of thin filaments at these lengths, and some of them form bundles that may extend the length of the sarcomere. In the middle of the sarcomeres, thick and thin filaments are totally segregated from each other. When the fibers are stretched to lengths beyond double overlap length, the myofilament patterns are conventional. The segregated pattern is reestablished when stretched fibers are allowed to shorten passively. The length-tension relationship of the SA fibers is described by a linear ascending branch, a plateau, and a linear descending branch. The ascending branch encompasses normal body lengths from slack length (Ls) with maximum double overlap to the length at which double overlap ceases (1.8 X Ls). The descending phase is comparable to that of other skeletal muscles. That is, tension decreases in proportion with the reduction in thick-thin filament interdigitation (2 X Ls to 3 X Ls).


DNA polymerase activity in muscle cultures.

Nuclei within myotubes do not synthesize DNA for replication. Accordingly, cultures of myotubes display low levels of DNA polymerase activity. The coincidental decline in DNA polymerase activity and increased formation of multinucleated myotubes during culture does not prove that the loss of capacity to synthesize DNA is a consequence of fusion. Tne experiments described demonstrate that myogenic cells prevented from fusing have low levels of DNA polymerase activity. This is consistent with the notion that, in myogenic cultures, there is a population of mononucleated cells, the myoblasts, which have withdrawn from the mitotic cycle before fusion.


Role of fatty acids in growth-promoting effect of serum albumin on hamster cells in vitro.

Dialyzed serum albumin had considerable growth-promoting effect on cultivated hamster cells. This effect was virtually lost on removal of the fatty acids, and it was completely restored by recombination of the fatty acid-free albumin with the isolated and purified fatty acids. The role of albumin itself appeared to be largely that of a carrier of fatty acids, protecting the cells against toxic effects of fatty acids in free solution. This conclusion was based on two observations: Fatty acids in the absence of albumin were growth-inhibitory except in extremely dilute solutions, and beta-lactoglobulin, a protein possessing, like albumin, the ability to bind and release fatty acids, could replace albumin in the presence of fatty acids with similar growth-promoting effect. Examination of individual molecular types of fatty acids showed that all unsaturated acids tested were growth-promoting, whereas the saturated acids were growth-inhibiting, with the exception of stearic acid in low concentrations. Although the possibility of a mitotic triggering effect was not excluded, the fatty acids presumably stimulated growth by providing substrate for cellular metabolism, since there was a direct relationship between the degree of growth stimulation and the duration of exposure of cells to the fatty acids.


Satellite DNA in differentiating chick tissues.

Embryonic chick DNA from different tissues was examined for differences which might indicate specific DNA amplification in somatic cells. The problem was approached by determining the DNA compositional heterogeneity and searching for possible variation in different tissues of the 12-day chick. Neural retina, muscle, and whole decapitated (general) chick DNA were analyzed in CsCl and Cs2SO4 density gradients. While overloaded CsCl gradients showed a main band (rho = 1.701 g/cm3) and a heavy shoulder (rho = 1.716 g/cm3), overloaded Cs2SO4 gradients displayed a main band (rho = 1.426 g/cm3) and a discrete heavy satellite (rho = 1.447 g/cm3). This satellite, comprising approximately 1% of the whole cell DNA, appeared to be of nuclear origin and not related to mitochondrial DNA, which was found to have a density of 1.426 g/cm3 in Cs2SO4. No differences were found in the densities of the main band or the satellite DNA in the DNA samples isolated from the different tissues. However, the method of DNA isolation was found to be of crucial importance when comparing satellite DNA's among different tissues.


Characterization of a guanine-sensitive mutant defective in adenylo-succinate synthetase activity.

A contingent auxotrophic mutant of CHO-Kl cell is described. This mutant grows in minimal medium. Its growth is inhibited by the exogenous addition of guanine at levels which do not affect the wild type parent. Adenine reverses the guanine effect. This mutant does not complement ade-H (defective in adenylosuccinate synthetase) and has been denoted as ade-HG because of its guanine sensitivity. Some partial revertants of ade-H are found to be also sensitive to guanine, suggesting a close relationship between the ade-H locus and the guanine sensitivity. Studies of 14C-hypoxanthine incorporation into nucleotides indicated that ade-HG has some adenylosuccinate synthetase activity whether it is pre-exposed to guanine or not. Early de novo purine synthesis in ade-HG, however, is greatly inhibited when pre-exposed to guanine. This inhibition of purine synthesis by guanine is reversible and its recovery is facilitated by adenine.


Constancy of the shift-up point in two temperature-sensitive mammalian cell lines that arrest in G1.

Two cell cycle-specific temperature sensitive (ts) mutants of mammalian cell lines, AF8 and K12, are known to arrest in G1 when shifted to the non-permissive temperature. We have determined the entry into S of both AF8 and K12 cells in five different growth conditions, namely: (1) quiescent sparse cultures stimulated to proliferative by serum; (2) quiescent dense cultures stimulated by serum; (3) quiescent sparse cultures stimulated by trypsinization and replating; (4) quiescent, dense cultures stimulated by trypsinization and replating; and (5) mitotic cells collected by mitotic detachment. In addition, for each cell line and for each different growth condition, we have determined the shift-up time, i.e., the time at which a shift-up to the nonpermissive temperature no longer prevents the entry of cells into S. In no case did K12 or AF8 enter S at the nonpermissive temperature. At the permissive temperature, the average time of entry into S varied in different growth conditions, and so did the shift-up time. However, in both cell lines, the distance of the average shift-up time from the average time of entry into S was remarkably constant, regardless of the growth conditions. i.e., 1.8 hours in K12 and 8.6 hours in AF8.


Neoplasms produced from C3H/10T 1/2 cells attached to plastic plates; saturation density, anchorage dependence and serum requirement of in vitro lines correlated with growth aggressiveness in vivo.

The C3H/10T 1/2 embryo cell line, which is nontumorigenic when inoculated subcutaneously in saline suspension, produces tumors when implanted subcutaneously attached to 1 X 5 X 10 mm plastic plates. Under these in vivo conditions there is direct selection for "spontaneous" transformants that have undergone the specific cellular alterations required for neoplastic behavior. This is in contrast to the conventional situation where transformants are obtained in vitro and are only secondarily tested in vivo for neoplastic behavior. Early passages of cell lines from four different C3H/10T 1/2 tumors explanted back in culture were quantitatively examined for tumorigenicity and for alteration in the properties of density inhibition, anchorage dependence, serum requirement, and plasminogen activator production. A fairly consistent quantitative relationship was found between the degree of growth aggressiveness in vivo and the degree of expression of these phenotypic markers of the transformed state in vitro during early passages of the cell lines after tumor explantation.


Regulation of megakaryocytes in W/Wv mice.

W/Wv mice were injected with antiplatelet serum to produce thrombocytopenia or with platelet transfusions to induce thrombocytosis. The responses of their platelets and megakaryocytes were followed to determine if proliferative abnormalities of the megakaryocytic system would be detected. W/Wv mice responded normally to the stimulation from thrombocytopenia with rebound thrombocytosis, macromegakaryocytosis, and macrothrombocytosis. The megakaryocytes of these mice became smaller than normal in response to post-thrombocytopenic rebound thrombocytosis but not to transfusion-induced thrombocytosis. Thus, endogenous thrombocytosis appeared to be a more potent suppressor of megakaryocyte growth than exogenous. These results failed to reveal an effective abnormality of the thrombocytopoietic regulatory system of W/Wv mice in spite of their intrinsically reduced numbers of megakaryocytes and the well known defect of stem cell proliferation. Thrombocytopoietic regulation appeared, therefore, to occur mainly at the committed, rather then pluripotential, stem cell level, and normal responses of the platelet system were observed in spite of severe abnormalities at the pluripotential stem cell level.


Erythroid stem cells in Friend-virus infected mice.

The erythropoietic stem cell compartment was studied in Friend-virus (polycythemic strain, FV-P) infected DBA/2 and NMRI mice with the CFUE and BFUE technique. Early after infection there was a depression in CFUE number in bone marrow and spleen, followed by an increase of the CFUE concentration, earlier and more pronounced in the spleen than in the marrow. Three days after FV-P infection an erythropoietin (Ep) independent CFUE population started to grow and replaced the normal Ep-dependent population within 8 to 12 days. The shift to Ep independency was not gradual. CFUE colonies of FV-P infected bone marrow cells were two to three times larger than control colonies after three days in vitro incubation. BFUE colonies increased in number during the first days of infection, but were totally lost after more than ten days. After velocity sedimentation of bone marrow cells of FV-P infected animals, however, the BFUE containing fractions showed normal BFUE colony growth and normal Ep sensitivity. In unfractionated bone marrow cell cultures BFUE colony growth could be observed later than ten days post infection when the cultures were refed with medium. It was therefore concluded that the loss of BFUE colony growth after FV-P infection was an in vitro artefact due to inadequate culture conditions.


Monosaccharides and monosaccharide derivatives in human seminal plasma.

Gas chromatography--mass spectrometry with an on-line data system was used to identify monosaccharides and monosaccharide derivatives in human seminal plasma. The carbohydrates were converted into the methoxime-trimethylsilyl derivatives before separation in open tubular glass capillary columns coated with SE-30. Twenty-one different compounds were detected in the seminal fluid, of which twelve have not been recognized before. Seventeen of the monosaccharides have previously been identified in urine. Similar patterns of sugars were found both in fertile and infertile individuals, including one with azoospermia. The compounds identified are, with the possible exception of D-ribose, present as free monosaccharides at the time of ejaculation, and they do not seem to be preformed by spermatozoa.


Quantitative analysis of 6,11-dihydro-11-oxo-dibenz[b,e] oxepin-2-acetic acid (isoxepac) in plasma and urine by gas--liquid chromatography.

A method is described for the quantitative analysis of 6,11-dihydro-11-oxo-dibenz[b,e]-oxepin-2-acetic acid (isoxepac) in plasma and urine. Isoxepac and internal standard was analysed by gas-liquid chromatography using a flame ionization detector. The method is accurate and precise over the range 0.1--30 microgram/ml. The method has been applied to the analysis of plasma and urine from both healthy volunteers and patients receiving therapeutic oral doses of isoxepac.


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

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


Studies on the metabolism of 2,4'-isobutylphenylpropionic acid (ibuprofen) by gas chromatography and mass spectrometry. Dialysis fluid, a convenient medium for studies on drug metabolism.

