Morphogenesis in Volvox: analysis of critical variables.

Inversion, the process by which Volvox embryos turn inside out, was analyzed by a combination of geometrical and experimental techniques. It was shown that simple geometric figures are adequate to represent cell shapes during inversion and that cell volumes remain constant as cell shapes change and the embryo inverts. The first stage of inversion, phialopore opening, results from the release of compressive forces as the embryo withdraws from its surrounding vesicle during a two-stage contraction of each cell around its radial axis. Premature phialopore opening occurs when withdrawal of the embryo from the vesicle is elicited artificially by exposure to either calcium ionophore or hypertonic solutions. The major event of inversion, generation of negative curvature, requires both microtubule-driven elongation of cells (to produce a classical "flask" shape) and cytochalasin-sensitive active migration of cytoplasmic bridges to the outermost ends of flask cells. Colchicine, cyclic GMP and isobutyl methyl xanthine (individually) block both normal elongation and bridge migration; cytochalasin D blocks bridge migration selectively. Flask cell formation and bridge migration are adequate to account for the negative curvature observed. An asymmetric bending of flask cell stalks along the ring of maximum curvature accounts for the fact that the embryo is not constricted in a "purse-string" fashion as negative curvature is generated. Inversion of the posterior hemisphere involves an elastic snap-through resulting from a combination of compressive stresses generated by inversion of the anterior hemisphere and the circumferential restraint imposed by cells at the equator. We conclude that the observed changes in cell shape and the migration of cytoplasmic bridges are the result of an ordered process of membrane-cytoskeletal interactions, and both necessary and sufficient to account for the morphogenetic process of inversion in Volvox.


Isolation of adenovirus type 5 host range deletion mutants defective for transformation of rat embryo cells.

A series of adenovirus type 5 (Ad5) deletion, insertion and substitution mutants, some of which are defective for transformation of rat cells, have been isolated. The mutants were selected as variants which lack the Xba I endonuclease cleavage site at 4 map units on the viral chromosome. The deletions range in size from 150-2300 bp and are located between 1.5 and 10.5 map units. The mutants can be propagated in 293 cells (Ad5-transformed human embryonic kidney cells), but are defective for growth in HeLa or human embryonic kidney cells. No viral DNA synthesis was observed in mutant virus-infected HeLa cells. All but one of the deletion mutants tested were defective for transformation of rat embryo and rat embryo brain cells.


Studies on microplasmodia of Physarum polycephalum V: electrical activity of different types of microplasmodia and macroplasmodia.

Results obtained by three different electrophysiological methods delivered mainly oscillating potentials (independently of the type of organization of Physarum polycephalum) with an average period length of 1 min 21 sec. This value is in good statistical agreement with corresponding data for the periodical oscillation of the shuttle streaming (1 min 47 sec) and coincides also with the radial tensiometric contractile activity (1 min 34 sec). The investigation favours the hypothesis that ion fluxes across the cell membrane and perhaps between intracellular compartments may act as a trigger mechanism for a still unknown oscillator-system which controls cell movement phenomena in the acellular slime molds.


Septate-like junctions between cells of the immune system in vivo.

Septate-like junctions between lymphocytes, lymphocytes and macrophages, and macrophages were observed in cellular capsules surrounding larvae of the tapeworm Taenia crassiceps after intraperitoneal inoculation into immunized mice. The morphology of the junctions found in vivo is similar to that reported for septate-like junctions between cells of the immune system in vitro. These findings support the possibility that septate-like junctions may be involved in functional interactions between cells of the immune system both in vivo and in vitro.


Growth in short term primary cell cultures derived from pregnancy-dependent mammary tumors.

The behavior of cells in primary cultures derived from autonomous and pregnancy-dependent mouse mammary tumors was studied. Despite initial growth both dependent and autonomous mammary tumors produced only short-term primary cultures. Initial plating density had a marked effect on growth with only cultures plated at greater than or equal to 2 X 10(5) cells/cm2 showing any short-termed growth. Time lapse analysis showed that the lack of growth was due to failures of cytokinesis and increased death rate and intermitotic time in cultures plated at less than 2 X 10(5) cells/cm2. Using continuous label autoradiographic techniques, a partial synchronous wave of DNA synthesis was observed with newly plated and restimulated cultures. DNA synthesis reached a peak 24-48 hrs. after plating or restimulation and then dropped to low values for the next few days. Attempts to maintain the initial high rate of DNA synthesis or to induce another round of DNA synthesis by enriched media, increased serum concentrations, or other types of serum and plasma were at best only partially successful. Important hormones necessary for growth of mammary tissue in vivo may be necessary for sustained growth in vitro.


Polyadenylate-protein complexes in resting and growing 3T3 cells.

The properties of the ribonuclease resistant cytoplasmic ribonucleoprotein particles were studied in contact-inhibited and serum induced proliferating 3T3 cells. The RNP particles were fractionated by oligo (dT)-cellulose chromatography and banded in CsSO4 gradients. The main RNP fraction, eluted with 25% formamide, contained the major ribonuclease resistant RNA sequences in both resting and growing cells. The protein component of this fraction had a molecular weight of about 72,000 in contact-inhibited cells and 81,000 in serum induced cells.


Effect of hormones on primary cell cultures from pregnancy-dependent mouse mammary tumors.

No hormone combination was successful in maintaining long term primary cultures of pregnancy-dependent mammary tumors. Insulin provided the most consistent dose dependent stimulatory effect on short term proliferation as measured by 3H-TdR incorporation into DNA. Insulin in combination with corticosterone and prolactin produced the greatest stimulatory effect in most tumors. Both insulin at 5 microgram/ml and insulin, prolactin and hydrocortisone induced a partially synchronous wave of DNA synthesis in restimulated cultures. The time course of the wave of DNA synthesis was different for different hormone treatments. Insulin as 5 microgram/ml caused an earlier wave of DNA synthesis than insulin, prolactin plus corticosterone.


Antithyroid and antiperoxidase activity of tropolone and 3-hydroxy-4-pyrone.

Tropolone (TR) and 3-hydroxy-4-pyrone were investigated for antithyroid activity following the finding that the 2-hydroxy-oxo pyridine, 3-hydroxy-4(1H)-pyridone (DHP, I), is goitrogenic. Both compounds inhibited the thyroidal uptake of radioiodine in rats and resembled the thioamide drugs in inhibiting the organic binding of iodine by the thyroid gland rather than the trapping of iodide, but were weaker binding inhibitors than 6-methyl-2-thiouracil (MeTU). Both compounds also inhibited the iodination of bovine serum albumin and thyroglobulin, catalyzed by thyroidperoxidase (TPO), lactoperoxidase (LPO), chloroperoxidase (CPO) and horseradish peroxidase (HPO) in vitro. The inhibitory effect of TR but not that of 3-hydroxy-4-pyrone was antagonized by ferrous ions. When fed to mice at levels of intake expected to produce goitre both compounds were toxic and caused severe liver damage. Thyroid enlargement was not observed in any of these feeiding experiments, but the thyroids of mice fed 0.1% TR showed moderate hyperplasia. It was concluded that both compounds are weakly goitrogenic. Hyperactivity was observed in the mice fed TR which may be associated with inhibition of catechol methyl transferase (COMT).


Accumulation of cadmium and lead in the gills of Mytilus edulis: X-ray microanalysis and chemical analysis.

The accumulation of Cd and Pb in the gills of the lamellibranch mollusc Mytilus edulis has been studied by electron microscopy, X-ray microanalysis, atomic absorption spectroscopy and radionuclide monitoring. The patterns of accumulation of the two elements differ markedly as do the sites of deposition whithin the gills. Lead is found extracellularly as crystalline deposits in the basal lamina which forms the capillary walls of the gill lamellae. The Pb is found associated with Ca in equiatomic ratios and occurs either as a mixed or complex carbonate. Cadmium is always associated with S and frequently with P in membrane bound vesicles within the cells of the gill epithelium and in the amoebocytes. The S is probably attributable to the presence of cysteine residues in a metal binding protein which can be extracted from the gills. Analysis of the metal binding protein shows that it binds Ag, Cd, Cu, Fe, Hg, Sn and Zn. Its amino acid composition is similar to that reported for eels and limpets but has a lower cysteine content than mammalian metal binding protein.


Mutagenicity testing on chinese hamster V79 cells treated in the in vitro liver perfusion system. Comparative investigation of different in vitro metabolising systems with dimethylnitrosamine and benzo[a]pyrene.

A comparative study of three in vitro metabolising systems was performed in combination with Chinese hamster V79 cells, at which point mutation to 6-thioguanine resistance was scored. The three metabolising systems used were: (1) rat liver microsomal fraction (S9-mix); (2) feeder layer of primary embryonic golden hamster cells, according to Hubermann's system; (3) in vitro perfusion of rat liver according to the system of Beije et al. As model substances dimethylnitrosamine (DMN) and benzo[a]pyrene (BP) was used. The liver perfusion was more efficient than S9-mix as an activating system of DMN, while the feeder layer of embryonic cells was unable to activate this compound. The activation of DMN with S9-mix was dependent on the presence of NADP. By exposing the target cells in the liver perfusion at different distances from the liver the biological half life of the active metabolite of DMN could be estimated to less than 5 s. With BP the three metabolising systems showed reversed results as compared with DMN--both the feeder layer cells and S9-mix activated BP, the feeder layer cells being most efficient. With liver perfusion, the perfusate itself was totally negative. Only the bile showed a week mutagenic effect. These results are in accordance with the notion that intact liver cells perform both an activation and a subsequent deactivation of BP. Because of the importance of hepatic bio-transformation in chemical mutagenesis and carcinogenesis it is emphasied that a liver perfusion system could be used in a testing protocol for genotoxic effects as a valuable tool in order to analyse the mechanism of action of mutagenic and carcinogenic compounds detected in other test systems, for instance bacterial/microsomal tests.


Effect of a single treatment with the alkylating carcinogens dimethylnitrosamine and methyl methanesulphonate on liver regenerating after partial hepatectomy. IV. Effect on methylase-mediated methylation of DNA.

The possibility that carcinogens may affect methylase-mediated methylation of replicating DNA was investigated. A system eminently suitable for this purpose is liver regenerating after partial hepatectomy, as one injection of dimethylnitrosamine (DMN) given during the ensuing period of increased DNA synthesis induces hepatocellular carcinoma. Methylation of DNA by DNA methylase normally occurs only in proportion to DNA synthesis. Therefore simultaneous measurements were made of synthesis (incorporation of [14C]adenine into DNA adenine, or of d[5-3H]cytidine into DNA cytosine), and of methylation (incorporation of [methyl-3H]methionine into 5-methylcytosine of DNA) in liver regenerating after partial hepatectomy. After treatment with DMN, the ratio of methylation: synthesis remained within the normal range. Methyl methanesulphonate (MMS), a compound which damages DNA in regenerating liver in a similar but not identical way to DMN and which does not induce tumors in liver even when given after partial hepatectomy, caused an increase in methylation in relation to synthesis. These experiments therefore do not support the view that altered DNA methylase activity is involved in carcinogenesis.


