A simple procedure for the isolation of L-fucose-binding lectins from Ulex europaeus and Lotus tetragonolobus.

L-Fucose-binding lectins from Ulex europeaus and Lotus tetragonolobus were isolated by affinity chromatography on columns of L-fucose-Sepharose 6B. L-Fucose was coupled to Sepharose 6B after divinyl sulfone-activation of the gel to give an affinity adsorbent capable of binding more than 1.2 mg of Ulex lextin/ml of gel, which could then be eluted with 0.1M or 0.05M L-fucose. Analysis of the isolated lectins by hemagglutination assay, by gel filtration, and polyacrylamide disc-electrophoresis revealed the presence of isolectins, or aggregated species, or both. The apparent mol. wt. of the major lectin fraction from Lotus was 35000 when determined on Sephadex G-200 or Ultrogel AcA 34. In contrast, the apparent mol. wt. of the major lectin fraction from Ulex was 68 000 when chromatographed on Sephadex G-200 and 45 000 when chromatographed on Ultrogel AcA 34. The yields of lectins were 4.5 mg/100 g of Ulex seeds and 394 mg/100 g of Lotus seeds.


Rapid and sensitive, colorimetric determination of the anomers of D-glucose with D-glucose oxidase, peroxidase, and mutarotase.

A modification, utilising mutarotase, of an enzymic, colorimetric system for determining D-glucose with D-glucose oxidase, peroxidase, and ABTS was satisfactory for the assay of the anomers of D-glucose in aqueous solution. The time required for a single assay is approximately 10 min, and the lower limit is 0.4 microgram of D-glucose. The method is applicable to the anomer analysis of D-glucose released by enzymic hydrolysis of D-glucosides.


Synthesis of derivatives of 3-amino-2,3-dideoxy-L-hexoses related to daunosamine (3-amino-2,3,6-trideoxy-L-lyxo-hexose).

The synthesis is described of 3-amino-2,3-dideoxy-L-arabino-hexose (10), methyl 2,3-dideoxy-alpha-L-lyxo-hexopyranoside(17), methyl 3-amino-2,3-dideoxy-alpha-L-ribo-hexopyranoside (21), methyl 2,3-dideoxy-3-trifluoroacetamido-alpha-L-xylo-hexopyranoside (26), and certain derivatives from methyl 4,6-O-benzylidene-2-deoxy-alpha-L-arabino-hexopyranoside (3). Conversion of 2-deoxy-L-arabino-hexose into 3 by modified, standard procedures, and on a large scale, gave a 75% yield.


Syntheses of 3',4',5,6-tetradioxyneamine and 5,6-dideoxyneamine.

3',4',5,6-Tetradoxyneamine (7) was synthesized from neamine by the reaction sequence N-acetylation, O-mesylation, treatment with sodium iodide-zinc dust, hydrogenation, and base hyrolysis, Alternatively, 7 was obtained by treatment of tetra-(N-methoxycarbonyl)neamine with sulphuryl chloride, followed by hydrogenation in the presence of palladium-on-charcoal and triethylamine, and removal of the N-protecting groups. 5,6-Dideoxyneamine (11) was synthesized from known 5,6-O-cyclohexylidene-tetra-(N-methoxycarbonyl)neamine by the reaction sequence 3',4'-O-acetylation, removal of the 5,6-O-cyclohexylidene group, 5,6-di-O-mesylation, treatment with sodium iodide-zinc dust, hydrogenation, and base hydrolysis. An alternative route involved treatment of 3',4'-di-O-acetyl-tetra-(N-methoxycarbonyl)-neamine (8) with sulphuryl chloride, hydrogenation of the product, and removal of the protecting groups to give 11.


High-pressure liquid-chromatographic assay of nucleotide-pool concentrations during polysaccharide biosynthesis in four ascomycetes.

High-resolution liquid-chromatographic methods developed for analyzing nucleotide pools at the nanogram level in four representative species of ascomycetes (Penicillium citrinum, Aspergillus niger, Fusarium moniliforme, and Cladosporium herbarum) were used to study polysaccharide biosynthesis. Nucleotides extracted from the mycelial mat were preseparated from interfering polysaccharides, glycoproteins, and nucleic acids on a column of Biogel P-2. Resolution of 18 nucleotides from each fungal species was accomplished on AS-Pellionex-SAX, pellicular anion-exchanger by using a high-pressure liquid chromatograph. Nucleotides were identified by comparing peak retention-times, by differential u.v. absorption with two detectors in series at selected wavelengths, and by acid or enzymic hydrolysis with product identification by liquid chromatography. Pyrimidine bases exceeded purines by at least three fold, and uridine nucleotides often constituted 60-80 mole percent of the total nucleotides; extractable cytidine nucleotides were negligible. Uridine 5'-(2-acetamido-2-deoxy-alpha-D-glucopyranosyl disphosphate) is the preponderant nucleotide throughout the growth cycles of all four species, amounting to 30-60% of all nucleotides present. For all four fungal species, a burst of nucleotide formation was observed after the first 48h (15-30 mumol/g tissue), with fluctuations that eventually fell to 0.1 mumol/g on the tenth day.


Cardiac catheterization experience in hospitals without cardiovascular surgery programs.

In order to study the cardiac catheterization experience in hospitals without cardiovascular surgery programs, data was collected from all 8 Washington "satellite" laboratories over a 5-year period. There were 5 deaths (0.13%) during the 3878 coronary arteriography procedures. Of the 5, 4 had severe left main coronary artery lesions. Of these patients, 2 died during the 24-hour follow-up period after an uncomplicated study. This mortality rate is remarkably good considering that the 5-year period includes the early experience of 7 laboratories. There were 7 myocardial infarctions (0.18%) and 6 strokes (0.15%). The average number of coronary arteriograms done per angiographer during 1976 was 65. The experience of the Washington State "satellite" cardiac catheterization laboratories proves that the immediate availability of cardiovascular surgery and large case loads per angiographer are not necessary in order to safely perform cardiac catheterization and coronary arteriographic studies. Additional studies should be undertaken to determine the appropriate distribution of cardiac diagnostic facilities.


Coronary artery fistula: estimation of shunt using 99m Tc-albumin particles.

A 60-year-old man presented with complaints on angina pectoris and was found to have a coronary artery fistula between his left main truck and main pulmonary artery. Particles of 99m Tc-Albumin were injected in the ostium of the left coronary artery, and differential radioactive counts were injected in the ostium of the left coronary artery, and differential radioactive counts were obtained over both lung fields and myocardium. The degree of left to right shunt was calculated at 56% of total left coronary artery flow. The patient underwent ligation of the fistula without any complications. This case report represents a new application of myocardial radioisotopic scanning.


Plain film identification of failure of interior vena caval-right atrial continuity.

The identification of absence of continuity of the inferior vena cava (IVC) with the right atrium assumes major importance at the time of right heart catheterization. The appearance of the IVC in the lateral chest radiogram is diagnostically different from the normal when discontinuity is present and 2 radiographic patterns have been recognized: 1) total absence of the IVC shadow; 2) straightened and posteriorly directed IVC shadow contrasting sharply with the anteriorly directed normally continuous IVC. This latter sign is considered reproducibly accurate in the identification of the IVC which fails to have continuity with the right atrium.


Atypical late cardiac tamponade after mitral valve replacement: case presentation with hemodynamic and echocardiographic observations.

A patient developed oliguria, peripheral edema, and dyspnea 5 days after mitral valve replacement. Chest roentgenogram and echocardiogram suggested pericardial effusion. Although pulsus paradoxus was absent, cardiac tamponade was suspected and subsequently confirmed during pericardiocentesis. Before pericardiocentesis right and left ventricular diastolic pressures were equal, as well as intrapericardial and right atrial pressures. The left ventricular cavity was reduced in size on the echocardiogram. Interventricular septal motion was normal. Pericardiocentesis normalized the hemodynamics and allowed the left ventricle to reexpand. Certain atypical features are tentatively explained as the combined effect of constrictive and effusive components caused by intrapericardial hemorrhage.


Longitudinal angulation in coronary arteriography: apparatus and evaluation.

When good visualization of the left coronary artery and proximal branches is difficult, the longitudinally angulated projection has proved useful. An accessory for achieving this angulation with a conventional angiographic table and without sacrificing the advantages of video monitoring and the protection of lead vinyl shielding is described.


Pediatric cardiac angiography using a 4 French catheter.

Thirty specially designed 4 French pigtail white Teflon catheters 50 cm in length were utilized for angiography using the retrograde arterial approach in 26 infants and children. Angiographic opacification was considered very satisfactory. Apart from one episode of temporary occlusion of the right coronary artery ostium, there were no other significant complications. The catheters were introduced percutaneously in 21 instances and distal pulses were palpable in 18 patients by the second day after catheterization. At postcatheterization testing with increasing volumes and flow rates, the first rupture occurred, delivering 13 ml at 13 ml/second. On testing an additional 15 catheters using fixed volumes of 5, 10, and 15 ml at increasing flow rates, rupture first occurred at 34, 22, and 18 ml/second, respectively. We conclude that this small catheter is relatively safe and satisfactory for the retrograde arterial approach in the study of infants and children.


Antegrade left heart catheterization.

Retograde arterial catheterization has been the standard approach to hemodynamic and angiographic evaluation of the left ventricle and systemic arterial system. An alternative approach--antegrade left heart catheterization--is reported that circumvents arterial puncture. During cardiac catheterization from the femoral vein in 18 children with normally related great arteries who had interatrial communications, a balloon-tipped catheter was looped in a "clockwise" fashion through the left atrium and left ventricle and into the ascending aorta. In 17 children the maneuver was rapidly accomplished without complications; it proved possible to perform in an infant with a hypoplastic left ventricular cavity, and was employed to perform a renal arteriogram. The major advantage of antegrade left heart catheterization is avoidance of complications related to arterial puncture. The antegrade approach also avoids right arm cut-down in children with coarctation of the aorta.


Unsuspected left-to-right shunt: is routine use of hydrogen platinum electrode system indicated in shunt detection?

Unsuspected left-to-right shunt was found in 6 of 1,500 adult patients undergoing diagnostic cardiac catheterization. In 3 patients were found unsuspected secundum atrial septal defects (1 associated with mitral stenosis and 2 with coronary artery disease). In 3 patients, unsuspected partial anomalous pulmonary venous drainage was found (1 associated with aortic valve disease and 2 with severe coronary artery disease). It is concluded that the incidence of unsuspected left-to-right shunt diagnosed during routine cardiac catheterization is very low. Careful physical examination and the application of conventional techniques during cardiac catheterization should suggest the possibility of such a shunt. Hydrogen platinum electrode system (HPES) should be reserved for patients in whom the right heart catheter takes an abnormal course or when the pulmonary artery oxygen saturation is high and the arteriovenous oxygen difference is small.