2,4'-Isobutylphenylpropionic acid (ibuprofen) has previously been demonstrated to yield four urinary metabolites, formed by omega 1-, omega 2- and omega 3-hydroxylation and by a further oxidation of the primary alcohol of the omega 1-hydroxylated metabolite to a carboxyl group. By synthesis and gas chromatography--mass spectrometry the suggested structure of the omega 3-hydroxylated metabolite was verified in the present study. Moreover, a new metabolite, 2,4'-carboxyphenylpropionic acid, was demonstrated to be present in substantial amounts in dialysis fluid from a nephrectomized patient. In such patients ingested drugs cannot be excreted in the urine, but are metabolized to end products. Thus, dialysis fluid may be a convenient medium for studies on drug metabolism.


High-pressure liquid chromatographic method for the simultaneous quantitative analysis of propranolol and 4-hydroxypropranolol in plasma.

A simple and rapid high-pressure liquid chromatographic procedure is reported for the simultaneous quantitative determination of propranolol and 4-hydroxypropranolol in plasma. Following an extraction the samples are chromatographed on a reversed-phase column and the components in the column effluent are detected by fluorescence monitoring. Using 1-ml plasma samples propranolol and 4-hydroxypropranolol concentrations at least as low as 1 ng/ml and 5 ng/ml, respectively, can be quantitated. The reproducibility of the method is satisfactory and no interference from endogenous plasma components or other drugs has been observed. A single plasma sample can be analyzed in approximately 20 min.


The estimation of quinidine in human plasma by ion pair extraction and high-performance liquid chromatography.

A rapid, sensitive, accurate method for determination of quinidine in plasma has been developed using ion-pair extraction and high-performance liquid chromatography. The method, which is capable of distinguishing between quinidine and dihydroquinidine, involves acidification of plasma with perchloric acid, extraction with methyl isobutyl ketone and chromatography of the carbonate-washed extract on a silica gel column with a mobile phase of methylene chloride-hexane-methanol--perchloric acid (60:35:5.5:0.1) followed by fluorometric detection. The procedure is sensitive to below 50 ng/ml (coefficient of variation 6.6%) and compares favourably with a standard spectrofluorometric method when tested with plasma from volunteer subjects.


Determination of amitriptyline and nortriptyline in human plasma by quantitative thin-layer chromatography.

A thin-layer chromatographic method for simultaneous determination of amitriptyline (AT) and nortriptyline (NT) in human plasma is described. Both substances are extracted from biological material by means of a single extraction. The extract is evaporated until dry and the residue quantitatively applied to a silica gel thin-layer plate. AT and NT are separated from interfering plasma components by chromatography. The spots are visualized by nitration, reduction and coupling with N-(1-naphtyl)ethylenediamine on the plate. The intensity of the azo-dyes formed can be measured densitometrically. Using 1 ml of plasma, the sensitivity limit was 0.5 ng/ml for both substances. About 10--15 plasma samples can be analysed per day. The method is applicable to pharmacokinetic studies after a single oral dose of 25 mg AT as hydrochloride in man.


Development of an electrochemical detector for high-performance liquid chromatographic assay of brain catecholamines.

An electrochemical detector that utilizes the reduction-oxidation phenomena of electrochemically active substances has been developed for high-performance liquid chromatography. The construction and application of the detector to brain catecholamines, particularly for norepinephrine and dopamine analysis, are described. The differences between this detector and those reported previously are discussed.


Determination of retinoic acid (13-cis- and all-trans-) and aromatic retinoic acid analogs possessing anti-tumor activity, in biological fluids by high-performance liquid chromatography.

A sensitive and specific high-performance liquid chromatographic assay was developed for the determination of 13-cis- and all-trans-retinoic acid in blood or urine with an overall recovery of 90 +/- 5.0% and a limit of detection of 10-20 ng/ml of sample. The method provides for rapid and simple quantitation of the compounds using 1 ml of blood. The assay was applied in the determination of blood levels of 13-cis-retinoic acid in the dog following intravenous and oral administration of 9.5 mg/kg and 2.0 mg/kg doses, and in man following a single 100-mg oral dose and following divided daily doses totalling 2 mg per kg of body weight. The assay is also applicable with minor modifications to the determination of a series of aromatic retinoic acid analogs of clinical interest as anti-tumor agents.


Studies on steroids. CXXVIII. Separation and determination of bile acids by high-performance liquid chromatography.

A method is described for the simultaneous determination of major bile acids by high-performance liquid chromatography without prior hydrolysis. A mixture of bile acids is divided into the free, glyco- and tauro-conjugate groups by thin-layer chromatography. Separation of each group into cholate, ursodeoxycholate, chenodeoxycholate, deoxycholate and lithocholate is attained in two stages on a muBondapak C18 column; first, 0.3% ammonium carbonate-acetonitrile (9:4) is used as a mobile phase for the separation of the last three compounds. Subsequently cholate and ursodeoxycholate are resolved by chromatography in 0.3% ammonium carbonate-acetonitrile (11:4).


Oxidation of 3beta-hydroxyandrostenes by the 3beta-hydroxy-steroid oxidase (cholesterol oxidase) from Brevibacterium sterolicum prior to their analysis by gas-liquid chromatography-mass spectrometry.

The 3beta-hydroxysteroid oxidase from Brevibacterium sterolicum has been applied to the oxidation of a number of 3beta-hydroxyandrostenes, including polar steroids containing up to three other hydroxylic groups. The substrates, products, and derivatives thereof have been examined by gas-liquid chromatography. Retention index increments for these conversions, and for parallel transformations of other steroids, show considerable regularities, and together with mass spectrometric data afford characteristic structural information.


Gas-liquid chromatographic method for the routine estimation of disopyramide in plasma or serum.

A rapid, sensitive and specific gas-liquid chromatographic method is presented for the routine monitoring of plasma concentrations of the anti-arrhythmic compound, disopyramide. The procedure involves extraction of the drug from alkaline plasma into ether, purification of the extract and gas chromatographic analysis using OV-101 liquid phase and flame ionization detection. The results demonstrate the accuracy and reproducibility of the method. Contrary to a previous report, it has been shown that delay in separating plasma from erythrocytes does not affect the disopyramide level in plasma.


High-pressure liquid chromatographic analysis of carbofuran and two non-conjugated metabolites in crops as fluorescent dansyl derivatives.

Carbofuran, and non-conjugated 3-hydroxycarbofuran and 3-ketocarbofuran were extracted from carrots, corn and potatoes with acetone and partitioned into hexane-methylene chloride. The organic extract was evaporated to a small volume for clean-up on a 2% deactivated Florisil column. All three carbamates were eluted with 15% acetone in hexane. The pesticide residues were hydrolysed to their corresponding phenols with 0.1 M sodium carbonate followed by derivatization with dansyl chloride in acetone. The derivatives were extracted and analysed by high-pressure liquid chromatography with fluorescence detection (excitation, 360 nm; emission, greater than 400 nm). Absolute recoveries for all three compounds were between 50 and 65% for spiked samples by the extraction method used. Detection limits approached 0.01 ppm in the foods studied.


Column chromatographic separation of deoxynucleotide monomethyl esters and related products of the reaction of n-methyl-n-nitrosourea with deoxynucleoside monophosphates.

Dowex 1 (HCOO-) column chromatographic procedures are described for the resolution of deoxynucleoside monophosphate monomethyl esters and other related products of the reaction of the carcinogen N-methyl-N-nitrosourea with deoxynucleoside monophosphates. These procedures provide convenient methods for the isolation and estimation of the products of these reactions.


Globin chain separation by SDS polyacrylamide gel electrophoresis. Simple screening method for elongated hemoglobin chains.

A simple method for the separation of hemoglobin chains from hemolysate or globin, by sodium dodecyl sulfate polyacrylamide gel electrophoresis, is described. The alpha, beta, and gamma chains can be clearly separated from each other. The alpha chain has the highest mobility, the beta chain has a slower mobility than the gamma chain, while the delta chain has about the same mobility as the beta chain. Hemoglobins with elongated chains can easily be detected by this method. Tak-beta, elongated by 11 residues, moves much more slowly than betaA but is much faster than alpha Constant Spring which is elongated by 31 residues. Screening of several individuals with slow-moving hemoglobins using this method led to the finding of a case with Hb Tak-beta thalassemia and other carriers of Hb Tak.


Direct gas chromatographic analysis of cyclic N-nitrosamines.

A rapid gas chromatographic technique has been developed for quantitative estimation of cyclic N-nitrosamines. Cumbersome clean-up procedures are unnecessary and quantitative estimation can be done by injecting hexane extract without further pretreatment. The detection limit for the procedures is ca. 0.5 ng.


Quantitative gas-liquid chromatographic method for the determination of indomethacin in biological fluids.

A sensitive and specific gas chromatographic method for the analysis of indomethacin has been developed. After extraction of the drug from blood or urine with ethylene dichrloride a derivative is formed with pentafluorobenzyl bromide. 5-Fluoro-indomethacin is used as an internal standard. The overall recovery of indomethacin is 85.1 +/- 2.3% and the method can detect 10 ng indomethacin in a 1 ml plasma or urine sample. No interference in the method is seen with some commonly used drugs. The method has been used to measure plasma indomethacin concentrations in 20 patients with rheumatoid arthritis, being treated with 25 mg indomethacin three times daily. The plasma concentration ranged from 168-596 ng/ml.


Determination of indomethacin in serum by an extractive alkylation technique and gas-liquid chromatography.

A method for determining indomethacin in serum has been developed, involving extractive alkylation, in which indomethacin is converted into its ethyl ester, and subsequent gas chromatographic determination of the ester. The method is specific and permits the determination of amounts down to 50 ng/ml in serum. The indomethacin concentration in serum was followed for 24 h after oral and rectal application and the results are compared with those reported in the literature.


Rapid and sensitive method for determination of haloperidol in human samples using nitrogen-phosphorus selective detection.

A sensitive gas-chromatographic method for quantitative analysis of haloperidol in human plasma is described. The use of nitrogen-phosphorus selective detection reduces the time required for analysis. Azaperone is used as the internal reference standard. The method is suitable for the determination of haloperidol plasma levels in patients treated with doses ranging from 1.2 to 200 mg/day. No interference from drugs needed in the associated antipsychotic therapy has been found. The simplicity, specificity and sensitivity of the method make it suitable for routine analysis of haloperidol plasma levels in psychotic patients undergoing chronic treatment.


Liquid chromatographic determination of daunorubicin and daunorubicinol in plasma from leukemic patients.

