Mitral valve prolapse. Two-dimensional echocardiographic and angiographic correlation. In order to define baseline descriptive criteria for the diagnosis of mitral valve prolapse with cross-sectional echocardiography, 49 patients undergoing catheterization were examined by a real-time, two-dimensional phased array echocardiographic imaging system. Angiography was used to separate patients into two distinct groups: 15 with normal mitral valve function and 34 with definite mitral valve prolapse. Systolic mitral leaflet and annulus motion were then observed in each patient and similarities and differences were noted between the two groups of patients. Correlative M-mode echocardiographic data were available in 37 patients. Certain two-dimensional echocardiographic findings restricted to the angiographically proven mitral valve prolapse group were defined: 1) posteriorly displaced coaptation of the leaflets, 2) systolic superior movement of one or both mitral leaflets above the level of the mitral ring, and 3) a systolic curling motion of the posterior mitral ring on its adjacent myocardium. One or more of these abnormalities were found in all 34 patients with angiographic mitral valve prolapse. When mitral valve prolapse does occur, the results of two-dimensional echocardiography would suggest that both leaflets are usually involved.


Distribution of coronary artery disease. Prediction by echocardiography. To assess the sensitivity of standard echocardiography in detecting ventricular motion abnormalities in patients with coronary artery disease (CAD) without prior myocardial infarction, 56 consecutive patients with a history of angina pectoris were studied during an angina-free period. In the 48 patients with adequate echocardiograms, the amplitude of septal and posterior wall motion in the high, mid, and low left ventricle was determined and used to predict prospectively in a blinded fashion the sites of angiographically-determined CAD. Twenty-eight of 35 patients (80%) with disease of the left anterior descending artery (LAD) had diminished interventricular septal motion (P less than 0.001) and 14 of 27 patients (52%) with disease of posterior vessels had diminished posterior wall motion on echocardiogram. When abnormalties of echocardiographic wall motion were compared with left ventriculography, the results were similar. Echocardiography may aid in predicting the presence and distribution of CAD, especially LAD disease.


Hemodynamic correlates of the normal aortic valve echogram. A study of sound, flow, and motion. The aortic valve echogram was recorded in open chest dogs simultaneously with aortic flow, acceleration of flow, aortic and left ventricular pressures, and intracardiac phonocardiograms. Comparison of echographic with hemodynamic data showed the following: The aortic valve started its opening with the onset of flow, at the same point that left ventricular pressure exceeded aortic pressure. Complete valve opening preceded peak aortic flow by an average of 43 msec and the cusps started to move toward closure while flow was still accelerating. Final closure of the valve was achieved at the time of zero flow and preceded the aortic second sound by 4-10 msec. The echographically determined "valve orifice area" correlated well with aortic stroke volume (r = 0.94). The intensity of the aortic first sound was related to peak acceleration of aortic flow. The intensity of the second sound was not related to the amplitude of cusp motion but correlated well with the aortic pressure at the time of closure and with peak flow deceleration.


Echocardiographic measurement of cardiac output using the mitral valve and aortic root echo. The current echo method for measuring cardiac output (CO) has various technical limitations and is based on the assumptions that the left ventricle is not dilated and that it contracts uniformly. We have taken a different approach to measuring CO by echo: CO = cross-sectional area of aortic root X left ventricular ejection time X mean aortic flow velocity X heart rate. Using the Fick method, 34 measurements of CO were made in patients with normal aortic and mitral valves, with simultaneous echo recordings from the aortic and mitral valves. The systolic closure slope of the anterior mitral valve leaflet was assumed to represent the mean aortic flow velocity. CO by Fick and the proposed echo method correlated well, both in patients with (r = 0.94) and those without (r = 0.87) asynergy and the overall correlation was strong (r = 0.90). Using the conventional echo and Fick methods, a much weaker association was found between the two measurements in those patients with asynergy (r = 0.44) as opposed to those without asynergy (r = 0.77).


The posterior aortic wall echocardiogram. Its relationship to left atrial volume change. The normal posterior aortic wall echocardiogram shows anterior motion during left ventricular systole and predominantly posterior motion in three phases during left ventricular diastole. In six patients undergoing simultaneous left atrial angiograms and posterior aortic wall echocardiograms, there was excellent correlation between the posterior aortic wall motion and the change in the left atrial angiographic area showing the value of the posterior aortic wall echocardiogram in describing the left atrial volume curve. Left atrial and left ventricular pressures were measured with manometer tip catheters and correlated with simultaneous posterior aortic wall and mitral valve echocardiograms in four patients with atrial septal defects. These echocardiographic, angiographic, and hemodynamic correlations, as well as other evidence reported in this paper suggest that a major portion of posterior aortic wall motion is related to left atrial events and describes the left atrial volume curve.


The isovolumic contraction time of the left ventricle. An echographic study. The echographic isovolumic contraction time (ICT) of the left ventricle (LV) was measured in order to assess alterations of LV performance in children with various forms of cardiac disease. The echographic ICT was defined as the interval between coaptation of the anterior and posterior mitral valve leaflets and aortic cusp opening. Four groups of patients were evaluated: normal (48); LV myocardial disease (20); patent ductus arterosus (PDA) (19); and aortic stenosis (AS) (15). In normal children ICT shortened with decreasing age and increasing heart rate. Isovolumic contraction time was more influenced by heart rate (HR) than age, but the relationship was still weak (r = 0.61). In patients with LV myocardial disease ICT was markedly prolonged, while in children with patent ductus arteriosus and aortic stenosis it was shortened as compared with normal children (P less than 0.01).