Forward and reverse mutations affecting the kinetics and apparent molecular weight of mammalian HGPRT.

Chinese hamster cells selected for resistance to 8-azaguanine following mutagenesis have hypoxanthine-guanine phosphoribosyltransferase (HGPRT; E.C. 2.4.2.8) with characteristics compatible with different mutations in the structural gene for that enzyme. Using immunopurification and SDS-polyacrylamide electrophoresis, mutants producing antigenically active forms of the enzyme can be analyzed for changes in the molecular weight of HGPRT. Enzyme subunits from mutants RJK3 and RJK39 are reduced in molecular weight by an estimated 4 and 2%, respectively. HGPRT activity is not detectable in RJK39. The enzyme from RJK3 is active but has altered substrate binding properties. Enzymes from two other mutants with altered kinetic properties, RJK44 and RJK47, have normal molecular weights. The genetic alterations of RJK44 and 47 are probably missense mutations, while RJK3 and 39 might contain either deletions or mutations causing premature peptide chain termination. Somatic cell hybridization between RJK39 and a revertant of that strain with HGPRT of normal molecular weight revealed that the revertant probably arose by intragenic mutation rather than extragenic mutation or suppression.


Catabolism of thymidine during the lymphocyte cell cycle.

Concanavalin A-stimulated lymphocytes degrade thymidine to beta-aminoisobutyric acid. Thymidine-catabolizing enzymes are active in the cells during G1, G2 and mitosis, but activity falls to very low levels just prior to the onset of S and remains low throughout the S period. The data suggest that cell pools of thymidine are regulated by degradation.


The isolation of a suppressible nonsense mutant in mammalian cells.

An HGPRT- cell line derived from mouse L cells has been shown to have the following properties: it is CRM+; the defective HGPRT molecules are altered in the carboxyterminal peptide; the mutant cells regain HGPRT activity when ochre-suppressor tRNA is microinjected into them, but not when amber-suppressor or wild-type tRNAs are injected. We conclude from these properties that this mutant cell line contains an ochre nonsense mutation (UAA) in the structural gene for HGPRT.


Regulation of flagellin synthesis in the cell cycle of caulobacter: dependence on DNA replication.

Synthesis of the two filament proteins (flagellin A and flagellin B) of the Caulobacter creascentus flagellum was measured during the cell cycle. Synchronous cells were pulse-labeled with 36S-methionine, and flagellin proteins were isolated from crude extracts by radioimmune precipitation. The results showed that both proteins are maximally induced during the G2 period and that their induction requires de novo transcription. Flagellin A, however, continues to be made in the progeny swarmer cells after flagellin B synthesis has stopped. This discoordination in flagellin A and B synthesis and the relative abundance of the two proteins may result in part from the longer functional half-life of the flagellin A messenger RNA. Analysis of temperature-sensitive DNA chain elongation mutants suggests that the periodicity of flagellin A and B synthesis in the cell cycle is controlled by a late cell cycle event, presumably the completion of chromosome replication.


Effects of androgens on the complexity of poly(A) RNA from rat prostate.

Poly(A)-containing RNA, isolated from rat ventral prostate, has been analyzed for its base sequence complexity. The kinetics of hybridization of total cellular poly(A)-containing RNA with its complementary DNA (cDNA) from normal and castrated animals are markedly different. RNA from normal animals consists of three abundance classes, about 36% comprises one or two highly abundant RNA sequences, 29% consists of about 24 sequences and the remainder is a scarce class of approximately 8200 sequences. In contrast, the hybridization kinetics of prostatic RNA from castrated animals demonstrate that there is a moderate abundance class of 53 sequences and a scarce class of about 7800 sequences, but that a class of abundant sequences is not present. Using normal prostatic cDNA as a probe, we showed that the abundant sequences were not absent but reduced 10 fold following a 3-day castration period and 100 fold after 100 fold after 7 days. Such heterologous hybridization experiments also suggest that there is significant sequence homology in the RNA sequences present in the prostate irrespective of the hormonal status of the animals. The major effect of testosterone appears to be the regulation of the abundance of specific RNA sequences.


Histone neighbors in nuclei and extended chromatin.

Histone neighbors in compact and extended chromatin have been investigated by cross-linking histones in nuclei and in nucleohistone extended with 6 M urea, using the bifunctional reversible reagent methyl-4-mercaptobutyrimidate (MMB). Similar histone dimers are found in both conformational states of chromatin. The dimers most frequently found are H2b-H2a, H2b-H3 and H3-H2a; dimers found less frequently are H3-H4, H3-H3 and H2b-H4. More H3-H3 is found in nuclei than in extended chromatin. H1 is found predominantly as poly-H1, although it can be cross-linked to H2b or H3. After reaction with MMB, native compact chromatin is no longer extendable in 6 M urea, which shows that the reagent is capable of linking together histones holding the chromatin in a compact conformation. Thus the histone propinquity in extended chromatin mimics and intimate histone associations in compact chromatin.


A physical map of the DNA regions flanking the rabbit beta-globin gene.

Rabbit liver DNA was cleaved with the restriction endonucleases Eco RI, Pst I or Kpn I. Resulting DNA fragments were denatured, separated by electrophoresis in agarose gels and subsequently transferred by blotting onto nitrocellulose filters. Filters were then hybridized with a plasmid containing a DNA copy of rabbit beta-globin messenger RNA (plasmid PbetaG1; Maniatis et al., 1976) which had been labeled by nick translation to a high specific activity with 32P. A very limited number of discrete rabbit DNA fragments was found which could form well matched hybrids with PbetaG1 DNA. Endonuclease Eco RI, which cleaves the rabbit beta-globin gene, produced two beta-globin DNA fragments, whereas endonucleases Pst I and Kpn I, which do not cut the gene, each generated only one fragment. Using double digests, these fragments could be ordered into a physical map of restriction endonuclease cleavage sites around the beta-globin gene on the rabbit genome. This map is consistent with the presence of a single copy of the beta-globin gene per haploid genome. The direction of transcription of the beta-globin gene within the physical map was determined.


Accumulation of alpha- and beta-globin messenger RNAs in mouse erythroleukemia cells.

The accumulation of alpha- and beta-globin mRNA sequences in murine erythroleukemia cells (MELC) treated with various inducers has been studied using specific alpha- and beta-globin complementary DNAs (cDNAs). In cells cultured with dimethylsulfoxide (Me2SO), hexamethylene bisacetamide (HMBA) or butyric acid, accumulation of alpha-globin mRNA is detectable after 16, 12 and 8 hr of culture, respectively. An increase in beta-globin mRNA sequences is not detected until 20-24 hr after culture. In cells exposed to hemin, both alpha- and beta-globin mRNAs are detectable by 6 hr of culture, and a constant ratio of alpha/beta-mRNA is maintained during induction. In maximally induced cells, the alpha/beta-globin mRNA ratios are approximately 1 in cells induced by Me2SO and HMBA, and 0.66 and 0.3-0.50 in cells induced by butyric acid and hemin, respectively. Thus different inducers of erythroid differentiation in MELC lead to different times of onset of the expression of alpha- and beta-like genes. In addition, the relative accumulation of alpha- and beta-globulin mRNAs in induced cells differs with various types of inducers.


Initiator RNA of discontinuous DNA synthesis in human lymphocytes.

A short RNA covalently associated with nascent DNA has been isolated after synthesis in vitro with labeled ribonucleaside triphosphates and the removal of DNA by DNAase digestion. The RNA migrates in polyacrylamide gels or chromatographs on DEAE-Sephadex columns as a relatively discrete oligonucleotide 8-11 nucleotides in length. The RNA is associated primarily with nascent DNA with stoichiometry of approximately one per DNA chain. The RNA has a triphosphate group at the 5' end and 2 or 3 deoxynucleotide residues at the 3' end that are not removed by DNAase. These results further support a role for the RNA as an initiator of discontinuous DNA synthesis. Examination of sequences present at the 3' end of the RNA using RNAase to effect transfer of 32PO4 from 32P-labeled DNA to covalently attached RNA indicates that a diverse, rather than unique, set of sequences are present in the RNA.


Detection of myosin heavy chain mRNA during myogenesis in tissue culture by in vitro and in situ hybridization.

A cDNA copy of purified chick embryonic skeletal myosin heavy chain mRNA (MHC mRNA) distinguished between myogenic and nonmyogenic cells compared by in vitro and in situ hybridization. The majority of cells in replicating mononucleate myogenic cell cultures contained no detectable MHC mRNA. Among the earliest cells to contain MHC mRNA were cells engaged in mitosis. A relatively large amount of MHC mRNA was found in postmitotic monucleate cells and myotubes, and we observed nucleolar localization of MHC mRNA in these cells.


Development of choline acetyltransferase and cholinesterase activities in enteric ganglia derives from presumptive adrenergic and cholinergic levels of the neural crest.

The cholinergic differentiation of enteric ganglia in embryos of chick and quail was studied with particular reference to cholinesterase and choline acetyltransferase activities. Differentiation during normal development was compared with that obtained after culture of the neural primordium or neural crest in direct association with aneural hindgut. Biochemically differentiated cholinergic ganglia developed in explants containing cells from either the 'vagal' (presumptive cholinergic) or 'truncal' (presumptive adrenergic) levels of the neural crest. Neither extra-intestinal migration of neural crest cell nor the presence of central preganglionic fibres is a prerequisite for enteric ganglion differentiation.


Hormonal control of milk protein synthesis in cultured mouse mammary explants.

SDS-urea polyacrylamide gel electrophoresis of mouse milk proteins revealed the presence of three major phosphoproteins (caseins) of m.w. 44,000, 26,000 and 22,000 daltons. By using an antiserum against crude casein fraction, an immunoprecipitation method was developed for the quantitative measurement of the rate of milk protein synthesis in the mouse mammary tissue. Cultivation of mammary explants with insulin, cortisol and prolactin resulted in the induction of milk protein synthesis as evidenced by the incorporation of [3H]amino acid and [32P]orthophosphate into immune precipitable materials. The present immunoprecipitation method coupled with a simplified explant culture technique provides a suitable procedure for the study of mouse mammary gland differentiation.


Altered histone-DNA interactions in rat liver chromatin containing 5-bromodeoxyuridine-substituted DNA.