Altered RNA transport without derepression in a rat kidney tumor induced by dimethylnitrosamine.

RNA/DNA hybridization in the presence of competing RNA was used to study transcription and transport of RNA from the nucleus to the cytoplasm in a rat renal tumor induced by a single intraperitoneal injection of dimethylnitrosamine (DMN). Neither repression nor derepression of transcription of repetitive DNA can be detected in this tumor, but transcripts of all active families of such sequences are found in the tumor cytoplasm. This is the pattern of RNA change found in the chemically-induced hepatocellular carcinomas and contrasts with the pattern of virus-induced renal tumors in which both altered transport and derepression of host repetitive sequences have been demonstrated. The alteration of RNA transport is nearly complete in the kidney only two days after the above treatment, but there is no such alteration detectable in the kidney after nine days of feeding a liver carcinogen which is not carcinogenic to kidneys. This data supports the significance and specificity of the loss of ability to restrict certain RNA sequences to the nucleus early during malignant transformation.


The effect of fluid volume loading on exclusion of interstitial albumin and lymph flow in the dog lung.

The excluded volume fraction for interstitial albumin (FE) was estimated in the lungs of seven mongrel dogs during steady state conditions following intravenous infusions of Ringer's solution, amounting to 0, 5, 10, and 15% of body weight (BW). We estimated the tissue blood volume with 51Cr red cells, extracellular space with 99mTc-diethylenetriaminepentaacetic acid (99mTc-DTPA), and the albumin pool with 125I human serum albumin. A prenodal tracheobronchial lymphatic was cannulated for recording lymph flow (QL), total protein (CL), and albumin [CL(A)] concentrations. From these measurements, we calculated the extravascular albumin content (QA) and 99mTc-DTPA space (VI) of lung tissue samples collected at the successive volume expansions. The apparent tissue concentration of albumin (CApp = QA/VI) decreased from a control value (mean +/- SE) of 0.89 +/- 0.06 to 0.46 +/- 0.04 g/dl following the 15% BW infusion, whereas CL(A) decreased from 1.43 +/- 0.16 to 0.50 +/- 0.07 g/dl for the same volume expansion. By assuming that pulmonary lymph represented tissue fluid, we calculated a control FE of 0.38 +/- 0.02 using the equation, FE = 1 - [CApp/CL(A)]. FE decreased following successive infusions to 0.28 +/- 0.03, 0.16 +/- 0.02, and 0.10 +/- 0.02. These data indicate a significant contribution by the decrease in FE to the total decrease in tissue albumin concentration as interstitial fluid volume increased. Somewhat unexpectedly, the mean steady state QL increased by only 2.1-fold following the 5% BW expansion, but did not further increase following subsequent volume expansions. This has been attributed to a nonlinear interstitial compliance, sequestration of interstitial fluid, or possible deterioration of the experimental preparation.


Evidence from bioassay studies indicating a role for adenosine in cardiac ischemic and hypoxic dilation in the dog.

Previous studies using bioassay demonstrated the presence of a vasoactive substance or substances in coronary sinus blood during myocardial reactive hyperemia which had, on bioassay, characteristics of adenosine and/or AMP. In the present studies, specific blockers were applied to an improved bioassay system to define more precisely the nature of the substance or substances and to determine whether it also appears during local cardiac hypoxia. In the anesthetized dog, coronary sinus blood was bioassayed in an isolated autologous kidney during reactive and hypoxic dilation. During reactive dilation, the bioassay kidney responded with a large resistance increase which was blocked by theophylline and adenosine deaminase and converted to a decrease by adenosine autoblockade. The same was true for hypoxic dilation, except in this instance, only adenosine deaminase reduced the response (40%). Theophylline and adenosine autoblockade eliminated responses of the bioassay organ to both exogenous adenosine and exogenous AMP, but adenosine deaminase was specific for adenosine. These results confirm that one or more vasoactive substances appear in sinus blood in vasoactive quantities during cardiac reactive dilation, and that the peak concentration correlates roughly with the peak flow. In addition, they show that this also is the case for hypoxic dilation. More importantly, they demonstrate that the substance almost certainly is adenosine in the case of reactive dilation, and that increased adenosine levels also are present during hypoxic dilation, but in addition, suggest that AMP sometimes appears in coronary sinus blood during severe cardiac hypoxia in the dog heart.


Coronary vasoactivity of adenosine in the conscious dog.

Intracoronary adenosine infusions in conscious dogs produced half-maximal coronary vasodilation at 0.57 +/- 0.18 (SD) microns and at 1.01 +/- 0.25 microns in open-chest dogs. In both preparations, adenosine at concentrations in the range found in cardiac muscle by direct analysis produced coronary vasodilation equal to that attained during a maximum reactive hyperemic response. The quantitative structure-activity relationship technique was applied to data on the coronary vasoactivity of 68 adenosine analogs to identify the chemical features of this molecule that determine its vasoactivity. These are: (1) the size of the purine base; (2) the inductive effect of C-2 substituent; (3) the electron-withdrawing effect of the C-6 substituent; (4) the glycosylic torsion angle; (5) the ability of the C-2' and C-3' hydroxyls to participate in hydrogen bonding; (7) the absence of sterically hindering groups in the vicinity of C-2' and, more importantly, C-3'; and (8) the inductive effect of the C-5' substituent. The hydrophobicity of these analogs did not correlate with vasoactivity, suggesting that the hydrophilicity of the ribose moiety overshadows any hydrophobic influence of the very weakly aromatic purine base.


Sensitization of isolated canine coronary arteries to calcium ions after exposure to cholesterol.

The abundance of membrane cholesterol is an important determinant of the functional properties of biomembranes. To determine whether arterial smooth muscle acquires altered contractile properties in a high cholesterol environment, isolated canine coronary arteries were exposed to cholesterol in stable aqueous solution. Cholesterol, 10(-12) to 10(-10) M, was an efficacious vasoconstrictor, as maximum contractions equaled those obtained with 15 mM KCl. Antiadrenergic interventions, including chemical sympathectomy in vivo with 6-hydroxydopamine and alpha- and beta-adrenergic blockade with phentolamine and L-propranolol (both 10(-6 M), did not significantly attenuate the contractions. However, responses to cholesterol were abolished completely by (+/-)-verapamil (10(-6) M). Cholesterol in picomolar concentration enhanced the constrictor effects of CaCl2 and KCl, both in the presence and absence of alpha- and beta-adrenergic blockade. Increases in tone in response to graded elevations in the CaCl2 concentration (0-2 mM) were augmented up to 1.5-fold by 10(-12) M cholesterol (P less than 0.01). Results indicate that cholesterol sensitizes isolated coronary arteries to external Ca2+ by a nonadrenergic mechanism. The findings are consistent with the hypothesis that acquisition of membrane cholesterol may alter the contractile properties of coronary arterial smooth muscle, a phenomenon that could play a role in the pathophysiology of atherosclerotic heart disease.


Brain adenosine production in the rat during 60 seconds of ischemia.

In rats, cerebral perfusion pressure were altered abruptly by aortic transection to determine the production by ischemic brain of adenosine and its metabolites, inosine and hypoxanthine. Brain samples were obtained after 0, 5, 10, 15, 30, and 60 seconds of ischemia. Also measured were ATP, ADP, AMP, phosphocreatine (PCr), lactate, and pyruvate. Blood pressure was monitored continuously, and arterial PO2, PCO2, and pH were measured just prior to induction of ischemia. Adenosine was elevated t 2.30 +/- 0.31 (SE) nmol/g at 5 seconds from a control value of 0.96 +/- 0.07. A significant elevation of adenosine continued to 60 seconds (5.50 +/- 1.24). Furthermore, inosine showed a progressive upward trend during the entire 60 seconds of ischemia, whereas no change in hypoxanthine occurred between the moment of transection (31.81 +/- 2.01 nmol/g) and 60 seconds of ischemia (34.72 +/- 2.93). PCr decreased by 1.24 mumol/g within the first 5 seconds. After the onset of hypotension, significant changes did not occur in AMP and ADP until 30 seconds, and in ATP and pyruvate until 60 seconds after aortic transection; lactate was elevated by 10 seconds. The rapid rise of cerebral adenosine within 5 seconds after the onset of ischemia supports a role for adenosine in the regulation of cerebral blood flow.


Sarcomere length-resting tension relation in single frog atrial cardiac cells.

It generally has been thought that the relatively high resting tension characteristic of cardiac tissue resides in structures (collagen, elastin) external to the individual cardiac cells, but the evidence to support this conclusion has been indirect, since the resting tension of intact single cardiac cells has not been determined previously. The purpose of the present investigation was to determine the resting tension (stress)-sarcomere length relationships of single intact frog atrial cells. For tension determinations, a single cell was attached between two poly-L-lysine coated glass beams; one beam served as a compliant calibrated cantilevered force beam, and length changes were imposed on the cell by movement of the other beam. Coventional bright-field light microscope techniques were used to view the cell, the sarcomere pattern within the cell, and the position of the force beam. The resting tension of the intact cell increased from a value of about 10 nN at a sarcomere length of 2.35 microns to a value of about 130 nN at a sarcomere length of 3.45 microns. Lagrangian and Eulerian resting stress-sarcomere length relationships were computed from the resting tension-sarcomere length relationships. The Lagrangian stress increased from a value of about 0.6 mN/mm2 at a sarcomere length of 2.35 microns to a value of about 7 mN/mm2 at a sarcomere length of 3.45 microns. These values of stress are about 8- to 30-fold less than those previously reported for intact frog atrial tissue and indicate that the resting tension of intact frog atrial preparations resides primarily in structures external to the individual cardiac cell.


Comparison of M-mode and cross-sectional echocardiography in infective endocarditis.

Cross-sectional and M-mode echocardiograms were performed on 23 consecutive patients with infective endocarditis. Both M-mode and cross-sectional echocardiography identified vegetations in 18 patients, 10 of whom required valve replacement within 1 month of presentation. Cross-sectional echocardiography alone identified a vegetation in one patient with a prosthetic valve. Neither technique identified vegetations in five instances. The size and shape of a vegetation on cross-sectional echocardiography did not accurately predict the need for early valve replacement of the incidence of major peripheral emboli.


Can cardiac tamponade be diagnosed by echocardiography? Experimental studies.