ST-segment variations after acute myocardial infarction. Relationship to clinical status. The degree of vectorcardiographic ST-segment elevation was employed as an index of myocardial ischemic injury in a study of 27 patients after acute myocardial infarction (AMI). The ST-segment vector magnitude (STVM) was derived from the continuously recorded modified Frank vectorcardiogram and was plotted serially by hours after onset of AMI. The STVM in normal subjects was 51.1 +/- 7.1 muV (mean +/- SE). A standard deviation of the pooled variance of 15.2 muV was obtained in a group of control patients and a change of more than 2 SD (greater than 30 muV) in an individual STVM was considered to be significant. The STVM progressively decreased in patients who survived without clinical complications while it remained elevated in those with congestive heart failure. A modest, sustained re-elevation of STVM was observed in patients who developed pericarditis, and a significant late average increase of 64 muV occurred in survivors with infarct extension. In contrast, STVM underwent a major increase in patients who died. In five of these six patients without associated pericarditis a mean increase of 164 muV was recorded in the last 5-12 hours of life. While death was clinically predictable in two patients with cardiogenic shock, it was not so for the four other patients who died. Thus, major increases in STVM frequently suggested significant new ischemic injury and were often premonitory to sudden death after AMI. The increases preceding death implied that not only ventricular extopy but also lethal conduction abnormalities after AMI might be ischemia-related.


Electrophysiologic properties and response to pharmacologic agents of fibers from diseased human atria. We used standard microelectrode techniques to record action potentials of human right atrial fibers obtained during cardiac surgery, and correlated these potentials with clinical and preoperative ECG data. Human atrial fibers were classified as follows: Group A (ten patients) had a maximum diastolic potential (MDP) of -71.4 +/- 5.1mV (mean +/- SD), and action potentials that were primarily fast responses. These atria were normal or slightly dilated. In group B (12 patients) MDP was 50.3 +/- 5.7 mV; action potentials were slow responses and the atria were moderately to markedly dilated. Atrial arrhythmias occurred in four group B and no group A patients. The ECG revealed a signficant difference (P less than 0.005) in P wave duration: group A, 89 +/- 3.0 msec; group B, 111 +/- 6.0 msec. Verapamil, 0.1 mg/L, markedly depressed only the action potential plateau of group A. Procainamide 1-100 mg/L had equivalent effects on fibers of both groups A and B, effects which were small at dosages of less than 40 mg/L. Procainamide did not depress slow response automaticity, but verapamil (0.1-1 mg/L) did.


The natural history of trifascicular disease following permanent pacemaker implantation. Significance of continuing changes in atrioventricular conduction. Seventy-two patients with trifasicular disease were followed for an average of 40 months following permanent pacemaker insertion. The indications for pacemaker insertion were either electrocardiographic evidence of complete heart block with a wide QRS escape complex or a pattern of bifasicular block with either periods of Mobitz type II atrioventricular (A-V) block or a documented history of syncope. The patients were then divided into three groups depending on subsequent change in A-V conduction. There were 31 (43%) patients with no change in A-V conduction, 17 (24%) with increasing A-V block, and 24 (33%) with decreasing A-V block. The characteristics of these three groups, including age and sex distribution, were compared and found to be similar. The incidence of previous transmural myocardial infarction as determined by electrocardiographic criteria was higher in the group with decreasing block. Survival curves showed a significantly decreased probablity of surviving for those with decreasing block compared to both those with increasing block and those with no charge in conduction (P less than 0.03). We conclude that the probability of long-term survival was less in the group with decreasing block. This finding may be related to the greater prevalence of coronary heart disease in the patients.


The hemodynamic effects of induced supraventricular tachycardia in man. The circulatory effects of supraventricular tachycardia (SVT) were studied in eight patients who reported disabling symptoms during paroxysms of the arrhythmia. Supraventricular tachycardia was induced in each patient by rapid atrial pacing or with atrial premature stimuli. Hemodynamic parameters in sinus rhythm and following the initiation of SVT were recorded and compared. The following mean values were observed in sinus rhythm (SR) and SVT. Heart rate (beats/min): SR 79, SVT 183; P-R interval (msec): during SR, 154; during SVT, 256; ratio of mean P-R intervals to mean R-R cycl lengths: SR 20%, SVT 76%; brachial artery pressures (mmHg): SR 141, SVT 99; cardiac index (L/min/m2): SR 3.6, SVT 2.2; pulmonary artery pressures (mmHg): SR 18/7, SVT 26/15; peak right atrial pressures (mm Hg): SR 4, SVT 17. Large waves appeared in the right atrium during SVT due to atrial contraction against closed tricuspid valves. Pulsus alternans were observed in each case during SVT. Despite the presence of chest pain during SVT, the coronary arteries were normally patent in four patients who underwent coronary arteriography.