The extractability of the different histone types from rat liver chromatin was studied following the incorporation of bromodeoxyuridine (BrUdR) in liver DNA. This was accomplished by a continuous application of 20 mumol BrUdR/ml/h 17--41 after partial hepatectomy. As a result, thymidine (TdR) replacement by BrUdR of about 80% in the newly-synthesized DNA strand of approx. 30% of total liver DNA was obtained; this causes remarkable changes in the histone--DNA interactions as determined from the release of histones from liver nuclei by ammonium sulfate and ethidium bromide (EB), respectively. In particular, the relative amounts of the two slightly lysine-rich histones H2A and H2B remaining on the BrUdR chromatin proved to be about 3-fold higher than those remaining on the control chromatin of TdR-treated animals. Similarly, histones H1 and H3 tend to bind closer to BrUdR-containing DNA. These results may be of interst with regard to the well-known selective effects of BrUdR on differentiation processes.


Action of 1,10-phenanthroline transition metal chelates and their constituents on the rat isolated diaphragm preparation.

The actions of two related series of fully co-ordinated, divalent 1,10-phenanthroline transition metal chelates have been investigated on the rat isolated diaphragm muscle-phrenic nerve preparation and, where possible, compared with those of their constituent metal ions and ligands. Each member of both series of chelates produced blockade of neuromuscular transmission, although mechanistically not of a uniform type, and several elicited varying degrees of muscle contracture. The kinetic reactivity of the metal chelate appeared to be an important factor determining the nature of the biological response and profound differences in response were observed between labile and inert chelates differing in some cases by only one electron in the 3d shell.


In vitro binding of beta-propiolactone to calf thymus DNA and mouse liver DNA to form 1-(2-carboxyethyl) adenine.

In vitro reaction of beta-propiolactone (BPL) with calf thymus DNA and mouse liver DNA followed by acid (HCL) hydrolyses of the BPL-reacted DNA's resulted in the isolation of a new compound, 1-(2-carboxyethyl)-adenine (1-CEA). The structure was assigned on the basis of ultraviolet spectra at acidic, alkaline and neutral pH and electron impact and chemical ionization mass spectra as well as chemical synthesis of 1-CEA from BPL and 2'-adenosine-5'-monophosphoric acid. The only other compound previously isolated from the in vitro and in vivo reactions of BPL and DNA was 7-(2-carboxyethyl)guanine (7-CEG) which we also identified as a product of our in vitro reaction. Under the conditions used the main product of alkylation was 1-CEA and the ratios of the concentrations of 1-CEA to 7-CEG was approx 3 : 1. The possible effect of the formation of 1-CEA on the structure of DNA and its role in chemical carcinogenesis is discussed.


Effect of various chemicals on the metabolism of benzo(a)pyrene by cultured rat colon.

The effect of various co- and anti-carcinogens of colon carcinogenesis on the metabolism of benzo(a)pyrene (BP) in cultured rat colon is reported. Rat colon enzymatically converted BP into metabolites which bind to cellular macromolecules i.e., DNA and protein. Activity of aryl hydrocarbon hydroxylase (AHH) activity and binding levels of BP to macromolecules were higher in the descending colon when compared to other segments. The major metabolites of BP, extractable with ethylacetate, were quinones, tetrols, 7,8-diol and a peak containing 9,10-dihydroxy-9,10-dihydrobenzo(a)pyrene and 7,8,9-trihydroxy-7,8-dihydrobenzo(a)pyrene. The binding levels of BP to DNA and protein in the explant was lowered by co-incubation with 7,8-benzoflavone (7,8-BF) (3.6 and 18.0 micron), a known inhibitor of AHH, and with disulfiram (100 micron), an anti-oxidant. The absence of vitamin A in the media also resulted in a lower level of BP binding to DNA and protein and in lower activity of AHH. Pretreatment with known inducers of AHH such as phenobarbital (PB) or benz(a)anthracene (BA), did not have any significant effect on the binding levels of BP to DNA or on the AHH activity. Of the bile acids investigated only taurodeoxycholic acid significantly increased the binding level of BP to DNA.


[Effect of dicarboxylic lecithins on liposomes].

We have investigated the effect of dicarboxylic lecithins on liposomes prepared from egg-lecithins and their incorporation into liposome preparation. In the presence of phospholipids, liposome cell permeability to glucose is five times higher than in controls. The incorporation of dicarboxylic lecithins into liposomes at a molar concentration of 50 molecules per cent reduces by 60% the amount of glucose trapped inside the liposomes. Measurements of ovolecithin-glutaryl-lecithin liposomes stability show that stable structures are formed at a molar concentration of 70 molecules per cent in the presence of cholesterol. On the contrary, the liposomes prepared in the absence of cholesterol are not stable at a molar concentration of 50 molecules per cent. Measurements of sedimentation of mixed dispersions of monocarboxylic lecithins and glutaryl-lecithins show that glutaryl-lecithins must be in the form of micelles to solubilize the phosphatidyl cholines.


Mechanism of alkylation by N-nitroso compounds: detection of rearranged alcohol in the microsomal metabolism of N-nitrosodi-n-propylamine and base-catalyzed decomposition of N-n-propyl-N-nitrosourea.

Metabolism of N-nitrosodi-n-propylamine by an isolated rat liver microsomal fraction yielded 17% isopropanol and 83% n-propanol (expressed as a percentage of total propanol formed). Base-catalyzed decomposition of N-n-propyl-N-nitrosourea yielded 39% isopropanol and 61% n-propanol. The values provide evidence for involvement of carbocations in both of these reactions.


The crystal structure of cholesteryl 17-bromoheptadecanoate.

Crystals of cholesteryl-17-bromoheptadecanoate (C44H77BrO2) are monoclinic (P21) with a = 7.663(2), b = 10.311(5), c = 55.96(2) A and beta = 103.10(3 degrees). These are two molecules in the asymmetric unit which have different conformations of the cholesterol side chain and about the ester bond. The molecules pack with regions of only steroid skeleta alternating with regions of hydrocarbon chains. Due to the packing requirements of the skeleta the carbon chains are forced into a hybrid type packing which contains features of the earlier known O perpendicular and T parallel subcells. The subcell (HS1) is orthorhombic with as = 10.3, bs = 7.5 and cs = 2.54 A. The molecular packing is such that the omega-bromine atoms do not continue the trans-carbon chains but adopt a gauche conformation.


Structural studies on branched fucosphingolipids of hog gastric mucosa.

A structural studies have been performed on new complex glycolipids extracted from hog gastric mucosa by 0.4 M sodium acetate in methanol-chloroform-water, and which were purified to homogeneity by DEAE-Sephadex and Florisil column chromatography and by preparative thin-layer chromatography in three solvent systems. Five branched fucolipids have been purified from this extract, three of which have been characterized previously [1] and remaining two were subject of this investigations. Based on the results of partial acid hydrolyses, oxidation with periodate and chromium trioxide, permethylation and serological activities following structures were proposed.


A study of some environmental influences in language development.

A study of the language development of a group of three year old children living on a housing estate is described. Various factors in their environment are examined in relation to their development. The only significant finding was that children possessing toys which encouraged creative play were more advanced in both verbal comprehension and expressive language. The role of language in intellectual development and possible implications for pre-school facilities are discussed.


Neural regulation of renin release: experimental evidence and clinical implications in arterial hypertension.

The assessment of the possible role played by the renin-angiotensin system in maintaining arterial hypertension requires an understanding of the mechanisms regulating renin release from the kidney. In addition to intrarenal mechanisms of control, recent investigation stresses the importance of sympathetic influences on the juxtaglomerular cells both in experimental animals and in man. Studies with beta-adrenergic blocking agents, interfering with sympathetic release of renin, and with peptides interfering with sympathetic release of renin, and with peptides interfering with the generation or action of angiotensin II, suggest that renin may play some pressor role in a number of cases of hypertension: a major one in the small group of patients with high plasma renin, and possibly a minor one in the large population of patients whose renin is stimulated by treatment with diuretics and vasodilators. On the whole, available evidence suggests that renin disturbances in essential hypertension are rarely primary. It is unlikely, therefore, that renin levels alone can suffice as guideline for treatment of hypertension. A more comprehensive approach should be based on a multifaceted profile, including but not limited to renin.


Tetralogy of Fallot: postoperative electrophysiologic studies.

Electrophysiologic studies performed during postoperative cardiac catheterization in 51 patients following repair of tetralogy of Fallot (TF) were compared with studies performed in a control group of 30 patients. These studies include His bundle electrograms, measurement of atrioventricular (A-V) conduction at progressively increasing atrial pacing rates, and evaluation of sinus node recovery time. More postoperative TF patients (20%) had prolonged H-V intervals compared to the controls (7%). With atrial pacing, no patient in the control group developed second degree A-V block below 160 beats/min, while 23% of the postoperative TF patients developed block below this level. Half of these patients had normal His bundle studies. Stress of the A-V conduction system by atrial pacing may unmask conduction abnormalities not present on the surface electrocardiogram or on His bundle electrogram. Two-thirds of the patients had some form of conduction abnormality on the basis of evaluation of one or more parameters but about half of the patients with a given abnormality had normal findings in other areas. The high incidence of electrophysiologic abnormalities following TF repair may have future prognostic significance. Systematic evaluation of atrioventricular conduction during preoperative and postoperative cardiac catheterization may be of value.


Observations on the development of spontaneous tele-kinesic communication in babies and young children.

Specialized body movements are universally employed to convey information between individuals and groups across some distance, near or far. The natural development of facial expressions, limb gestures and whole body movements, for the purpose of interpersonal communications in young children, is briefly discussed.


Playgroups-their effects on the language and socialization of children.

This paper attempts to evaluate the effects of pre-school playgroups on child development by reporting the views of first year infant school teachers.


Modification and abolition of re-entry within the His-Purkinje system in man by diphenylhydantoin.

The phenomenon of macrore-entry (Re) within the His-Purkinje system (HPS) was consistently observed in 10 of 19 patients during retrograde refractory period studies. Effects of intravenous infusion of diphenylhydantoin (DPH) on Re were studied in these 10 patients 10 minutes after completion of infusion (mean plasma level equal to 17.0 microgram/ml). Diphenylhydantoin modified determinants of Re in seven patients (group I) and abolished Re in the remaining three patients (group II). In group I, DPH shortened the critical V1 V2 from 310.0 +/- 30.5 to 292.9 +/- 25.6 msec (P less than 0.025) and critical V2 H2 intervals for Re from 201.4 +/- 18.4 to 185.0 +/- 13.8 msec (P greater than 0.05). In group II, DPH abolished Re in two of three patients by precluding attainment of critical V2 H2 intervals whereas Re was abolished in the remaining one patient despite attainment of critical V2 H2 intervals (vs control). For both groups, DPH significantly shortened functional and effective refractory periods of the HPS (P less than 0.001 and less than 0.01, respectively) without significantly affecting the effective refractory period of the ventricular muscle. Diphenylhydantoin either completely abolished or significantly shortened the retrograde gap zones in the HPS. It is concluded that diphenylhydantoin significantly shortens His-Purkinje system refractoriness, abolishing Re in the patients with higher degree of improvement in refractoriness.


