The postantennal organ: a specialized unicellular sensory input to the protocerebrum in apterygotan insects (Collembola). A postantennal organ occurs in three different families in Collembola. In spite of considerable diversity in their outer structures, the organs manifest many similarities in their internal structure (pore system with pores of about 50-100 A diameter, a single sensory cell with two branching ciliary outer segments, reduction of the outer receptor lymph cavity and occurrence of an electron dense secretion around the dendrites). A tendency towards an enlargement of the outer surface of the organ and towards an incorporation of the sensory cell into the protocerebrum appears to be correlated with adaptations to a strict euedaphic life. An appropriate stimulus for the organ cannot be derived from the structure unequivocally, however, it could be hygro-, chemo- or thermosensitive. Perforated surface and number of pores in the postantennal organ correspond to the values found in insect sensory hairs.


The effect of dibromodulcitol on the template activity of DNA chromatin and nuclei from Yoshida sarcoma cells. It has been shown earlier that after in vivo administration, dibromodulcitol (DBD) reacts with DNA and to a greater extent with chromosomal proteins of Yoshida sarcoma cells. The present experiments were designed to show if the binding of DBD to the chromatin elements of Yoshida sarcoma cells causes any changes in RNA synthesis using either DNA or chromatin as template in bacterial RNA polymerase system. During 4 to 24 h following in vivo administration, DBD reduces the template activity of dna without detectable single-strand breaks in the template DNA in alkaline sucrose gradients. Using chromatin as template the same dose of DBD produces no or very slight inhibition of RNA synthesis. Measuring the DNA-dependent RNA synthesis in nuclei isolated from Yoshida cells of treated rats, the dose of DBD which markedly inhibited the template activity of DNA, resulted in a significant stimulation of the nuclear RNA synthesis. The increased RNA synthesis was not due to an inhibition of ribonuclease activity. The observed alterations of the transcriptive properties of chromatin and nuclei produced by DBD are interpreted as being due to a modification of the whole nucleoprotein structure caused by the interaction of DBD with both DNA and chromosomal proteins.


Kinetic studies of benzpyrene and hydroxybenzpyrene metabolism. The kinetics of benzypyrene (BP) metabolism were examined in liver microsomes, and in accordance with the results of Hansen and Fouts [9] exhibited curcilinear Lineweaver-Burk plots. The problem was exacerbated in microsomes of 3-methylcholanthrene (MC-ms) treated rats. The Km for BP, measuring hydroxybenzypyrene (OHBP) appearance was about 0.3 muM in MC-treated adult rats and about 1.0 muM in untreated rats. These values were obtained using a substrate range of 0.2-2.0 muM benzpyrene, 20 mug of microsomal protein/ml and a 3 min assay time. With longer assay times and with higher microsomal protein concentrations curvilinear reciprocal plots were obtained. This was found to be due to a combination of three factors, namely non-specific binding of BP to the microsomes, rapid depletion of substrate, and further metabolism of hydroxy products. At 100 mug microsomal protein/ml about 50% of added BP was non-specifically bound to the microsomes in the range of 0.2-2.0 muM BP. Addition of albumin to the medium (1 mg/ml) greatly enhanced the BP hydroxylase activity but only slightly increased the amount of BP remaining in the medium after sedimentation of the microsomes by centrifugation. 3-OHBP, one of the phenolic products of BP metabolism was found to be metabolized to a non-fluorescent products(s); the Km for this compound was similar to that for BP. Differences were seen in the Vmax rates of BP disappearance and OHBP appearance. Disappearance of BP is several fold faster than OHBP appearance and has a larger Km. The latter may be due to the need to use higher amounts of protein and to allow depletion of enough substrate to make measurements significantly reproducible or the higher Km may reflect a composite value for different routes of BP metabolism.


Hepato- and pneumotoxicity of pyrrolizidine alkaloids and derivatives in relation to molecular structure. 62 pyrrolizidine alkaloids and derivatives have been screened for acute and chronic hepato- and pneumotoxicity by the single dose method previously described. This procedure is satisfactory for the compounds of medium to high hepatotoxicity but failed to detect toxicity in certain other compounds of known, low hepatotoxicity. New findings significant in relation to hepatotoxicity are as follows: (i) On a molar basis, diesters of heliotridine and retronecine are about 4 times as toxic as the respective mono-esters and heliotridine esters are 2-4 times as toxic as retronecine esters. (ii) Crotanecine esters are less toxic than retronecine esters, and the 6,9-diester madurensine, 2-4 times less toxic than the 7,9-diester anacrotine (the difference being ascribed to there being only one reactive alkylating centre in the toxic metabolite from madurensine). (iii) Hepatotoxicity was confirmed for 7-angelylheliotridine but not observed for 9-angelyheliotridine and 7- and 9-angelylretronecine. (iv) Other significant compounds failing to induce hepatotoxicity were 9-pivalyl- and 7,9-dipivalyheliotridine, the alpha- and beta-epoxides of monocrotaline, 7-angelyl-1-methylenepyrrolizidine and the methiodides of monocrotaline and senecionine. The following compounds are readily converted by rat liver microsomes in vitro into dehydroheliotridine (or dehydroretronecine): 7- and 9-angelyheliotridine, 7- and 9-angelylretronecine, 7,9-dipivalylheliotridine and otosenine. 7,9-Divalerylheliotridine, the alpha- and beta-epoxides of monocrotaline, and retusamine yield pyrrolic metabolites more slowly. The preparation and characterisation of several alkaloid derivatives are described. Chronic lung lesions were produced by most compounds which gave chronic liver lesions, although a higher dose was required in some instances. This requirement may sometimes mean that chronic lung lesions cannot be induced because of the intervention of acute or peracute deaths. Apart from this factor, structure activity requirements for pneumotoxicity are the same as for hepatotoxicity, consistent with their being both caused by the same toxic metabolites.


A study of the change in DNA synthesis of S phase cells treated with N-methyl-N-nitrosourethane: a study using Amoeba proteus as a single cell model. The present experiments using Amoeba proteus as a single cell model show that DNA synthesis continues during and after exposure of S phase cell to N-methyl-N'-nitrosourethane (MNU). At sublethal dose levels which caused long division delays, division and growth abnormalities and mutations, the amount of [3h] thymidine ([3h]Tdr) incorporated was decreased by 20-30%; at dose levels which killed all S phase cells it was inhibited by up to 90%. There was a direct correlation between the dose of MNU used and the degree of inhibition of [3H]Tdr incorporated. The effect was rapid, mainly taking place within 20 min of treatment. Amoeba heterokaryons (HKs) were used to examine the rate of DNA synthesis of treated and untreated nuclei in the same cytoplasm, i.e. where the nuclei would have the same [h]tdr intake, the same thymidine kinase (TK) activity and the same endogenous precursor pools. Direct comparison of the nuclear DNA synthetic activity in this way revealed less difference between treated and untreated nuclei than comparisons made using the nuclear grain counts from treated and untreated amoebae. This suggested that much of the decrease in [3H]Tdr incorporation by MNU-treated S phase cells was due to a change in the cytoplasm and/or the cell membrane, rather than to nuclear damage. Thus MNU-treated nuclei were able to synthesize DNA at a near normal rate when they could draw on the resources of untreated cytoplasm, while the rate of DNA synthesis of control nuclei decreased when they occupied cytoplasm which had been exposed to high doses of MNU. These studies suggest that nuclear sites of damage were only involved when lethal doses of MNU had been used.


The formation of poly A-containing RNA in rat liver after administration of 3-methylcholanthrene. The incorporation of [32P] orthophosphate into liver poly A-rich cytoplasmic RNA was investigated in rats which were pretreated with 3-methyl cholanthrene (3MC) or with the vehicle, corn oil. This incorporation was markedly increased within 6 h after administration of the polycyclic hydrocarbon and reached a maximum by 24 h, i.e., 33-fold increase. The poly A-rich RNA fraction was examined by gel electrophoresis and the specific activity of individual species was shown to be elevated.


A study of the growth inhibition of Lactobacillus casei by N-alkyl homologs of N-nitro-N-nitrosoguanidine. N-methyl-(MNNG), N-ethyl-(ENNG), and N-propyl-(PNNG) derivatives of N'-nitro-N-nitrosoguanidine inhibited the growth of Lactobacillus casei in the order of potency, MNNG greater than ENNG greater than PNNG. L-Cysteine, gluathione, and dithioerythritol reversed the inhibition on a molar basis. The -SH compounds accelerated the loss of the N-nitroso group in vitro, yielding non-inhibitory N-alkyl nitroguanidines. The significance of the loss of the nitroso group and the size of the N-alkyl group is discussed.