Thallium 201 myocardial imaging in patients with pulmonary hypertension. The appearance of the right ventricular myocardium on thallium 201 myocardial perfusion images was evaluated in patients with chronic pulmonary hypertension and compared to patients without pulmonary hypertension. Four groups of patients were studied: 1) eight normals, 2) five patients with angiographically documented coronary artery disease and normal pulmonary artery pressures, 3) ten patients with moderate to severe pulmonary parenchymal or vascular disease and documented pulmonary hypertension and 4) eight patients with chronic left ventricular dysfunction and pulmonary hypertension discovered during cardiac catheterization. The right ventricular free wall was visualized on the thallium 201 myocardial perfusion image in only one of eight normals (group 1) and in only one of the five patients with coronary artery disease (group 2) and measured 0.5 cm and 0.9 cm in thickness, respectively. In patients with documented pulmonary hypertension the right ventricle was visualized on low contrast thallium 201 myocardial perfusion image in all patients. The apparent right ventricular free wall thickness measured from the ungated thallium 201 myocardial perfusion images was 1.7 +/- 0.3 cm in group 3 and 1.5 +/- 0.2 cm in group 4. Right ventricular hypertrophy was detected by electrocardiography in only five of ten patients in group 3 and only one of eight patients in group 4. Thallium 201 myocardial perfusion imaging appears to be a useful technique for assessing the effects of chronic pulmonary hypertension on the right ventricular myocardium.


Spontaneous resolution of massive congenital tricuspid insufficiency. Four cases of congenital tricuspid insufficiency (CTI) are presented in whom spontaneous improvement occured. There was no evidence of Ebstein's disease in three of the four. All presented with similar clinical and laboratory findings including massive cardiomegaly, mild to moderate congestive heart failure, and moderate to severe desaturation. Catheterization data demonstrated severe TI and right-to-left atrial shunting. During the period of follow-up, up to 3.5 years, all symptoms have spontaneously disappeared. Clinical and angiocardiographic evidence for minimal residual TI have remained. Right atrial and right ventricular volume measurements were markedly abnormal initially and returned to normal or near normal. These measurements may be a useful method for quantitating the severity of tricuspid insufficiency. The course of the three infants with massive CTI (without Ebstein's disease) differs from previous reports and may be important both in formulating future management plans and in defining the etiology of the disorder.


Right ventricular volume characteristics in ventricular septal defect. Right and left ventricular volume characteristics were determined from biplane cineangiocardiography in 37 patients with isolated ventricular septal defects. Patients were divided into three categories as determined by the degree of left-to-right shunt: small shunt-less than 35% of pulmonary blood flow (N=9); moderate shunt-35-49% (N=8), and large shunt-greater than 50% (N=20). Right ventricular (RV) end-diastolic volume was increased above normal in 15 of 20 studies performed in patients with large left-to-right shunts and averaged 159 +/- 10% of normal (P less than 0.001). In contrast, only one of the patients in the small shunt group and only half of the patients in the moderate shunt group showed increases in RV end-diastolic volume. The increase in RV volume was proportional to the corresponding increase in left ventricular end-diastolic volume, with the right ventricle ranging from 48 to 116% of LV end-diastolic volume (average 83%). Right ventricular ejection fraction was normal in all patient groups. Right ventricular outpur was increased commensurate with the increases in the RV end-diastolic volume. These data indicate that substantial augmentation in RV end-diastolic volume does occur in patients with isolated ventricular septal defects and large left-to-right shunts. These data can be explained by the significant diastolic and "isovolumic" shunting from left ventricle to right ventricle which occurs in these patients.


Quantitative analysis of pulmonary vascular disease in complete transposition of the great arteries. Forty autopsy cases of complete transposition of the great arteries (TGA) and 22 autopsy cases of ventricular septal defect (VSD) were analyzed histologically for evidence of vascular damage due to pulmonary vascular disease (PVD). Positive correlations were generally observed between an index of pulmonary vascular disease (IPVD) and blood pressure of pulmonary circulation. No significant difference in IPVD was found between TGA and VSD in the first five months of life, when cases of each disease were compared at similar blood pressure levels. After that age, however, IPVD was much higher in TGA, and particularly severe PVD in this disease was demonstrated histologically. Morphometrical analysis of the pulmonary artery revealed hypertrophy of the muscular cost in response to elevated blood pressure. However, the progress of medial hypertrophy was retarded in TGA in the first five months, and medial thickness in arteries of cases of TGA older than five months was only 70% of that in VSD at the same blood pressure levels. Suppression of this process of reinforcement of the arterial wall in response to the stress of high pulmonary pressure was regarded as one of the important factors precipitating severe pulmonary vascular disease in transposition of the great arteries.


Results of a two-year health education campaign on dietary behavior. The Stanford Three Community Study. A two-year, bilingual mass-media health education campaign was carried out in two communities with a third serving as control. In one of the two treated communities, personal couseling of high risk individuals ("intensive instruction") took place. Dietary behavior, before and after the campaigns, was assessed using a dietary questionnaire which estimated the average daily consumption by participants of cholesterol, saturated fat, and polyunsaturated fat. Both intensive instruction and the mass-media campaigns led to significant reductions (20-40%) in cholesterol and saturated fat consumption in both men and women, with intensively instructed men tending to outperform men exposed to mass-media alone. In general, the improvements in the treated communities were maintained over the two years of the study, whereas the initial small drops in the control community at the end of the first year were followed by recidivism during the second year. Polyunsaturated fat consumption fell slightly in all three communities so that increases in P/S ratios in the two treated communities were of only modest degree. Mean changes in plasma cholesterol concentration for the various cohorts under study were higly correlated with those which would have been predicted on the basis of the self-reported changes in dietary behavior. The results indicate that mass-media health education campaigns can lead to significant changes in dietary practices in the general population with potentially important public health implications.