The purpose of this study was to determine if respiratory variation and/or absolute size of echocardiographically measured right or left ventricular internal dimension at end-diastole (RVIDd or LIVIDd) are accurate indicators of the presence or severity of cardiac tamponade. We measured RVIDd or LVIDd by echocardiography in nine closed-chest, spontaneously breathing dogs in control and during hypotensive tamponade. With tamponade, the end-expiratory RVIDd and LVIDd were significantly smaller than control. Inspiratory increases in RVIDd and decreases in LVIDd were exaggerated during tamponade. Because of the wide range and overlap of RVIDd and LVIDd, no single expiratory value or amount of respiratory change indicated the presence or severity of tamponade. We conclude that if serial echocardiograms show a pericardial effusion, a decreasing end-expiratory RVIDd and LVIDd and an increasing percentage change in ventricular diameter with inspiration, a progressive degree of tamponade should be suspected However, a single echocardiogram cannot accurately predict the presence or severity of tamponade.


Dynamic exercise echocardiography.

To evaluate left ventricular (LV) reserve, we developed a method of dynamic exercise echocardiography (DEE). Forty-six healthy persons and 47 cardiac patients performed bicycle ergometer exercise in the supine position. A special table was used on which the subjects could be firmly attached at shoulder level to prevent bodily movements which might disturb the recordings. In 83% of the subjects in whom a clear echocardiogram was obtained at rest, a clear echocardiogram was also obtained during dynamic exercise. During exercise, cardiac output estimated from the echocardiogram and that from the dye-dilution method showed an excellent correlation. The changes of the mean velocity of LV circumferential shortening during exercise permitted discrimination between older and younger healthy men, and also between healthy subjects and those with either mild or severe LV dysfunction. We conclude that DEE is useful for evaluating LV reserve.


Assessment of left ventricular ejection fraction and volumes by real-time, two-dimensional echocardiography. A comparison of cineangiographic and radionuclide techniques.

Five different algorithms for determining left ventricular (LV) ejection fraction (EF) and volumes from two-dimensional echocardiographic examination (TDE) were compared with standard methods for obtaining EF and volume from x-ray cineangiography (cine) and EF from radionuclide ventriculography (RVG) in 35 patients. Although all methods correlated positively, the degree of correlation varied with the algorithm used. For EF determination, TDE algorithms (especially those using multiple planes of section) were superior to unidimensional algorithms commonly used with M-mode echocardiography. The best algorithm (modified Simpson's rule) correlated well enough with cine EF (r = 0.78; SEE 0.097) and RVG EF (r = 0.75; SEE 0.087) to make clinically useful estimates. TDE volumes also correlated meaningfully with cine end-diastolic and end-systole volumes (r = 084; n = 70) but were associated with a large standard error of the estimate (43 ml) and offered less advantage over unidimensional volume estimates. Quantitative application of TDE appears to be a useful noninvasive method of evaluating LVEF, but is not as useful for estimating LV volumes.


Pathologic basis of thallium-201 scintigraphic defects in patients with fatal myocardial injury.

Using a quantitative, computer-aided circumferential profile technique, we have shown that thallium-201 scintigrams with large defects can identify a group of patients with a high mortality after acute myocardial infarction. To determine whether high-risk thallium scintigrams predict poor survival because of a critical loss of myocardium, we correlated infarct size in 24 autopsied patients with the extent of thallium defect in three views. Of 13 patients with large defects (computer score greater than or equal to 7.0) eight (62%) had greater than 25% loss of left ventricular (LV) myocardium, but five (38%) had smaller infarcts (4--24% of LV myocardium), suggesting that part of the scintigraphic defect was related to ischemia without necrosis. Eight of the nine patients with loss greater than or equal to 25% LV myocardium had large defects. In 10 of 11 patients with small defects (computer score less than 7.0), infarcts involved less than 20% of LV myocardium. Although scintigrams with large defects predicted a critical loss of myocardium in over 60% of our patients, they included an important second group, in which the scintigraphic defect appeared to reflect a small infarct and a large surrounding area of reversibly ischemic myocardium.


The effects of coronary artery disease on the ventricular fibrillation threshold in man.

The ventricular fibrillation threshold (VFT) was measured in 28 patients at the time of cardiac surgery. The VFT was measured with a 100 Hz train of 24 rectangular pulses positioned across the ST segment and T wave. Current was applied to the epicardial surface of either ventricle with a bipolar electrode probe. In six patients, the normal right VFT was 24.3 +/- 5.2 mA, and in 10 patients the normal left VFT was 33.6 +/- mA (p less than 0.05). In 12 patients with greater than or equal to 75% obstruction of the left anterior descending coronary artery, the left VFT was 18.6 +/- 6.9 mA. This value was significantly less than the left VFT in patients without coronary artery disease (p less than 0.001). This study shows that the VFT can be measured in man and that coronary artery disease reduces this parameter.


Effect of submaximal exercise on vulnerability to fibrillation in the canine ventricle.

The risk of instantaneous death due to ventricular fibrillation was compared in resting and exercised dogs. Three weeks before testing, all dogs had bipolar left ventricular stimulating electrodes implanted and a reversible snare was placed around the anterior descending coronary artery. The dogs were randomly assigned to either an exercise (13 dogs) or a control (12 dogs) group. We measured ventricular fibrillation thresholds (VFTs) in all dogs before and after inducing ischemia by tightening the snare while the dogs stood at rest. The next day, nonischemic and ischemic VFTs were redetermined for control dogs at rest and for the exercise group during a treadmill run. No statistically significant changes were noted within and between groups in nonischemic or in ischemic VFTs at rest. In five exercise dogs, spontaneous ventricular fibrillation occurred during the first 8 minutes of the ischemic run, For the eight other exercise dogs, running increased the mean drop in VFTs during coronary occlusion by 23% (p less than 0.01). These data suggest that moderate dynamic exercise may greatly enhance the risk of ventricular fibrillation and sudden death in the presence of myocardial ischemia. In the absence of ischemia, exercise does not appear to increase vulnerability to ventricular fibrillation.


Evaluation of warning arrhythmias before paroxysmal ventricular tachycardia during acute myocardial infarction in man.

In order to determine the relationship of paroxysmal ventricular tachycardia (PVT) to any antecedent (premonitory) ventricular arrhythmias during the early phases of acute myocardial infarctions, 24-hour Holter monitoring was begun on 52 male patients an average of 12.6 hours after the onset of prolonged chest pain that was documented as acute infraction. Twenty-four patients had PVT and 28 did not. We analyzed in detail the incidence of frequency of premature ventricular complexes (PVCs), prematurity and pairing during the 10 minutes immediately preceding PVT from a continuous 10-minute rhythm strip. There was no positive correlation between PVT and the number or complexity of PVCs in the 10 minutes immediately before ventricular tachycardia. These findings suggest that there is no consistent pattern or frequency of ventricular arrhythmia that could be identified as premonitory for PVT during the immediate pre-PVT period, even during the acute phase of myocardial infarction in man.


Suppression of chronic ventricular arrhythmias with propranolol.

The antiarrhythmic efficacy of propranolol was evaluated in 32 patients with chronic high frequency ventricular arrhythmias in a placebo-controlled protocol. After a placebo control period, propranolol was begun and the dosage increased sequentially until arrhythmia suppression was achieved, side effects appeared, or a maximum dosage of 960 mg/day was reached. Computerized analysis of ambulatory recordings was used to quantify the arrhythmias. Twenty-four patients had 70--100% arrhythmia suppression at plasma levels ranging from 12--1100 ng/ml (end of dosing interval). Eight patients in this group had frequent episodes of ventricular tachycardia that were totally suppressed at or below the dosage that produced greater than or equal to 70% suppression of ventricular ectopic depolarizations (VEDs). A biphasic dose-response curve was seen in five patients who responded with a decrease in arrhythmia frequency in the lower ranges of dosages but had increased frequency of ectopic rhythms as the dosage was increased above the optimal level. Only one-third of patients responded at doses less than or equal to 160 mg/day. However, with dosages of 200--640 mg/day, an additional 40% responded. Propranolol appears to control ventricular arrhythmias safely and effectively in many patients. The finding that the antiarrhythmic effect in many patients required plasma concentrations greater than those that produce substantial beta-adrenergic blockage raises a question whether blockade of cardiac beta receptors can directly account for all of the antiarrhythmic actions of propranolol.


Characteristics of ventricular function in single ventricle.

Funtional characteristics of the single ventricle were studied by means of biplane angiocardiography in 34 patients. Group 1 consisted of 14 patients with normal or increased pulmonary vascular marking on chest film and no pulmonary stenosis. Group 2 included 20 patients with severe angiographic pulmonary stenosis and decreased pulmonary vascular marking. Ventricular volume parameters were calculated according to Simpson's rule and were compared with normal values. The sum of the normal left and right ventricular volumes was assumed to be 100%. In group 1, ventricular end-diastolic volumes averaged 143 +/- 11% and were significantly (p less than 0.001) larger than end-diastolic volumes in group 2 (81 +/- 4%). The presence or absence of severe pulmonary stenosis affecting pulmonary blood flow was a main factor regulating the ventricular chamber size in single ventricle. In both groups, the ejection fraction of a single ventricle was significantly lower than that of a normal left or right ventricle. Ventricular size and function in patients with a single ventricle should be carefully assessed before ventricular septation surgery.


Platelet suppressant therapy in patients with prosthetic cardiac valves. Relationship of clinical effectiveness to alteration of platelet survival time.

Platelet survival time (SURV) has correlated with thromboembolism in patients with prosthetic cardiac valves. Sulfinpyrazone increases SURV. SURV (autologous labeling with 51Chromium) was measured in 126 patients who had aortic or mitral valve replacement. These patients were followed prospectively. Ninety-four with shortened SURV received sulfinpyrazone; 32 with normal SURV were not treated with platelet suppressants. Eighty-seven patients were anticoagulated with warfarin--67 with shortened SURV and 20 with normal SURV. Eleven patients have had thromboembolism, and all had shortened SURV (2.4 +/- 0.08 days; average half-time +/- SEM; normal 3.7 +/- 0.03 days; n = 26) none had an increase of SURV with sulfinpyrazone (2.3 +/- 0.09 days). Of 83 patients with shortened SURV who did not have embolism, sulfinpyrazone increased SURV in 59 (71%) 2.6 +/- 0.05 to 2.9 +/- 0.06 days). Of 35 patients with shortened SURV who failed to increase SURV with sulfinpyrazone, 11 (31%) had embolism; none of 59 (0%) with an increase of SURV with sulfinpyrazone had thromboembolism. These results suggest that patients with thromboembolism after prosthetic cardiac valve replacement have shortened SURV and that patients treated with slufinpyrazone who have thromboembolism do not have an increased SURV.