Dimensions of parental involvement in pre-school programmes.

This report describes an approach towards the clarification of the role of parent involvement in the decision-making process and assessment of preschool programmes. A model-placing involvement on a continuum is developed using as its endpoints high and low levels of parent involvement. The implications of this distinction in the decision-making process are examined from the viewpoint of practical consequences for all those concerned with maximizing the effects of various pre-school programmes. Finally, an example of a case of programme evaluation which actively sought high levels of parent involvement in the development of a behaviour rating scale is presented and an extension of the model to other aspects of a child's life is suggested.


Entrainment and interruption of atrial flutter with atrial pacing: studies in man following open heart surgery.

To examine the question of why the pacing rate and duration of atrial pacing are crucial factors in the successful interruption of atrial flutter, studies were performed on 30 patients in the period following open heart surgery. In each patient the diagnosis of atrial flutter was made using a pair of wire electrodes placed on the right atrial epicardium at the time of operation and brought out through the anterior chest wall. The same electrodes were used for atrial pacing. Pacing faster than the spontaneous rate of the atrial flutter which failed to interrupt the atrial flutter was associated with transient entrainment of the atrial flutter up to the atrial pacing rate. Atrial flutter was interrupted successfully when the atria were paced at a rate which was too fast for the atrial flutter to follow. This was heralded by the conversion of previously negative flutter waves to positive atrial complexes in ECG lead II. When pacing the atria at a constant rate, 2-22 seconds with a mean of 10 seconds were required to interrupt the atrial flutter.


The use of Glasgow hospital beds by children under the age of one year.

A study of computer-based hospital discharge data for Glasgow infants showed a 50% rise in the number of discharges per 1000 births from 1968 to 1972. This was attributable to increased rates for infections of the alimentary and respiratory systems. There was no evidence of a rise in disease prevalence nor a fall in mortality rates. It is hypothesized that hospital resources may be used inappropriately to meet demand when the actual need is for primary care services.


Transthoracic ventricular defibrillation in the 100 kg calf with unidirectional rectangular pulses.

The effectiveness in reversing ventricular fibrillation of 30 seconds duration of unidirectional rectangular-wave shocks having pulse widths of 0.5 through 64 msec, pulse amplitudes of 35, 50, 70, 100, and 140 amp, and pulse energies of 109 through 1,660 J was studied in 3,303 transthoracic fibrillation-defibrillation episodes in 100 kg calves. A total of 38 animals were used in the study. Postdefibrillation electrocardiograms were recorded. Families of curves of percent successful defibrillation vs pulse duration, percent successful defibrillation vs pulse energy, duration of postdefibrillation complete block or standstill vs energy, and time required for a return to normal sinus rhythm vs energy were derived. The most effective waveform studied (70 amp--8 msec--862 J) yielded defibrillation on the initial attempt in 93% of 120 episodes. In general, the duration of complete block or standstill and the time required for a return to normal sinus rhythm increased with increasing pulse current and pulse energy.


A comparison of unipolar and bipolar electrograms for cardiac pacemaker sensing.

Simultaneous unipolar and bipolar electrograms were recorded and compared from 49 pacemaker patients with bipolar endocardial electrodes. Average bipolar depolarization signal voltage equalled that of unipolar but showed greater variation. Bipolar and unipolar slew rates were equal in both mean and variance. The proximal pole voltage had little effect on the bipolar result in 8% of the cases, tended to cancel the tip voltage in 49% of the cases and augmented the tip voltage in 43% of the electrograms. The average bipolar R wave duration was 28% less, the T wave amplitude 34% less, and the ST-segment elevation 37% less than the unipolar values. By consistently attenuating the undersirable T waves and ST elevations, while leaving the depolarization signal unaffected, the bipolar electrode offered the advantage of a superior signal-to-noise ratio for sensing depolarization. In one case, however, the bipolar signal was so small as to cause a clinical sensing failure.


Real-time cross-sectional echocardiographic diagnosis of coarctation of the aorta: a prospective study of echocardiographic-angiographic correlations.

Real-time cross-sectional echocardiographic sector scan examinations were performed from a suprasternal notch location to image aortic anatomy in 15 children (ages 1 day to 21 years) who were subsequently shown at cardiac catheterization to have coarctation of the descending thoracic aorta. The resulting echocardiographic images of the ascending, transverse and descending aorta imaged juxtaductal coarctation in all 15 patients. Echocardiography predicted discrete coarctation of the aorta in eight, isthmic hypoplasia in two, hourglass type coarctation deformities in three and longer segment coarctation in four patients. Catheterization and angiography confirmed all of these anatomic observations. The control group, 100 patients with congenital heart disease but without angiographic coarctations, included four patients with right-sided aortic arch and six patients with dextrocardia. The ascending, transverse and descending aorta were adequately imaged in 94 of these and no descending aortic abnormalities were noted. This study shows the potential utility of cross-sectional echocardiography for the noninvasive precatheterization or preoperative assessment of coarctation of the aorta in children and critically ill newborns.


Cross-sectional echocardiographic visualization of the stenotic pulmonary valve.

Real-time, cross-sectional echocardiograms of the pulmonary valve were recorded in 22 patients with valvular pulmonary stenosis (VPS) (14 mild, eight moderate or severe) and 25 normal subjects. Normally during systole the pulmonary leaflet echoes moved rapidly apart and in the fully opened position lay parallel and in close apposition to the margins of the pulmonary artery. In 20 of 22 patients with VPS in whom the pulmonary valve was recorded the systolic configuration of the leaflets, opening pattern of the leaflet echoes, and presence of presystolic doming served to differentiate the stenotic valve from normal. In contrast M-mode recordings of the pulmonary valve were possible in only 12 of these 22 cases (seven mild and five moderate or severe) and suggested VPS in only the five cases with moderate or severe stenosis. Cross-sectional echocardiography offers a direct, noninvasive method for visualizing the stenotic pulmonary valve and should be improvement over the indirect M-mode data.


Right ventricular compression as a sign of cardiac tamponade: an analysis of echocardiographic ventricular dimensions and their clinical implications.

We have identified pericardial effusion by echocardiography in 174 patients. Seventeen had cardiac tamponade which was not always clinically obvious. Right ventricular narrowing or compression, occurring in the minor axis at end diastole and end expiration, to 7 +/- 2 mm or less, was strongly associated with tamponade in patients with effusion. Right ventricular compression and signs of tamponade abated with pericardiocentesis. One patient was in tamponade without obvious right ventricular narrowing. Nonetheless, he demonstrated serial increases in right ventricular dimensions which paralleled hemodynamic improvement. Diminished left ventricular end-diastolic dimension, "swinging heart," electrical alternans, reciprocal respiratory variations in right and left ventricular end-diastolic dimensions and variation in amplitude of the mitral D-E slope were nonspecific for tamponade. Evaluation of right ventricular dimensions may be clinically useful to diagnose and monitor cardiac tamponade.


The wavefront phenomenon of ischemic cell death. 1. Myocardial infarct size vs duration of coronary occlusion in dogs.

Irreversible ischemic myocardial cell injury developes in an increasing number of cells as the duration of coronary occlusion is prolonged. The present study quantitates myocardial necrosis produced by 40 minutes, 3 hours, or 6 hours of temporary circumflex coronary occlusion (CO) followed by 2 to 4 days of reperfusion, or by 24 or 96 hours of permanent circumflex ligation in pentobarbital anesthetized open chest dogs. After 40 minutes of ischemia, myocyte necrosis was subendocardial but with increasing duration of coronary occlusion, irreversible injury progressed as a wavefront toward the subepicardium. Transmural necrosis was 38 +/- 4% after 40 min, 57 +/- 7% after 3 hours, 71 +/- 7% after 6 hours and 85 +/- 2% after 24 hours of ischemic injury. These results document the presence of a subepicardial zone of ischemic but viable myocardium which is available for pharmacologic or surgical salvage for at least three and perhaps six hours following circumflex occlusion in the dog.


Infarct size reduction by propranolol before and after coronary ligation in dogs.

Coronary occlusion in the dog results in irreversible myocardial cell injury which develops first in subendocardial areas of severe ischemica and subsequently spreads into mid and subepicardial areas of moderate ischemia. The effect of propranolol on this progression of ischemic injury was evaluated. Three groups of dogs were studied: 1) untreated, 2) treated with propranolol before and throughout coronary ligation, and 3) treated with propranolol beginning three hours after ligation. Dogs were sacrificed 24 hours after coronary ligation and necrosis was quantitated from histologic sections of transmural slices through the posterior papillary muscle. Propranolol reduced infarct size by preventing necrosis in peripheral (subepicardial) areas of moderately ischemic myocardium. Pretreatment with propranolol reduced necrosis from 85 +/- 3% (untreated) to 52 +/- 4% (P less than 0.05). Delayed propranolol therapy was about half as effective as pre-treatment and reduced necrosis to 71 +/- 3% (P less than 0.05). Propranolol also limited microvascular injury so that perfusion defects, detected with the dye thioflavin S, were smaller in treated dogs.


New criteria to enhance the predictability of coronary artery disease by exercise testing in asymptomatic subjects.

Thirty-seven subjects (10 asymptomatic and 27 symptomatic) who had undergone maximal treadmill exercise tests and coronary arteriography were selected to determine whether the predictive value of ST-segment depression as a marker of coronary artery disease (CAD) in asymptomatic subjects could be enhanced. All subjects had greater than or equal to 2 mm ST-segment depression during testing. Three of the ten asymptomatic subjects had significant CAD (predictive accuracy 30%) and all had greater than or equal to 2 mm ST depression in the one-minute recovery tracing. ST-segment depression resolved by one minute in all seven subjects without CAD. All 27 symptomatic subjects had CAD (predictive accuracy 100%) and 26 of 27 had greater than or equal to 1 mm (21/24 greater than or equal to 2 mm) ST depression in the one-minute recovery tracing. The age, maximum heart rate and exercise tolerance did not differ significantly between the symptomatic and asymptomatic subjects. Thus the predictive accuracy for CAD in asymptomatic subjects is not enhanced by increasing the degree of ST depression required for a positive exercise test. The predictive value may be enhanced if ST depression persists for greater than or equal to 2 minutes into recovery. These data suggest that by using new criteria accurate identification of asymptomatic patients with CAD by exercise testing may be practical.


"Reverse coronary steal" induced by coronary vasoconstriction following coronary artery occlusion in dogs.