Bilateral ductal origin of the pulmonary arteries. Systemic-pulmonary arterial anastomosis as first stage in planned total correction. Absence of continuity between the heart and the pulmonary arteries has the potential for total correction. When the pulmonary arteries are narrow, a palliative first stage systemic-pulmonary arterial anastomosis serves to enlarge the pulmonary arteries so that, with time, they become of adequate caliber for total correction. In a case with bilateral ductal origin of pulmonary arteries, palliative anastomosis between the aorta and right pulmonary artery was performed as the first stage in a program planned for ultimate total correction.


Alveolar rhabdomyosarcoma involving the heart. An echocardiographic, angiographic and pathologic study. A 12-year-old girl, who had had an above-knee amputation of the left leg for a soft tissue sarcoma six years prior to this admission, presented with syncope, angina, and signs of aortic stenosis. Echocardiography demonstrated a mass arising from the interventricular septum with obstruction of the left ventricular outflow tract and aortic valve. Cardiac catheterization and angiography confirmed these findings. At operation, a tumor was found involving the interventricular septum. Histologically, it was an alveolar rhabdomyosarcoma, and it appeared similar to the previous tumor. The patient was treated by partial surgical resection, cytotoxic drugs and localized radiation therapy and has been followed by serial echocardiography.


Chronic hypersensitivity lung disease with recurrent episodes of hypersensitivity pneumonitis due to a contaminated central humidifer. A child with a 4-year history of acute and chronic respiratory symptoms of unknown aetiology was investigated for hypersensitivity pneumonitis. Lung disease due to inhalation of material from a contaminated central humidifier was suggested by the clinical history, the presence of precipitating antibodies in the serum against the humidifier water, a pulmonary response to challenge with the humidifier water, and marked improvement after removal of the humidifier. No fungi were cultured from the humidifier nor were antibodies against a number of fungal antigens identified by radioimmunoassay inhibition techniques. Antigenic material was found in the humidifier water and the household water prior to its reaching the humidifier. This antigenic material was not found in laboratory tap water supplied from the same general source (Lake Michigan) but from a different pumping station. Three of the child's siblings gave histories suggestive of a single concurrent episode of acute hypersensitivity pneumonitis and one sibling had a history suggestive of chronic hypersensitivity lung disease. No association could be found between HLA-haplotypy and disease in the patient and the siblings.


The effect of a new anti-allergic drug ICI 74, 9I7, given by aerosol, on nasal stenosis induced by allergen. Nasal airways resistance was measured in ten patients with allergic rhinitis during intranasal application of an extract of grass pollen. Pretreatment with placebo did not inhibit the increase in nasal airways resistance, whereas ICI 74,917 administered from a pressurized aerosol gave almost complete protection. ICI 74,917 was well tolerated and no evidence was obtained of local hyposensitization during the period of the study.


Oral aspirin challenges in asthmatic patients: a study of plasma histamine. Under carefully controlled conditions, seven aspirin-intolerant asthmatic patients were challenged with oral aspirin and experienced respiratory tract reactions with a decline in forced expiratory volume in 1 sec (FEV1) ranging from 26 to 64%. Venous blood samples, which were collected during the challenges, showed a rise in plasma histamine in all seven patients. The increase in plasma histamine occurred at the onset of their respiratory reactions and those patients with the most severe asthmatic responses were found to have the highest and most prolonged levels of plasma histamine. The aspirin-intolerant asthmatic patients were able to ingest Maalox or sodium salicylate without untoward effects, decline in FEV1 values or changes in plasma histamine levels. Ten non-asthmatic individuals and eight out of ten asthmatic control patients were able to ingest aspirin without any reactions or changes in their plasma histamine levels. However, two asthmatic control individuals, with severe asthma requiring treatment with moderate dosages of corticosteroids, were found to have elevated pre-challenge plasma histamine levels which increased during their ASA challenges despite the absence of respiratory reactions or changes in FEV1 values. It is possible that these two individuals were unsuspected aspirin-intolerant asthmatics. These studies demonstrate that asthmatic reactions to acetylsalicylates are associated with release of histamine into plasma in the subgroup of asthmatic patients with the aspirin-intolerance syndrome. Such a finding suggests that histamine may be one of the mediators of bronchospasm in aspirin-induced asthma.


Value of lymphoblast transformation test in cow's milk protein intestinal intolerance. Lymphoblast transformation tests were carried out in the presence of alpha-lactalbumin and beta-lactoglobulin. In patients with cow's milk protein intestinal intolerance in seventeen of forty-five (37.8%) the lymphoblast transformation tests were positive. Sensitization in the first month of life seemed to favour lymphoblast transformation. In control children in only four of forty-three (9.5%) the lymphoblast transformation tests were positive, the difference from intolerant patients being significant 0.01 less than P less than 0.001. Lymphoblast transformation tests were negative in the seven children with active coeliac disease. Although a negative test does not exclude cow's milk protein intolerance, lymphoblast transformation tests can be considered a useful aid in diagnosis because of its specificity.