A survey of the response of different strains of mice to substances metabolised by microsomal oxidation; hexobarbitone, zoxazolamine and warfarin. Sixteen strains of mice were compared with respect to their hexobarbitone sleeping time and their zoxazolamine paralysis time. The strains were A2G, CBA, CE, C3H, C57BL, C57L, DBA, F/st, ICFW, NMRI, NZB, Schneider, Simpson, SM, TO and 129/rr. All the strains except 129 Rr were also tested for survival on a diet containing 0.05% racemic Warfarin. There was highly significant interstrain correlation between hexobarbitone sleeping time and zoxazolamine paralysis time (r = 0.72) and between hexobarbitone sleeping time Warfarin survival (r = 0.68). There was a significant correlation between zoxazolamine paralysis time and Warfarin survival (r = 0.56). The correlations can be explained if: (1) there is a genetically determined interstrain variable which is some common component of the microsomal mixed-function oxidase systems involved in the hydroxylation of the three substances; (2) the anticoagulant action of Warfarin is caused more by a hydroxylated metabolite of Warfarin than by Warfarin itself. Phenobarbitone pretreatment shortened hexobarbitone sleeping times and zoxazolamine paralysis times, but its effect was greater in those strains with longer initial hexobarbitone sleeping times and zoxazolamine paralysis times. Piperonyl butoxide pretreatment lengthened hexobarbitone sleeping times, but had no effect on zoxazolamine paralysis times. Warfarin survival was unaltered by pretreatment with either phenobarbital or piperonyl butoxide.


Inhibition by caffeine of post-replication repair in Chinese hamster cells treated with cis platinum (II) Diamminedichloride: the extent of platinum binding to template DNA in relation to the size of low molecular weight nascent DNA. Treatment of Chinese hamster lung V79-379A cells with the anti-tumour agent cis platinum (II) diamminedichloride, (cis Pt(II)), resulted in an immediate recuction in the rate of DNA synthesis. Sedimentation of newly synthesised DNA through alkaline sucrose gradients revealed it to be approximately the same size as that obtained from untreated cells. In contrast, in the presence of 0.75 mM caffeine, the rate of DNA synthesis rapidly returned to control levels, although sedimentation analysis showed the DNA synthesised in cis Pt(II)-treated cells to be of lower molecular weight than in untreated cells. The reduction in molecular weight was directly proportional to the initial dose of the platinum compound. Furthermore, the results of separate binding studies suggested that at several levels of reaction the new DNA was synthesised up to a size approximately equal to the interplatinum distance in the template strand. This has been interpreted as being the result of the formation of a gap in the daughter DNA strand opposite every DNA-platinum product in the template strand. If caffeine was removed from the culture medium, there was a rapid increase in the molecular weight of the nascent DNA strands. However, if caffeine remained in the medium, the DNA remained of lower molecular weight than in untreated cells. It is proposed that this effect of caffeine is the result of the inhibition of a post-replicative DNA repair process which allows the eventual synthesis of a continuous DNA strand on a template containing unexcised lesions. It is further proposed that inhibition of this post-replicative DNA repair process provides a molecular basis for the previously observed potentiation by caffeine of cis Pt(II)-induced chromosomal aberrations and lethality.


The relationship of myocardial infarct size and prognosis. Patients with cardiogenic shock were arbitrarily divided into groups, those in whom shock appeared within 6 hours after the onset of acute infarction and those in whom the symptoms appeared more than 6 hours after the onset of acute infarction. The patients with more rapidly developing shock had larger areas of necrosis (average 48%) than the slow-onset group (average 28%). The former group had more sites of total occlusion of the epicardial arteries (3.5 vs 1.6) than the slow-onset group. By postmortem X-ray examination less collateral flow was visible in the rapid-onset shock patients than the slow-onset ones. These observations suggest that slow-onset shock is more likely to respond to presently available therapeutic interventions than the cases with rapid-onset shock.


Relations between enzymatically estimated myocardial infarct size and early ventricular dysrhythmia. The relation between ventricular dysrhythmia during the first 20 hours after hospitalization for acute myocardial infarction and enzymatically estimated infarct size was studied prospectively. The duration of dyshythmia was measured by the total time during which the average premature ventricular depolarization (PVC) rate was above 25 PVCs/hr. This index was significantly correlated with infarct size index (ISI) and the strength and significance of the correlation increased when patients with previous, remote myocardial infarction were excluded. An initially expected correlation between PVC frequency and average heart rate was not observed. In a subset of patients selected to include the entire range of ISI, median PVC coupling interval did not correlate with ISI or heart rate. Our results confirm the general reduction of PVC frequency with time after hospital admission. An analysis of total lidocaine administered for therapeutc purposes and dysrhythmia evaluated by retrospective computer analysis disclosed a moderately strong and significant correlation. These findings indicate that persistence and severity of ventricular dysrhythmia in the first 20 hours after hospitalization for myocardial infarction are related to enzymatically estimated infarct size.


Significance of ventricular arrhythmias during myocardial ischemia. The scoring of premature ventricular contractions (PVC) after myocardial infarction provides little accurate information about infarct size or about other ventricular arrhythmias, although it might be of some immediate prognostic value. Continuous recording with computer analysis and differentiation of serious ventricular arrhythmias has shown in untreated patients that the incidence of PVC does not reflect that of other ventricular arrhythmias. Ventricular tachycardia and R-on-apex T PVC continue without significant decrease in incidence up to 12 hours after the onset of symptoms. Primary ventricular fibrillation is rare after about 5 hours and stands on its own in significance.


Studies on the subcellular pathophysiology of ischemia. The loss of ability to synthesize adenosine triphosphate (ATP) by mitochondria in ischemic cells even in a favorable medium correlates with the loss of cell viability. The early lesion at the molecular level needs further investigation but appears to involve an increased permeability of the mitochondrial membrane possibly promoting proton leak and obviating oxidative phosphorylation. The nature of this leak could involve changes in phospholipid-protein interactions, especially since the early release of free fatty acids and changes in phospholipid composition occur.


Glucose-insulin-potassium, free fatty acids and acute myocardial infarction in man. In patients with stable coronary artery disease, a GIK solution may increase arterial glucose and the arterial-coronary sinus difference of glucose across the myocardium. In the same patients, GIK may reduce arterial free fatty acids as well as the arterial-coronary sinus difference of free fatty acids across the myocardium. As the arterial values of free fatty acids fall, so does the arterial-coronary sinus difference of free fatty acid across the myocardium, defining a myocardial threshold for free fatty acids of approximately 200 mEq/L. Glucose-insulin-potassium may lower free fatty acid values to near or below myocardial threshold in patients with acute myocardial infarction despite recurrent pain. Preliminary data suggest that GIK in the concentration and infusion rates used in this study may favorably influence survival in patients with acute myocardial infarction.


Lysosomes in myocardial infarction: studies by means of cytochemistry and subcellular fractionation, with observations on the effects of methylprednisolone. To evaluate lysosomal involvement in myocardial infarction, coronary artery thrombosis was induced by ligation in 16 dogs. Biopsies of infarcted and normal left ventricles were studied by ultrastructural cytochemistry and subcellular fractionation (0.25 M sucrose) from 30 min to 96 hrs post injury. Normal myocardium contained few "classical" (residual body) lysosomes: instead, acid phosphatase and aryl sulfatase were localized to longitudinal and to lateral sac elements of the sarcoplasmic reticulum. In postnuclear (450 X gm, 10 min) supernates, lysosomal acid phosphatase and beta-glucuronidase were divided 60:40 between sedimentable (98,000 X gm, 15 min) and non-sedimentable fractions of normal endocardium and epicardium (studied separately). At 2 hrs post infarction, ischemic muscle showed: 1) loss of membrane-bound acid  phosphatase and aryl sulfatase; 2) mitochondrial damage; 3) loss of glycogen and disappearance of I but not A bands; and 4) entry into cells of colloidal lanthanum (= loss of plasma membrane integrity. Total lysosomal hydrolase did not increase until 6-5 hrs post infarct. At 2 hrs, significant increments (32 +/- 7%) were found in nonsedimentable acid phosphatase and beta-glucuronidase of endocardium (P less than 0.005 vs. normal) but the epicardium. In dogs given methylprednisolone (50 mg/k) 30 min post infarct, ultrastructural cytochemistry showed retention of lysosomal enzymes within endocardial sarcoplasmic reticulum and no significant redistribution of enzymes into non-sedimentable fractions (vs. eight paired, infarcted, untreated controls). Data show early disruption of lysosomes in myocardial infarction and their protection by steroid given after the acute insult.