The phenomenon of "coronary steal," i.e., the shunting of blood from ischemic to normally perfused areas of myocardium, has been described as an effect of the administration of several vasodilating agents. This study was performed to ascertain whether the reverse situation can be induced, i.e., whether vasoconstriction of the vessels supplying the nonischemic zone could increase the collateral flow to the ischemic area. In 16 open chest dogs, 15 and 30 min after occlusion of the left anterior descending coronary artery, epicardial electrograms were recorded and regional myocardial blood flow (RMBF) was measured with radiolabeled microspheres. Methoxamine was infused intravenously between 17 and 30 min, the mean arterial pressure being kept constant. The results indicate that while the coronary arterial flow to the normal myocardium fell from 90.6 +/- 4.3 to 77.7 +/- 3.2 ml/min/100 g (P less than 0.01), the collateral blood flow to the ischemic area increased from 21.4 +/- 3.5 to 41.0 +/- 4.2 ml/min/100 g (P less than 0.01), and thereby reduced acute myocardial ischemic injury. This favorable redistribution of blood flow might be considered a "reverse coronary steal."


Prognostic efficacy of early clinical categorization of myocardial infarction patients.

To assess the prognostic validity of "low risk" classification at 24-36 hours as the basis for early transfer to an intermediate coronary care unit, Killip and Hutter/Sidel criteria for risk classicication were applied to 410 patients with acute myocardial infarction (MI) and prospectively compared with respect to mortality and morbidity. Six percent of "low risk" patients by the Killip classification subsequently died and were therefore misclassified. No patients in the group classified as "low risk" by the Hutter/Sidel criteria died; however, more than two and a half times as many potentially "low risk" patients were excluded from this group. With respect to morbidity, 35 of 87 patients (40%) classified as "low risk" by Hutter/Sidel criteria developed 43 major complications during their hospitalization. Nine of these patients developed 11 of the 43 complications (26%) on day six or later, and for five of these patients, this was their first major complication during hospitalization. This significant incidence of morbidity in "low risk" patients, despite their low mortality rates as a group, must be taken into account in developing intermediate coronary care units.


The use of "fresh" unstented homograft valves for replacement of the aortic valve: analysis of 6 1/2 years experience.

Between August 1969 and January 1976, 561 patients underwent homograft replacement of the aortic valve (AVR). Isolated AVR was performed in 339 patients, ranging in age between 18 months and 74 years. The valves were sterilized in antibiotic solution and preserved at 4 degrees C in tissue culture medium. There were 11 early deaths (3.2%) and 23 late deaths (6.8%). Actuarial analysis showed 88% survival at 5 years and 85% at 6 years. Valve failure occurred in 13 patients (3.8%) due to prolapse of one cusp in five patients (1.5%), infective endocarditis in three and degeneration of the graft in five (1.5%). Degenerative valve failure was encountered after the fourth year with an incidence of 3.5% of patients at risk, and occurred only in grafts from donors over the age of 70 years. Diastolic murmurs were present in 22% of patients followed up for more than one month and increased very slightly with time. The clinical result was judged to be good or excellent in approximately 90% of the surviving patients.


Mean velocity of circumferential fiber shortening in prolapsed mitral leaflet syndrome.

In 26 patients with mitral valve prolapse, ventricular function was evaluated by mean velocity of circumferential fiber shortening (MVCF) as measured along the basilar, middle and apical axes. Significantly increased rates of MVCF were found in patients with mitral prolapse along the basilar axis (1.75 +/- 0.23 circ/sec) and middle axis (2.09 +/- 0.34 cir/sec) (P less than 0.025 and P less than 0.05, respectively). Patients with mitral valve prolapse and regurgitation demonstrated a significant increase in MVCF along the basilar axes (1.72 +/- 0.15 cir/sec) (P less than 0.05). Asynergy apperars to have a negative effect on the MVCF along the middle axis. The MVCF was found not to be related to clinical findings, symptoms or electrocardiographic changes. The mechanism for the increase in MVCF in patients with mitral valve prolapse remains unsettled.


Urinary kallikrein in normal renin essential hypertension.

The relationship between urinary kallikrein, urinary aldosterone, and plasma renin activity (PRA) was studied in hypertensive patients and normal subjects. Kallikrein was measured by a radiochemical esterolytic assay. Nine white males with normal renin, mild essential hypertension (25 +/- 5 [SD] yr; blood pressure [BP] 143 +/- 7 / 95 +/- 5 mm Hg) and six white normal males (23 +/- 3 yr; BP 115 +/- 15 / 70 +/- 6 mm Hg) were placed on a one-week diet consisting of 400 mEq Na+, 80 mEq K+ diet and a one week diet of a 10 mEq Na+, 80 mEq K+ diet. During salt restriction, PRA, urinary aldosterone, and urinary kallikrein progressively rose. (Urinary kallikrein on day 7: normals 18.3 +/- 13.7 esterase units [EU] per 24 hr; hypertensives 22.7 +/- 12.5 EU/24 hrs). There were no significant differences between the normals and hypertensives in PRA, aldosterone, or kallikrein excretion. After sodium balance was achieved, during salt loading, the PRA, aldosterone and kallikrein values were similar in both normals and hypertensives. (Urinary kallikrein on day 7: normals 5.0 +/- 5.2; hypertensives 7.9 +/- 4.4 EU/24 hrs.) Abnormalities in urinary kallikrein in hypertensives were not found when young white males with normal renin essential hypertension were compared to age-matched white male normal subjects. PRA appears related to urinary kallikrein excretion in this type of patient.


The aortic closure sound in pure aortic insufficiency.

The second sound in aortic insufficiency has been described as accentuated, normal, or moderately diminished. A study of intracardiac phonocardiograms was performed to evaluate its intensity and to eliminate extracardiac factors. Pressure and intracardiac sound measurements were made in 28 patients undergoing diagnostic cardiac catheterization. Recordings were obtained above the aortic valve and within the left ventricle in 14 patients with normal aortic valves and 11 patients with aortic insufficiency uncomplicated by aortic stenosis. The amplitude of the aortic closure sound in the patients with pure aortic insufficiency, 1000 +/- 100 dynes/cm2, was significantly lower than in those patients with normal aortic valves, 3100 +/- 200 dynes/cm2 (P less than 0.001). The results indicate, therefore, that the presence of aortic insufficiency causes a diminished amplitude of the aortic closure sound. These results are supportive of the theory that the second heart sound is caused by diastolic vibrations of the closed aortic cusps. Diminished valvular vibrations and sound would occur in pure aortic insufficiency if the valve is unable to properly tense during diastole, or if the rate of development of the driving pressure is diminished.


Relationship between plasma lipid concentrations and coronary artery disease in 496 patients.

The relationship between fasting plasma cholesterol and triglyceride concentrations and the frequency and extensiveness of coronary artery disease (CAD) was studied in 496 subjects evaluated for chest pain by coronary arteriography at The Methodist Hospital. One hundred six of the patients had no CAD while 390 had 25% or greater stenosis of one or more major vessels. Ninety-one percent had 75% or greater stenosis of at least one major vessel. Mean age for the group with CAD was 55.7 +/- 8.7 and without disease 49.4 +/- 11.6 (P less than 0.01). Both cholesterol and triglyceride concentrations were higher (P less than 0.001) in the group with CAD. Mean cholesterol concentration in males increased from 195 +/- 36 mg/dl in the group without CAD to 219 +/- 41 in the group with three vessel disease and in females from 207 +/- 40 to 252 +/- 42. A progressive increase in triglyceride values was also detected but was less consistent. At the level of 25% and greater obstruction, the partial correlation coefficients between the number of vessels involved and the cholesterol and triglyceride concentrations, respectively, were +0.201 and +0.181.


Clinical primary pulmonary hypertension: three pathologic types.

Clinically, there is a group of patients with elevated pulmonary arterial pressure in whom the underlying cause is not apparent. The pulmonary arterial wedge pressure is not elevated. For such cases, the designation of primary pulmonary hypertension may be made clinically. From the clinical categorization of primary pulmonary hypertension, three distinct pathologic entities emerge, namely 1) plexogenic pulmonary arteriopathy, 2) recurrent pulmonary thromboembolism, and 3) pulmonary veno-occlusive disease. The plexogenic type is characterized initially by pulmonary arterial vasoconstriction with medial hypertrophy. Secondary proliferative intimal lesions, including the plexiform lesion, develop. Recurrent pulmonary thromboembolism is characterized by the presence of arterial thrombi of varying ages involving the microscopic-sized pulmonary arteries. Thrombi may be embolic in nature or may develop in situ. Pulmonary veno-occlusive disease is characterized by obstructive lesions of pulmonary veins and venules. The clinical presentation of the three pathologic types may be so similar that definitive diagnosis depends upon histologic examination of the lung from tissue obtained either by biopsy or at necropsy.


Methysergide-induced heart disease: a case of multivalvular and myocardial fibrosis.

Methysergide (Sansert) is known to cause mitral and aortic valvular fibrosis and dysfunction, but has generally not been known to damage right heart valves or the myocardium, and cardiac fibrosis has not been considered to be a risk if therapy is intermittently interrupted. The woman who is the subject of this case report developed catheterization-proven severe tricuspid and moderate aortic and mitral regurgitation during noncontinuous therapy with methysergide. In addition, right ventricular endomyocardial biopsy revealed extensive endocardial and intramyocardial fibrosis.


Urinary mandelic acid: identified in normal individuals following a single oral load of phenylethylamine.

1. An increased urinary mandelic acid was identified by gas-liquid chromatography in the urine of 3 healthy subjects following the oral ingestion of phenylethylamine. 2. The pathway for the formation of mandelic acid from phenylethylamine was suggested to involve the intermediate phenylethanolamine. The basic similarity of the metabolism of phenylethylamine and tyramine was suggested by the excretion of 4.5% of phenulethlamine as mandelic acid which correlated well to a reported 7.2% of p-tyramine excreted as p-hydroxy mandelic acid.


The measurement of volatile chromium in biological materials.

Chromium is an essential trace element in mammals since dietary chromium deficiency results in glucose intolerance due to decreased sensitivity to insulin. In humans, both adults and children with glucose intolerance have been improved by treatment with chromium. Furthermore, chromium deficiency has been implicated as a causative factor in hypercholesterolemia and atherosclerosis. However, little is known of the metabolism of chromium in humans, primarily because of analytical difficulties. The biologically active form of chromium is the "glucose tolerance factor" (GTF) which is a co-ordination complex of trivalent chromium with nicotinic acid and certain amino acids. At physiological pH, ionic chromium as a simple inorganic salt is insoluble in water, but trivalent chromium forms stable complexes with ascorbic acid, amino acids and other substances present in blood and tissue. Chromium is present in serum, bound to protein and also as dialysable or ultrafiltrable chromium (free chromium). The free chromium includes G.T.F. and other coordination complexes and represents the metabolically active form of the element; the ratio free/protein bound chromium in serum varies within the individual according to the diet and the metabolic state.