An application of non-linear regression analysis to tests of liver function. Serum samples from 42 cases of acute hepatitis were analysed for up to 10 tests of liver function. The results of these tests were then analysed by standard statistical methods. A curve fitting exercise was performed and regression coefficients and comparative data were calculated for each of six curve types, relating to each pair of liver function tests. The correlation coefficients found were then tested for significance. From the 216 correlation coefficients tested eleven were found to be highly significant (p less than 0.001) and of these, seven were of non-linear regression curves. Examples have been given of the use of regression equations in assessing the relative sensitivities of different tests at levels on the borderline of the normal range.


Elevation of serum creatine kinase in severe hypokalemic hyperaldosteronism. The association between hypokalemia and increased serum creatine kinase has been investigated. Two patients were found who had severe hypokalemic hyperaldosteronism who had elevation of serum creatine kinase activity. The CPK activity returned to normal values after fluid and potassium replacement. In a prospective study of 129 patients with hypokalemia which did not include any patients with hyperaldosteronism, 12 were found to have increased CPK activity but each of these had some other severe disease process which could account for the increased CPK. Three other patients with K+ (2.2 MEq/1 did not have increased CPK. It is concluded that patients with severe hypokalemia such as that occurring in hyperaldosteronism with muscle weakness may have elevated CPK directly related to their hypokalemia. In other patients with hypokalemia, 10% may have increased CPK but this is related to other disease processes.


Plasma and urinary aldosterone measurement in healthy subjects with a radioimmunoassay kit not requiring chromatography. We examined the application of a radioimmunoassay kit, not requiring chromatography, to the measurement of aldosterone in healthy human subjects. Plasma aldosterone, urinary aldosterone, and plasma renin activity measurements were performed on samples from 47 subjects under conditions in which dietary sodium intake, posture, and times of sample collection were rigidly controlled. Nomograms are presented to illustrate the continuous, rather well defined, inverse relationship between 24 h sodium excretion and aldosterone measurements in plasma and urine. Mean plasma and urinary aldosterone values for subjects on normal sodium diets were 0.332+/-0.211 nmol/liter and 36.6+/-18.6 nmol/24 h and agreed well with previously reported values. A significant (p less than 0.05 increase in 24 h urinary aldosterone, plasma aldosterone and plasma renin activity resulted with decreased dietary sodium intake and conversely, a significant (p less than 0.05) decrease in each hormone occurred with increased dietary sodium intake. Rising from the supine to upright position resulted in a 230% increase in plasma aldosterone concentrations and a 284% increase in plasma renin activity.


Estimation of amitriptyline and its metabolites in serum and urine by GLC using nitrogen-specific detector. 1. A gas-chromatographic procedure for the estimation of amitriptyline and its metabolites in serum and urine using a nitrogen-specific detector is described. Specially cleaned glassware and purified solvents are used for the extraction of serum to further minimize extraneous peaks. Trimethylamine is added to serum before extraction to improve the recovery of drugs. Urine is refluxed at pH approximately 1 to hydrolyze the conjugates and to convert hydroxymetabolites to corresponding dehydro compounds. Serum is not hydrolyzed. 2. Two internal standards, one a tertiary amine similar in structure to amitriptyline and the other a secondary amine similar in structure to nortriptyline, are added to the specimen prior to extraction to obviate the need for accurate measurements of volumes during extraction and analysis. Urine and serum are washed with organic solvents at acidic pH to remove neutral and acidic impurities. Secondary bases are converted to their acetyl derivatives. 3. In the serum of a patient who is on amitriptyline therapy or who has ingested an overdose of amitriptyline, nortriptyline, a pharmacologically active metabolite is also measured. However, detection or estimation of hydroxymetabolites in serum is not clinically relevant. Hydroxylation index of an individual patient is determined by measuring the ratio of nortriptyline to its hydroxymetabolite in urine. 4. Amitriptyline and nortriptyline can be estimated in serum at a lower level of 10 and 20 ng/ml respectively. The procedure is linear over a wide range of amitriptyline and its metabolites. The use of an electronic integrator allows the estimation of different compounds with 100 fold difference in their concentration from the same chromatogram.


Iterpretation of clinical chemical data with the aid of automatic data processing. I summarize conventional applications of computers in clinical laboratories and discuss the possible role of the computer in providing assistance in interpretation of laboratory data through the use of data bases. The data bases include effects of drugs on the results of laboratory tests, factors influencing references values, and conversion factors for converting data from conventional units to SIunits. Reported diagnostic applications of the computer are discussed with reference to both clinically oriented and laboratory oriented programs.


Determination of phenprocoumon, an anticoagulant, in human plasma. Phenprocoumon is extracted from acidified plasma, the organic phase evaporated, and part of the residue, in ethanol, is quantitatively applied to a thin-layer plate. After separation, the quantity of phenprocoumon is assayed by fluorescence densitometry in situ. Results are reproducible to about 2.5%. The lower limit of detection is 0.1 mg/liter, which makes the method fully applicable to human plasma, because therapeutic concentrations range from 1 to 3 mg/liter. Seven determinations can be made within 3 h. For toxicological purposes, a qualitative analysis can be done in a shorter time, because the phenprocoumon spots are visible under ultraviolet light at 254 nm.