Evaluation of the echocardiogram as an epidemiologic tool in an asymptomatic population.

An asymptomatic adult population of 196 men and women was studied with the echocardiogram to derive age- and sex-specific "normal" values for a number of clinically used echocardiograhic variables. The results are in general agreement with previously published normal values. Body position during the examination, age and sex influence the echocardiographic results; body surface area correction normalized most of these effects. The prevalence of occult abnormalities determined by the echocardiogram is 7%; the most common finding was mitral valve prolapse. Inter- and intraobserver variability was assessed. The interobserver differences found on analysis are statistically, but not clinically , significant. The echocardiogram appears to be a suitable tool to use in epidemiologic studies to detect selected cardiac abnormalities, but is limited for this purpose because some subjects in such a population cannot be adequately examined.


Electrophysiologic studies in patients with ventricular inversion and "corrected transposition".

We evaluated the intracardiac conduction intervals using His bundle recordings in 40 patients with ventricular inversion and 1-transposition of the great arteries. Twenty-nine subjects had 1:1 atrioventricular (AV) conduction. In 15 of those with normal PR intervals and QRS durations, the conduction intervals were not different from those of subjects with normal hearts. In the 14 patients with first-degree AV block, the block was located between the sinus node and AV node in four, between the low right atrium and bundle of His in seven, and below the common bundle of His in four. In 11 subjects with complete AV block, the stie of block was above the site of the His potential in four, below in two and within the His bundle in one. In four patients we could not record a His potential and thus could not localize the site of block. Complete block below the His recording site was associated with syncope in one patient and sudden death in another. His bundle recording is a safe technique for studying the conduction system in children with ventricular inversion and 1-transposition of the great arteries.


Depressed responsiveness to vasoconstrictor and dilator agents and baroreflex sensitivity in conscious, newborn lambs.

The effects of vasoconstrictors and vasodilators were compared in conscious, newborn lambs and adult sheep instrumented with electromagnetic flow probes on the ascending aorta and catheters in the thoracic aorta. Methoxamine, angiotensin II, norepinephrine, nitroglycerin and isoproterenol were administered intravenously to evaluate their effects on arterial pressure, cardiac output and systemic vascular resistance (SVR). The difference in response between adults and newborns was most apparent with methoxamine. Methoxamine, 400 micrograms/kg, i.v., which increased mean arterial pressure by 57 +/- 6% and SVR by 278 +/- 27% in newborn lambs, caused greater increases (p less than 0.01) of 81 +/- 8% and 1418 +/- 141%, respectively, in the adults. Responses also differed significantly between newborn and adult animals to norepinephrine, angiotensin II, nitroglycerin and isoproterenol. In a second group of animals in which smaller amounts of methoxamine and isoproterenol were injected directly into the terminal aorta, changes in terminal aortic flow and resistance were examined. Again, both vasoconstrictor and vasodilator responses were more marked in adults than in newborns. Finally, the sensitivity of the arterial baroreceptor reflex was evaluated by comparing the regression of pulse interval (PI) on systolic arterial pressure (SAP) after an intravenous dose of methoxamine in conscious, adult and newborn animals. The PI/SAP slopes in adult sheep, 45.4 +/- 3.5 msec/mm Hg, were significantly greater (p less than 0.01) than in newborn lambs, 11.7 +/- 2.2 msec/mm Hg.


Determination of His-Purkinje refractoriness in man with His bundle pacing.

Consistent His bundle pacing was accomplished in one of 60 consecutive patients. Intracardiac electrophysiologic studies in this patient revealed rapid atrioventricular nodal conduction (AH = 55 msec). While the relative refractory period of the His-Purkinje system as a whole determined by His bundle pacing (using the extrastimulus technique) was identical to that determined by atrial pacing, changes in right bundle branch refractoriness differed. An atrial extrasystolic interval of 370 msec resulted in right bundle branch block, whereas direct His pacing at a shorter extrasystolic interval (360 msec) failed to produce a bundle branch block pattern. The effective refractory period of the His-Purkinje system could not be determined by atrial pacing because of atrial refractoriness, but was obtained by His bundle pacing. Theoretically, direct His bundle pacing may be of value in determining His-Purkinje system refractoriness when this parameter is unobtainable due to either atrial or atrioventricular nodal refractoriness. This technique appears to have limited clinical usefulness, however, because of the very small success rates. Finally, His-Purkinje system refractoriness may differ, depending on site of stimulation, presumably due to differing inputs into the His-Purkinje system.


Prevalence of asthma: a comparative study of urban and rural Xhosa children.

An epidemiological study was undertaken to determine the prevalence of asthma in young urban and rural black (Xhosa) children. One thousand three hundred and seventy five children were studied, 694 from a Cape Town african township and 671 from a rural area in Transkei. The exercise tolerance test which required free range running at maximum effort for 6 min was used to identify asthmatic subjects. A fall of 15% or more in the post-exercise FEV1 and PEFR values was regarded as a positive result. Twenty-three children were found to be asthmatic, twenty-two from the city area, but only one from the country, giving a prevalence figure for asthma of 3.17% in the first group and 0.14% for the second. Possible reasons for these differences are discussed. The exercise tolerance test was found to be a useful tool for epidemiological studies of asthma.


Asthma, IgE and environment in northern Nigeria.

Sixty-four asthmatics and seventy-five control subjects from rural villages and various urban settings have been investigated. Rural asthmatics had a significantly higher IgE level than both urban asthmatics and village controls. Urban asthmatics had similar IgE levels to urban controls. There was no relationship between IgE levels and the results of skin tests for asthma allergens. Stool parasites were found as frequently in asthmatics as in controls. Our findings do not support the theory that parasitic infestation, high IgE levels or rural living protect from asthma.


Bronchial asthma in the southern Maldives.

A whole population survey was performed in Addu Atoll, southern Maldives to establish the prevalence of bronchial asthma. Assessments of atopic eczema, allergic rhinitis, conjunctivitis and intestinal infection were made, and serum specific IgE titres to a number of allergens were measured. Asthma is a common condition in the southern Maldives, and there is no evidence that the IgE produced by intestinal helminths prevents the condition.


Serum IgE in healthy infants fed breast milk or cow's milk-based formulas.

A group of ninety-five infants was followed from birth to one year of age, and the total serum IgE concentration was studied. The infants were fed on breast milk, home-prepared cow's milk formula, or proprietary infant milk formula. Solid foods were introduced at 3.5 months of age. The breast milk group had lower IgE than the cow's milk group up to 4 months of age, i.e. until the end of exclusive milk feeding. The formula group had intermediate IgE values. The difference was not due to the presence of IgE antibodies to cow's milk in bottle-fed infants, since no such antibodies could be detected. After the introduction of solid foods the differences between the groups disappeared. Our data suggest that exclusive breast milk feeding can reduce total serum IgE concentration in early infancy. Reference values for unselected infants and for infants with no atopic manifestations are reported.


Natural history of allergy to Hymenoptera.

One hundred and nineteen patients who had a generalized reaction to a bee sting and who subsequently had a history of being re-stung before desensitization were studied. The reaction, in these 119 patients was as follows: 44.5% had an improved grade of reaction, 42.9% had the same grade of reaction and 12.6% had a worse grade of reaction. Of these 119 patients, 39.5% had only a local reaction to their re-sting. Those patients who were re-stung within 2 weeks (anergic period) or over 5 years after a generalized reaction to a sting had significantly improved response. Results of pretreatment RAST to venoms of hornet, yellow jacket, wasp, honey bee and to phospholipase A appeared to correlate with the clinical signs and symptoms of the previous sting in 75% of cases.


Haemagglutinating antibodies to cat dander in relation to exposure and respiratory allergy.

Cat epithelial extract was coupled with chromic chloride to red cells and used as an antigen in a haemagglutination assay. Agglutinins coexisted with IgE type antibodies in 44% of patients with respiratory allergy and sensitivity to cat dander but occurred in only 1% of asthmatic patients sensitized to other allergens or in controls. The agglutinins were detected in six out of 104 cat fanciers, and three of those with agglutinins had asthma. The agglutinins were of IgG type and were heterogeneous in specificity.


Treatment of superficial cancers by combination hyperthermia and radiation therapy.

Clinical studies are being carried out to evaluate whether radiation combined with hyperthermia evokes better tumor control than that achieved with radiation alone. Thus far, 54 patients with various superficial cancers have been treated with combination hyperthermia and radiation. The heating methods used have been temperature regulated water bath immersion and radiofrequency inductive heating. Hyperthermia alone has caused significant, though transitory, tumor regression in 10 out of 19 patients. Selective heating of tumor tissues has been achieved in 20 out of 24 lesions following radiofrequency inductive hyperthermia. The overall local tumor control rate after the combined therapy was 78 percent compared to 26 percent after radiation alone.


A model program for education and training of clinical chemists.

Some general guidelines can help in the assessment of clinical chemistry training and training programs. A clinical chemist must be, first of all, a good chemist, but to this must be added other knowledge and skills. A program that begins with a bachelor's degree in chemistry equivalent to the American Chemical Society's standards is proposed for the clinical chemistry technologist. The model provides more flexibility as the education proceeds toward the doctoral and postdoctoral levels and as the opportunities and responsibilities become increasingly more diverse. An essential portion of the training at all levels is experience in a clinical laboratory. It is strongly recommended that this be started early and be continued throughout the program. Certification by an independent agency (National Registry in Clinical Chemistry or American Board of Clinical Chemistry) is strongly recommended as a final step in all plans.


Hemoglobinuria and hematuria: accuracy and precision of laboratory diagnosis.

Five methods of assessing blood in urine were studied and compared: sedimentation count, hemacytometry, "Clini-lab" reagent strip and "N-Multistix" and "ChemStrip-8" dipsticks. The minimum sensitivity of two commercially available urine dip-stick procedures for blood was established. The study includes method association, assay variation among and within technologists, accuracy, precision, and sensitivity. Our results demonstrate generally poor association between results by the dip-stick procedures and the hemacytometer or sedimentation count. Results by the last two procedures also showed very poor association with each other.


Column chromatography and immunoassay compared for measuring the isoenzymes of aspartate aminotransferase in serum.