An improved filter-disc assay for 3H-DNA and serum deoxyribonucleases.

1. The filter-paper disc assay for tritium-labeled nucleic acids was modified to reduce washing and manipulation. Evaluation of the procedure indicated greatly enhanced counting efficiency for tritium, complete removal of nuclease digestion products and retention of even nanogram levels of radioactive DNA on the discs in the presence of protein. The modified assay was quite sensitive and had a coefficient of variation of 5.2%. Quite low concentrations of DNase I and endogenous human serum nuclease activity were readily measured by the improved technique, 22 sera yielding a mean value equivalent to about 0.5 ng DNase I/ml serum. We are now using the disc assay to follow serum DNA and DNase levels in patients with autoimmune disorders and various types of cancers.


The use of equilin as an internal standard to quantitate estriol in pregnancy urine.

The assay of pregnancy urine estriol by gas chromatography is most often carried out by adding cholesterol as an internal standard at the end of the extraction procedure. The reason for this is that cholesterol does not contain a phenolic ring, and would be lost during the extraction procedure for estriol. This does not allow for corrections of recovery loss, which would be the case if the internal standard was a phenolic steroid. This paper describes the utilization of equilin (delta 1,3,5,7-estratetraen-3-01-17-one) as the internal standard. The sample is hydrolyzed to remove sulfate and glucuronide groups and then the equilin is added. If equilin is added before hydrolysis, variable destruction takes place. Derivative studies indicate that room temperature silylation can be used with bis (trimethylsilyl)-trifluoroacetamide/trichlorosilane/trimethylsilylimidazole, 3:3:2 by weight. Results on control material containing a low, critical pregnancy concentration of estriol were as follows--listed value 5.0 mg/1, found 5.3 mg/1, co-efficient of variation %, 8.9% for 17 runs on different days.


Clinical pharmacology of anti-epileptic drugs: a summary of current information.

This compendium represents what we believe to be the most current and reliable pharmacological data on anticonvulsant drugs. The information presented is derived from determinations of the drugs in plasma or serum by gas--liquid chromatography in studies of the efficacy of anti-epileptic agents. We present information on the limitations of therapeutic concentration ranges, half-lives, active and inactive metabolites, and structure/activity relationships of anticonvulsant drugs. This report provides answers to many of the questions clinicians direct to anticonvulsant-monitoring laboratories. Information on other pharmacoloical variables supplements this review in the interest of the clinical investigator.


Fluorescent assay of total serum cholesterol, with use of gas-liquid chromatography to study saponification efficiency.

We describe a fluorescent determination of total cholesterol in serum for which the accuracy and precision are comparable to that for the method of Abell-Kendall, a method of generally accepted accuracy. By the use of quality reagents and the rigorous exclusion of water, the strong fluorophor that develops on reacting concentrated sulfuric acid with cholesterol can be used to quantitatively determine the total cholesterol in serum. We used gas-liquid chromatography to monitor the extent of saponification of the cholesterol esters, because we found them to have fluorescent efficiencies that differed from that of free cholesterol. Sodium methoxide in methanol/methylene chloride (1/3 by vol) was shown by gas-liquid chromatography to very effectively saponify the cholesterol esters in serum.


High-performance liquid-chromatographic analysis for tryptophan in serum.

Concentrations of total tryptophan were determined rapidly and sensitively in 50 microliter of serum by a reversed-phase partition version of high-performance liquid chromatography. For determination of total tryptophan, sample preparation requires only precipitation of the serum protein with trichloroacetic acid and removing excess trichloroacetic acid with a 1,1,2-trichlorotrifluoroethane(Freon)/tri-N-octylamine solution. Tryptophan in serum samples was detected by ultraviolet and fluorescence spectrometry. No interferences from the naturally occurring constituents of serum were observed. Elution time for tryptophan is 15 min, the limit of detection is 1 pmol.


Colorimetric determination of phospholipids by use of molybdophosphate, and its application to amniotic fluid.

We studied the reaction between molybdophosphoric acid and lecithin. The resulting complex was isolated and shown to contain molybdophosphate and lecithin in the molar ratio 1/3. It is insoluble in water but soluble in chloroform, and the reaction is specific for molecules containing electrophilic nitrogen, indicating good specificity in biological fluids for nitrogen-containing phospholipids. In solution the complex may be reduced, yielding molybdenum blue. We applied the reaction to the measurement of phospholipid concentrations in amniotic fluid. As compared with procedures involving either digestion or the determination of lecithin/sphingomyelin ratio, the present technique is faster and its analytical precision exceeds that of the lecithin/sphingomyelin ratio determination. Although results by the three methods do not correlate well, the predictive value of the present method appears to be comparable with the other two.


Methaqualone metabolites in human urine after therapeutic doses.

We measured five principal metabolites of methaqualone in the urine of seven volunteers after single and multiple doses of the drug. Urine, collected for up to 72 hours after the last dose, was analyzed for methaqualone and its principal metabolites by high-resolution capillary-column gas chromatography. The major biotransformation of methaqualone under therapeutic conditions occurred through benzylic and para-hydroxylation of the o-tolyl moiety. Methaqualone itself was present in concentrations of no more than 1 mg/liter, if it could be detected at all. The observed physiological effects ant total urinary excretion of metabolites reflected the cumulative nature of the parent drug when it was administered in multiple doses. No clear relationship was found between appearance of a specific metabolite and time after ingestion of the drug, although higher amounts of 2-methyl-3-(2'-hydroxymethylphenyl)-4(3H)-quinazolinone were noted in those individuals who tolerated the drug less well.


Improved colorimetry of urinary 3-methoxy-4-hydroxyphenylacetic acid (homovanillic acid).

Homovanillic acid is an important metabolite of dopamine, and a high proportion of patients with neuroblastoma excrete increased amounts of it in their urine. When this diagnosis is suspected, both homovanillic acid and vanilmandelic acid should be measured, because such a combined measurement reportedly leads to detection of 95% of cases. Although rapid and reliable chemical methods are available for vanilmandelic acid, the same is not true for homovanillic acid. We report here a colorimetric method for homovanillic acid that reasonably fills this void and that can be used in most clinical laboratories. In addition, we present normal values determined for children of various age groups.


Design of quality-control specimens for use with a small multi-channel analyzer.

Specimens having linear relationships between concentration and instrument response for seven analytes were prepared for use with a small multi-channel analyzer. The specimens had inter-specimen and inter-constituent relationships that facilitated the performance evaluation of all channels simultaneously with each individual specimen. Techniques are described for use of the specimens in various aspects of quality control.


Specific serum quinidine assay by high-performance liquid chromatography.

The cardioactive drug quinidine has a narrow therapeutic index; consequently, determination of serum quinidine concentrations can be important. We describe a relatively rapid and specific assay for quinidine in serum by high-performance liquid chromatography. It is suitable for use in patient monitoring or pharmacodynamic studies. Alkalinized serum is extracted with benzene, which is evaporated under nitrogen and reconstituted with methanol; an aliquot is chromatographed. Quinidine is separated from its metabolites and dihydroquinidine (a contaminant in quinidine raw materials). The retention time for quinidine is 4 min 10 s, for dihydroquinidine it is 5.5 min. Results for patients' sera by this assay method and the double-extraction method of Cramer and Isaksson [Scand. J. Clin. Lab. Invest. 15, 553 (1963] correlate well (r = .975).


Removal of an endogenous antigen from an antibody to increase its effective affinity constant, as illustrated by triiodothyronine assay.

Antisera to triiodothyronine were shown to contain large quantities of the hormone, well in excess of normal circulating concentrations. Extracting the triiodothyronine from the antiserum with alkaline ethanol yielded an antibody of increased affinity. Use of this antibody in a radioimmunoassay resulted in a fourfold increase in sensitivity as compared with the unextracted material.


Binding proteins from fish sera and intrinsic factor compared in vitamin B12 radioassay.

We compare serum proteins from rainbow trout, chinook salmon, coho salmon, and oyster toadfish with intrinsic factor as binding proteins in a simplified radioassay for B12. Regression analysis of B12 values, determined in 21 serum samples, shows good correlation (r greater than .975) between results for the fish sera and intrinsic factor. The accuracy of the five assays, as evaluated by analytical recovery of B12 added to pooled human serum, ranges from 90 to 110%. Intra-assay precision ranges from 2.6% for coho salmon serum to 5.5% for intrinsic factor, Ionic strength and variations in pH influence binding of [57Co]vit B12 to the fish sera. Maximum binding occurs from pH 6 to 10 at an ionic strength of 0.1 for all sera. The sera are stable for longer than two years when stored at -20 degrees C. Important advantages of fish sera are their high binding capacity (typical assay dilutions range from 1500-fold for trout serum to more than 50 000-fold for chinook salmon); high affinity for B12 (K greater than 10(12) liter/mol); their relative constant binding characteristics as compared to commercial intrinsic factor preparations; and the finding that the accuracy of radioassays with use of fish sera is not significantly affected by the amount of B12 or human serum proteins present.


Electrophoretic patterns for serum glycoproteins reflect the presence of human breast cancer.

We describe a group of glycoproteins that are synthesized and released by human breast tumors maintained in organ culture and similar glycoproteins released by a human breast carcinoma cell line (BT-20). The electrophoretic mobility of these glycoproteins on cellulose acetate is consistent with increased glycoprotein-staining material present in the alpha2- to beta-globulin region of serum glycoprotein electropherograms from patients with breast cancer. Moreover, after mastectomy, this glycoprotein material in serum decreases to concentrations seen in a control population of patients with benign breast lesions. Patients with proven metastatic breast cancer have patterns reflecting their clinical status: those who respond to treatment have glycoprotein electropherograms similar to the group of patients with benign breast lesions, while those who do not have increased amounts of alpha2- beta-glycoprotein. We believe serum glycoprotein measurements in breast-cancer patients reflect the presence of glycoproteins that are released by the malignant cells and enter the circulation.


Electrophoretic assay of specific estrogen receptors: a contribution to methodology.

Experimental evidence is presented that supports the use of the cold agar-gel electrophoretic method for the clinical quantitation of specific estrogen-binding protein present in some human mammary carcinomas. It is necessary to dilute tumor extracts to avoid interference by serum-borne, non-relevant hormone-binding proteins such as albumin, which migrates to the same anodal region as does the binding protein. Dilution to 2.5 mg or less of total protein per milliliter circumvents such interference while still permitting reliable quantitation of the binding protein. Seventy-two mammary carcinomas were compared for binding-protein content by both the cold agar-gel electrophoresis and a single-point dextran-coated charcoal assay. The correlation coefficient (0.96) indicated excellent agreement between results by the two methods. In addition results are presented which indicate that the preparation of tumor extracts for electrophoresis does not require the use of an ultracentrifuge.