Use of cholesterol oxidase for assay of total and free cholesterol in serum by continuous-flow analysis. Idescribe an assay for total cholesterol in serum, with use of the AutoAnalyzer ii (Technicon), in which cholesterol esters are saponified by alkali, cholesterol is held in aqueous micellar solution with a surfactant (Triton X-100) and oxidized by cholesterol oxidase, and the hydrogen peroxide produced is measured by chelation with Ti4+ and xylenol orange. An assay for free cholesterol in serum, based on similar principles, is also described, and the two can be run simultaneously on a dual-channel AutoAnalyzer II. Standard solutions of cholesterol in isopropanol have poorer carryover characteristics than sera, and therefore do not reach the same continuous-flow steady state as sera of equivalent concentrations. Consequent potential calibration errors are avoided by using micellar solutions of cholesterol containing albumin for standardization. The formation of cholesterol peroxide in solutions of cholesterol in isopropanol is demonstrated, and this constitutes another potential source of error in the calibration of enzymic cholesterol assays. In analyzing patients' sera, results of the total cholesterol assay correlate well with those of a mechanized method in which cholesterol esterase and cholesterol oxidase are used; an automated Abell method, calibrated with solutions of cholesterol in isopropanol, gave slightly higher values. Determinations of the ratio of free cholesterol to total cholesterol by our automated cholesterol exidase assays given values that agree well with published results in which digitonin precipitation is used.


Microcalorimetric determination of glucose in reference samples of serum. Ireport a detailed series of microcalorimetric measurement of glucose concentrations in five reference samples of serum. The method utilized (a) calibration in the actual medium of analysis and (b) a correction for interferences owing to the nonspecificity of the enzyme hexokinase. The microcalorimetric results are compared with the tentative results of analyses obtained by the spectrophotometric hexokinase/glucose-6-phosphate dehydrogenase method and by isotope-dilution mass spectrometry. In most cases, the microcalorimetric results appear to be in agreement with the results obtained by these latter two methods. A discussion of the basis of the microcalorimetric measurements is presented.


Automated method for L-carnitine determination. Because of renewed interest in a possible connection between carnitine, lipid disorders, and myopathy, an automated method of analysis is desirable. Deproteinization of serum by use of membrane filter cones and automated assay with a bichromatic analyzer (the ABA-100) substantially increases efficiency without sacrificing the specificity and accuracy of the original manual enzymatic method. The described procedure allows for analysis of 80 speciments a day and is thus suitable for screening of selected populations. Normal values found in blood sera of adults were in the range of 25.0-73.8 mu mol/liter and the method has sufficient sensitivity to accurately measure concentrations as small as 10 mu mol/liter.


Determination of serum retinol (vitamin A) by high-speed liquid chromatography. We report a fast and simple high-speed liquid-chromatographic assay for serum retinol. Only 100 mul of serum is required. The lower detection limit is 50 mug/liter; linearity was demonstrated up to 1.50 mg/liter. On analyzing a serum pool eight times, a CV of 2.5% was obtained. Values by this method are compared with results obtained by a flurometric method [Clin. Chem. 16, 766(1970)].


Automated method for determination of mercury in urine. I describe a sensitive, specific, automated method for determination of mercury in urine by the "flameless atomic absorption" technique. One can analyze 20 samples per hour, with less than 0.5-ml samples of urine. The detection limit is 0.01 mumol/liter (2mug/liter) and the mean coefficient of variation 12%. The procedure is being used to monitor mercury excretion by workers exposed to mercury.


Amino acid composition of human nail, as measured by gas-liquid chromatography. We describe a quantitative method for determination of histidine and 16 other amino acids in a nail hydrolysate. This is based on the formation of trifluoroacetyl amino acid methyl esters and gas-liquid chromatographic analysis with use of two columns of mixed stationary phases. Histidine is determined as a triple derivative Nalpha-trifluroacetyl-Nim-carbethoxy methyl histidinate. The specificity and reproducibility of the method are illustrated. As little as 1 mg of amino acid per gram of nail can be determined, and 15 hydrolysates can be completely analyzed in 10 working days. Data obtained from multiple analyses of 10 nail hydrolysates by the method described closely parallel those obtained in another laboratory, in which an amino acid analyzer was used.


Direct enzymatic determination of urea in plasma and urine with a centrifugal analyzer. A direct enzymatic micromethod (sample volume, 3mul) has been adapted to the centrifugal analyzer (ENI-GEMSAEC) for measurement of urea in plasma and urine. The method is based on urease (urea amidohydrolase, EC3.5.1.5)/glutamate dehydrogenase [l-glutamate:NAD(P)+oxidoreductase (deaminating), EC1.41.3] coupled reactions, and uses a two-point fixed-time (t(1)=20s,t(2)=50s)kinetic scheme for monitoring the rate of comsumption of NADH at 340 nm. Sensitivity and precision of the method are excellent,and results compare well with those from a commonly used continuous-flow method.


Comparison of procedures for extracting free amino acids from polymorphonuclear lekocytes. We studies five methods for extracting amino acids from human polymorphonuclear leukocytes. Both the use of cell lysis and of a deproteinizing agent interfere with quantitative determination of the amino acids, basic amino acids being the most sensitive to the extraction procedure. Among the methods used, disruption of the cells by freezing-thawing is the best method for extracting all the amino acids. Taurine is the only amino acid extracted in the same amount by all the methods studied, and it represents half of the intracellular pool.