We compare a column-chromatographic method and a homogeneous immunoassay method for separately measuring the mitochondrial and cytoplasmic isoenzymes of aspartate aminotransferase. Analytical recovery for the two methods averaged 102% (SD, 2%) and 103% (SD, 4%), respectively, for 11 pools prepared by adding the purified isoenzymes to serum and 102% (SD 8.9%) and 89% (SD, 8.1%) for 26 unaltered specimens of human serum. In comparing the results of the immunoassay method (y) to the chromatographic method (x), our measurements agreed closely for the mitochondrial (y = 0.947 X + 7, r = 0.9991, standard error of estimate = 2.9 U/L) and cytoplasmic (y = 0.92x-6, r = 0.9995, standard error of estimate = 2.1 U/L) isoenzymes in pools prepared from the purified isoenzymes. Similar measurements of the 26 human serum specimens yielded the following results for least-squares evaluation; cytoplasmic isoenzyme y = 1.03x-11, r = 0.994, and standard error of estimate = 6.0 U/L; mitochondrial isoenzyme y = 0.75x+0, r = 0.927, and standard error of estimate = 3.9 U/L.


Improved radioimmunoassay for 11-deoxycortisol (compound S) in plasma.

We describe a radioimmunoassay for 11-deoxycortisol (Compound S) that involves preliminary chromatography on Sephadex LH-20, to isolate 11-deoxycortisol from crossreacting structurally related steroids. Pools with means of 3.4, 12.9, and 69.9 micrograms/L gave intra-assay coefficients of variation of 9.7, 9.6, and 11.4%, respectively; pools with means of 4.6, 27, and 95 micrograms/L gave inter-assay CV's of 10.1, 9.7, and 6.2%, respectively. A normal range (mean +/- 2SD) of 0.5 to 12.0 micrograms/L was obtained. This procedure is useful for the specific determination of 11-deoxycortisol, commonly determined in blood as part of the metyrapone test for assessing pituitary corticotropin reserve.


Determination of 4-O-methylated catecholamine metabolites in urine by mass fragmentography.

We describe the determination of the 4-O-methylated catecholamine metabolites 4-methoxy-3-hydroxyphenylacetic acid (iso-HVA), 4-methoxy-3-hydroxyphenylmandelic acid (iso-VMA) and 4-methoxy-3-hydroxyphenylethylene glycol (iso-MOPEG) in urine with use of mass fragmentography and deuterated internal standards. Normal values, expressed in terms of creatinine, are given as a function of age. Urinary excretion values during neuroblastoma, pheochromocytoma, and parkinsonism treated with L-DOPA, and during the intravenous administration of dopamine are discussed. For normal persons, the mean percentages (+/- SD) of 4-O-methylated metabolites relative to their 3-O-methylated analogs were 6.29 +/- 2.79% (iso-HVA/HVA), 0.70 +/- 0.57% (iso-VMA/VMA), and 0.77 +/- 0.53% (iso-MOPEG/MOPEG). We observed no significant changes in these parameters for the patients studied and conclude that the determination of 4-O-methylated catecholamine metabolites for these patients does not add information to the results obtained from their quantitatively more important 3-O-methylated analogs.


Arylesterase in serum: elaboration and clinical application of a fixed-incubation method.

A sensitive, specific, and simple method for determining serum or urine arylesterase (EC 3.1.1.2) is described. The enzyme acts on phenyl acetate to release phenol, which produces a stable indophenol dye with 4-aminoantipyrine and potassium ferricyanide. Arylesterase, a thiol enzyme, is reactivated by 2-mercaptoethanol and by cysteine, but not by reduced glutathione. Calcium is indispensable to stabilize and to activate (Km = 0.85 mmol/L) the enzyme; complete protection is achieved at CaCl2 20 mmol/L. Magnesium acts as a weak (Ki = 116 mmol/L), lanthanum as a potent (Ki = 5 mumol/L) competitive inhibitor. The activity is measured in diluted sera at phenyl acetate 4.0 mmol/L (Km = 1.12 mmol/L), pH 7.8 and 25 degrees C. The normal range extends from 53 to 186 kU/L, and four isoenzymes are present in sera from healthy adults. Arylesterase decreases in hepatic disorders, especially in cirrhosis and carcinoma of the liver, with reduction of the penultimate fraction in polyacryalmide gel electrophoresis.


Chemical inhibition used in a kinetic urease/glutamate dehydrogenase method for urea in serum.

We describe a fixed-time-interval, kinetic inhibition method, with use of a competitive inhibitor (l) of the urease/glutamate dehydrogenase reaction to increase the "apparent" Michaelis constant by a factor of (1 + [l]lKl). This allows greater flexibility in selecting an appropriate sample dilution for kinetic determinations of urea in serum (i.e., [S]lKm ratio). Nine compounds were screened as potential inhibitors for this study. Adding 5 mmol of hydroxyurea per liter increases the "apparent" Michaelis constant for the coupled enzyme reaction by 10-fold. We used a sample dilution of 21-fold vs. dilutions of 141- to 350-fold for previously reported kinetic methods. Mean analytical recovery with this method was 100.2%. Reaction rate vs. urea concentration was linear, and complete recovery extended to 30 mmol of urea per liter. Of 22 potential interferents, only fluoride (250 mmol/L) and bilirubin (1 mmol/L, or 580 mg/L) caused greater than 5% interference. We discuss precision and effects of specimen dilution, and compare results for 100 specimens with those by a manual Berthelot-indophenol method, a manual diacetyl monoxime method, and a diacetyl monoxime method adapted to continuous-flow analysis.


DuPont aca III performance as tested according to NCCLS guidelines.

We evaluated the analytical performance of 10 representative channels on the new microprocessor-controlled DuPont aca III according to the guidelines (draft documents PSEP-2, 3, and 4) proposed by the Instrument Evaluation Subcommittee of the National Committee for Clinical Laboratory Standards. These guidelines were used for the experimental design and data analysis for the precision and accuracy testing, the latter by comparison with an aca II, with results of atomic absorption spectroscopy for calcium, and with the National Glucose Reference Method. From a 20-day replication study, we estimated within-run, between-run/within-day, between-run/between-day, and total standard deviations at three concentrations for each method. From duplicate analyses of 100 samples on the aca III, aca II, and other methods, we estimated the bias from the regression line at specific concentrations and total error from a tolerance limit about the regression line. Analytical performance of the aca III was judged acceptable because these estimated errors were small.


Metabolic balance of aluminum studied in six men.

The metabolism of aluminum was followed in patients who were receiving strictly controlled diets, either unaltered or supplemented with aluminum-containing antacids. Aluminum was determined by atomic absorption spectrophotometry with a graphite furnace. All food, water, and medications were analyzed, as were aliquots of urine and stool collected throughout six-day periods. Patients were usually studied for five consecutive six-day periods on the same diet and aluminum intake. This study included six patients who were studied for seven to 21 such six-day periods. During control periods, when the patients were receiving less than 5 mg of aluminum per day, the balance was usually slightly negative, but when the diet was supplemented with antacids to contain 1--3 g of aluminum per day, an average positive balance of 23 to 313 mg of aluminum per day was observed for the total time on the same intake. Consecutive six-day periods while a subject was receiving antacids might show either positive or negative balances, but the average retention for 18--84 days on antacids was always positive. Thus the potential effects of the storage of aluminum in the tissues must be considered in the chronic administration of antacids.


The use of immobilized enzyme reactors in continuous-flow analyzers for the determination of glucose, urea, and uric acid.

We describe the use of immobilized enzymes in assay methods for the determination of glucose with glucose oxidase, uric acid with uricase, and urea with urease in serum samples. The enzyme reactor tubes were adapted to continuous-flow analyzers (Technicon AA II, SMA 12/60, and SMAC) used in routine laboratory determinations, and results with their use were compared to those from assays involving soluble enzymes. We substituted the reactors for the free enzyme reagents in the respective channels of the SMA 12/60 and SMAC, without modifying the parameters of the remaining channels. We compared assay sensitivity, precision, and carryover for immobilized and conventional liquid enzymes. Immobilized enzyme reactors provide accurate, reliable, convenient, and economical alternatives to the use of free enzyme reagents in these systems.


Rapid analysis for iodotyrosines and iodothyronines in thyroglobulin by reversed-phase liquid chromatography.

We describe a 10-min reversed-phase "high-pressure' liquid-chromatographic procedure for measuring tyrosine, monoiodotyrosine, diiodotyrosine, 3,5-diiodothyronine, 3,5,3'-triiodothyronine, 3,3',5'-triiodothyronine, and thyroxine. Resolution and quantitation of a mixture of these amino acids were excellent on LiChrosorb (Altex) RP-8 with isocratic elution (1.5 mL/min) with acetonitrile/water/glacial acetic acid (50/49/1 by vol). As little as 100 ng of each iodoamino acid could be detected and quantitated with a conventional 1-cm, flow-through spectrophotometric (254-nm) detector coupled to a 10-mV strip-chart recorder. Analyses for monoiodotyrosine, diiodotyrosine, 3,5,3'-triiodothyronine, and thyroxine in hog and beef thyroglobulin hydrolysates (sequential digestion with pronase and aminopeptidase) agreed well with results by anion-exchange chromatography and by competitive radioassays. To prevent interference by tryptophan in the analysis for diiodotyrosine, we batch-separated the iodoamino acids by anion-exchange chromatography before the procedure. The procedure we describe seems generally useful for detection and quantitation of thyroid hormones, thyroid hormone metabolites, and iodotyrosines.


Spurious hyperbilirubinemia in uremic patients on propranolol therapy.

Chronic, low-grade, unexplained increases of total serum bilirubin concentration were observed in 16 of 132 (12%) chronic dialysis patients. Cessation of antihypertensive medication in four patients resulted in disappearance of hyperbilirubinemia. Propranolol was the only antihypertensive drug common to all 16 patients. Daily dosage varied among the patients, but the mean dose was 308 mg (+/- 51 SEM). Serial determinations of sera from individual patients given different doses and from the group as a whole demonstrated a linear relationship between propranolol dose and apparent total serum bilirubin concentration with continuous-flow analysis. When serum specimens from uremic patients receiving propranolol were treated with diazotized fulfanilic acid and examined spectrophotometrically, an absorbance peak distinct from but overlapping that of bilirubin was consistently demonstrated. The material producing the peak disappeared when the drug was stopped, did not dialyze, and was not reproduced by the in vitro addition of propranolol to uremic serum. We postulate that a metabolite(s) of propranolol is retained in uremic serum and interferes with the bilirubin determination.


Radiochemical method for measuring plasma clearance and urinary excretion of pteroylglutamic acid.