Effects of hyperosmotic mannitol in reducing ischemic cell swelling and minimizing myocardial necrosis. Impaired metabolism interferes with the active extrusion of intracellular sodium and results in intracellular edema. In the brain and regionally in the kidney, elevation of extracellular osmolality is accompanied by a reduction of ischemic cell swelling and improvement of reflow of blood after arterial occlusion. Studies were therefore performed to examine the effect of elevation of extracellular osmolality on ischemic myocardial physiology and by morphologic examination on the extent of acute injury and subsequent necrosis. Under conditions of controlled hemodynamics, administration of hyperosmotic mannitol resulted in improvement of function of the canine heart with regional ischemia, a lessening of the extent of ischemic injury assessed by electrocardiographic ST segment mapping, and improved total and collateral blood flow. Metabolic studies under conditions of controlled hemodynamics revealed that hyperosmotic mannitol reduced the myocardial oxygen requirement of the ischemic heart. Mannitol dilated large collateral conductance vessels in addition to improving blood flow through the region of myocardial ischemia. Under conditions of ischemia induced by a prolonged reduction in coronary perfusion, hyperosmotic mannitol attenuated the progressive rise in vascular resistance. Direct morphologic examination of areas of myocardium subjected to total interruption of blood flow followed by reflow of blood revealed swelling of both myocardial and capillary endothelial cells early during the reflow period. The extent of swelling was substantially reduced with elevation of the extracellular osmolality with mannitol. Simarilty, osmolality elevation strikingly reduced the extent of eventual myocardial necrosis following prolonged periods of reflow of blood.


Influence of collateral flow on the ischemic tolerance of the heart following acute and subacute coronary occlusion. Acute occlusion of the circumflex branch of the left coronary artery was produced in chronically instrumented conscious dogs. Tracer microspheres were used to measure during an established time period, the distribution of collateral flow within the infarcting myocardium. For up to 2 hours after coronary occlusion the amount and distribution of the collateral flow remained unchanged. Two to 4 hours after coronary occlusion the subendocardial flow fell to almost zero and the subepicardial flow rose. Between 6 and 48 hours subepicardial and total collateral flow rose markedly. A no-reflow phenomenon is responsible for the decline of collateral flow in the subendocardium. Evidence for this hypothesis was provided by releasing the artery 1,2, 4 and 6 hours after occlusion. The amount of subendocardium that could not be reperfused was small after 1 hour and large after 6 hours of occlusion. When the total collateral flow was very low, the subepicardium was not able to be reperfused and a transmural myocardial infarction developed. We conclude that the time delay between onset of ischemia and the appearance of a no-reflow phenomenon depends upon the amount of collateral flow. The occurrence of a no-reflow phenomenon in the subendocardium increases the amount of flow to the subepicardium which increases its chances of survival. Beyond the sixth hour after occlusion the total amount of collateral flow increases which is interpreted as a reduction of collateral resistance by passive caliber changes of the collateral vessels. DNA-synthesis that signal active caliber changes through cellular proliferation were always detected 24 hours after complete occlusion of a coronary artery regardless whether the time between onset of stenosis until complete occlusion was varied between 36 hours and 5 days. When the time to complete occlusion was 4 days, myocardial infarction was prevented due to growth-transformation of pre-existing collaterals. Four phases of collateral reactions in acute coronary occlusion were observed: redistribution of available collateral flow in favor of the subepicardium (t = 1 to 4 hours after occlusion), 2) increase of total collateral flow due to passive "stretch" of collateral vessels (t = 4 to 24 hours after occlusion), 3) radial growth of collateral vessels due to active cellular proliferation, (t = 24 hours to 5 days) 4) cellular proliferation to ensure a normal wall thickness in growth'transformed collaterals (t = 5 days to 20 days after coronary occlusion). In subacute coronary occlusion the first phase does, of course, not apply.


Analysis of regional myocardial function, dimensions, and wall thickness in the characterization of myocardial ischemia and infarction. Disorders of contractile function constitute one of the earliest events to follow acute coronary occlusion, and occur in the central ischemic region within approximately 10 seconds. Measurements of regional myocardial dimensions and function in the central, border, and normal zones around an area of ischemia allow assessment of such changes, and when these disorders are persistent after permanent coronary occlusion they may offer an indirect means for assessing the severity and extent of myocardial ischemia and infarction. Recent experimental studies that used pairs of miniature ultrasonic crystals implanted within the subendocardium of open-chested and chronically instrumented, unanesthetized dogs indicate that functional function may be studied simultaneously in these regions; a holosystolic bulge rapidly develops in the central zone of ischemia while hypokinesis  is apparent in marginal zones bordering the central region. It has been shown that function in the marginal zone reflects the balance between oxygen supply and demand and may be favorably or unfavorably influenced by acute therapeutic interventions. This finding suggests that the extent of an ischemic region may be altered by such therapy.Our studies further indicate that regional changes in dynamic wall thickness closely parallel the characteristics of shortening of nearby subendocardial segments, indicating that measurement of wall thickness alone may be useful for characterizing regional function. Studies in chronically instrumented animals also have established that the miniature crystals are useful for measuring regional dimensions and function over prolonged periods of time; for example, reduction in end-diastolic dimensions that reflex tissue loss over a 3- to 4-week period after coronary occlusion is substantially greater in the central ischemic regions than in the marginal zones. It is proposed that persistent changes in myocardial function and progressive alterations changes in dimensions over time offer indirect measures of the extent and severity of ischemic damage and infarction. With the development of improved echocardiographic or other clinically applible methods, such measurements may be a useful tool for assessing the effects of therapy on myocardial infarct size.


Biochemical markers of ischemic injury.

Biochemical markers of ischemic injury. Enzymatic estimation of infarct size entails assumptions requiring analysis and modification during evolution of the method. Myocardial creatine phosphokinase (CPK) depletion reflects infarct size under many experimental conditions, and release of CPK into the circulation has been correlated with histologically demonstrable necrosis. Use of a one compartment model relating serum CPK changes to myocardial CPK depletion is a simplification requiring estimates of CPK disappearance rate (kd) as one parameter. Conformity of kd to first order kinetics and its lack of variance despite marked hemodynamic perturbations, myocardial infarction per se, or successive daily determinations have been documented. In patients with myocardial infarction, enzymatic estimates of infarct size correlate with morbidity and mortality, functional cardiac impairment, severity of clinical manifestations, frequency of ventricular dysrhythmia, and morphologically estimated infarct size. Improved estimates result from MB CPK analyses which avoid difficulties due to noncardiac CPK in the circulation and from individualization of values of kd. The discriminant power of enzymatic estimates as an index of prognosis has been demonstrated by discriminant function analysis. Prediction of infarct size enzymatically requires additional assumptions and conventionally employs an empirical algorithm rather than a physiological, deterministic model. Although predictions can probably be improved by incorporation of parameters reflecting processes governing CPK release and disappearance, the need for a prolonged interval during which observations are required prior to prediction limit the applicability of this approach to evaluation of therapeutic interventions whose effects are likely to be maximum when early implementation is possible.


Second degree block during reciprocal atrioventricular nodal tachycardia. Of 67 patients with reciprocal atrioventricular (A-V) nodal tachycardia consecutively studied by programmed electrical stimulation of the heart, nine patients showed second degree block toward the ventricle and one patient toward the atrium during tachycardia. In four patients the occurrence of block was critically related to the prematurity of the test stimulus initiating the tachycardia. In three patients block developed following increase in rate of tachycardia. In two patients block could be elicited by introducing premature ventricular stimuli during tachycardia. Our observations indicate that different mechanisms may be responsible for second degree block during reciprocal supraventricular tachycardia. The finding of second degree block during reciprocal supraventricular tachycardia excludes a tachycardia with A-V conduction over the A-V node - His pathway and V-A conduction over an accessory A-V pathway.


Prospective observations in patients with chronic bundle branch block and marked H-V prolongation. Eighteen of 388 patients with chronic bundle branch block, studied electrophysiologically and followed prospectively, had H-V intervals of 80 msec or greater. Five patients were functional class I, five class II, seven class III, and one class IV. Follow-up ranged from 103 to 1919 days (mean 711 +/- 118). Three patients needed permanent pacing for the following indications: sino-atrial block, sinus bradycardia post-cardiac surgery, and 2 degrees block distal to the His bundle. Six patients died, three suddenly, and three nonsudden. The five initially asymptomatic patients are alive and without pacemakers (mean follow-up 732 +/- 139 days). Although marked H-V prolongation was associated with high morbidity and mortality in this small series, this was only in patients with symptomatic heart disease. Asymptomatic patients (five patients) had a benign clinical course. Prophylactic pacing would probably not modify clinical course in the former group, and is probably not indicated in the latter group. Longer follow-up will be needed for definitive prognostication.