Cholesterol oxidase-based determination, by continuous-flow analysis, of total and free cholesterol in serum. We describe a continuous-flow, automated determination of total cholesterol in serum, which is based on enzymatic hydrolysis of cholesterol esters, oxidation of cholesterol by cholesterol oxidase, and colorimetric measurement of liberated perioxide with 4-aminoantipyrine, phenol, and peroxidase. Free cholesterol is determined with the same AutoAnalyzer II manifold and reagents, except that cholesterol esterase is omitted from the reagent. Cholesterol-in-serum materials that have been assayed by an established method are used for calibration. We found this approach to be necessary because primary cholesterol standards in organic solvents are incompatible with the aqueous reagent. Results of the enzymatic total cholesterol method correlated well with those by an AutoAnalyzer II method which involves an extraction with isopropanol and the Liebermann-Burchard color reaction (total cholesterol, g/liter, yenz= 0991xlb +0.05;r=0.996). Results of the enzymatic free cholesterol procedure agreed satisfactorily with one in which free cholesterol is precipitated as the digitonide and subsequently analyzed colorimetrically with the Liebermann-Burchard reaction (free cholesterol, %, yenz = 0.982xdig -0.7;r= 0.956).


Kinetic determination of hexosaminidases for Tay-Sachs heterozygote screening. We describe a method for continuous kinetic measurement of hexosaminidase activity, and have applied it to detection of heterozygotes for Tay-Sachs gene. In contrast to existing single-point methods, a ph of 4.5, which is optimal for hexosaminidase activity on the substrate (4-methylumbelliferyl-N-acetyl-beta-d-glucosaminide) is maintained while the increase in fluorescence produced by 4-methylumbelliferone, the reaction product, is being determined. Under the conditions described the reaction follows zero-order kinetics, and activity is linearly related to serum dilution. There is a fairly narrow but practicable range of optimal substrate concentration. The presence of substrate in concentrations exceeding 1 mmol/liter results in progressive quenching of fluorescence and a decrease in apparent enzyme activity. The procedure is standardized with 4-methylumbelliferone in a solution of the substrate. This method is adaptable to use with automated multiple-point discrete-sample devices and especially with the fluorometric kinetic analyzers now being developed.


Gas-chromatographic analysis for 1,2:5,6-dianhydrogalactitol, an anti-neoplastic agent, in plasma. We describe a gas chromatographic method for measuring submicrogram quantities of dianhydrogalactitol, a promising antineoplastic agent (currently undergoing clinical trials in humans), in plasma. The drug is first extracted from blood plasma by saturating the aqueous phase with solid potassium carbonate and extracting with isopropanol/chloroform (9/1 by vol). It is then converted to the corresponding n-butaneboronic ester by reaction at room temperature with butaneboronic acid and chromatographed on an SE-30 (3%) column, with flame ionization detection. Practicality of the method for monitoring drug distribution was demonstrated by administering dianhydrogalactitol in therapeutic doses to a dog and monitoring its concentrations in blood for the next 2 h.


Clinical usefulness of measuring prothrombin time as a routine admission test. A questionnaire indicating the presence of a history or physical findings consistent with liver desease or bleeding disorders was completed by house officers on 301 admissions to a Veterans Administration medical service. Each patient had prothrombin time determined on admission. Only two of 107 patients for whom the prothrombin time was measured as a screening test had an abnormal results and one of these was normal when repeated. Of 73 patients with a history of alcoholism but no other pertient case history or physical examination results, only one had an abnormal prothrombin time. Of the remaining 121 patients, who had a pertinent history or physical examination, 41 had an abnormal admission prothrombin time. We conclude that measurement of prothrombin time adds little additional information to that obtained by history and physical examination in screening for liver disease and coagulation defects unless the patient has specific clinical evidence of liver disease, anticoagulation, or other conditions predisposing to bleeding disorders.


A faster radioimmunoassay of progesterone in plasma, with use of polyethylene glycol as precipitant. We report a reliable radioimmunoassay for plasma progesterone in which polyethylene glycol ("Carbowax 6000") is used to separate antibody-bound and free hormone. Initially, progesterone is concentrated by putting the sample through a column of LH-20 Sephadex. Procedural losses in each sample are not monitored. We compared the reliability and practicality of this method with that of another method in which procedural losses are monitored for each sample; accuracy and precision were similar. Our method saves technical assay time and scintillation counter space and time without sacrificing reliability.


High-pressure liquid-chromatographic determination of 5-(4-hydroxyphenyl)-5-phenylhydantoin in humanurine. We describe a sensitive and precise high-pressure liquid-chromatographic method for measurement of total 5-(4-hydroxyphenyl)-5-phenylhydantoin, a metabolite of phenytoin, in urine. An aliquot of urine, containing 5-(4-methylphenyl)-5-phenylhydantoin as an internal standard, is processed and chromatographed. The metabolite and internal standard are identified from their retention times and quantitated from their relative response factors. The metabolite is separated from normal urine constituents and internal standard in less than 8 min. The sensitivity of the method is such that after the usual dose it can be measured in 0.5 ml of urine; the lower limit of detection is 300 ng.