Serum creatinine assay by use of 3,5-dinitrobenzoates: a critique.

Methyl-3,5-dinitrobenzoate and 3,5-dinitrobenzoyl chloride are demonstrated to react with creatinine through their conversion to 3,5-dinitrobenzoate. Analytical recovery of creatinine added to serum is improved by an "acid-supernate" modification, which is also much less susceptible to a combination of known interfering agents than is the picrate procedure. The "neutral-supernate" procedure shows negligible interference by cephalothin as compared to commonly used picrate procedures. Replacement of tetramethylammonium hydroxide by sodium hydroxide was also tested. The interrelationships between dimethyl sulfoxide concentration, recoveries of creatinine, and slopes of correlations (r greater than .992) of various dinitrobenzoate and picrate methods are critically assessed. Because of its precision, greater specificity, and other criteria, we recommend the methyl-3,5-dinitrobenzoate (or 3,5-dinitrobenzoic acid) neutral-supernate method for routine applications.


Use of protein containing magnetic microparticles in radioassays.

We describe a radioassay method that involves the use of magnetic protein microparticles composed of a water-insoluble protein matrix containing magnetically responsive material. We define two different types of particles according to the mechanism of action: (a) The substrate is sorbed nonspecifically by the protein matrix of the particle or by a second substance such as charcoal or ion-exchange resin incorporated within the protein matrix of the particle. These particles are useful for separating free from bound substrate. Examples of these are albumin magnetic microparticles for use in a total thyroxine radioassay and triiodothyronine uptake test, or albumin magnetic microparticles containing charcoal for use in a vitamin B12 radioassay. (b) The substrate is sorbed specifically by a binding protein incorporated within the matrix of the particles. The binding protein can include antibodies or other specific nonimmune proteins. Particles of this type are useful in solid-phase radioassays. These particles are exemplified by albumin magnetic microparticles containing sockeye salmon serum, used in a solid-phase B12 radioassay. We discuss the methods for the preparation of both types of magnetic microparticles and their use in radioassays. We describe a unique inexpensive magnetic separation rack, which provides simple, fast, and reproducible separation of the magnetic microparticles from their suspending medium during the assay.


Radioimmunoassay for flupenthixol in plasma.

We describe a radioimmunoassay for the neuroleptic drug flupenthixol, suitable for routine monitoring of its concentration in blood. Antibodies for the assay were raised in a sheep against a 7-carboxyflupenthixol/ovalbumin conjugate. The resulting assay, with [3H] flupenthixol as the label, is capable of detecting 2.0 microgram of flupenthixol per liter, in a 100-microliter plasma sample. The antiserum shows no cross reactivity with tricyclic drugs and low interference from the major metabolites of flupenthixol. Concentrations in plasma after a single oral dose of flupenthixol have been followed in one volunteer. Peak values were reached after 3 h. Determinations of flupenthixol after fortnightly intramuscular depot injections of the sustained-release preparation, flupenthixol decanoate, showed the extent of fluctuations during this period.


New method for determining thrombin-clottable fibrinogen.

We describe a new method for determination of thrombin-clottable fibrinogen, which eliminates the systematic error caused by occlusion of other serum proteins in the fibrin clot and reduces the sensitivity to high concentrations of fibrin degradation products. Essentially, the method consists of densitometric quantitation of the fibrin band after a standard electrophoresis run of plasma, thrombin fixation of the fibrinogen, and removal of the non-clotted proteins by washing in saline. The procedure shows good precision and gives results that are accurate, significantly correlate with results for the classical thrombin clotting method (r = 0.92, P less than .001), and are not affected by fibrin degradation product concentrations up to 900 mg/liter. These characteristics make the method especially valuable in establishing fibrogen concentration in patients who are undergoing thrombolytic therapy.


Development, validation, and application of a single-tube radioimmunoassay for cholic and chenodeoxycholic conjugated bile acids in human serum.

A single-tube radioimmunoassay for both cholic acid and chenodeoxycholic acid conjugates in serum is based on the use of [1-(14)C]glycocholic acid and [H-3H]glycochenodeoxycholic acid as tracers. The assay was shown to be specific, sensitive, accurate, and precise (CV = 13% at low concentrations, 5.5% at higher concentrations) when used for serum samples from subjects and patients with increased bile acid in the serum because of liver disease, and results correlate well with those by gas chromatography (r = .99).


A solid-phase enzyme immunoassay for serum ferritin.

We describe an enzyme immunoassay for human serum ferritin in which antibody adsorbed on polystyrene tubes is used. Adsorbed gamma-globulins against human ferritin were first allowed to react with ferritin and a second antiferritin antibody, labeled with alkaline phosphatase, was added. The amount of bound enzyme/antibody conjugate was proportional to the ferritin titer in the assay. This method offers stable reagents that can be kept for many months at 4 degrees C. The average values for ferritin in normal men and women were, respectively, 58 and 43 microgram/liter. The lowest detectable concentration was 5 microgram/liter.


Determination of 14CO2 in breath and 14C in stool after oral administration of cholyl-1-[14C]glycine: clinical application.

Twenty patients with intestinal bacterial overgrowth and 20 control subjects were investigated for bile acid deconjugation, by measuring 14CO2 in the breath after cholyl-1-[14C]glycine administration. 14CO2 output/24h was 11.0 +/- 5.2% (mean +/- SD) in controls and 54.2 +/- 14.0% (mean +/- SD) in bacterial-overgrowth patients (P less than .001). 14CO2 excretion rate in 12h, when normalized to 100% of the dose at the 12th hour, gave an even finer discrimination between the two groups (no false responses). 14C in stool, analyzed in 20 malabsorption patients and 20 controls by two different techniques, was 6.6 +/- 4% and 31.38 +/- 21.7% (mean +/- SD), respectively. Results by the two different techniques described here correlated well (r = .99). Bile acid malabsorption was in reasonable agreement (r = .67) with percentage of "chenoid" (chenodeoxycholic acid plus ursodeoxycholic acid) in the stool by gas-liquid chromatography; a poorer correlation was observed when "chenoid" plus "choloid" (cholic acid plus its epimers) were plotted vs. -4C in stool (r = .57, n = 15).


Removal of interference by erythromycin, phenazopyridine, and methenamine mandelate in the Porter-Silber reaction.

Erythromycin, phenazopyridine, methenamine mandelate, acetazolamide, and ascorbic acid reportedly interfere with the Porter-Silber reaction in the determination of urinary 17-hydroxycorticosteroids. Sodium bisulfite, added to the urine after hydrolysis with glucuronidase, removed almost all nonsteroidal interferences in urine from healthy adults medicated with the first three drugs, but such studies proved that acetazolamide and ascorbic acid do not interfere with the Porter--Silber reaction, whether or not sodium bisulfite is added.


Methotrexate assay by enzymatic inhibition, with use of centrifugal analyzer.

We present a method for measuring methotrexate by enzymatic inhibition, with use of the centrifugal analyzer. In the presence of dihydrofolate reductase (EC 1.5.1.3), dihydrofolic acid is converted to tetrahydrofolate. NADPH acts as a coenzyme. Methotrexate inhibits the action of dihydrofolate reductase. Increasing concentrations of methotrexate depress the conversion of NADPH to NADP+, and the reaction may be followed at 340 nm. Precision, accuracy, and sensitivity are satisfactory. A comparison to the radioimmunoassay method (x) showed the following least-squares regression: y = 0.9813x + 5.6 microgram/liter;  r = .9812; Sy = 6.3 microgram/liter. Some interference was noted from bilirubin, lipermia, and hemoglobin. Up to 60 patients' samples per hour may be estimated by this technique.


Hemoglobin interference with Du Pont Automatic Clinical Analyzer procedure for calcium.

Hemoglobin in concentrations of approximately 2.5 g/liter or greater significantly increases values for apparent calcium in serum as determined with the Du Pont aca, an interference attributable to the 575-nm absorption band of oxyhemoglobin being uncompensated by the 577--600 nm differential absorption reading of the calcium o-cresolphthalein complexone complex.


Plasma alkaline phosphatase assay: interconversion of results by two methods.

Two methods for measuring plasma alkaline phosphatase activity are compared: one makes use of phenyl phosphate, carbonate-bicarbonate buffer, and continuous-flow methodology; the other of p-nitrophenyl phosphate, diethanolamine buffer, and reaction-rate analysis. Results by the methods correlate well (r = 0.98) over a wide range of values (up to 10-fold the upper limit of normal). A factor can therefore be applied to convert results by one method into those that would be obtained by the other. The possibility that the presence of different proportions of isoenzymes in the plasma will affect this factor is considered. We have used the new method, with a conversion factor, as the routine method of alkaline phosphatase measurement in a clinical chemistry laboratory, with no problems.


Determination of urinary total phenolic compounds with use of 4-aminoantipyrine: suggested screening test for hyperthyroidism and for catecholamine-producing tumor.

We describe a method for determining those urinary total phenolic compounds that are tyrosine analogs or metabolites, such as thyroxine and catecholamines. The urine sample, 4-aminoantipyrine in carbonate-bicarbonate buffer, and potassium ferricyanide solution are mixed and the quinoneimine dye that forms is measured at 500 nm. Some cases of hyperthyroidism, diabetes mellitus, nephrosis, obesity, hypertension, or catecholamine-producing tumor showed above-normal values, so that this determination seems useful as a screening test for these disorders.


Method for measuring increased plasma hemoglobin in the presence of erythrocytes.

Despite its many drawbacks the classic benzidine technique is the method most widely used for determining plasma hemoglobin. An additional disadvantage, reported here, is that the method measures hemoglobin in erythrocytes as well as in plasma. Thus, if plasma specimens are accidentally contaminated with erythrocytes, apparent free hemoglobin in plasma will be artifactually high. Although the present method lacks the sensitivity of the benzidine technique, it self-corrects for the presence of erythrocytes. We have found it particularly useful for specimens of rodent (rat mouse) blood, where some cells appear to resist centrifugation, where the small sample sizes frequently result in accidental contamination of plasma with cells, and where normal values may be higher than those for human plasma.


Simultaneous measurement of mitochondrial and microsomal cytochrome levels in human liver biopsy.