Dissociation and delayed conduction in the canine right bundle branch. Bipolar electrograms were recorded from five to six sites on the septal right bundle branch (RBB) using a multielectrode patch in ten mongrel dogs. Antegrade activation was recorded along the right bundle during sinus rhythm and during right atrial pacing at varying heart rates. Retrograde activation was produced by pacing from right ventricular epicardium. Right bundle conduction velocities varied from 1.4 to 3.3 m/sec (mean 2.0 m/sec) for antegrade conduction and from 1.8 to 2.7 m/sec (mean 2.1 m/sec) for retrograde activation. Varying degrees of conduction delay were elicited by premature stimulation and mechanical pressure to the right bundle. The conduction delays occurred over small segments of the right bundle branch with regeneration of normal propagation velocity distal to the region of block. The conduction delays were associated with fragmentation of the RBB electograms with initiation of delayed activation waves which traveled variable distances antegrade and retrograde. These data show that stress-induced longitudinal dissociation may produce delayed activation waves within the right bundle branch which may simulate re-entrant arrhythmias.


Anatomical configuration of the His bundle and bundle branches in the human heart. The relationships among the His bundle, the origin of both bundle branches, and the interventricular (IV) septum were examined histologically in 32 human hearts, and the entire bundle branch systems were delineated in 13 of these. The His bundle in five hearts traversed the right IV septal crest, and the LBB origin was a very narrow stem (maximum 1.5 mm in cross-section) crossing from right to left through the inferior margin of the membranous septum. Proximal LBB anatomy was extremely variable, demonstrating multiple fiber groups which fanned out over the entire left septal surface. The LBB did not divide into two discrete divisions without multiple interconnections. The RBB formed an obtuse angle with the His bundle in 27 of 32 hearts. In those five hearts with "right-sided His bundles," the right bundle branch was a direct continuation. The clinical, electrophysiologic, and electrocardiographic implications of these anatomical observations are discussed.


Interobserver variability in coronary angiography. Four experienced coronary angiographers (two radiologists and two cardiologists) independently assessed the location and degree of coronary artery stenosis, and the location and degree of left ventricular wall motion abnormalities in 20 coronary angiograms. Marked interobserver variability was noted in quantifying percent coronary artery stenosis and degree of left ventricular wall motion abnormalities. For example, in only 13/20 (65%) of the coronary angiograms did all observers agree about the significance of a stenosis (defined as greater than 50% in diameter luminal narrowing) in the proximal or mid left anterior descending coronary artery. In 3/20 (15%) angiograms there was disagreement by at least one observer about the significance of lesions noted in the main left coronary artery. The ventricle was divided into five segments and the degree of wall motion abnormality graded into six categories of increasing severity from normal to dyskinesis. There was a 42% mean disagreement among all four observers where a disagreement between observers was defined as any difference in grading wall motion abnormalities. Interobserver variability reveals a significant limitation of coronary angiography.


Intervention ventriculography. Comparative value of nitroglycerin, post-extrasystolic potentiation and nitroglycerin plus post-extrasystolic potentiation. The comparative value of nitroglycerin (TNG), post-extrasystolic potentiation (PESP) and their combination (TNG + PESP) to unmask asynergic residual contraction was examined, each patient serving as his own control. Twelve of 13 hypokinetic zones improved both with TNG and PESP. One remained unchanged with either. Of 15 akinetic zones, four improved with both TNG and PESP, while ten remained unchanged. One akinetic zone, although improved with TNG, remained unchanged with PESP. Four dyskinetic zones did not change with either. Six asynergic zones responding to TNG alone demonstrated further augmentation with TNG + PESP. However, none of 13 TNG unresponsive zones improved with TNG + PESP. Thus, TNG, PESP, and TNG + PESP are each equally capable of unmasking asynergic residual contractile ability.


Myocardial LDH isozyme distribution in the ischemic and hypoxic heart. Small myocardial specimens were obtained from 12 patients undergoing coronary reconstructive surgery and from 12 patients undergoing surgical correction for cyanotic congenital heart defects. The specimens were analyzed for LDH isozyme distribution. A control analysis was performed on myocardial specimens obtained at the time of surgical correction for acyanotic congenital heart defects in seven patients with normal coronary arteries. There was a 42% increase in the proportion of A subunits in the hearts of coronary patients as compared to controls. This represented a shift toward an anaerobic isozyme distribution. There was no change in the percentage of A units from the hearts of cyanotic patients as compared to acyanotic hearts of the same age. Cardiac muscle from patients with coronary vascular disease had an altered LDH subunit composition. Such an alteration was not present with chronic systemic hypoxia. These deficiencies may or may not be related to differing local metabolic responses to the two conditions. However, in the clinical situations, ischemic heart muscle may be oxygen deprived to the point of lactic acid production while hypoxic heart muscle usually is not. Consequently, these findings may represent a compensatory cellular mechanism which provides for continued energy production during chronic ischemia by enhancing glycolysis.


M-mode echocardiography in the evaluation of patients for aneurysmectomy. In order to determine whether echocardiography could be useful in predicting surgical mortality of aneurysmectomy, preoperative condensed M-mode echocardiographic scans were taken from both mid (standard position) and low (nearer apex) intercostal spaces and/or from the subxiphoid area in eighteen patients who were sent to surgery for aneurysmectomy. Eleven of the eighteen patients survived aneurysmectomy. All eleven had mid left ventricular dimensions less than 3.3 cm/m2 and low dimensions of 3.8 cm/m2 or less. Of the seven patients who died, the mid and low left ventricular dimensions exceeded 3.3 cm/m2 and 3.8 cm/m2, respectively, with one exception. The combination of abnormal mitral valve closure, a dilated mid dimension and lack of normal motion in opposing wall segments was only seen in six nonsurvivors. Echocardiography can provide information concerning the state of the left ventricle in patients with ventricular aneurysms and these findings may be helpful in predicting surgical mortality for aneurysmectomy.


Echo-phonocardiographic and contrast studies in conditions associated with systemic arterial trunk overriding the ventricular septum: truncus arteriosus, tetralogy of Fallot, and pulmonary atresia with ventricular septal defect. Echocardiography was performed in 62 patients--20 with truncus arteriosus, 14 with tetralogy of Fallot, and 28 with pulmonary atresia with ventricular septal defect. Features common in all three groups were: large single systemic arterial trunk overriding the ventricular septum, mitral-semilunar continuity, large right ventricular dimension, and normal septal motion. Indocyanine green contrast flow patterns were similar and were most helpful in detecting an associated atrial septal defect and in studying the timing and pattern of right-to-left shunting. Echo-phonocardiographic studies revealed that the ejection click coincided with the point of maximal opening of aortic or truncal valve. Left atrial dimension was measured in 55 patients and was normal or small in 41 patients and large in 14 patients. Our observations indicate that left atrial dimension provides a good index of pulmonary flow and can help differentiate those patients with increased pulmonary flow and can help differentiate those patients with increased pulmonary flow (truncus arteriosus) from those patients with reduced pulmonary flow (tetralogy of Fallot and pulmonary atresia with associated ventricular septal defect).


Noninvasive stress testing. Methodology for elimination of the phonocardiogram. Measurement by systolic time intervals (STI) of cardiac responses requires extremely careful recording during actual stress test performance. Previous work indicated no significant changes in the pulse transmission time (PTT) during exercise and other challenges. Since external STI depend on the carotid pulse offset by the PTT as an aortic curve equivalent, stable PTT implies that timing of the carotid upstroke (CARu) and the carotid incisura (CARIN) would respectively track the pre-ejection period (i.e., the externally calculated onset of the aortic upstroke) and the aortic incisura which is externally timed by the aortic component of SII (IIA). In ten subjects, STIs were recorded at supine rest, sitting, standing, during prompt and sustained squatting and during isometric and dynamic exercise. The results demonstrated the tracking of both points: regression slopes and correlation coefficients were close to 1.00 for each series and for each subset. Coefficients of correlation (r) and of determination (r2) were uniformly high for all challenges except isometric handgrip (IHG). Since left ventricular ejection time is obtained directly from the pulse curve, with the exception of IHG, STI responses during stress testing can be measured without a phonocardiogram.