A radiochemical procedure is described for specific determination of pteroylglutamate in serum and urine. This method depends on denaturation of methyltetrahydrofolate with peroxide and measurement of the residual folate by a ligand-binding radioassay. The binding determinant for the radioassay is a folate-binding protein, partially purified from chronic myelogenous lekemia cells, that has low affinity for the reduced folates and thus will preferentially measure residual pteroylglutamate rather than any nondenatured residual methyltetrahydrofolate. We used this assay to measure the clearance from plasma and the urinary excretion of pteroylglutamate and a small fraction of serum folate that is stable to this oxidation procedure. The plasma clearance after intravenous injection is characterized by an initial rapid distribution phase followed by a second, slower metabolic phase; after about 2 h all of the administered pteroylglutamate has been cleared from the blood. The peak concentration of total folate in serum 1--2 min after administration of pteroylglutamate exceeded the sum of the endogenous stable and baseline serum folate, indicating that a reduced labile folate was released from the liver and perhaps from other tissues. This reduced folate had a slower metabolic clearance rate and was excreted to some extent in urine. Only 2.3 and 7.9% of the pteroylglutamate administered to two normal subjects was excreted as stable folate.


Measurement of acetate in human blood by gas chromatography: effects of sample preparation, feeding, and various diseases.

We measured acetate concentrations in whole blood, serum, and plasma by a modification of a previously described method involving vacuum distillation and gas chromatography. The mean acetate concentration of fresh venous plasma from 27 normal subjects was 51 +/- 5 mumol/L (95% confidence limits ranged from 0 to 103 mumol/L). The acetate concentrations of serum and plasma incubated for 2 h at either 4 degrees C or 27 degrees C were the same. The acetate concentration of whole blood incubated at 27 degrees C was significantly greater than that of blood incubated at 4 degrees C. This change may have resulted from the production of acetate by erythrocytes or from the hydrolysis of acetate esters. Storage of plasma at -20 degrees C for 24 h significantly increased acetate concentrations from 26 +/- 6 mumol/L to 63 +/- 4 mumol/L. After the subjects consumed a standard breakfast, venous plasma acetate concentrations increased from 58 to 97 mumol/L at 30 min. Acetate concentrations in arterial plasma exceeded those in venous plasma. Plasma acetate concentrations were not significantly altered in patients with malignancy or diabetes mellitus, but severe liver disease and severe acidosis were both associated with increased acetate concentrations. These preliminary observations suggest that plasma acetate concentrations may be altered in several disease states.


Measurement of 4-hydroxyphenylacetic aciduria as a screening test for small-bowel disease.

We evaluted measurement of urinary 4-hydroxyphenyl acetic acid as a potential screening method for small-bowel disease and bacterial overgrowth syndromes in 360 unselected acutely ill infants and children. Control data were obtained on 120 healthy children, ages 1.5 to 15 years, from a general medical practice, 48 healthy infants, ages one to five years, from local day nurseries, and 150 healthy babies, ages less than one to eight days. Comparative data were from 300 acutely ill hospitalized babies and children, ranging in age from less than one day to 15 years and without clinical evidence for small-bowel disease and bacterial overgrowth syndrome. No false-negative results and only 2% false-positive results were observed. Among the 10 patients whose urinary excretion of the analyte was considered to be abnormal were patients with Giardia lamblia infestation, ileal resection with blind loop, and other diseases of the small intestine associated with bacterial overgrowth. We conclude that measurement of 4-hydroxyphenylacetic acid excretion is useful in screening for such diseases.


Increase in dialyzable calcium associated with therapy with lithium.

We measured total and dialyzable calcium concentrations in consecutive sera submitted for routine lithium analysis. Of 98 samples from 61 different individuals, six (6.1%) total calcium results and 32 (33%) dialyzable calcium results were above the respective reference intervals. By comparison, when both total and dialyzable calcium were measured on 50 different apparently healthy volunteers, no results were outside either reference interval (2.20--2.58 mmol/L for total and 1.30--1.47 mmol/L for dialyzable calcium). These increases were not due to age or sex differences between the patients and controls. From the dialyzable calcium data, there appears to be an even higher incidence of mild hypercalcemia in patients receiving oral lithium salts than is indicated by the total calcium concentration alone.


Adsorption of organic acids from amniotic fluid and urine onto silica gel before analysis by gas chromatography and combined gas chromatography/mass spectrometry.

We describe a method for adsorbing organic acids from amniotic fluid and urine specimens onto a column of silica gel before analysis by gas chromatography and by combined gas chromatography/mass spectrometry. Analytical recoveries of individual organic acids by our adsorption method compare favorably with those obtained by a more laborious manual extraction with ethyl acetate. Results for specimens of amniotic fluid and urine show that our method gives better recoveries of most of the organic acids. Furthermore, because citric acid is also extracted in our method without extracting significant amounts of sulfate or phosphate, the method is a suitable alternative to methods involving the use of DEAE-Sephadex for concentrating these acids.


Washington University Case Conference. Multiple myeloma and hypercalcemia?

We present a case of multiple myeloma with high values for total calcium. The case discussion focuses on the role of the laboratory in diagnosis and monitoring of multiple myeloma and on the causes of the associated hypercalcemia. We present evidence for calcium binding to the paraprotein as a cause for the "hypercalcemia" in this case and we document a difference in the results for total calcium among various methods when this patient's serum was analyzed.


Three different radioiodination methods for human spleen ferritin compared.

We compared three different methods for radioiodination of human spleen ferritin. Tracers prepared by direct oxidative iodination with use of Chloramine T or 1,3,4,6-tetrachloro-3 alpha, 6 alpha-diphenylglycoluril (Iodogen) were structurally altered by the labeling procedure, as was made apparent by gel filtration and electrophoresis on polyacrylamide gel. Tracers prepared by conjugation to radioiodinated N-succinimidyl-6-(4-hydroxyphenyl)propionate retained their structural integrity, as assessed by physicochemical methods. Such tracers bound more than 90% in antibody excess, yielded self-displacement curves parallel to unlabeled ferritin standards, and retained their immunoreactivity for nine weeks. By contrast, immunological activity and stability of tracers prepared by the oxidative methods were less satisfactory. Tracers prepared by using lodogen were unsuitable for use in the ferritin radioimmunoassay because of shallow standard-inhibition curves and poor binding in the absence of added standard. Thus, we found the conjugation procedure to be the most suitable of the three methods for preparation of spleen-ferritin tracers.


Evaluation of the DuPont aca amylase method.

The dilution/activity curve for the DuPont aca kinetic amylase (EC 3.2.1.1) method is linear to approximately six times the upper limit of the expected range. Total reaction time on the aca is 4.3 min; however, there is a nonlinear rate of change in absorbance for at least the first 8 min, which is consistent with a lag phase. The within-run and day-to-day coefficients of variation were 2.4% at 100 U/L and 3.6% at 114 U/L, respectively. There was no interference by marked hemolysis (hemoglobin 4 g/L) or by glucose concentrations of 10--50 g/L. Comparisons by the split-sample method between the Amylochrome procedure and the aca procedure gave correlation coefficients of 0.992 for serum and 0.989 for urine. A second comparison, between the AutoAnalyzer I method and the aca, gave correlation coefficients of 0.998 for serum and 0.909 for urine. The expected range for serum was 18--106 U/L and for urine 2--33 U/2 h.


Modification of the DuPont aca as exemplified by a new procedure for neonatal total bilirubin.

Hemoglobin interferes with determination of total bilirubin by the diazo procedure with the DuPont aca. We compared results by that procedure with those by a manual direct spectrophotometric two-wavelength method. Because the manual method was not inhibited by hemolysis as was the DuPont aca diazo procedure, we reprogrammed our DuPont aca to measure the absorbance of diluted serum samples at 452 and 577 nm with use of DuPont absorbance packs as the sample pack and cuvette. Results correlated well with those by the manual spectrophotometric method, and hemolysis no longer interfered. The method is primarily intended for use with serum from neonates.


Fluorometric measurement of theophylline.

We have developed a simple, sensitive procedure in which theophylline in a solution of cerium(IV) acts as an oxidizable compound to produce fluorescent cerium(III) when combined with a microsolvent for extraction at pH 7.9. This method provides excellent sensitivity and specificity because other xanthines and phenobarbital, secobarbital, amobarbital, and phenytoin do not interfere. Day-to-day reproducibility (coefficient of variation, 6%) is attained for theophylline concentrations of 10 and 20 mg/L. Results correlate well with liquid chromatography and enzyme immunoassay procedures. Total analysis time for a single sample is 10 min.


Comparison of theophylline concentrations in venous blood and capillary blood in asthmatic children.

Twenty children, five to 16 years old, diagnosed as having asthma, were studied to compare the serum concentrations of theophylline obtained by standard venipuncture with capillary concentrations obtained by finger lancet. The mean theophylline concentration in venous samples was 9.27 mg/L and in capillary samples was 9.26 mg/L. Comparison by the paired t-test showed no statistical difference between kinds of samples. When surveyed as to their preference, nine patients preferred the venous method, five the capillary method, and six had no preference.


A commercial digoxin radioassay modified for use as an emergency ("stat") procedure.

A recent "stat" procedure recommended by the manufacturer for use with a commercial digoxin radioassay involves doubling the amount of antibody used in the regular procedure, to compensate for the decrease in binding that results from use of a shorter (10 min) incubation. This has two obvious disadvantages: increased material cost and decreased sensitivity because of the increase in the binding capacity of the assay. In our modified procedure, we kept the binding capacity constant by decreasing the volume of the incubation mixture without increasing the amount of antibody. Total binding and the rate of binding on 10-min incubation are 50.4 and less than 1.3% per minute, respectively. Results by our modified stat procedure compare well with those by the manufacturer's suggested regular procedure in terms of sensitivity, accuracy, and precision, and are more sensitive and economical than those by the manufacturer's suggested stat procedure.


Evaluation of the BMC glucose oxidase/peroxidase-4-aminophenazone-phenol procedure for glucose as adapted to the Technicon SMAC.

We evaluated the analytical performance of Trinder's glucose oxidase (EC 1.1.3.4)/peroxidase (EC 1.11.1.7) 4-aminophenazone-phenol method for the quantification of serum glucose as adapted to the Technicon SMAC. Our results correlated well with those by the routine SMAC glucose oxidase/peroxidase 3-methyl-2-benzothiazolinone hydrazone-N,N-dimethylaniline method (y = 1.02x - 49.4; r = 0.99) and the glucose oxidase oxygen-rate method (y = 0.99x + 14; r = 0.99) with the Beckman Glucose Analyzer. Sample-to-sample interaction was less than 1%. Ascorbic acid or uric acid in concentrations as high as 200 mg/L were without demonstrable effect on results for glucose. Intra- and inter-assay precisions (CV) were 1.6 and 2.3%, respectively. The upper limit of linearity was about 5 g/L. Adaptation of the Trinder method for glucose to the SMAC is simple and provides an analytically acceptable and economical alternative to the methods ordinarily used with the SMAC.


Myocardial infarction?--a conflict between electrocardiographic changes and biochemical data.