A method is given for the determination of daunorubicin and its main metabolite, daunorubicinol, in plasma from leukemic patients after administration of daunorubicin as the free drug or as a complex with DNA. Daunorubicin and daunorubicinol are extracted from 2 ml of plasma (pH 8.1) using a mixture of chloroform and 1-heptanol (9:1). After re-extraction into phosphoric acid (0.1 M), the separation is performed as reversed phase liquid chromatography on a LiChrosorb RP-2 (5 micrometer) column with a mobile phase of acetonitrile-water, acidified with phosphoric acid. The precision, by quantitation with a photometric detector, was better than 2% within the range 20 ng/ml to 200 ng/ml. Some determinations of plasma levels of daunorubicin and daunorubicinol are presented.


Clean-up procedure for the extraction of soil samples in the determination of 2,3,7,8-tetrachlorodibenzo-p-dioxin.

This paper describes the method which was developed in relation to analytical work connected with microbial and physico-chemical degradation experiments on 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Soil samples are best extracted with methanol plus methylene chloride. Microbial preparations are extracted with light petroleum after boiling with methanolic potassium hydroxide. The clean-up consists of a sulphuric acid treatment and chromatography on a multilayer column (Celite + H2SO4/silica gel) followed by alumina column chromatography. The clean-up procedure proved to be suitable for soil samples and microbial preparations even when large quantities of organic matter (hydrocarbons, oils, surfactants) were present.


Application of ion-exchange and lipophilic-gel chromatography to the purification and group fractionation of steroidal spirolactones, isolated from biological fluids.

Chromatography of steroidal spirolactones on DEAE-Sephadex A-25 under selected pH conditions allowed efficient separation of these compounds from other steroids and many of the endogenous components of human urine. The spirolactones were recovered in high yield, mostly over 90%. Lipophilic-gel chromatography provided a useful method for group fractionation of mixtures of these spirolactones with high recoveries (generally over 90%), unaffected by the presence of endogenous material from normal human urine.


Simple gas-liquid chromatographic method for the measurement of disopyramide in blood-plasma or serum and in urine.

A simple method has been developed for the measurement of disopyramide in blood-plasma or serum at the concentrations attained during therapy. A relatively small (200 microliter) sample volume is made basic and extracted with 50 microliter of chloroform containing an internal standard, and the extract is analysed directly by gas-liquid chromatography with flame-ionization detection. The instrument calibration is linear and passes through the origin of the graph. Neither solvent transfer nor evaporation steps are used in the extraction procedure, which takes less than 3 min to complete, and urine specimens may be analysed by an analogous technique. No interference from either endogenous sample constituents or other drugs has been observed, although a simple back-extraction procedure is described which eliminates potential interference from a small number of basic and neutral drugs.


Analysis of chlormethiazole, ethchlorvynol and trichloroethanol in biological fluids by gas-liquid chromatography as an aid to the diagnosis of acute poisoning.

A simple method has been developed whereby chlormethiazole, ethchlorvynol and trichloroethanol can be simultaneously detected and measured in biological fluids. The procedure is based upon the rapid extraction of a small (50-microliter) sample volume with an equal volume of chloroform containing an internal standard, followed by the gas-liquid chromatographic analysis of this extract. Specimens of blood plasma or serum, urine and gastric contents can be used, and no interference from either endogenous or exogenous sources has been observed. The method is suitable for the measurement of the plasma concentrations of these compounds attained after overdosage.


Rubella virus hemagglutination with human and animal erythrocytes: effect of age and trypsinization.

Sensitivity to rubella virus hemagglutination differs considerably with newborn as opposed to adult erythrocytes (RBC) in several animal species. In humans, cord RBC are 16 times more sensitive to hemagglutination, whereas hemagglutination titers with newborn RBC are 6 to 16 times higher in chickens, mice, and rabbits. This higher sensitivity is, however, short-lived, and adult RBC values are observed 7 days after birth in mice. Trypsinization has no effect on newborn RBC, but increases sensitivity of adult RBC to that of neonatal RBC and tends to diminish differences among individuals. On the other hand, trypsinization does not further enhance the very high sensitivity of pigeon, duck, and goose RBC or the low sensitivity of both newborn and adult guinea pig RBC. Human cord RBC proved suitable for use in hemagglutination-inhibition tests, since they gave titers comparable to those obtained with other types of RBC and offered some advantages: by comparison with animal RBC, they did not require adsorption to remove nonspecific agglutinins; by comparison with adult human RBC, they did not need trypsinization.


New medium for selection and presumptive identification of the Bacteroides fragilis group.

A medium, Bacteroides fragilis bile-esculin (BBE) agar, was designed for the selection and, presumptive identification of the B. fragilis group. BBE agar contains bile, esculin, ferric ammonium citrate, hemin, and gentamicin in a Trypticase soy agar base. Growth in the presence of 20% bile and esculin hydrolysis, detected by blackening of the medium, provide presumptive evidence for the identification of the B. fragilis group. In addition to stimulating the growth of many strains of the B. fragilis group, hemin provides the option of testing isolates for catalase production. Gentamicin and bile prevent the growth of most organisms other than the esculin-positive bacteroides that can tolerate bile. Of 160 clinical isolates of the B. fragilis group tested on BBE agar, 159 grew well on the medium and 157 blackened it. Other anaerobes, Enterobacteriaceae, and enterococci either failed to grow on BBE agar or did not produce the characteristic morphology and blackening associated with isolates of the B. fragilis group. In a clinical laboratory trial, 687 specimens from patients were inoculated onto BBE agar plates. The B. fragilis group was recovered from 81 (11.8%) of these specimens in 24 to 48 h. Use of BBE agar in the clinical laboratory enables earlier recovery and identification of this important pathogen.


Evaluation of the card test for diagnosis of human brucellosis.

The relative efficacy of the card test in the diagnosis of human brucellosis was evaluated by comparison with four other tests: the standard tube agglutination test, centrifugation agglutination test, 2-mercaptoethanol degradation technique, and 2-mercaptoethanol centrifugation agglutination test. A total of 1,701 serum specimens from persons with various degrees of potential exposure to brucella organisms or cross-reactive antigens were used in this study. In comparison with standard tube agglutination results, the card test had a sensitivity of 95.3% and a specificity of 84.1%. The card-test antigen was nonreactive with serum specimens from tularemia patients and cholera vaccinees. Our data indicated that this test measures both immunoglobulin G and immunoglobulin M agglutinins. The value of the card test in the presumptive serological diagnosis of clinical brucellosis in humans appears to be low; however, the card test may be of value in serological surveys to delineate high-risk populations.


Polycythemia vera. The in vitro response of normal and abnormal stem cell lines to erythropoietin.

Bone marrow cells from two glucose-6-phosphate dehydrogenase (G-6-PD) heterozygotes with polycythemia vera were cultured to determine whether progenitors which wre not of the polycythemia vera clone were present, and, if present, which cell lines contributed to the increase in erythroid colonies observed in response to added erythropoietin (ESF). To accomplish this, the G-6-PD isoenzyme activity of individual erythroid colonies was determined. All of the erythroid colonies analyzed in cultures without added ESF, contained the G-6-PD isoenzyme type characteristic of the abnormal clone. With higher ESF concentrations in the culture, however, there was an increase in the colonies that were not of the polycythemia vera clone. Analysis of the ratio of the various types of colonies indicated that normal and polycythemia vera cells are capable of responding to ESF in vitro. In selected patients, this technique permits analysis of the ratios of normal to abnormal cells during the course of the disease, in response to therapy and during late complications, such as myelofibrosis or leukemic transformation.


Effects of coronary artery reperfusion on myocardial infarct size calculated from creatine kinase.

The effects of coronary artery reperfusion at 1 and 3 h after occlusion on infarct size (IS) in the conscious dog were compared with a second group of dogs that were not reperfused (24 h occlusion). Infarct size was calculated from creatine kinase (CK) appearing in blood samples (IS(s)) and myocardial CK depletion (IS(m)), and determined from gross and histological inspection of the pathological tissue (IS(p)). Under both conditions, IS(m) correlated well with IS(p). In dogs with 24-h coronary occlusions, IS(s) correlated well with IS(m) (IS(s) = 14.26 + 1.18 x IS(m), r = 0.92). In reperfused dogs, the relationship remained linear but was altered (IS(s) = 15.33 + 2.07 x IS(m), r = 0.89). The slope was significantly greater, P <0.05, than that observed for dogs that were not reperfused, suggesting that more CK appeared in serum per gram of infarct. Similarly, significantly different relationships were observed in the reperfused and nonreperfused dogs, when IS(s) was compared with IS(p). Moreover, the configuration of the serial blood CK curve was changed significantly by reperfusion. In dogs with a 24-h occlusion, CK rose gradually to a peak at 11.4+/-0.5 h. In dogs reperfused at 3 h, CK rose sharply at 3 h and reached a peak at 6.8+/-0.5 h, significantly earlier (P <0.01) than occurred in dogs reperfused at 1 h, i.e., when the peak occurred at 4.2+/-0.4 h. The rapid appearance of CK in blood after reperfusion at 1 and 3 h suggested a washout phenomena. Thus, reperfusion alters the shape of the serial blood CK curve and results in a different linear relationship between calculated and measured infarct size, resulting in greater recovery of CK in blood per unit of infarcted myocardium.


125I-insulin binding to cultured human lymphocytes. Initial localization and fate of hormone determined by quantitative electron microscopic autoradiography.

Morphologic and biochemical studies indicate that the initial action of insulin is binding to a cell surface receptor. Whether further translocation of the hormone, or a product of the hormone, occurs is unclear and has not been investigated by direct means. To determine the fate of 125I-insulin bound to its receptor, we have examined the distribution of radioactivity by quantitative electron microscopic autoradiography. Cultured lymphocytes of the IM-9 cell line were incubated with 0.1 nM 125I-insulin at 15 degrees and 37 degreesC for incubation periods extending from 2 to 90 min. At 15 degreesC, grains localize to the plasma membane and there is no translocation as a function of time. At 37 degreesC, grains predominantly localize to the plasma membrane but there is a small shift in distribution to a distance of 300-700 nm from the plasma membrane. This small additional band component of irradiation extends to approximately to10--15% of the cell radius. When a morphometric analysis is applied to grains extending 300 nm and beyond from the plasma membrane, we find no preferential localization to any intracellular organelle. We interpret these data to indicate that in the cultured lymphocyte, labeled insulin initially localizes to the plasma membrane but as fuanction of time and increasing temperature there is a small but definite translocation of the hormone or a product of the hormone to a hihgly limited aea of the cell periphery.


Abnormal erythrocyte membrane protein pattern in severe megaloblastic anemia.