The incidence of bacteremia in pediatric patients following tooth extraction. Procedures which produce bacteremias may lead to bacterial endocarditis in the susceptible patient. Recent work has suggested that bacteremia does not occur in children following extraction of teeth as it does in adults. One hundred and seven children were divided into four groups. Group I, which consisted of children who had nondiseased primary teeth extracted, had 35% positive blood cultures. Group II consisted of children who had diseased primary and permanent teeth removed. The incidence of positive blood cultures was 53%. Group III, which consisted of patients who had extractions of nondiseased permanent teeth, had a 61% incidence of positive blood cultures. Group IV served as a negative control. Bacteremias do occur in children following the extraction of normal and diseased primary and permanent teeth. Therefore, the susceptible pediatric patient who is to undergo a dental extraction procedure must be given prophylactic antibiotics.


Q fever endocarditis in Queensland.

Q fever endocarditis in Queensland. Thirteen patients with proven Q fever endocarditis and three additional patients with probable endocarditis are reviewed. The most helpful diagnostic test is the demonstration of a high complement fixing antibody titre to Phase 1 antigen of Coxiella burneti. The macroscopic pathology of the aortic valve is described and includes aneurysmal pockets in the aortic wall and valve annulus which are demonstrable angiographically. Evidence is presented that the infection may be controlled by prolonged tetracycline therapy and that this is accompanied by a falling antibody titre to Phase 1 antigen. Valve replacement is undertaken only for symptomatic and hemodynamic indications. The combined tetracycline therapy and valve replacement have produced a fall in titres with eradication of infection and palliation of the cardiac disability in all patients followed for long periods.


Platelet hyperaggregability in idiopathic recurrent deep vein thrombosis. Studies of platlet function were performed in 30 patients with idiopathic recurrent venous thrombosis. Evidence of platelet hyperactivity was found in 14 patients who exhibited spontaneous platelet aggregation and in 13 patients who had evidence of circulating platelet aggregates. No other differences in clinical characteristics or coagulation parameters could be elucidated between these two subgroups. In nine patients who had 51Cr-labeled platelet survival studies, there was a good correlation between the platelet hyperactivity and shortened platelet survival. Spontaneous platelet aggregation was inhibited both in vivo and in vitro by aspirin. In addition, the circulating platelet aggregates were normalized by combination treatment with aspirin and dipyridamole. The results indicate that the idiopathic recurrent venous thrombosis patient population could be differentiated by the platelet function tests into subgroup I, in which no platelet abnormality could be detected, and subgroup II, where platelet hyperaggregability was present. These results indicate that the role of platelets should be considered in the pathogenesis of recurrent venous thrombosis and therapy with agents which inhibit platelet aggregation may be beneficial in the patients with platelet hyperaggregability.


Heparin kinetics in venous thrombosis and pulmonary embolism. The response to a standard dose of heparin was studied in 20 patients with venous thromboembolism. The heparin regimen consisted of intravenous injection of 70 units per kg, followed after 90 minutes by a maintenance dose of 400 units per kg per 24 hours given by continuous infusion. Plasma heparin activity and the activated partial thromboplastin time (APTT) were measured at intervals to determine clearance of the initial injection and the response to maintenance dose. Large inter-individual variations were found in the anticoagulant effect and these were due in part to differences in heparin clearance and in part to differences in the APTT response to given amounts of heparin (heparin effect index). The heparin half-life was 63 +/- 15 minutes when plasma heparin activities were used for this calculation and 84 +/- 71.5 minutes when the APTT was used. These results are similar to values previously reported in normal volunteers. Four of the 20 patients had pulmonary embolism and in these heparin half-life was significantly shortened (P less than 0.005).


Impedance plethysmography using the occlusive cuff technique in the diagnosis of venous thrombosis. Impedance plethysmography using the cuff technique has been compared with venography in 346 consecutive patients with suspected venous thromboembolism. The limbs were classified according to the venographic results as no thrombosis, proximal (popliteal, femoral, or iliac) vein thrombosis, and calf thrombosis. A discriminant analysis was performed. The impedance plethysmographic result was normal in 386 of 397 limbs which were normal on venography, a specificity of 97%, and abnormal in 124 of 133 limbs which showed proximal vein thrombosis, a sensitivity of 93%. Seventy-three of 88 limbs with calf vein thrombi and a normal impedance plethysmographic result. The sensitivity in 29 limbs with asymptomatic proximal vein thrombosis was 83%. Impedance plethysmography is an accurate method for detecting proximal vein thrombosis but has limitations which include the possibility of false positive results due to arterial insufficiency and muscle tension.


Recurrence risks in children having one parent with a congenital heart disease. The risk of recurrence of a congenital cardiovascular malformation in a child having one parent with congenital heart disease has been determined for each of the seven most common anomalies presently compatible with survival to reproductive age. The range of risk is 2.5% to 4.3% depending on the lesion. This is within the range of expectation for the model of multifactorial inheritance previously used to predict recurrence in other first-degree relatives of probands (siblings and parents) with congenital heart disease. The cardiovascular abnormality occurring in the child was most often the same as in the parent or was a closely related variant of it.


The effects of delta-9-tetrahydrocannabinol (cannabis) on cardiac performance with and without beta blockade. Systolic time intervals (STI) were measured in ten healthy male volunteers before and after intravenous (i.v.) administration of 25 mug/kg delta-9-tetrahydrocannabinol (delta-9-THC). Mean +/- SEM heart rate increased 32 +/- 7 beats/min, while systolic and diastolic blood pressures were unchanged after delta-9-THC. Total electromechanical systole lengthened 17 +/- 4.2 msec, left ventricular ejection time (LVETc) prolonged 24 +/- 4.0 msec and pre-ejection period (PEP) shortened 17 +/- 5.1 msec after delta-9-THC. All of these changes were significant (P less than 0.01). In nine other subjects who underwent prior beta adrenergic blockade, similar but less marked changes were noted in heart rate, blood pressure, and STI after delta-9-THC. The shortening of PEP after delta-9-THC was only 9 msec (NS) in beta blocked subjects. Thus, delta-9-THC significantly increased heart rate, shortened PEP and prolonged LVETc without any change in afterload. Beta adrenergic blockade prevented significant shortening of PEP and blunted other responses. These findings suggest that delta-9-THC enhanced cardiac performance. Partial inhibition of this effect was achieved with prior beta adrenergic blockade.


Infantile cardiomyopathy with histiocytoid change in cardiac muscle cells. Report of six patients. Clinical and pathologic findings are presented in 14 patients (six newly reported, eight described previously), all children ranging in age from 6 to 24 months, with a clinicopathologic syndrome termed "infantile cardiomyopathy with histiocytoid change in cardiac muscle cells." This syndrome is manifested clinically by severe, eventually fatal cardiac arrhythmias, and is characterized pathologically by cardiac hypertrophy and by a distinctive type of focal degeneration of the muscle cells, which lose their myofibrils, undergo marked mitochondrial hyperplasia, become rounded in shape and enlarged, and resemble histiocytes. Evidence is presented to support the conclusions that these manifestations are those of a cardiomyopathy, that cardiac hypertrophy precedes the onset of the clinical features, that the focal degeneration is likely to be a cause rather than a consequence of the arrhythmias, and that the latter develop only in the late stages of the disorder. The etiology of this cardiomyopathy remains unclear.


Real-time observation of ruptured right sinus of Valsalva aneurysm by high speed ultrasono-cardiotomography. Report of a case. A two-dimensional echocardiographic manifestation of a ruptured right sinus of Valsalva aneurysm is described in this case report. The ultrasono-cardiotomograms demonstrated the aneurysm cut longitudinally and protruding into the outflow tract of the right ventricle. The configuration of the aneurysm was consistent with the contrast medium-filled sinus demonstrated by angiocardiography. In systole, the aneurysmal sinus moved anteroinferiorly together with the aortic wall. The echocardiogram revealed an abnormal two-layered horizontal echo in the right ventricle which showed the aneurysm when the ultrasonic beam passed through the interventricular septum near the aortic root and mitral leaflets. During the operation, the protruding sinus and a small interventricular septal defect in the membranous septum under it were observed through the opened right ventricle. After the closure of the aneurysm, the two-dimensional echocardiogram no longer showed an abnormal configuration.