We present a case of hypovolemic shock accompanied by electrocardiographic changes classically associated with acute myocardial infarction. Prompt therapeutic intervention, which included correction of the hypovolemic shock, resulted in stabilization of the patient's clinical course, increased activities of the cardiac-specific enzymes in serum were not documented. Serial electrophoretic determinations of the isoenzymes of serum lactate dehydrogenase did not show the characteristic changes associated with myocardial infarction. Accurate determination of the serum isoenzymes provided valuable diagnostic information which, accompanied by the patient's clinical improvement, militated strongly against the occurrence of myocardial infarction.


Automated analysis of hydroxyproline with elimination of non-specific reacting substances.

A modified urinary hydroxyproline method is presented which utilizes an automated technique, Technicon instrumentation and Ehrlich's reagent. Acid hydrolysates are neutralized automatically. Non-specific reacting substances are determined simultaneously with each sample and subtracted as a blank. This method has been found to be superior to the use of charcoal-resin as an absorbent. After blank correction, the mean hydroxyproline excretion of normal and osteoporotic patients are similar. In all groups studied, including Paget's disease, the blanks reresent a significant fraction of total reacting substances and show wide variation between samples. Multiple samples from patients over a two year period show little variation in hydroxyproline excretion when corrected for non-specific reacting substances.


A new sensitive method for assay of histidase in human skin and detection of heterozygotes for histidinemia.

A new sensitive method was developed for assay of histidase in the stratum corneum of human skin with [14C] histidine as substrate. With this method histidase could be detected using less than 1 mg of stratum corneum. Skin histidase was markedly reduced in seven infants with histidinemia. The enzyme activities in thirteen parents of the patients were about 50% of the normal values, and even the highest values in the parents did not overlap the lowest normal values. Thus heterozygotes in the autosomal recessive form of histidinemia can be distinguished from normal persons by their skin histidase activity measured by this sensitive assay method.


Automated determination of total plasma cholesterol: a serum calibration technique.

The automated (AutoAnalyzer II) determination of cholesterol in nine serum pools in the concentration range 3.465-8.871 mmol/l, gave results that were approximately 10% higher than reference values when the analyses were based on unesterified cholesterol standards containing the same amount of water as the sample extracts (1963 analyses in 12 laboratories during a 12 month period; automated value = 0.032 + 1.10 X (Reference value)). A serum calibration procedure was successful ilues, and was equally effective in correcting the values observed for aliquots of 368 fresh-frozen plasma samples analyzed in each of the 12 laboratories during a 38 month period.


Comparison of platelet count and platelet protein methods for determination of platelet MAO activity.

Platelet monoamine oxidase (MAO) activity in 10 normal volunteers was studied as a function of platelet protein or electronically-determined platelet counts. Comparisons of the two methods were made for samples assayed on the same day as well as one week later. The MAO activities resulting from both methods were significantly correlated and reproducible but the results of the platelet count method were, in most instances, slightly but significantly more reliable than the platelet protein method. The relevance of these results to the controversy concerning platelet MAO activity in schizophrenia is discussed.


A new enzymatic method for the determination of glycollate in urine and plasma.

An enzymatic assay for the measurement of glycollate in urine and plasma is described. Gycollic acid oxidase (glycollate:oxygen oxidoreductase, EC 1.1.3), extracted from spinach leaves, is used in the enzymatic oxidation of glycollate to produce glyoxylate and hydrogen peroxide. In the presence of peroxidase, the hydrogen peroxide oxidatively couples sulphonated 2,4-dichlorophenol and 4-aminophenazone to form a soluble purple quinoneimine dye. Interfering substances are removed from plasma by deproteinisation and from urine by adsorption onto activated charcoal before analysis. Glycollic acid oxidase also catalyses the oxidation of lactic acid which therefore has to be determined separately. The mean normal urinary glycollate for adults is 0.19 mmol/24 h with no difference between males and females. The mean normal plasma glycollate for adults is 0.17 mmol/l, but higher in males than in females.


The urinary excretions of iron and chromium by children with burns and scalds.

There is a wide variation in the urinary output of chromium by children who have been burned or scalded. It seems possible that when the excretion of chromium is increased this may be related to an increased plasma concentration of glucose which may follow injury, and when the excretion of chromium is reduced this may be related to a low body content of chromium. During the first seven days after injury there is a significant increase in the excretion of iron. Likewise the iron/chromium ratio is also raised over the first seven days. The overall loss of iron during the first twelve days or thereabouts is probably not sufficient to require nutritional supplements over this period.


Acquired pyruvate kinase deficiency. The effect of maleic acid upon human erythrocyte pyruvate kinase.

1. Maleic acid is shown to be able to bind the thiol compound 2-mercaptoethanol. This is fully consistent with the data of Morgan and Friedman (1938). 2. Human erythrocyte pyruvate kinase dissolved and quantitated in Tris-maleate shows a loss of positive homotropic interactions, as compared to the same preparation in Tris-HCl. Hill coefficients (n) of n = 1.0-1.2 and n = 1.6-1.8 are obtained in Tris-maleate and Tris-HCl respectively. Half saturation [S] 0.5 and Vmax remain unchanged. Pyruvate kinase in Tris-maleate is slightly more stable to heating at 60 degrees C than in Tris-HCl. Incubation of the enzyme in Tris-maleate for one h with high concentrations of dithiotreitol restores the positive homotropic interactions. 3. It is proposed, that the abnormalities of the pyruvate kinase of some patients with acquired pyruvate kinase deficiency, obtained from a study in Tris-maleate, may partly be induced by the buffer itself.


Fatty acid differences amongst leukemia cell types, with special reference to hairy cell leukemia: a preliminary report.

Fatty acid analyses of malignant cells from hairy cell leukemia patients revealed a profile which was rich in arachidonate and relatively low in linoleate and oleate. This profile was distinct from that of mononuclear cells derived from patients with other lymphoproliferative diseases or myeloproliferative disorders, and may reflect the unique membrane morphology of the hairy cell.


A sensitive fluorescent assay for N-acetyltransferase activity in human lymphocytes from newborns and adults.

We have developed a simplified assay for the enzyme N-acetyltransferase, based upon the loss of fluorescence after acetylation of the substrate p-aminobenzoic acid. This method is sufficiently sensitive to permit the quantitation of N-acetyltransferase activity in 10(5) human lymphocytes. Using this method, we have compared the level of N-acetyltransferase activity in lymphocytes from adult peripheral blood and from cord blood samples.


Aggregation-induced stability of dexamethasone receptors in rat kidney.

The stability of the dexamethasone-receptor complex of rat kidney cytosol was studied using homogenizing media of varying pH and composition. It was found that the complex is less stable with buffers usually employed for the study of steroid receptors such as Tris, EDTA etc., whereas the stability increases considerably with a solution composed of monothioglycerol 12 mmol/l and glycerol 5% in distilled water. Sepharose 4-B column chromatography revealed that the increased stability was associated with a larger form (probably an aggregate) of the receptor-steroid complex.


Pacemakers, cardiac mapping and EEG in the community hospital: a concise review of background and basic technical considerations.

The heart is a pump. The objective of cardiac pacing is to improve the cardiac output. The most popular method of cardiac pacing (RV) is not physiologic and results in reduced stroke output when compared to that of normal sinus or atrial paced rhythm. Recognition of pacemaker induced low output states and pacemaker aggravated neurological symptoms has quickened interest in physiologic atrial pacing. The increased complexity of atrial pacing has been discouraging in the past, but technical advances in electrode and pacemaker design are promising. Selection of the optimal pacing site appears critical and requires the determination of the focal electrical potential and stimulating threshold before electrodes are positioned. With minimal expense, the EEG can be adapted for this type of cardiac mapping in the community hospital.


Acute subdural hematoma and the periodic lateralized epileptiform discharges.

Focal motor seizures associated with periodic lateralized epileptiform discharges (PLEDs) were observed in two patients with acute subudral hematoma. Following the surgery, one patient continued to exhibit PLEDs but clinical seizures were absent PLEDs recurred in the second patient due to inadequate anticonvulsant medication. Autopsy study in one patient suggests that underlying cerebral contusion was not responsible for the occurrence of PLEDs.


Visual evoked potentials in Reye's syndrome.

Visual evoked potentials (VEPs) induced by flash stimulation in a child who was recovering from Reye's syndrome with complaints of poor left-eye vision were recorded. Ophthalmological examination disclosed intact visual fields and normal visual acuity. Analysis of VEP's, however, showed a grossly abnormal configuration of wave forms, marked prolongation in latency and reduction in amplitude when the left eye was stimulated. Follow-up observation 3 weeks later showed parallel improvements in VEPs and subjective complaints. Thus, from VEP analysis, it may be possible to detect subtle changes in the visual system that have diagnostic and prognostic value.


Non-protein-bound oestrogens in plasma and urinary excretion of unconjugated oestrogens in men.

Unconjugated oestrone and unconjugated oestradiol-17 beta were measured in 24 h urine specimens from twenty normal men. The concentrations of non-protein-bound oestrone and non-protein-bound oestradiol-17 beta, in samples of blood obtained from these subjects during the time in which the urine was collected, were measured by equilibrium dialysis (using a correction for plasma dilution). The mean excretion of unconjugated oestrone (0.44+/- 0.36 nmol/24 h, mean +/- SD) was significantly greater (Pless than 0.01) than that of unconjugated oestradiol-17 beta (0.20 +/- 0.13 nmol/24 h). The concentrations of non-protein-bound oestrone and non-protein-bound oestradiol-17 beta in plasma were 5.52 +/- 2.69pmol/1 and 2.42 +/- 0.73 pmol/1, respectively. There was no correlation between the quantity of unconjugated oestrone excreted and the concentration of non-protein-bound oestrone in plasma (r=0.27) nor between the quantity of unconjugated oestradiol-17 beta in the urine and the concentration of non-protein-bound oestradiol-17 beta in plasma (r=0.05). Therefore in normal men, estimation of unconjugated oestrone or oestradiol-17 beta in urine provides no guide to the concentration of the corresponding non-protein-bound oestrogen in plasma.


Amniotic fluid concentrations of 3,3',5'-tri-iodothyronine (reverse T3), 3,3'-di-iodothyronine, 3,5,3'-tri-iodothyronine (T3) and thyroxine (T4) in normal and complicated pregnancy.