The erythrocyte membrane protein pattern of patients with megaloblastic anemia was determined by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. In severe megaloblastic anemia, secondary either to folic acid or vitamin B12 deficiency, the erythrocyte membrane protein pattern was grossly abnormal, lacking bands 1, 2 (spectrin), and 3 and having several diffuse, faster migrating bands. After adequate vitamin replacement therapy, the erythrocyte membrane protein pattern returned to normal. In mild megaloblastic anemia, secondary either to folic acid of vitamin B12 deficiency, and in severe iron deficiency anemia, the erythrocyte membrane protein pattern was normal. Erythrocyte membrane protein pattern of normal membranes did not change after mixing with abnormal membranes before polyacrylamide gel electrophoresis in sodium dodecyl sulfate. Protease activity extracted from membranes of megalocytes was not different from normal. These findings indicate that the erythrocyte membrane protein pattern is abnormal in severe megaloblastic anemia and that this abnormality is not secondary to increased activity of the endogenous erythrocyte membrane proteinase.


Renin-angiotensin system inhibition in conscious sodium-depleted dogs. Effects on systemic and coronary hemodynamics.

The role of the renin-angiotensin system in the regulation of the systemic and coronary circulations during sodium depletion was studied in conscious normotensive dogs by i.v. administration of teprotide (0.5 mg/kg), an angiotensin-converting enzyme inhibitor, and saralasin (0.05-5 mug/kg per min), an angiotensin-receptor antagonist. Sodium depletion was produced by administering a low sodium diet and furosemide for 5 days. Administration of both teprotide and saralasin lowered systemic arterial blood pressure and total peripheral vascular resistance. Simultaneously, cardiac output increased, but left ventricular end-diastolic pressure, dP/dt, and dP/dt/P did not change significantly. Furthermore, both agents reduced diastolic coronary vascular resistance and increased coronary blood flow, but did not affect myocardial oxygen consumption, left ventricular work, or myocardial efficiency. These systemic and coronary vasodilator effects of teprotide and saralasin, however, were not observed in normal dogs on a regular sodium diet; in this group, the only effect noted was a slight increase in arterial pressure during saralasin infusion. Arterial plasma concentration of norepinephrine did not differ between normal and sodiumdepleted dogs, nor did it change significantly after teprotide administration. These results suggest that, during salt depletion, angiotensin II exerts an active vasoconstrictor action on the systemic and coronary vessels, but has no significant effects on myocardial contractility or energetics. It also appears likely that the increase in cardiac output observed in sodiumdepleted dogs after angiotensin inhibition was caused, at least in part, by the decrease in systemic arterial pressure.


A micropuncture study of renal phosphate transport in rats with chronic renal failure and secondary hyperparathyroidism.

Micropuncture studies were carried out in rats to determine changes in tubular transport of phosphate which occur in chronic renal failure and secondary hyperparathyroidism. Rats underwent subtotal nephrectomy (NX) and were fed a low calcium, high phosphorus diet for 3--4 wk. Other groups consisted of normal control animals, normal rats infused with sodium phosphate to raise filtered load of phosphate, subtotal NX rats parathyroidectomized (PTX) on the day of experiment, and normal PTX rats infused with sodium phosphate. It was found that filtered phosphate/nephron is markedly increased in subtotal NX rats due to high single nephron filtration rates, proximal tubular fluid plasma phosphate ratios are less than 1.0, and fractional reabsorption of phosphate is decreased in the proximal tubule. More phosphate was present in the final urine than in surface distal convoluted tubules. Acute PTX in subtotal NX rats resulted in a striking increase in proximal phosphate reabsorption, and urinary phosphate became approximately equal to that remaining in surface distal tubules. Phosphate loading in normal rats reduced fractional reabsorption in the proximal tubule, but urinary phosphate was not greater than that at the end of surface distal tubules. Acute PTX in normal phosphate-loaded animals had no significant effect on proximal tubular phosphate reabsorption. These observations suggest that phosphate homeostasis in chronic renal failure is acheived by inhibition of proximal phosphate reabsorption, counteracting a greatly enhanced intrinsic capacity for reabsorption. In addition, the large amount of urinary phosphate is consistent either with secretion by the collecting ducts or with a disproportionately high contribution by deep nephrons. The changes in phosphate transport are mediated by parathyroid hormone and are completely abolished by acute removal of the hormone.


The acute effects of antiglomerular basement membrane antibody upon glomerular filtration in the rat. The influence of dose and complement depletion.

Recent studies from this laboratory have revealed that single nephron filtration rate (sngfr) decreases significantly within 1 h of the administration of large doses of complement-fixing antiglomerular basement membrane antibody (AGBM Ab) in plasma-expanded Munich-Wistar rats. This reduction in sngfr was due to decreases in nephron plasma flow (rf) and the glomerular permeability coefficient (LpA) utilizing direct evaluation of all pertinent pressures, flows, and permeabilities. With identical micropuncture techniques, we have determined (a) the respective influences of rpf and LpA upon sngfr by examining the effects of differing doses of AGBM Ab, and (b) the specific effect of complement fixation upon the reduction in sngfr. In normal rats, low dose (1.4 microgram/g body wt) AGBM Ab decreased sngfr from 57.9 +/- 3.4 to 50.8+/- 3.9 nl/min per g kidney wt (kw) (P less than 0.001), and this was due to a 10% reduction in rpf and a decrease in LpA FROM 0.069 +/- 0.014 in control to 0.041 +/- 0.007 nl/s per g kw per mm Hg (P less than 0.02). At the high dose (2.3 microgram/g body wt), sngfr fell dramatically from 58.4 +/- 4.0 to 7.6 +/- 3.8 nl/min per g kw (P less than 0.001), and this effect upon filtration was the result of an 86% reduction in rpf and a decrease in LpA from 0.092 +/- 0.020 to 0.007 +/- 0.004 nl/s per g kw mm Hg (P less than 0.001). Therefore, at lower doses sngfr fell primarily as a result of a 40% reduction in LpA and a 10% decrease in rpf; however, at the high dose massive reductions in both rps and LpA led to the large decrease in sngfr. In complement-depleted rats, receiving identical doses, low-dose AGBM Ab no longer reduced the sngfr, but a reduction in LpA persisted (other factors compensating to maintain sngfr). At the high dose, complement depletion ameliorated the reduction in sngfr (55.1 +/- 2.4 to 37.2 +/- 3.4 nl/min per g kw mm Hg) by nearly eliminating the vasoconstriction but only partially diminished the reduction in LpA (0.097 +/- 0.020 to 0.032 +/- 0.004 nl/s per g kw mm Hg, P less than 0.05). Complement depletion prevented the migration of polymorphonuclear leukocytes (present in larger numbers after the high dose of AGBM Ab) into the capillary and eliminated vasoconstriction. Complement depletion resulted in a lesser effect of high-dose AGBM Ab upon LpA than in normal rats, and this is likely due to lesser polymorphonuclear leukocyte effects upon capillary surface area. The persistent reduction in LpA observed in complement-depleted rats correlated with separation of the endothelial cell from the glomerular basement membrane after AGBM Ab, AGBM Ab diminished glomerular ultrafiltration by decreasing LpA and altering the endothelial surface of the glomerular membrane, and this effect is not totally dependent upon the fixation of complement.


Characterization of a carrier-mediated transport system for taurine in the fetal mouse heart in vitro.

Cardiac taurine levels are elevated in hypertension and congestive heart failure. A possible mechanism for this increase in taurine is an alteration of its uptake. We sought to identify and characterize a carrier-mediated transport system for taurine in the mammalian myocardium utilizing the fetal mouse heart in organ culture. Hearts from fetuses of 16-19 days gestational age used in these studies had an endogenous taurine content of 14.1+/-0.5 nmol/mg tissue. The uptake of [(3)H]taurine was linear for up to 8 h. Taurine was accumulated against a concentration gradient as demonstrated by a net increase in taurine concentration when hearts were incubated in 0.5 mM taurine. [(3)H]Taurine uptake was saturable, K(m) = 0.44 mM, temperature dependent, and required sodium. The close structural analogues, hypotaurine and beta-alanine, reduced [(3)H]taurine uptake by 87% when present in 100-fold excess. The alpha-amino acids alanine, alpha-aminoisobutyric acid, glycine, leucine, and threonine did not inhibit uptake. Other taurine analogues tested were guanidinotaurine, guanidinopropionic acid, gamma-aminobutyric acid, 2-aminoethane phosphonic acid, aminomethane sulfonic acid, 3-aminopropane sulfonic acid, N-acetyltaurine, and isethionic acid. We conclude that a carrier-mediated transport system for taurine exists in the fetal mouse heart based on the demonstration of (a) temperature dependence, (b) saturability, and (c) structural selectivity of the uptake process. Transport was demonstrated to be mediated by a beta-amino acid uptake system. In addition, taurine uptake was observed to be sodium dependent, energy dependent, and capable of accumulating taurine against a concentration gradient.


Influence of diets high and low in animal fat on bowel habit, gastrointestinal transit time, fecal microflora, bile acid, and fat excretion.

Epidemiological observations and animal experiments suggest that large bowel cancer is related to serveral factors. Among them, high dietary intakes of animal fat, the presence in the colon of relatively high levels of bile acids, specific patterns of intestinal microflora, slow transit through the gut, and low stool weights. Under metabolic conditions we have observed the effect on these variables of dietes containing 62 or 152 g/day of fat mainly of animal origin in six healthy young men over 4-wk periods. No change attributable to the diet was observed in the subjects' bowel habit, fecal weight, mean transit time through the gut, or in the excretion of dry matter. Total fecal bile acid excretion was significantly higher on the high fat diet (320 +/- 120 mg/day) than on the low fat diet (139.7) +/- 63 mg/day) t test = 7.78 P less than 0.001 as also was the total fecal fatty acid excretion, 3.1+/-0.71 and 1.14+/-0.35 g/day, respectively t test = 11.4 P less than 0.001). The fecal microflora including the nuclear dehydrogenating clostridia were unaltered by the dietary changes as was fecal beta-glucuronidase activity. Dietary changes which increase animal fat intake clearly influence fecal bile acid excretion in a way that would favor the development of large bowel cancer if current theories prove to be true. Dietary fat however has no effect on overall colonic function so other components of the diet must be responsible for the observed associations of bowel cancer with slow transit and reduced fecal bulk.


Loss of central nervous system component of dopaminergic inhibition of prolactin secretion in patients with prolactin-secreting pituitary tumors.