De subitaneis mortibus. XVII. Multifocal stenoses due to fibromuscular dysplasia of the sinus node artery. The sinus node artery was focally narrowed by fibromuscular dysplasia in two examples of sudden unexpected death reported here. Although both cases had additional histological abnormalities in the conduction system of the heart, the more striking feature was the focal fibromuscular dysplasia. These findings are discussed in relationship to a large number of similar examples of focal fibromuscular dysplastic narrowing of the sinus node artery observed in other victims of sudden unexpected death, considering some possible mechanisms for lethal electrical instability of the heart and also the possible pathogenesis of such fibromuscular dysplasia.


Use of polyethylene glycol in separating bound from unbound ligand in radioimmunoassay of thyroxine. In most rapid radioimmunoassay methods, absorbents are used that disturb the equilibrium of the ligand-antibody interaction; thus the separation process must be rigidly timed. We show here that polyethylene glycol (mol wt. 6000) abolishes this constant. We measured gamma-globulin precipitation by polyethylene glycol (150g/liter) in a thyroxine radioimmunoassay system involving use of a sheep anti-thyroxine antiserum, quantitatively and qualitatively, with either normal human serum or completely precipitated at pH 6-9 at 4 and 25 degrees C, but incompletely if salt concentration was high (1 mol/liter). Specificity of gamma-globulin precipitation increased with increasing pH and temperature. All gamma-globulin-bound thyroxine was precipitated, and also some not bound to gamma-globulin. This was taken into account by including "blank" tubes (no antibody, but with normal sheep serum) in all assays. Because this reaction is predominantly entropic and temperature independent, the entire procedure can be done at 37 degrees C and room temperature without rigid timing. The assay requires 10 mul of serum, its reproducibility is 4-8% (CV) in the euthyroid and hyperthyroid range, and its accuracy is close to 100%.


Simultaneous quantitation of 4-hydroxy-3-methoxymandelic (vanilmandelic) and 4-hydroxy-3-methoxyphenylacetic (homovanillic) acids in human urine. We describe a simultaneous analysis for vanilmandelic and homovanillic acids in urine. An aliquot of a final extract, containing phenylpropriopionic acid as external standard, is dried, an internal standard (resorcinol) is added, and the mixture is silylated and chromatographed. The two compounds are identified from their characteristic retention times and from their elution behhavior in relation to four different marker compounds (the external and internal standards and two other naturally occurring compounds, p-hydroxyphenylacetic and hippuric acids) and are quantitated from their relative response factors. Within-run precision (CV) varied from 2 to 14% for vanilmandelic acid and 1 to 18% for homovanillic acid. The ratio of the amount of phenylpropionic acid extracted to the amount of added resorcinol found is a useful quality-control index. Values for vanilmandelic acid obtained by this method and by the technique of Pisano et al. [Clin. Chim. Acta 7, 285 (1962)] correlated well (r = 0.844).


Use of enzyme-loaded erythrocytes in in-vitro correction of arginase-deficient erythrocytes in familial hyperargininemia. The capacity of arginase-deficient erythrocytes of patients with familial hyperargininemia to produce urea and to catabolize arginine can be increased in vitro by introducing human liver arginase into their erythrocytes. The results of this study on a specific human model show that it is possible to change the metabolic function of a genetically defective erythrocyte by incorporating exogenous human enzyme. The in vivo application of enzyme-loaded erythrocytes for enzyme replacement therapy of inborn metabolic errors in humans must await in vivo studies on animal models.


Evaluation of methods of coenzyme activation of erythrocyte enzymes for detection of deficiency of vitamins B1, B2, and B6. We describe optimized, ultraviolet spectrophotometric procedures for determination of erythrocyte transketolase, glutathione reductase, and aspartate aminotransferase activity, and their activation by their respective coenzymes--thiamine pyrophosphate, flavin-adenine dinucleotide, and pyridoxal-5-phosphate--as tests for vitamin B1, B2, and B6 deficiency. With these procedures we have investigated healthy subjects on normal and vitamin-supplemented diets, and a series of (mainly) alcoholic hospital in-patients. The enzyme procedures described have good precision and can be readily carried out in the routine laboratory. Abnormal transketolase activation correlated well with clinical evidence of vitamin B1 deficiency.


Comparison of results for 13 clinical laboratory determinations with three automated analytical systems. We evaluated the Abbott Bichromatic Analyzer-100 (ABA-100) for use in the routine clinical chemistry laboratory by examining 13 different determinations that can be performed on the instrument. Results with the Du Pont "aca" and Technicon continuous-flow systems were compared to the ABA-100 in terms of upper limits of linearity, inter-run coefficient of variation, and results for samples from patients. The upper limits of linearity for the methods on the ABA-100 exceeded all of those for the continuous-flow systems, except for urea nitrogen. Precision of the ABA-100 was as good as or better than that of the aca for all determinations, except for glucose in a normal control serum and creatine kinase and creatinine in an above-normal control serum.


Nonchromatographic radioimmunoassay of unconjugated estriol in plasma, with polyethylene glycol as precipitant. We describe a rapid, reliable radioimmunoassay for unconjugated estriol in plasma. Polyethylene glycol (Carbowax 6000) is used to separate antibody-bound and free steroid. The assay is sensitive (25 pg for standards), precise, and accurate. At high and low concentrations of estriol, intra-assay coefficients of variation were 7.1% and 7.6%, respectively, and inter-assay coefficients of variation were 7.2% and 10.0 %, respectively. Free [3H] estriol is not precipitated by polyethylene glycol. This radioimmunoassay of estriol, with a highly specific antiserum and with polyethylene glycol as the antibody precipitant, is a reliable one-day assay that is practical both for the clinical laboratory and the obstetrician.


Simple fluorometric determination of aldosterone in urine without use of isotopes or chromatography. Aldosterone 18-glucuronide in urine is hydrolyzed by adjusting the pH to 1.0 and allowing the mixture to stand overnight at room temperature. The free aldosterone is then extracted into dichloromethane, which is washed with carbonate to remove acidic compounds and evaporated. The residue is partitioned between a nonpolar organic phase and an aqueous phase, and the aldosterone oxidized to the 13-carboxylic acid derivative with Benedict's qualitative glucose reagent. Neutral compounds are extracted from this oxidation mixture with dichloromethane at pH 7.5, the mixture is acidified, and the oxidized aldosterone extracted into dichloromethane. After washing with pH 3.5 buffer, this extract is evaporated and the oxidized aldosterone determined fluorometrically via a two-stage reaction with sulfuric acid/water (85/15 by vol) and methanol containing ferric chloride.


Evaluation of contemporary methods for serum CO2. We evaluated and inter-compared several newer methods for CO2 analysis--titrimetric (Van Slyke), automatic titrator, continuous-flow/indicator, and coupled-enzyme procedures--with respect to accuracy, precision, specificity interferences, and management criteria. All methods studied appeared to be clinically acceptable; but precision decreased (standard deviations increased) for the last two methods in above-normal ranges.


Modified colorimetric method for procainamide in plasma. We describe a modified colorimetric method for determining procainamide in plasma by use of a diazotization and coupling reaction with N-(1-naphthyl)-ethylenediamine dihydrochloride (Marshall's reagent). The sensitivity of the assay is increased twofold over the originally reported colorimetric method, and the lower limit of detection for the assay of a 1.0-ml plasma sample has been decreased to 0.5 mg/liter.


Fluorometry of plasma amino nitrogen, with use of fluorescamine. We describe a fluorometric procedure, with use of fluorescamine, for determining plasma amino nitrogen in a 10-mul sample of deproteinized plasma. Values for apparently healthy human subjects agree well with those obtained by spectrophotometry. The simplicity and speed of the assay recommends it as a screening procedure for detecting aminoacidopathies.


Method for monitoring plasma progesterone concentrations in pregnancy. We present a time-series model for monitoring concentrations in plasma of hormones produced in the placenta, progesterone being chosen as an example. The model, which is based on the assumption that variations in plasma progesterone concentration in pregnant subjects mainly reflect variations in the growth rate of the placenta, was applied to eight series of progesterone values measured during pregnancy in eight subjects. In the model, which was found to fit the data, it is assumed that progesterone concentration is proportional to the size of the placenta and that the growth rate of the placenta varies at random, with a mean value alpha. The variation of alpha was of the same magnitude among and within the subjects. If the average of many subjects alpha values is used, a single subject may be used as her own reference, based on only one previous observation. When two observations are available, an individual's own alpha value may be estimated and used for the prediction. The predictive power of the new method was found to be far superior to the conventional method in which a single sample reference material is used. Furthermore, one need not know the gestational age in order to use the method.