The mitochondrial and microsomal cytochromes in patients' livers obtained from needle biopsies have been analyzed using a sensitive spectrophotometric method. To determine simultaneously the concentration of these cytochromes from hemoglobin-contaminated liver, difference spectra of liver homogenates were taken at the temperature of liquid nitrogen before and after photolysis of carbon monoxide molecules from various hemoprotein-CO complexes. This method utilizes a characteristic property of hemoprotein-CO complexes, in which the reassociation of CO molecules with photolyzed hemoglobin, cytochrome P-450 and cytochrome alpha3 is temperature-dependent. The results in 69 cases with a variety of liver diseases have revealed that the concentrations of cytochromes alphaalpha3, P-450 and beta5 showed a huge variation in the injured livers as compared with those in the normal controls. The significance of the measurement of these cytochrome concentrations and their ratios is discussed.


Measurement of creatine kinase Z in human sera using a DEAE-cellulose mini-column method.

A DEAE-cellulose mini-column method has been developed which allows for the quantitation in human serum of creatine kinase Z, a sub-band of creatine kinase first described by Lim ((1975) Clin. Chem. 21, 975, Abstract 181) and Sax et al. (Sax, S.M., Moore, J.J., Giegel, J.L. and Welsh, M. (1976) Clin. Chem. 22, 87). We have shown that creatine kinase Z is rather unstable in nature, and converts to a form which electrophoreses with creatine kinase MM on agarose gel electrophoresis. CK-Z is not present in normal human serum. CK-Z is present in human heart extracts, in patients with myocardial infarcts and in patients with skeletal muscle trauma. In infarct patients CK-Z levels paralleled changes in the CK-MB levels. CK-Z ranged in activity from 8.8-67.2 I.U. whereas CK-MB ranged from 29.6-121.6 I.U. in infarct patients. CK-Z and CK-MB activity were measured at or close to the peak rise in total CK activity.


Rise of unconjugated sex hormones in human urine on storage.

Levels of unconjugated testosterone, 5alpha-dihydrotestosterone, oestradiol and progesterone in human urine were measured before and after storage at various temperatures. Specific radioimmunoassays were used for all measurements. Levels did not change at -20 degrees C, and no change was detected at 4 degrees C for up to 5 days. Testosterone showed an average increase of 100% in 2-3 days at 18 degrees C and 5alpha-dihydrotestosterone about 200%. At 37 degrees C, the rise was two to three times faster still. The addition of enzyme inhibitors, bacteriostatic agents and various salts had no effect on the increase of the unconjugated androgens. Adjustment of the pH of samples to 2 caused about 4-fold rise of the androgens within 10 min, but at pH 8.6 there was no significant effect. Unconjugated oestradiol and progesterone levels showed a rise in a few samples at 18 degrees C for three days. At 37 degrees C both hormones showed a rise after 18 h in about two-thirds of samples, about 2-fold for progesterone and 6-fold for oestradiol.


Removal of background interference in nephelometric determination of serum proteins.

By precipitation of beta-lipoprotein by a dextran sulphate and calcium chloride mixture the background light scattering in continuous flow nephelometric systems can be reduced to such a level that it can be neglected in practice. No loss of IgG, IgA, IgM, orosomucoid or transferrin on precipitation could be demonstrated. The supernatant could not be used for immunoelectrophoresis or radial immunodiffusion in analysis of these proteins since significantly lower values were obtained for some of them than in native serum.


Comparison of methods for the determination of total and free tryptophan in plasma.

A comparison is made between two fluorimetric techniques for the determination of total and free plasma tryptophan. Comparison is also made between methods for the separation of the unbound and bound fraction of plasma tryptophan using ultrafiltration, Centricones and small-scale equilibrium dialysis.


Properties of monoamine oxidase (MAO) in human blood platelets, plasma, lymphocytes and granulocytes.

The properties of monoamine oxidase in plasma, platelets, lymphocytes and granulocytes have been studied using cells prepared from a single small (about 20 ml) sample of blood. The three substrates, 5-hydroxytryptamine, tyramine and benzylamine, have been used to obtain a more complete picture of blood monoamine oxidase than was previously possible. Measurement of Michaelis constants, use of selective inhibitors, and activity against the three substrates distinguished three types of activity. The monoamine oxidases in platelets and lymphocytes are very similar, being most active with tyramine or benzylamine as substrate and inhibited by low concentrations of deprenil. The enzymes in plasma and granulocytes are similar in their relatively high activity against 5-hydroxytryptamine and in their inhibition by semicarbazide and cuprizone with tyramine or benzylamine as substrates. They differ in their affinities for 5-hydroxytryptamine and their activity against tyramine. The activity in platelets, plasma, lymphocytes and granulocytes has been measured in a group of 15 normal subjects using three substrates.


Serum alkaline phosphatase isoenzyme patterns in patients with chronic renal failure.

1. Using acrylamide gel electrophoresis the serum alkaline phosphatase (ALP) isoenzyme patterns of 204 patients with chronic renal failure have been examined for periods up to 18 months in length. 2. Of those with elevated serum ALP activity the bone isoenzyme was largely responsible. The presence of increasing amounts of the bone isoenzyme even if the total serum ALP activity remains within the normal reference range should also indicate bone pathology. 3. Intestinal alkaline phosphatase was the major serum alkaline phosphatase in 15% of patients on regular haemodialysis and 10% of uraemic patients not on dialysis. The overall incidence of detectable intestinal alkaline phosphatase in those with normal serum ALP activity was 36%. 4. With those patients whose serum ALP activity changed significantly during the investigation this usually reflected changes in the amount of the bone isoenzyme. Patients with abnormal amounts of the intestinal isoenzymes in their serum usually showed little variation in serum ALP activity over the period of the study.


Seasonal variations in the composition of urine from normal subjects: a longitudinal study.

The volume, pH and composition of 24-h urine samples, collected by 13 healthy male adults, were followed over a period of one year. Significant and systematic variations in urine pH, calcium, phosphate, oxalate, uric acid, potassium and magnesium were observed. A significant but non-sinusoidal variation in sodium excretion was found but there were no significant changes in urinary volume, creatinine or hydroxyproline. Many of the observed changes could be attributed to variations in the pattern of food consumption throughout the year but calcium, phosphate and oxalate were exceptions in that seasonal variations in these parameters appeared to be due to the effects of sunlight (or vitamin D) rather than to the diet.


Electrophoretic patterns of serum albumins collected from different blood vessels.

Rabbit sera collected from different blood vessels, e.g. vena renales, vena mesenterica, vena portae, vena hepatica and aorta, were electrophoresed in an urea-containing polyacrylamide gel. The albumin fraction was separated into 5-6 sub-bands. The profile of these sub-bands (electrophoretic pattern) of the sample from one blood vessel differed from that of another blood vessel. Especially, the electrophoretic pattern of serum collected from the renal vein 2h after deprivation of food differed from that of other blood vessels. Free fatty acid concentrations of each sample were also measured, and differences in these levels were observed in sera collected from different blood vessels. However, the fatty acid concentrations in serum from the renal vein were not low enough to permit detection of any abnormality in electrophoretic pattern in the albumin. This suggests the possibility of decreased concentration of lysolecithin in the renal vein which binds to albumin and changes the electrophoretic mobility of albumin, as do the free fatty acids.


Pyruvate kinase: diagnostic value in neuromuscular disease.

Pyruvate kinase activity was examined in the sera of a group of patients with neuromuscular disease and in carriers, and compared with that of creatine kinase. The following observations were made: 1. Pyruvate kinase activity was elevated in all 14 patients with Duchenne muscular dystrophy, with very high values generally correlating inversely with age or disease duration. Elevated values of pyruvate kinase were usually, but not invariably, associated with elevated values of creatine kinase. 2. Almost all patients with other muscle diseases and those with neural atrophy had modest elevations in pyruvate kinase activity. 3. ten of 17 individuals were identified as carriers of muscle disease by using both pyruvate kinase and creatine kinase while eight and nine, respectively, were detected using either assay alone. 4. When frozen stored EDTA-plasma was used for pyruvate kinase estimation, higher levels, as compared with the corresponding sera or fresh plasma, were found in controls and carriers but not in Duchenne muscular dystrophy patients. Frozen stored EDTA-plasma should, therefore, not be used for diagnostic purposes.


Development of radioimmunoassays for the measurement of aldosterone in unprocessed plasma and simple plasma extracts.

Inexpensive and rapid radioimmunoassay techniques for the measurement of aldosterone in unprocessed plasma and simple plasma extracts are described. The use of low pH (pH 5.0) and merthiolate to minimise plasma protein binding and the use of aldosterone-free plasma in the standards allows the measurement of aldosterone in 50 microliter of unprocessed plasma, which has been found useful in the diagnostic screening and classification of hyperaldosteronism. Despite quantitative recovery of added (+)-aldosterone and high specificity, the aldosterone content of unprocessed plasma is overestimated, probably by the presence of a water-soluble compound which closely resembles aldosterone. The use of a simple preliminary dichloromethane extraction procedure gives an excellent correlation with values obtained after chromatography. Values are given for chosen normal people and people with benign essential hypertension, using both assay procedures in three different physiological contexts.


A semi-automated measurement of urinary catecholamines using high-speed ion-exchange column chromatography.

Semi-automated measurement of urinary catecholamines, specific for dopa, noradrenaline, adrenaline and dopamine and other metabolites has been developed. Steps included alumina extraction, separation by high-speed ion-exchange column chromatography and fluorescent development by trihydroxyindole (THI) method without interference by other fluorescent substances. Recovery, reproducibility and sensitivity were satisfactory. alpha-Methyldopa, a metabolite of alpha-methyldopa, and isoproterenol were separated completely without interfering with the measurement of noradrenaline, adrenaline and dopamine.


Production and characterization of an antiserum against pancreatic glucagon.

To produce a carboxy-terminal specific antiserum against pancreatic glucagon, glucagon was coupled mainly via its amino-terminal histidine to thyroglobulin, using the amino group reactive pentandial at pH 7.0 for the conjugation procedure. After repeated immunization of rabbits, one high titer antiserum was obtained with a combining site recognizing a part of the carboxy-terminal portion of glucagon as judged from the cross-reaction curves with glucagon fragments, duck pancreatic glucagon and gut glucagon-like immunoreactive substances. There was no cross-reaction with secretin, vasoactive intestinal peptide, and gastric inhibitory peptide. In spite of strong immunoreactive similarities with the antiserum 30K (Unger) this antiserum results in determinations of a somewhat higher amount of plasma immunoreactive glucagon (IRG). The difference was found to depend on a higher content of IRG with a molecular size close to immunoglobulin IgG. A patient with agamma-globulinemia had no measurable IRG of molecular size close to that of IgG, indicating a possible competitive binding of some immunoglobulins to the antiserum.