The administration of l-dopa suppresses prolactin (PRL) secretion in normal subjects and in patients with hyperprolactinemia, although it is not known whether this effect, which requires the conversion of dopa to dopamine, is mediated peripherally or through the central nervous system. To distinguish between these effects, 10 normal subjects (6 male, 4 female) and 8 patients with hyperprolactinemia associated with pituitary tumors were given l-dopa, 0.5 g alone, or 0.1 g after a 24-h pretreatment with carbidopa, 50 mg every 6 h, which produces peripheral dopa decarboxylase inhibition. Similar degrees of PRL suppression were observed in normal subjects (basal plasma PRL 13+/-2 ng/ml) after l-dopa alone (48+/-4%) and after l-dopa plus carbidopa (58+/-6%). In patients with pituitary tumors and elevated plasma PRL (73+/-14 ng/ml), l-dopa alone led to PRL suppression comparable with that in normal subjects (47+/-6%). However, l-dopa plus carbidopa resulted in only minimal suppression of plasma PRL (19+/-4%) which was significantly less than after l-dopa alone (P < 0.001). Urinary homovanillic acid excretion, which reflected peripheral dopa decarboxylation was similar in controls and tumor patients after l-dopa both alone and after carbidopa pretreatment. Comparable suppression of PRL levels in response to a dopamine infusion (4 mug/kg per min for 3 h) was observed in controls and tumor patients. The results indicate that although peripheral conversion of exogenous dopa to dopamine can suppress PRL secretion, in normals, the central nervous system conversion of dopa to dopamine in the presence of peripheral dopa decarboxylase inhibition is sufficient to account for its PRL-suppressive effects. In contrast, patients with tumors, while retaining peripheral dopaminergic inhibitory effects on PRL secretion, exhibit a marked reduction of central dopaminergic inhibition of PRL secretion.


The physical chemistry of cholesterol solubility in bile. Relationship to gallstone formation and dissolution in man.

We determined the maximum solubilities of cholesterol in aqueous conjugated bile salt-egg lecithin-cholesterol systems as a function of several physical-chemical variables including those of physiological importance employing phase equilibria techniques. Equilibration rates are influenced by time and the method of sample preparation in that metastable supersaturation is readily induced at high bile salt: lecithin ratios, and equilibrium saturation by dissolution is achieved sluggisly at low bile salt:lecithin ratios. Equilibrium values for cholesterol saturation vary with the bile salt species, bile salt: lecithin ratio, temperature, ionic strength, and, in particular, with the total concentration of biliary lipids. Within physiological bile salt:lecithin ratios at 37 degreesC the influence of bile salt type and ionic strength is small, whereas the effects of bile salt:lecithin ratio and the total lipid concentration are major factors. We plotted on triangular coordinates a family of cholesterol solubility curves for each total lipid concentration (0.30--30 g/dl) and computed fifth-degree polynomial equations for each curve. With both the curves and the polynomial equations the "per cent cholesterol saturation" of fasting gallbladder and hepatic biles from patients with and without gallstones was calculated and both methods gave similar values. These results deomonstrate that by employing cholesterol saturation values appropriate to the total lipid concentration (range 0.2--24.9 g/dl) of individual biles, all cholesterol stone patients have supersaturated gallbladder biles, (mean, 132% [normal weight individuals], and 199% [morbidly obese individuals]). With controls and pigment stone patients the mean values were 95 and 98%, respectively, and in both approximately 50% of biles were supersaturated. Fasting hepatic biles were significantly more supersaturated than gallbladder biles (means 228--273%). Cholesterol monohydrate crystals were found in the majority of gallbladder (83%) and hepatic (58%) biles of cholesterol gallstone patients but were not observed in pigment stone patients or controls. We conclude that of the several factors in addition to the bile salt:lecithin ratios which can influence the cholesterol saturation of bile the total lipid concentration is the predominant determinant physiologically. Our results demonstrate that (a) metastable supersaturation is frequent in both normal and abnormal biles, (b) cholesterol gallstone patients have supersaturated gallbladder and hepatic biles without exception, and (c) the predominant driving force for cholesterol precipitation appears to be the absolute degree of cholesterol supersaturation.


Sustained effect of glucose-insulin-potassium on myocardial performance during regional ischemia. Role of free fatty acid and osmolality.

To evaluate the influence of glucose infusate administered with insulin and potassium on left ventricular function during 4 h of ischemia, as well as mechanism of action, four groups of intact anesthetized dogs were studied. Acute regional ischemia was induced with a balloon tip catheter in the left anterior descending artery and infusates were begun after 20 min of ischemia. A threefold increase of plasma glucose concentration was associated with improved left ventricular function during ischemia, compared to animals receiving isovolumic saline. There was a significant decline of left ventricular end-diastolic pressure associated with elevation of stroke volume and ejection fraction to control levels, as determined by indicator dilution. In a separate subgroup studied by cineangiography, shortening of the ischemic anterior wall, after an initial decline, was increased in response to glucose but there was no evidence of extension of injury. Ischemic tissue exhibited a smaller gain of water as well as Na+ per gram dry weight as compared to ischemic controls. On precordial electrocardiogram mapping there was a significant decrease in the sigmaST (sum of ST elevation) as well as NST (number of ST segment elevations), but the reduction of R wave amplitude was not different from controls. To further evaluate long-term effects, eight controls and six treated animals underwent myocardial ischemia and were sacrificed after 4 mo. Calculated area and weight of scar, as well as degree of wall thinning, were similar in both groups. The glucose-treated animals had a significant decrease of plasma FFA in contrast to controls which manifested a significant rise. To examine the postulate that the decrease in FFA was important to therapeutic action, a third group was infused with Intralipid (Cutter Laboratories, Inc., Berkeley, Calif.) and heparin, simultaneously with the glucose infusate, to effect an elevation of plasma FFA during ischemia. Changes in myocardial function and electrolyte composition, as well as precordial electrocardiogram mapping, were similar to that of animals receiving glucose alone. Because serum osmolality was increased approximately 40 mosmol during the glucose infusion, the potential role of hyperosmolality was assessed by infusion of 20% mannitol during acute ischemia in a fourth group. After a transient small increase, there was a moderate decline in function by 4 h, suggesting that the response to glucose is not dependent upon extracellular osmolality. Thus, it is concluded that during the initial hours after the onset of myocardial ischemia the glucose infusate improves ventricular performance without evidence of arrhythmia induction or intensification of ischemic injury. Evolution of irreversible necrosis appears to be delayed rather than prevented under the circumstances of this study.


Abnormal plasminogen. A hereditary molecular abnormality found in a patient with recurrent thrombosis.

A patient who suffered a recurring thrombosis over the last 15 yr has been investigated. The only abnormality found in this patient was a significantly depressed level of plasminogen activity in plasma. In spite of the depressed plasminogen activity, the patient was found to have a normal level of plasminogen antigen concentration. It was calculated that the activity per milligram of plasminogen of the patient was approximately one-half the values of normal subjects. The same discrepancy between biological activity and antigen concentration was found in the other members of the kindred. A niece was found to have practically no plasminogen activity but possessed a normal concentration of plasminogen antigen. Both her parents were found to have approximately half the normal plasminogen activity and normal antigen levels. These studies suggested that the molecular abnormality was inherited as an autosomal characteristic, and the family members who had half the normal levels of activity with normal plasminogen antigen were heterozygotes whereas the one with practically no plasminogen activity was homozygote. Subsequent studies showed that the pattern of gel electrofocusing of purified plasminogen of the heterozygotes consisted of 10 normal bands and 10 additional abnormal bands, each of which had a slightly higher isoelectric point than each corresponding normal component. This indicates that plasminogen of the heterozygote is a mixture of normal and abnormal molecules in an approximately equal amount, which was substantiated by active site titration of purified plasminogen preparations obtained from the propositus and a normal individual. The gel electrofocusing pattern of the homozygote consisted of abnormal bands only. The defect is a hereditary abnormality of plasminogen.


Effect of hypoxia on myocardial relaxation in isometric cat papillary muscle.

Myocardial relaxation is an important energy-dependent process. Hypoxia, unlike ischemia, has not been shown to impair myocardial relaxation. This difference may be because (a) the traditional index to assess isometric muscle relaxation (half time to relaxation or RT((1/2))) reflects both changes in developed tension as well as relaxation and (b) the relaxation process is highly sensitive to temperature and previous papillary muscle studies have been conducted under hypothermic conditions. The present study examines the effect of hypoxia on the relaxation process of 31 isometrically contracting kitten papillary muscles at hypothermic (29 degrees C) and euthermic (38 degrees C) conditions using RT((1/2)), the peak rate of tension fall (-dT/dt) and -dT/dt normalized for tension ([peak -dT/dt]/T and max [-dT/dt per T]). Hypoxia at 29 degrees C resulted in a fall in RT((1/2)) from 278+/-11 (SEM) to 230+/-17 ms (P < 0.01) and no change in (peak -dT/dt)/T and max (-dT/dt per T). However, at 38 degrees C, hypoxia impaired relaxation as reflected in a prolongation of RT((1/2)) from 101+/-6 to 126+/-8 ms (P < 0.01) in spite of a substantial fall in peak tension. Moreover, (peak -dT/dt)/T decreased from -15.4+/-0.7 to -11.0+/-0.8/s (P < 0.01) and max (-dT/dt per T) decreased from -25.1+/-1.8 to -13.8+/-0.9/s (P < 0.01). In conclusion, the present study demonstrates that hypoxia impairs the relaxation process of cardiac muscle.


The mechanism of 5-methyltetrahydrofolate transport by human erythrocytes.

The mechanism involved in 5-methyltetrahydrofolate uptake by human cells is poorly understood. To more clearly elucidate this physiologically important process, transport of the vitamin was studied in human erythrocytes. 5-methyltetrahydrofolate uptake was found to increase with reticulocytosis, but measurable incorporation occurred in erythrocyte suspensions depleted of reticulocytes, leukocytes, and platelets, indicating uptake by mature erythrocytes. Incubation of erythrocytes with increasing concentrations of [(14)C]5-methyltetrahydrofolate resulted in increasing uptake but decreasing percentage incorporation, consistent with saturation of a carrier system. Both influx and efflux phases of uptake were temperature dependent, with almost no transport at 4 degrees C. Uptake of [(14)C]5-methytetrahydrofolate was effectively inhibited by unlabeled 5-methyltetrahydrofolate, 5-formyltetrahydrofolate, and methotrexate, but not by pteroylglutamic acid. Prior incubation with 5-formyltetrahydrofolate increased uptake of [(14)C]5-methyltetrahydrofolate, and extracellular 5-formyltetrahydrofolate enhanced efflux of [(14)C]5-methyltetrahydrofolate. Nearly total depletion of ATP increased uptake of [(14)C]5-methyltetrahydrofolate, but efflux was unchanged. Column chromatography of membrane-free hemolysate after incubation with [(14)C]5-methyltetrahydrofolate showed 95% of radioactivity corresponded to marker radioisotope, and no other peak was noted. Thus peripheral erythrocytes incorporate 5-methyltetrahydrofolate by a saturable, temperature-dependent, substrate-specific process which is influenced by counter-transport. This mechanism is qualitatively similar to the carrier-mediated transport of folate compounds previously described in other cell types. Therefore, human erythrocytes should be useful for detailed characterization of this membrane carrier system.