Adaptation of blank-corrected methods for measurement of total and conjugated bilirubin and uric acid to the centrifugal analyzer. The blank-corrected determination in serum of total and conjugated bilirubin and of uric acid has been adapted to the centrifugal analyzer. Bilirubin is measured by a modified Jendrassik and Grof procedure; uric acid is determined kinetically by using a reaction based on reduction of ferric ion, including a uricase-treated blank. Sensitivity and precision of both methods are excellent, and results compare well with those obtained by commonly used procedures.


Clinical laboratory evaluation of the Perkin-Elmer KA-150 Enzyme Analyzer. We evaluated the Perkin-Elmer KA-150 Enzyme Analyzer in a clinical laboratory situation according to a rigid evaluation protocol. The factors evaluated for five commonly assayed enzymes were as follows: (a) rate of analysis of patient samples, (b) analytical linearity, (c) overall precision (within-run and between-day), (d) carry-over from a sample with activity near 1000 U/liter, (e) error detection at low and high concentrations, (f) correlation of values with those obtained by methods used in our laboratory and (g) normal-value ranges. We find the KA-150 to be an effective and rapid enzyme analyzer.


Electrophoretic pattern of amylase isoenzymes in serum and urine of normal persons. We separated and measured amylase isoenzymes in the serum and urine of 3036 normal persons by electrophoresis on a thin layer of polyacrylamide gel. We wished to establish the normal pattern of these isoenzymes and to evaluate the usefulness of this method of electrophoresis in clinical diagnosis. Results for patients with hyper- or hypofunctioning pancreas and salivary glands suggested that essentially all the isoamylases in human serum and urine are derived from the salivary glands and the pancreas, and revealed that isoamylases of more than 98% of normal persons consisted of two major isoenzymes and two to three minor ones. Although these observations indicate that data on changes in the proportion of amylase activity of each isoenzyme can be useful in clinical medicine, the following points should be remembered: (a) quantitative differences in the isoenzyme pattern were observed, depending upon the condition of the samples; (b) because the proportion of isoamylase activity in serum of different normal persons differs, seriatim determination of amylase isoenzymes is necessary; and (c) because five different genetically controlled types of isoamylases were observed in normal persons, genetic investigations are also necessary.


Development of an aqueous temperature-indicating technique and its application to clinical laboratory instrumentation. We describe a new technique for monitoring intra-cuvette temperatures in temperature-controlled spectrophotometric cells by use of two solutions, one for which absorbance is highly sensitive to temperature and one for which absorbance is insensitive to temperature. The first solution, cresol red in tris(hydroxymethyl)aminomethane buffer, is used to construct an absorbance-vs.-temperature calibration curvae. The second solution, cresol red in sodium hydroxide, is used for optical calibration of the spectrophotometric system to be tested. The combination of these two procedures allows accurate estimation of temperature. We show how this technique is applied to temperature measurement in several types of laboratory instrumentation.


Preparation of optically clear lyophilized human serum for use in preparing control material. We describe a simple, rapid process--which includes the specific precipitation of pre-beta and beta-lipoproteins with dextran sulfate and divalent metal ions--for preparing an optically clear human serum that retains its clarity upon reconstitution with water after having been frozen and lyophilized. Such serum contains the normal constituents of human serum, except for the removed lipoproteins. The process causes no apparent interference with results of analyses for 22 of the more commonly measured constituents. Fresh or aged pooled serum or blood-bank plasma containing acid-citrate-dextrose or citrate-phosphate-dextrose are equally suitable as raw materials. This stabilized serum is an excellent matrix for use in preparing standards and quality-control material for assay of components of human serum.


Automated separation and measurement of lactate dehydrogenase isoenzymes. We describe a totally automated, computer-controlled system for separating and measuring the activity of lactate dehydrogenase (EC 1.1.1.27) isoenzymes. These isoenzymes in tissue extracts were separated on disposable DEAE-cellulose mini-columns. Resolution was complete except that LD-1 was not resolved from LD-2.


High-pressure liquid-chromatographic separation of lactate dehydrogenase isoenzymes. The isoenzymes of lactate dehydrogenase (EC 1.1.1.27) were separated with excellent resolution in less than 1 h by high-pressure anion-exchange chromatography. These isoenzymes were determined in some tissue extracts and high-activity serum samples by this technique.


Expression of random analytical error as a precentage of the range of clinical interest. I propose that random method error be expressed as a percentage of the range(s) of clinical interest (upper range limit minus lower range limit), in addition to its usual forms of presentation. The interpretation of this percent random error is simple because a given percentage error implies the same reliability, regardless of test or range of clinical interest. Two different upper limits for percent random analytical error are suggested, based on the medical application of the test and the statistical properties of various amounts of error. When the purpose of the test is to distinguish an upper medically defined limit from a lower medically defined limit, a maximum random error equivalent to 60% of the difference between the limits is proposed. If the purpose of the test is to monitor values in the neighborhood of a normal range or therapeutic range, an upper limit for random error equal to 20% of the difference between the limits of the range is suggested.


Performance studies on the Technicon "SMAC" analyzer: Precision and comparison of values with methods in routine laboratory service. We present performance studies on the Technicon "SMAC" analyzer. Precision was estimated from control data during routine operation and from blind duplicates of sera from patients. Patients' specimens were analyzed with the SMAC and the SMA 12/60 during initial evaluation studies. Comparisons were also made with the Du Pont aca and other methods during routine service. Performance is judged relative to medical usefulness requirements, by using previously developed criteria and recommendations for decision levels and allowable errors.


On the design of clinical chemistry quality-control sera. Quality-control specimens having quantitave interspecimen relationships were prepared by (a) making admixtures of two serum pools and (b) making various dilutions of the admixtures. The specimens were analyzed for many constituents to illustrate how the data obtained could be manipulated and used. The inter-related specimens permit the reduction of quality-control data to a few target values and should be particularly useful for the evaluation of multi-channel analyzers, and for use in inter-laboratory proficiency surveys. Aspects of the design of quality control and proficiency test specimens are also discussed.


Comparison of three procedures for isolating human ferritin, for use as a standard in an immunoradiometric assay. We evaluated three methods for isolating ferritin for use as a standard, with respect to purity of the products, ease of preparation, and yield. Examination of the respective products by gel filitration on Sephadex G-200 and Sepharose 6B suggested that the preparations isolated by ammonium sulfate and cadmium sulfate precipitation (Method 1) and by ultracentrifugation (Method 2) were homogeneous, while the product of a procedure including precipitation with ammonium sulfate (Method 3) contained significant amounts of nonferritin protein. The ratios of ferritin as measured by immunoradiometric assay to the amount of protein in the product indicated the ferritin prepared by Method 1 to be the most highly purified. Methods 1 and 2 were both comparatively simple. Although the yield from Method 1 was lowest, it is probably the method of choice, on the basis of the ease of obtaining a highly purified product. The most appropriate method for estimating protein in the isolated preparations appears to be that of Lowry et al.


Fast, economical technique for separating serum from blood samples. We evaluated the effectiveness with which polystyrene beads separate serum from blood. Paired blood samples were analyzed for 23 different serum constituents. One specimen of each pair was processed by the conventional separation method, the other by a single centrifugation with the polystyrene beads. Differences between the assay means were evaluated by the z-test, and the data pairs were compared by regression analysis. No statistically significant differences were found.


Evaluation of a new enzymatic kit for serum triglycerides. We evaluated a new enzymatic kit for serum triglycerides (triacylglycerols) in two laboratories. The procedure involved both enzymatic hydrolysis of the triglycerides and conversion of the glycerol, which was linked to an indicator reaction. There was excellent agreement with the mechanized procedure of Kessler and Lederer and with a manual method. The test requires 50 mul of serum and can be completed in about 50 min. Inter- and intra-laboratory variation, linearity, accuracy, and specimen storage were studied.


Serum iron determination in patients receiving therapy with iron dextran ("Imferon"). Imferon (Lakeside Labs., Inc.), an iron dextran complex, is widely used for parenteral therapy with iron. Colorimetry and atomic absorption spectroscopy give different values for serum iron concentration in patients receiving such therapy. Values determined by atomic absorption spectroscopy were usually much higher than those obtained by either a manual or an automated colorimetric procedure, and also were higher than the total serum iron-binding capacity of the same sera. This difference is attributed to circulating iron being present both as iron dextran as as transferrin-bound iron. We conclude that atomic absorption spectroscopy should be used to determine serum iron in patients receiving Imferon.