Amniotic fluid concentrations of 3,3',5'-tri-iodothyronine (rT3), 3,3'-Di-iodothyronine (3,3'-T2), 3,5,3'-tri-iodothyronine (T3) and T4 were studied in 384 women during normal and complicated pregnancy. An inverse correlation was observed between decreasing rT3 and increasing 3,3'-T2 concentrations in amniotic fluid with gestational age. The mean rT3 level in normal pregnancy was 2.81 nmol/1 at 12-20 weeks and decreased significantly to 1.06 nmol/1 at 36-42 weeks of gestation. The mean 3,3'-T2 concentration was 49.1 pmol/1 at12-20 weeks increasing to 119 pmol/1 at 36-42 weeks. The mean T4 value of 3.83 nmol/1 at 12-20 weeks was about half that of later periods. The T3 concentration in a random sample of 45 amniotic fluids ranged from less than 28 to 370 pmol/1 (mean 102 pmol/1). The mean rT3, 3,3'-T2 and T4 values measured in patients with intra-uterine malnutrition, gestation diabetes, tocolysis, placental insufficiency and rhesus incompatibility at 31-40 weeks of gestation were not significantly different from those in uncomplicated pregnancy. Significantly decreased rT3 and T4 concentrations were found in toxaemia. From the results obtained in complicated pregnancy it may be concluded that measurements of iodothyronines, especially rT3, in amniotic fluid have insignificant diagnostic value in the recognition of intra-uterine lesions with the probable exception of fetal hypothyroidism. The analysis of the dependence of iodothyronine concentrations on the gestational age showed a maximum of rT3 and T4 levels between 20 and 30 weeks of pregnancy. This marked rise of iodothyronine concentrations in amniotic fluid at mid-gestation may be due to the onsetting maturation of the hypothalamic-pituitary-thyroid control system of the fetus.


Decreased blood pressure in response to an angiotensin II antagonist in Addison's disease.

The synthetic angiotensin II antagonist, 1-sarcosine, 8-isoleucine angiotensin II was infused in two patients with untreated Addison's disease. Blood pressure decreased following infusion of this angiotensin II antagonist. Re-infusion of the antagonist after cortisol replacement therapy for one month caused a slight increase in blood pressure. Addison's disease is one of the conditions in which the renin-angiotensin system is involved in the maintenance of blood pressure.


Prolactin concentrations in normal menstrual cycles and conception cycles.

Plasma prolactin concentrations were measured daily throughout twenty-three menstrual cycles from regularly ovulating women. In five of the cycles conception occurred spontaneously. The frequency distribution of prolactin concentrations was calculated and an appropriate linear transform obtained. Means and 95% confidence limits were determined by inspection of the logarithmic probability plots and these gave mean concentrations (and ranges) of 302 (106-871) mU/1 for the nonconception cycles and 178 (58-550) mU/1 for the conception cycles. Distribution of prolactin concentrations was log-normal in all cases, although the population containing the conception cycles was distinct from the population of non-conception cycles. It is suggested that the normal range found in ovulating women is 80-800 mU/1 but that for conception to occur levels should be slightly lower and approximate to the range 60-600 mU/1. Thus the lower limit of normal is about 60 mU/1 and the upper limit about 800 mU/1. Any concentration over 800 mU/1 should be designated as hyperprolactinaemia.


Effect of human growth hormone therapy on head circumference in children with hypopituitarism.

Head circumference was measured before and during hGH therapy in fourteen children with isolated growth hormone deficiency (IGHD) and in twenty-one children with multiple pituitary hormone deficiencies (MPHD). In both groups there was a retardation in growth of the neurocranium, more marked in the children with IGHD, which was less than the retardation in linear height. In the group with IGHD, initiation of hGH therapy before a chronological age of 5 and a bone age of 3 had been reached led to a rapid catch-up in cranial growth with normalization of the head size. In older children the induction of head growth was similar to that achieved by the long bones but without a true catch-up phenomenon. In three adults with hereditary IGHD without therapy or with irregular treatment during late puberty, the head circumference was below normal range. In view of the possible role played by development of brain tissue upon cranial size, the importance of early diagnosis and initiation of therapy in infants and young children with a deficiency of hGH is stressed.


Serum T3 and T4 levels in patients with anorexia nervosa showing transient hyperthyroidism during weight gain.

Serum thyroxine (T4) and triiodothyronine (T3) levels were measured in a group of thirty-three patients with anorexia nervosa (AN) and compared with twenty-five control women presenting with hirsutism and twenty-one patients with primary myxoedema. T3 levels in the AN patients were significantly lower than in the control subjects and in the patients with myxoedema while T4 levels were significantly higher than in the patients with hypothyroidism but significantly lower than in the control group. Seventeen anorexia patients had further T3 and T4 levels measured following an arbitrary 25% weight gain and both levels had increased significantly. For individual patients, the absolute rise in T3 levels was significantly correlated with the rate of weight gain. Thirteen patients had serial T3 and T4 levels measured during their periods of weight gain. Eight of these subjects showed a gradual rise in T3 levels from subnormal or low normal levels to values in the upper normal range. Four subjects showed a distinct and self limiting overshoot of T3 levels and, associated with this, the patients had clinical features of mild hyperthyroidism.


Disturbed mineral metabolism in hyperthyroidism: good correlation with tri-iodothyronine.

Mineral metabolism is frequently disturbed in hyperthyroidism. In a group of seventy-two patients with hyperthyroidism, we observed an increase in serum diffusible calcium in 50% of the cases, elevated inorganic phosphorus in 30% and elevated alkaline phosphatase in 44% of the cases. Correlations existed between the values of diffusible calcium, inorganic phosphorus, alkaline phosphatase and certain indices of thyroid function (T4, FT41, T3, FT3I), of which that with T3 was the best (P less than 0.001). Our results suggest that the magnitude of the disturbances of mineral metabolism depends on the severity of the hyperthyroidism and that it is the T3 level that constitutes the best index of that severity.


Plasma levels of somatostatin-like immunoreactivity-independence of kidney function.

Plasma levels of somatostatin-like immunoreactivity (SLI) and gastrin were determined by radioimmunoassay in patients with varying degrees of renal dysfunction. The plasma SLI was not increased in patients with end-stage renal failure as compared to healthy controls. In patients with varying degrees of renal insufficiency no significant correlation was found between the plasma SLI and kidney function as estimated by the 51Cr-EDTA-clearance rate. In uraemic patients a significant inverse correlation was found between plasma SLI and plasma gastrin.


Ectopic functioning adrenocortico-myelolipoma in longstanding Nelson's syndrome.

A 26-year-old man with the Nelson's syndrome is described, in whom an abdominal tumour developed, accompanied by recurrent Cushing's syndrome 11 years after total adrenalectomy. Upon removal the tumour was a myelolipoma containing islands of adrenocortical-like elements. On incubation of the tumour with radioactive precursors, its capacity to produce cortisol in vitro was shown. The name adrenocortico-myelolipoma is suggested for this tumour.


The effect of somatostatin on metabolic and hormonal changes during and after exercise.

The effect of intravenous somatostatin on blood levels of metabolites and hormones has been examined in normal subjects who performed a 30-minute period of bicycle exercises at 70% maximal exercise capacity. The results have been compared with control studies in the same subjects. Measurements were made of blood levels of lactate, glucose, free fatty acids, glycerol, acetoacetate, 3-hydroxybutyrate, insulin, glucagon, growth hormone (hGH) and prolactin. Growth hormone and glucagon release were suppressed during exercise with somatostatin and there was a subsequent elevation during recovery. There was slight post-exercise depression of insulin, but no alteration of plasma prolactin secretion. Blood glucose was reduced during exercise with somatostatin and increased during recovery. The elevation of ketone bodies after exercise was greater in the investigation with somatostatin, but there were no significant changes in other metabolites. Somatostatin, although causing inhibition of hGH release, appeared to have no significant effect upon fatty acid mobilization during exercise.


Mineralocorticoid replacement in Addison's disease.

Eight patients with treated Addison's disease were studied whilst receiving different doses of fludrocortisone together with a constant intake of glucocorticoid. Plasma renin activity (PRA), blood pressure, pulse rate and plasma potassium and urea concentrations were measured after 2-week periods on each dose. In two patients, PRA measurements indicated that mineralocorticoid replacement therapy had been inadequate. In four others, PRA remained normal throughout the study, even after fludrocortisone had been discontinued, suggesting that the drug was unnecessary for the maintenance of normal sodium balance in these patients. Asymptomatic fludrocortisone overdosage was indicated by a low plasma potassium concentration, but not by PRA measurements which failed in this study to distinguish between adequate and excessive mineralocorticoid replacement.


Propranolol, triiodothyronine, reverse triiodothyronine and thyroid disease.

Propranolol alone was given to sixteen hyperthyroid, and concomitantly with thyroxine therapy to ten hypothyroid patients. Following treatment of the hyperthyroid group for 1-2 weeks there was a significant decrease in serum triiodothyronine (T3) which correlated with the plasma propranolol steady state concentration. The serum reverse T3 (rT3) rose significantly. Weight loss ceased in this group while weight gain occurred in patients who had a marked fall in serum T3. One patient with T3 toxicosis went into remission. The reduction in serum T3 was maintained in six patients receiving propranolol for more than 1 month. In the hypothyroid group the mean serum T3 level achieved with 0.15 mg thyroxine per day was significantly lower than in a control group who did not receive propranolol. In five patients following propranolol withdrawal there was a significant rise in T3, a fall in rT3 and TSH, and weight loss. Propranol may therefore have a clinically significant and direct action on the peripheral conversion of thyroxine to T3 and rT3.


Screening for neonatal hypothyroidism by thyroxine and thyrotrophin radioimmunoassays using dried blood samples on filter paper.

A routine and automatized methodology for thyroxine (T4) and thyrotrophin (TSH) radioimmunoassay (RIA) using dried blood samples on filter paper is described. T4-RIA was performed on one single dot (5 mm diameter equivalent to 4 microliters of serum) while two dots were necessary for TSH-RIA. Reference filter papers were introduced in each assay for quality control. In a preliminary study on 4,155 neonates, samples generally obtained between the 5th--7th day gave a mean 'dot-T4' of 97.95 +/- 36.04 nmol/l and a mean 'dot-TSH' of 10.19 mU/l +/- 8.25, corresponding to 2.47 mU/l of serum. Within an 18-month period (November 1976-April 1978), a total of 16.522 neonates have been screened allowing detection of three cases of congenital hypothyroidism (incidence 1 : 5507), two cases of congenitally low TBG and thirty-three cases of transient hypothyroidism.


Effect of somatostatin on abnormal growth hormone and prolactin secretion in patients with the carcinoid syndrome.

Growth hormone (GH) secretion has been studied in two patients with the carcinoid syndrome during glucose loading and growth hormone-release inhibiting hormone (GHRIH, somatostatin) infusion. Both patients had elevated fasting GH levels which were not suppressed by glucose; GH levels fell rapidly during GHRIH infusion. One patient also had hyperprolactinaemia with galactorrhoea and the prolactin (PRL) levels were unaltered by GHRIH. The association between carcinoid tumours and abnormalities of GH and PRL secretion is discussed.