3,3'-Diiodothyronine production, a major pathway of peripheral iodothyronine metabolism in man.

3,3'-Diiodothyronine (3,3'-T(2)) has been detected in human serum and in thyroglobulin. However, no quantitative assessment of its clearance rate (CR), production rate (PR), or of the importance of extrathyroidal sources of 3,3'-T(2) relative to direct thyroidal secretion is yet available. This study examines these parameters in seven euthyroid subjects, and in eight athyreotic subjects (H) eumetabolic due to thyroxine therapy (HT(4)) (n = 5) or triiodothyronine replacement (HT(3)) (n = 3). A highly specific radioimmunoassay for the measurement of 3,3'-T(2) in whole serum was developed. Serum 3,3'-T(2) concentrations were (mean +/- SD) 6.0+/-1.0 ng/100 ml in 13 normal subjects, 9.0+/-4.6 ng/100 ml in 25 hyperthyroid patients, and 2.7+/-1.1 ng/100 ml in 17 hypothyroid patients. The values in each of the latter two groups were significantly different from normal. 3,3'-T(2) was detected regularly in normal concentrations in 11 hypothyroid patients eumetabolic by treatment with synthetic T(4), in 10 eumetabolic patients suffering from nonthyroidal systemic illness, and in 2 subjects with elevated serum T(4)-binding globulin. The 3,3'-T(2) CR was assessed from data acquired from the (125)I-3,3'-T(2) constant infusion technique. The 3,3'-T(2) PR was calculated from CR and serum concentration of 3,3'-T(2) determined by radio-immunoassay. In the HT(4) subjects the 3,3'-T(2) CR averaged 840+/-377 liters/day and 3,3'-T(2) PR 33.9+/-12.5 mug/day. These results were not significantly different from those in the control group: 3,3'-T(2) CR 628+/-218 liters/day and 3,3'-T(2) PR 39.8+/-19.8 mug/day (all corrected to 70 kg body wt). In addition to 3,3'-T(2) PR, T(3), and reverse triiodothyronine (rT(3)) PR were determined in three of the HT(4) subjects. In each case studied, the 3,3'-T(2) PR was close to the combined triiodothyronine (T(3) + rT(3)) PR. The mean molar ratio of T(2) PR/(T(3) + rT(3)) PR was 1.08+/-0.10. The results obtained in the HT(4) subjects indicate that the production of 3,3'-T(2) is a major route of T(4) metabolism. The combined studies of 3,3'-T(2), T(3) and rT(3) PR in the HT(4) subjects indicate that both T(3) and rT(3) are major precursors of 3,3'-T(2). In the HT(3) subjects, the conversion of T(3) to 3,3'-T(2), determined as the molar ratio of 3,3'-T(2) PR to T(3) PR, ranged from 0.36 to 0.92, providing further evidence that T(3) is a precursor of 3,3'-T(2). From the close agreement between the mean values for 3,3'-T(2) PR in the euthyroid and HT(4) group it is concluded that most, if not all of the 3,3'-T(2) produced in normal humans is derived by extrathyroidal conversion from T(3) and rT(3).


Urinary excretion of elastin peptides containing desmosin after intratracheal injection of elastase in hamsters.

The intratracheal injection of pancreatic elastase results in an acute loss of elastin from the lungs of hamsters and the development of emphysema. We used measurements of the unique covalent cross linking amino acids of elastin, desmosine and isodesmosine, to quantitate elastin. Direct measurements on the lungs estimated an average loss of elastin of 57% after elastase injection. Elastin breakdown products were also quantitated in the urine and feces after injection. An average of 8.8 nmol of desmosines was recovered from the urine of each hamster. This amount represented the desmosines from 61% of the elastin lost from the lungs. Desmosine and isodesmosine existed in the urine in peptide fractions that ranged from 9 to 27,000 daltons with an average of 13,000. Only trace quantities of desmosines could be detected in feces. Desmosines injected intraperitoneally were completely recovered in the urine, and radioactive tracer studies failed to reveal in vivo catabolism of injected desmosines. These results suggest that measurement of urinary desmosines holds promise for the study of elastin turnover.


Pulmonary vascular effects of fat emulsion infusion in unanesthetized sheep. Prevention by indomethacin.

Pulmonary diffusing capacity and arterial blood Po(2) decrease in humans when 10% fat emulsion is infused. To study its effects on the pulmonary circulation and lung fluid balance, we infused 0.25 g/kg x h of a 10% fat emulsion (Intralipid, Cutter Laboratories, Inc., Berkeley, Calif.) into an awake sheep lung lymph preparation. The emulsion caused a sustained increase in pulmonary artery pressure to approximately twice base line with little change in left atrial pressure. Pa(O2) decreased an average 13 torr and lung lymph flow increased two- to threefold. Lymph/plasma total protein concentration fell as lymph flow increased; the magnitude of the lymph/plasma protein decrease was similar to that reported previously when lung vascular pressures were mechanically elevated. Heparin infusion (loading dose = 4,000 U, maintenance dose = 2,000 U/h) cleared the serum of triglycerides but did not alter the response to fat emulsion. Indomethacin infusion (loading dose = 5 mg/kg, maintenance dose = 3 mg/kg x h) blocked the rise in pulmonary artery pressure, the increase in lung lymph flow, and the fall in Pa(O2). Neither extravascular lung water nor [(14)C]urea lung vascular permeability surface area products were altered by fat emulsion infusion. We conclude that fat emulsion infusion in sheep increases lung microvascular filtration by increasing vascular pressures, but has no effect on vascular permeability. Since the effects are blocked by indomethacin, they may be prostaglandin mediated.


Relationship between para-aminohippurate secretion and cellular morphology in rabbit proximal tubules.

Previous studies in the mammalian proximal tubule have suggested that para-aminohippurate (PAH) secretion is approximately threefold greater in the straight segment, or pars recta, than in the convoluted segment, or pars convoluta. However, the possibility that the site of maximal PAH secretion might be related better to particular tubule segments as identified by cell type had not been explored. In addition, the presence or absence of differences in PAH secretion between morphologically identical regions of superficial (SF) vs. juxtamedullary (JM) proximal tubules has not been examined. These issues were studied using a combination of histologic methods and measurement of [(3)H]PAH secretion in isolated perfused tubules. Measurements of microdissected SF and JM proximal tubules from young and adult rabbits revealed that SF proximal tubules were slightly but significantly longer than JM tubules ([young rabbits: SF, 8.69+/-SE 0.14 mm vs. JM, 7.97+/-SE 0.13 mm; P < 0.01] [adult rabbits: SF, 10.61+/-SE 0.28 mm; JM, 9.17+/-SE 0.19 mm; P < 0.001]). Light and electron microscopy revealed three sequential segments (S(1), S(2), and S(3)) along the length of SF and JM proximal tubules as defined by cell type. PAH secretion was measured in each of these three segments by the isolated perfused tubule technique. Net PAH secretion in fmol/mm per min in SF proximal tubules was: S(1), 281+/-SE 21; S(2), 1,508+/-SE 104; S(3), 318+/-SE 46. Corresponding values in JM proximal tubules were 353+/-SE 31, 1,391+/-SE 72, and 188+/-SE 23. Net PAH secretion did not differ between comparable segments of SF and JM proximal tubules. It is concluded that differences in PAH secretion along the proximal tubule correlate best with cell type rather than the arbitrary division of the proximal tubule into pars convoluta and pars recta according to its external configuration. Evidence of functional heterogeneity between comparable segments of SF and JM proximal tubules was not observed.


Human leukocytic pyrogen induces release of specific granule contents from human neutrophils.

The ability of highly purified human leukocytic pyrogen (LP) to induce neutrophil lysosomal protein release is described. Human peripheral blood neutrophils isolated by Ficoll-Hypaque and dextran sedimentation were exposed to purified human LP. The specific granule-associated proteins, lysozyme and lactoferrin were selectively released, whereas primary granule (beta-glucuronidase) and cytoplasmic (lactic dehydrogenase) enzyme markers were not. Optimum release was observed after 45 min in the presence of Ca++ and Mg++. Cytochalasin B (5 microgram/ml) had no effect on LP-induced lysosomal enzyme release. Since the pyrogenicity of LP is dependent on prostaglandin synthesis, the effect of two potent inhibitors of prostaglandin synthesis on lysozyme release was studied. Both indomethacin and naproxen failed to inhibit specific granule protein release. These observations suggest that the concommitance of fever, elevated serum or urine lysozyme and hypoferremia may, in part, be explained by the interaction of LP and peripheral blood neutrophils.


Intestinal secretion induced by vasoactive intestinal polypeptide. A comparison with cholera toxin in the canine jejunum in vivo.

The effect of vasoactive intestinal polypeptide (VIP) on intestinal water and electrolyte transport and transmucosal potential difference was investigated in the dog jejunum in vivo and compared to secretion induced by cholera toxin. Isolated jejunal loops were perfused with a plasma-like electrolyte solution. VIP (0.08 mug/kg per min) was administered directly into the superior mesenteric artery by continuous infusion over 1 h. From a dye dilution method, it was estimated that a mean plasma VIP concentration of 12,460 pg/ml reached the loops. VIP caused secretion of water and electrolytes; for example, chloride: control, 8 mueq/cm per h absorption; VIP, 92 mueq/cm per h secretion. A marked increase in transmucosal potential difference (control, -1.0 mV; VIP, -5.9 mV, lumen negative) occurred within 1 min after starting VIP infusion. Analysis of unidirectional fluxes showed increased plasma-to-lumen flux of sodium and chloride and decreased lumen-to-plasma flux of sodium. Chloride and bicarbonate were actively secreted against an electrochemical gradient. Although sodium secretion occurred down an electrochemical gradient, flux ratio analysis suggested a component of active sodium secretion. VIP caused a slight increase in protein output into the loops; light microscopy revealed capillary dilatation and closed intercellular spaces. The effect of VIP was readily reversible. Except for the delayed onset of secretion, the effect of cholera toxin was qualitatively similar to VIP; however, capillary dilatation and increased protein output were not noted with cholera toxin.