Glycylprolyl beta-naphthylamidase activities in serum, liver and kidney of rats in chronic carbon tetrachloride intoxication. Following chronic carbon tetrachloride intoxication in rats, the activity of glycylprolyl beta-naphthylamidase increased in serum and decreased in liver, suggesting that the enzyme is released from liver in hepatic injury. In contrast, the enzyme activity in kidney did not decrease when the enzyme level in liver decreased significantly.


Photometric determination of nitrogen. Wet incineration followed by formation of indophenol blue with salicylate/hypochlorite. Ammonia in wet incineration residues has been analyzed photometrically by a modified Berthelot reaction using a water-free digestion mixture. Phenol is substituted by salicylate in the dye-producing reaction. The method can be adapted for any biological material with a minimum content of 0.04 mug N/sample. Nonprotein nitrogen may be determined in the supernatant of TCA precipitates.


[Methodological and first clinical investigations using a new pancreatic function test]. The synthetic peptide N-benzoyl-L-tyrosyl-p-aminobenzoic acid was tested in vitro and in vivo for its chymotrypsin specificity. Comparative kinetic tests with bovine chymotrypsin and human pancreatic juice, as well as clearance tests with p-aminobenzoic acid (PABA), made this peptide seem suitable for an indirect pancreatic-function test. A testing scheme was set up for clinical use which has yielded promising preliminary results. While in 31 out of 32 healthy subjects there were normal PABA excretion rates in the urine collected over six hours, the test correlated well in 15 out of 16 cases of definite pancreatic disorder. The percentage dose excretion of PABA in the urine collected over six hours gave good correlations with the one-hour amount of chymotrypsin in the pancreozymin-secretin-test and with the concentration of chymotrypsin in the feces. These preliminary results make this test appear an appropriate screening method for pancreatopathy.


Use of small samples of urine to monitor testicular response to human chorionic gonadotropin. The increased urinary excretion of androsterone and aetiocholanolone, 17-oxosteroids, and oestrogens which occurs after the administration of human chorionic gonadotropin to normal men, was followed by measuring steroid to creatinine ratios in small samples of urine. For androsterone + aetiocholanolone, values of the ratio correspond closely with 24 h output (correlation coefficients: 0.91, 0.92, and 0.91 for first morning, mid-morning, and mid-afternoon urine specimens.) Comparable correlation coefficients for the 17-oxosteroids in urine were 0.90, 0.95, and 0.91; and for oestrogens, 0.88, 0.87, and 0.86.


The sulphite precipitation method for fibrinogen measurement; its use on small samples in the presence of fibrinogen degradation products. A technique based on precipitation by sodium sulphite (Na2SO3) has been developed for measuring the fibrinogen concentration of small (100 mul) samples. This technique offers technical advantages over the thrombin-clotting method, especially when measuring dilute solutions. Measurements made with the sulphite technique on fibrinogen solutions on plasma samples were not significantly different from those obtained with the thrombin-clotting method.


Calculation of three hours calcium absorption from a double isotope test; a simplified method. For the rapid evaluation of calcium absorption in human subjects several isotopic tests have been proposed. Among them, the double isotope test based on the concurrent oral and intravenous administration of calcium isotopes seems the most reliable. However, the computations involved in this test, a deconvolution of the two specific-activity functions obtained, are quite extensive. By simulating these functions we developed a simple formula to calculate the absorption in the first three hours of the test. Applied to the data of 79 patients the result differed less than the experimental error from the absorption computed with the more elaborate deconvolution method.


Simultaneous isothermal determination of diphenylhydantoin and phenobarbital serum levels by gas-liquid chromatography following flash-heater hexylation. A rapid, accurate, precise, and sensitive procedure for the simultaneous determination of diphenylhydantoin and phenobarbital under isothermal conditions involves on-column hexylation of the compounds. After extraction of serum samples, the evaporated residues are dissolved in tetrahexylammonium hydroxide. An aliquot of the resulting solution is introduced directly into a gas chromatograph where conversion to hexyl derivatives and subsequent separation takes place. The hexylated derivatives are identified by their retention times relative to appropriate internal standards. Concentrations are obtained from standard curves plotting relative peak height versus concentration of drug.


Beta2-microglobulin in human colostrum and milk: effect of breast feeding and physico-chemical characterization. A statistically significant increase in beta2-microglobulin concentration in babies' sera after birth was accompanied by a decrease in beta2-microglobulin concentration in sera of nursing and non-nursing mothers; the amount by which babies' sera concentrations increased was not correlated with the decrease in serum or milk concentrations in their mothers. These results suggest that breast feeding does not affect the concentration of beta2-microglobulin in babies' sera. Furthermore, there was no relationship between serum beta2-microglobulin concentration of mothers and their babies at either point of observation. In all instances, however, the beta2-microglobulin concentration was significantly higher in infants' sera than in mothers' sera.


Low serum bilirubin in chronic renal failure. Relation to haem metabolism. In renal failure serum bilirubin values are lower than normal. When renal failure is moderate this decrease is not due to a reduced-red-cell mass or to low levels of serum albumin. In splenic fine-needle aspirates from patients with renal failure, conditions for haem degradation were normal as indicated by normal activities of haem oxygenase and biliverdin reductase. Nor were the in vitro activities of these enzymes significantly depressed in the presence of ultrafiltrates of uraemic sera or of high concentrations of creatinine, urea or methylguanidine. The capacity of plasma from patients with renal failure to bind bilirubin was less than that of normal plasma, which at least partially explains the observed low values of serum bilirubin. There remains the possibility that in renal failure some bilirubin is removed from the plasma by a hitherto unknown route.


Comparative calcium ion determinations in plasma and whole blood with a new calcium ion analyzer. A new Ca2+ analyzer has been used for the determination of Ca2+ in plasma and whole blood from normal subjects and patients with disorders of calcium metabolism. The results were nearly identical: the equation of the regression line was y=1.02x-0.037 and the coefficient of correlation r=0.998. The possibility of using whole blood for measurement with concomitant simplification of the anaerobic sample preparation is an obvious advantage.


Bilateral renal dysplasia in three siblings: report of a survivor. Three siblings, born nonconsectively to an unrelated couple, have had bilateral dysplastic kidneys. The first child had associated skeletal anomalies, the second, ureteral atresia, and the third, no other anomalies. The occurrence of dysplastic kidneys in three siblings is compatible with an autosomal recessive inheritance pattern. In addition the variety of presenting syndromes in the children suggests that variable pathogenetic mechansims were responsible for the appearance of dysplastic kidneys. The third child is alive with kidneys that markedly decreased in size over the neonatal period but is growing well at 41 months of age indicating that the presence of bilateral dysplastic kidneys does not necessarily preclude a favorable prognosis.


Possible cobalt toxicity in maintenance hemodialysis patients after treatment with cobaltous chloride: a study of blood and tissue cobalt concentrations in normal subjects and patients with terminal and renal failure. The myocardial cobalt concentration in a patient who died 3 months after treatment with cobalt was 25-80 times greater than the concentration in control samples. Blood cobalt concentrations in maintenance hemodialysis patients who had been treated 13-20 months previously with cobaltous chloride were significantly higher than those in maintenance hemodialysis patients who had not received cobalt. Prospective studies of blood cobalt concentrations in maintenance hemodialysis patients and normal subjects after the administration of cobaltous chloride were carried out. It was found that prolonged elevation of blood cobalt concentrations occurred in both normals and maintenance hemodialysis patients, but that the blood cobalt concentrations were much higher in the dialysis patients. The urinary excretion of cobalt following the administration of a single dose of cobaltous chloride was studied in two normal subjects. Cobalt metabolism and toxicity are discussed. In view of the limited therapeutic gains to be expected and because of the lack of information regarding the long term significance of elevated blood cobalt concentrations, it is concluded that cobalt should not be used in the treatment of the anemia of patients with sever renal failure.


Reversible renal failure from isolated granulomatous renal sarcoidosis. A patient presented with a 6 month history of progessive renal failure. Serum creatinine was 7.1 mg/100 ml, creatinine clearance 4 ml/minute and hematocrit 22%. Skin tests for tuberculosis, trichophyton, SK/SD and candida were all unreactive and all cultures were negative or showed normal growth. A renal biopsy revealed non-caseating epithelial granulomas and foreign body multinucleated giant cells. No deposits or micro-organisms were seen on electron microscopy. Bone marrow and lymph node biopsies were unremarkable and no lung pathology was present. Therapy with systemic prednisone produced a dramatic improvement with serum creatinine diminishing to 1.3 mg/100 ml, creatinine clearance increasing to 81 ml/minute and hematocrit improving to 39%. A search of the literature has failed to reveal similar reports of reversible renal failure from isolated granulomatous renal sarcoidosis.