The role of glucagon deficiency in the Houssay phenomenon of dogs.

Plasma glucose, immunoreactive glucagon (IRG), and insulin were measured in hypophysectomized dogs receiving cortisol and thyroid replacement therapy. 4 wk after hypophysectomy mean fasting plasma glucose levels had declined from 90+/-2 mg/100 ml to 64+/-2; fasting and arginine-stimulated insulin and IRG levels were, respectively, approximately 50% lower and unchanged. 12 wk or more after hypophysectomy, despite lower plasma glucose levels, fasting and arginine-stimulated IRG levels were significantly below control dogs. Hypophysectomized and shamhypophysectomized dogs were subjected to total pancreatectomy. Postoperatively, in the sham-hypophysectomized, depancreatized dogs fasting glucose levels ranged from 300-500 mg/100 ml on 8-10 U/day of insulin; IRG levels averaged 215+/-29 pg/ml. The hypophysectomized, depancreatized dogs required 0-4 U/day and fasting glucose levels under 100 mg/100 ml were not uncommon, even without insulin; fasting IRG levels averaged 63+/-4 pg/ml (P < 0.001). During arginine infusion in sham-hypophysectomized, depancreatized dogs, IRG levels rose from 215+/-60 pg/ml to a peak of 404+/-112 pg/ml; in hypophysectomized, depancreatized dogs, the base line IRG averaged 44+/-8 and the peak 110+/-25 pg/ml (P < 0.05). IRG levels in the venous effluent of the gastric fundus, the major source of nonpancreatic glucagon, reached a peak of 4,898+/-959 pg/ml in the sham-hypophysectomized, depancreatized group during arginine infusion and only 219+/-128 pg/ml in the hypophysectomized, depancreatized group. In three hypophysectomized, depancreatized dogs, a replacement infusion with glucagon for 10 h promptly increased hyperglycemia by 80-180 mg/100 ml and worsened glycosuria, evidence of a hepatic response to glucagon replacement. It is concluded that hypophysectomy somehow decreased both the hypersecretion of gastric IRG and the severe hyperglycemia that otherwise follows pancreatectomy. The hypophysectomized, depancreatized animal, therefore, has combined insulin and glucagon deficiency, and the latter may contribute to reduced severity of its hyperglycemia.


Demonstration of the insulin receptor in vivo in rabbits and its possible role as a reservoir for the plasma hormone.

Based on studies of the interaction of insulin with its receptors in vitro, we calculated that a receptor compartment should be measurable directly in vivo. For this purpose, rabbits were injected intravenously with a labeled insulin that has low affinity for receptors in combination with a radioiodinated insulin that has high affinity for receptors. Plasma concentrations of labeled insulins were measured at selected intervals after injection. Apparent volumes of distribution were calculated by extrapolation of plasma distribution were calculated by extrapolation of plasma disappearance curves; high affinity insulins consistently distributed into spaces that were two-three times greater than those of the low affinity insulins. Injections of unlabeled pork insulin before tracer insulins decreased the distribution space of the high affinity insulin in a dose-dependent manner while having little or no effect on the distribution space of the low affinity labeled insulin. When unlabeled insulin was injected after the tracer insulins, there was an immediate rise in the plasma concentration of the high affinity insulin with only a slight change in the plasma concentration of the low affinity insulin. These results demonstrate that high affinity insulins distribute into a body compartment which has many properties of the insulin receptor previously studied in vitro. This receptor compartment: (a) recognizes insulins based on their biological potencies; (b) is saturated by elevated concentrations of insulin; and (c) insulin bound to receptors is in equilibrium with free hormone in plasma. Further, the bound to free ratios for hormone, calculated from these data, suggest that in vivo greater than 50% of the extrapancreatic insulin is bound to receptors during normal physiological states.


Inhibition of parathyroid hormone secretion by 25-hydroxycholecalciferol and 24,25-dihydroxycholecalciferol in the dog.

We studied the effects of vitamin D metabolites on parathyroid hormone (PTH) secretion. Test materials were injected into the cranial thyroid artery of the dog, and immunoreactive PTH was measured frequently in serum samples from the inferior thyroid vein and the femoral vein. This model for the study of secretion had previously been validated with the use of known modulators on PTH secretion. In control experiments, injection of 100% ethanol, the vehicle in which cholecalciferol (D(3)) metabolites were suspended, resulted in no change in PTH secretion. Likewise, native vitamin D(3), in doses ranging from 250 to 1,250 ng had no effect on PTH secretion. 25-Hydroxycholecalciferol, 25-(OH)D(3), in doses of 125-240 ng, caused complete suppression of PTH secretion. When 24,25-dihydroxycholecalciferol, 24,25-(OH)(2)D(3), was injected in doses of 50-250 ng, suppression of PTH secretion was again complete; in doses of 5 ng, injection of this metabolite resulted in significant but incomplete suppression of secretion. In doses of 50-250 ng, 1,25-(OH)(2)D(3) strongly stimulated PTH secretion, but in a dose of 5 ng this metabolite had no effects. Injection of equal doses of 1,25-(OH)(2)D(3) and 24,25-(OH)(2)D(3) resulted in significant suppression of PTH secretion. Hypocalcemia-induced stimulation of PTH secretion was suppressed by 24,25-(OH)(2)D(3) while hypercalcemia-induced suppression of PTH secretion was stimulated by 1,25-(OH)(2)D(3). In all experiments showing suppression of PTH secretion, peripheral PTH decreased. Arguments are presented for considering the suppressive effects of D(3) metabolites as physiologic modulators. However, this stimulating effect of 1,25-(OH)(2)D(3) occurred only in pharmacologic doses and hence probably has no physiologic relevance.


Lymphocytes binding C-reactive protein during acute rheumatic fever.

Lymphocytes binding C-reactive protein (CRP) were studied in 31 patients with acute rheumatic fever and 30 controls who were children. Marked elevations in both proportions and absolute numbers of CRP-binding lymphocytes were recorded in rheumatic fever (P less than 0.001). No clear correlation was noted between plasma CRP as quantitated by radioimmunoassay and proportions or numbers of CRP-binding cells. Double-labeling experiments indicated that 60-80% of CRP-binding lymphocytes also showed Fc receptors reacting with fluorescein-conjugated IgG aggregates. Passage of lymphocytes over Ig--anti-IgG columns, removed cells bearing surface Ig but not CRP-binding lymphocytes. Studies of T-cell subpopulations indicated no overlap between Tmicron- and CRP-binding cells; however about half of Tgamma-cells showed concurrent CRP binding. "Active" T-cell rosetting cells did not bind CRP. A 12-15-h incubation of lymphocytes at 37 degrees C in 5% CO2-air showed persistence of CRP binding in substantial proportions of cells particularly in acute rheumatic fever. CRP-binding lymphocytes may represent a marker for immunologically committed cells in acute rheumatic fever.


Bacterial adherence in the pathogenesis of endocarditis. Interaction of bacterial dextran, platelets, and fibrin.

The role of dextran in the pathogenesis of bacterial endocarditis was investigated by studying the adherence of dextran producing oral streptococci to the constituents of nonbacterial thrombotic endocarditis (NBTE) in vitro and in vivo. The adherence of Streptococcus sanguis to fibrin and platelets was determined in an in vitro assay system simulating nonbacterial thrombotic endocarditis. Adherence was increased when the organisms were grown in sucrose-supplemented media (adherence ratio X 10(4), 177 +/- 6 in 5% sucrose vs. 140 +/- 7 in 0.5% sucrose, P less than 0.001), and decreased by incubating the organisms in dextranase (adherence ratio X 10(4), 117 +/- 16, P less than 0.001), an effect which was nullified by heat inactivating this enzyme (adherence ratio X 10(4), 192 +/- 7, P less than 0.001). The amount of dextran produced in broth by three different oral streptococci correlated directly with the adherence observed to fibrin and a fibrin-platelet matrix in vitro (P less than 0.001). These organisms adhered more readily to a fibrin-platelet matrix than to fibrin alone (adherence ratio X 10(4), 455 +/- 30 vs. 177 +/- 6, respectively, P less than 0.001). The role of dextran formation was also examined in vivo in rabbits with preexisting NBTE. After injection of 10(7) S. sanguis, 12 of 17 animals developed endocarditis. In contrast, when the organisms were pretreated with dextranase (an enzyme that removes dextran from the bacterial cell surface), the same inoculum resulted in endocarditis in only 5 of 19 animals (P less than 0.05). In addition, a fresh strain of S. sanguis that produced high levels of dextran (1,220 +/- 50 microgram/ml) and adhered avidly to fibrin (adherence ratio X 10(4), 220 +/- 11) produced endocarditis in 12 of 18 rabbits after injection of 10(7) organisms. Another isolate of the same strain that had been passed repeatedly in the laboratory produced less dextran (400 +/- 30 microgram/ml), adhered poorly to fibrin (adherence ratio X 10(4), 140 +/- 7), and produced endocarditis in only 3 of 14 rabbits under identical conditions (P less than 0.05). This study demonstrates that dextran production is important in the adherence of oral streptococci to the constituents of NBTE and may play a role in the pathogenesis of bacterial endocarditis by oral streptococci.


Somatostatin-like immunoreactivity in rat blood. Characterization, regional differences, and responses to oral and intravenous glucose.

Somatostatin-like immunoreactivity (SLI) has been demonstrated by radioimmunoassay (RIA) in rat serum using an antiserum specific for somatostatin and cross-reacting maximally with the biologically important area on the peptide. The RIA has a sensitivity of 35 pg/ml. SLI dilutes in parallel with synthetic somatostatin standard in the RIA and shows characteristics similar to synthetic somatostatin on Sephadex G-25 (f) gel chromatography eluting largely as a single peak with 1 M acetic acid. Significant regional differences in serum SLI are present. A positive gradient was found in paired samples from aorta (mean+/-SEM, 0.304+/-0.024 ng/ml) and portal vein (0.495+/-0.047 ng/ml) consistent with the known presence of somatostatin in gut and pancreas, and a negative gradient was noted between paired samples from portal vein (0.523+/-0.076 ng/ml) and hepatic vein (0.290+/-0.048 ng/ml) indicating hepatic clearance. No significant differences were demonstrated between aorta and confluence of cerebral venous sinuses or between aorta and inferior vena cava (IVC). After intragastric glucose, a significant and marked elevation of portal SLI was observed, maximal at 5 min (0.416+/-0.137 vs. 1.55+/-0.30 ng/ml at 5 min). A significant biphasic elevation of portal SLI also occurred after intravenous glucose. After both routes of glucose administration, the patterns of portal SLI followed closely those of portal glucose and insulin. By contrast, IVC SLI failed to reflect these changes.Thus, SLI in the rat shows chromatographic similarity with synthetic somatostatin. Regional differences in serum levels are marked; the highest concentrations being found in the portal venous effluent of pancreas and gut. Furthermore, glucose causes elevation of portal SLI in a pattern similar to portal insulin and glucose and without concomitant elevation in IVC. This differential elevation of SLI after glucose is consistent with a hormonal action within the portal system as a direct effect of somatostatin on the liver has previously been demonstrated. In addition, the liver is important in the clearance of portal SLI, possibly to prevent extraportal effects in response to gut and pancreatic stimulation. Finally, it is clear that regional sampling of serum for SLI measurement may be critical in the investigation of the putative physiological roles for somatostatin.


Biochemical heterogeneity in glutathione synthetase deficiency.

Two different clinical syndromes are associated with glutathione synthetase deficiency, one presenting with hemolytic anemia and 5-oxoprolinuria, the other with isolated hemolysis. We have differentiated these disorders on an enzymatic basis. In 5-oxoprolinuria, all cell types examined have grossly deficient enzyme activity and glutathione content. In contrast, in the nonoxoprolinuric variant, erythrocytes have decreased enzyme activity and glutathione content, whereas nucleated cells maintain substantial levels of both. The enzyme in this disorder is unstable in vitro and has shortened survival in intact erythrocytes. Nucleated cells appear able to maintain sufficient enzyme activity and concentrations of glutathione to suppress overproduction of 5-oxoproline.


Bicarbonate secretion by rabbit cortical collecting tubules in vitro.

We previously reported that rabbit renal cortical collecting tubules can secrete bicarbonate in vitro (i.e., there can be net transport from bath to lumen, causing the concentration in the lumen to increase). Net bicarbonate secretion was observed most often when rabbits had been pretreated with NaHCO(3) and were excreting alkaline urine before being killed for experiments. The purpose of the present studies was to elucidate the mechanism involved by testing the effects of ion substitutions and drugs on collecting tubules that were secreting bicarbonate. Acetazolamide inhibited net bicarbonate secretion, suggesting that the process is dependent upon carbonic anhydrase. Net bicarbonate secretion also decreased when sodium in the perfusate and bath was replaced by choline, but not when chloride was replaced by nitrate or methylsulfate. Ouabain had no significant effect. Amiloride caused net bicarbonate secretion to increase. The rate of net secretion did not correlate with transepithelial voltage. The results are compared to those in turtle urinary bladders that also secrete bicarbonate. There are no direct contradictions between the results in the two tissues, i.e., in turtle bladders acetazolamide also inhibited bicarbonate secretion and ouabain had no effect. Nevertheless, it seems unlikely that net secretion of bicarbonate by collecting tubules involves specific exchange for chloride, as has been proposed for turtle bladders, because replacement of chloride by other anions did not inhibit bicarbonate secretion by collecting tubules. It was previously shown that the collecting tubules in vitro also may absorb bicarbonate, especially when the rabbits have been treated with NH(4)Cl and are excreting acid urine before being killed. The effects of drugs on net bicarbonate secretion found in the present studies are compared to their previously reported effects on net bicarbonate absorption and the possibility is discussed that bicarbonate absorption and secretion are independent processes, as was previously proposed for turtle bladders.


The critical role of iron in host-bacterial interactions.

The ability of potential pathogens to acquire iron in a host is an important determinant of both their virulence and the nature of the infection produced. Virulent gram-negative bacteria are capable of acquiring sufficient iron from the host because their virulence (for chick embryos) is unaffected by exogenous iron. Avirulent mutants which are apparently limited in their ability to acquire iron could be isolated from the virulent strains. The lethality of these mutants was significantly enhanced by exogenous iron. Reduction of the relatively high serum iron saturation of chick embryos (to levels more closely approximating those in man) by pretreatment with iron-binding proteins or endotoxin inhibits the lethality of some virulent bacteria. Those bacteria whose virulence was reduced include the Shigella, Vibrio cholerae and strains of Neisseria gonorrhoeae, all of which are nondisseminating pathogens in the normal human host. Pathogens which produce septicemic and disseminating infections such as Neisseria meningitidis, Haemophilus influenzae type B, Escherichia coli possessing K-1 antigen, Pseudomonas aeruginosa and Salmonella typhimurium and disseminating strains of N. gonorrhoeae were, in general, unaffected by reduced serum iron saturation. These disseminating bacteria appeared to produce greater quantities of compounds (siderophores) which stimulated microbial growth in low-iron media than did the nondisseminating pathogens. Thus, the gram-negative bacteria tested can be divided into four major classes according to their responses to modifications in iron levels in the chick embryo model and these results correlate with the nature of the infections which they typically produce in man.


Comparative responses of tracheal spirals and parenchymal strips to histamine and carbachol in vitro.

The responses of isolated guinea pig tracheal spirals and parenchymal strips to histamine and carbachol were compared. The parenchymal strip, a 1.5 x 1.5 x 20-mm strip cut from the periphery of the lung, constricted at a lower dose and had a larger maximal response to histamine than to carbachol. In contrast, the response of the tracheal spiral to equimolar doses of histamine or carbachol was the same. The responsiveness of both muscle strips to histamine was decreased by treatment with the H1 receptor antagonist mepyramine (0.1 micrometer), and the response to carbachol was blocked by treatment with atropine (0.1 micrometer). Indomethacin (3 micrometer), cimetidine (1 micrometer), propranolol (10 micrometer), and N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) (4 micrometer) did not alter the differential response of the two strips to histamine and carbachol. The differential response of parenchymal strips with many, few, or no conducting airways and blood vessels was identical, suggesting that the contractile element is alveolar duct smooth muscle or alveolar contractile elements. This differential pharmacologic response in vitro is consistent with the in vivo observation that histamine causes more peripheral airway constriction than does acetylcholine.


Neurodiagnostic abnormalities in patients with acute renal failure.

Neurological abnormalities are a major cause of morbidity in patients with renal failure. The pathophysiology of these neurological changes is unclear, and the effects on them of dialysis and return of renal function have not been well studied. Studies were done in 31 patients who had acute renal failure (ARF), all of whom were either treated with dialysis within 5 days or did not survive. Studies on these patients included the electroencephalogram (EEG), motor nerve conduction velocity, and plasma Ca(++) and parathyroid hormone (PTH) levels. Studies were done at the time ARF was diagnosed, after stabilization on dialysis, during the diuretic phase of ARF, and 3 mo after recovery from ARF. In 16 patients with acute or chronic renal failure who did not survive and in nine patients without renal disease who died, measurements were made in brain of content of Na(+), K(+), Cl(-), Ca(++), Mg(++), and water. In patients with ARF for less than 48 h, despite the fact that there were only modest increases in plasma urea and creatinine, there were striking abnormalities in the EEG. The percent EEG power < 5 Hz+/-SE was 41+/-8% (normal = 2+/-1%), whereas the percent of frequencies > 9 Hz was only 22+/-6% (normal = 62+/-3%). These changes were unaffected by dialysis, but became normal with return of renal function and remained normal at 3 mo follow-up. The motor nerve conduction velocity was unaffected by either ARF or dialysis. In patients with ARF, the brain Ca(++) was 46.5+/-3.2 meq/kg dry wt, almost twice the normal value of 26.9+/-1.0 meq/kg dry wt (P < 0.001). The plasma PTH level was 3.2+/-0.6 ng/ml (normal < 1.5 ng/ml, P < 0.01). The increased brain Ca(++) was not related to an increased plasma (Ca(++)) (PO(4) (---)) product (r(2) = 0.14, P > 0.05). There was a small but significant decrement in brain Na(+) (P < 0.05), but brain water, K(+), and Mg(++) were unaffected by ARF.Thus, in patients with ARF for less than 48 h, the EEG is grossly abnormal and there are elevated levels of PTH in plasma. The PTH appears to have a direct effect on the brain, resulting in an increased brain Ca(++) content. The EEG abnormalities are unaffected by dialysis, but they become normal with return of renal function and remain normal after 3 mo follow-up. Thus, PTH may be a major uremic toxin, demonstrating evidence for central nervous system toxicity when there are only minimal abnormalities of other biochemical markers of ARF.


Increased adrenal sensitivity to angiotensin II in low-renin essential hypertension.

Studies were undertaken to determine if the dissociation of aldosterone and plasma renin activity in low-renin essential hypertension is due to altered adrenal responsiveness to angiotensin II. The responsiveness of the adrenal glands to angiotensin II was determined by infusing graded doses of angiotensin II into normal subjects and into patients with essential hypertension and measuring changes in levels of plasma aldosterone in response to the infusion. To minimize the influence of endogenous angiotensin II and ACTH, supplemental sodium and dexamethasone were given before the infusions. Levels of plasma aldosterone and plasma renin activity were determined in normal subjects and in the same patients after the combined stimuli of furosemide and upright posture, a maneuver used to increase the level of endogenous angiotensin II. To determine if the changes in levels of plasma aldosterone during infusion of angiotensin II were due to alteration of the metabolic clearance of aldosterone, the metabolic clearance of aldosterone was measured before and during the infusion of angiotensin II. After sodium loading, dexamethasone treatment, and supine posture, levels of plasma aldosterone of normal subjects and patients with essential hypertension were suppressed equally. In response to the infusion of angiotensin II, the levels of plasma aldosterone of patients with low-renin essential hypertension were significantly higher than those of normal subjects or of patients with normal-renin essential hypertension. After furosemide and upright posture, levels of plasma aldosterone of patients with low-renin essential hypertension were significantly higher than those of patients with normal-renin essential hypertension, despite a blunted response in plasma renin activity of the patients with low-renin essential hypertension. Decreases in metabolic clearance of aldosterone during infusion of angiotensin II were similar in patients with normal-renin essential hypertension and in patients with low-renin essential hypertension and accounted for only a small fraction of the marked increase in levels of plasma aldosterone of patients with low-renin essential hypertension. It is concluded that patients with low-renin essential hypertension have increased adrenal sensitivity to angiotensin II. This increased sensitivity may explain the dissociation of aldosterone and plasma renin activity in low-renin essential hypertension.