Proficiency testing for blood-gas quality control. We have conducted a voluntary, community blood-gas proficiency testing program, with use of tonometered human blood, for 32 analyzers located in 16 laboratories. Instruments initially showed inaccuracies as large as -30.8 to +17.3% for po(2), and -14.0 to +42.9% for pco(2), but inaccuracy and imprecision decreased in most laboratories during the program. For a typical 15-week period, mean group precision (CV) was 4.3 To 5.1% for po(2) from 6.92 to 33.3 Pa (52 to 250 mmHg), and 4.0 to 6.9% for pco(2) from 2.0 to 6.8 Pa (15 to 51 mmHg). This program can detect increasing imprecision or inaccuracy caused by analyzer deterioration, and can identify interlaboratory or interinstrument bias and problems not detected by participant quality-control programs. Participants have used the proficiency information in discussing data quality with clinicians, promoting internal control and maintenance programs, and justifying instrument purchases. We believe that proficiency-testing documentation of variability in blood-gas analysis may help to establish realistic patient-care protocols.


Results for serum cholesterol and triglycerides by gas-liquid chromatography, as compared with a continuous-flow technique. We assessed the usefulness of gas-liquid chromatography forthe routine and reference measurement of cholesterol and triglycerides (triacylglycerols). Compared with results obtained by an AutoAnalyzer II method, correlation was good with both serum specimens (triglycerides, r = 0.88; cholesterol, r = 0.90) and lipoprotein fractions isolated by ultracentrifugation (triglycerides, r = 0.98; cholesterol, r = 0.98). However, none of the comparisons was completely free of analytical bias, and in general the AutoAnalyzer method underestimated the cholesterol value and overestimated triglycerides. The obvious advantage of the AutoAnalyzer is its greater analytical speed, but the chromatographic method appeared to be more accurate and precise and to suffer less from interfering substances, and in addition gave information about the cholesterol ester and triglyceride composition. We expect gas-liquid chromatography to be of major importance in the calibration of other analytical methods.


Gas-chromatographic analysis for therapeutic concentration of imipramine and disipramine in plasma, with use of a nitrogen detector. We describe a method for the simultaneous gas-chromatographic analysis of imipramine and desipramine (the active N-desmethyl metabolite) at therapeutic concentrations in human plasma, with use of a nitrogen detector. Imipramine is measured as the unchanged base and desipramine as its N-tribluoroacetyl derivative. Promazine is the internal standard. The lower limit of sensitivity is 5 mug/liter for each drug, and the within-day coefficients of variation for the determination of imipramine and desipramine, respectively, are 6.0% and 3.3%. We measured concentrations of these drugs both in patients receiving therapeutic doses of imipramine and in those who had ingested an overdose.


Enzymatic, fluorometric assay of alpha-amylase in serum. An enzymatic, fluorometric method for kinetic assay of serum alpha-amylase (1,4alpha-D-glucan 4-glucanohydrolase, EC 3.2.1.1) is described. A soluble starch is used as substrate, in tris(hydroxymethyl)methylamine buffer. All measurements are made in Pyrex cuvettes at 37 degrees C, with a reaction volume of 1.16 ml. The assay is based on the following reaction sequence: (see article) The rate of appearance of fluorescence of NADH (lambdaex = 365 nm, lambdaem = 460 nm), developed in the indicator reaction (4), is measured and equated to the activity of alpha-amylase in serum. A calibration plot of the change of fluorescence per min vs. enzyme concentration shows a good proportionality in the range of 0.50-5.0 kU/liter.


Comparison of results by three kit methods for assay of serum gastrin. We evaluated and compared Nuclear International, Schwarz/Mann, and Squibb kit methods for measurement of serum gastrin with respect to intra-run precision, analytical recovery of added gastrin, and values obtained for healthy fasting adults and for selected patients. As a result of this study, the Schwarz/Mann kit was chosen for use in our laboratory.


Modified determination of total and inorganic mercury in urine by cold vapor atomic absorption sectrometry. In this procedure a single-beam spectrophotometer is used without background correction. By the method of Magos [Analyst (London) 96, 847 (1971)] mercury in undigested urine is complexed to L-cysteine in acid solution. At high pH and in the presence of stannous ions, mercury ions are reduced to elemental mercury. The mercury vapor is partitioned above the reagent solution in a specially designed chemical reduction apparatus similar in principle to that used by Kubasik et al. [Clin. Chem. 18, 1326 (1972)]. The vapor is then flushed by air through an "absorption" cell, where the absorption of the mercury line at 253.7 nm is measured. The value obtained for inorganic mercury subtracted from that for total mercury gives a value for organic mercury. CV's for the inorganic mercury procedure at 40 and 5 mug/liter concentrations were 3.1% and 7.5%, respectively. The detection limit is 0.82 mug/liter. The CV for the total-mercury procedure (20 mug/liter) was 2.6%. Mean analytical recoveries of organic and inorganic mercury were 96.5% and 101%, respectively. We investigated storage conditions for urine and compared results by the present technique with those by activation analysis. Our method is a convenient way to screen individuals who have been exposed to a mercury hazard.


Use of values for calcium and protein in serum, and of a derived index obtained from a probability population sample. We measured serum protein and calcium concentrations in 2340 individuals between 10 and 96 years of age from 900 families chosen by probability methods to give a representative population. These values were used to calculate an index, based on a regression analysis of serum protein on calcium, which was then treated as a new variable. Age-sex specific reference values and frequency distributions are presented for this index as well as for protein and calcium calculated by both parametric and nonparametric methods.


Interference by acetaminophen in the glucose oxidase-peroxidase method for blood glucose determination. Acetaminophen, p-aminophenol, and oxyphenbutazone interfere with the glucose oxidase/peroxidase method for glucose. Structurally related compounds that lack a free phenolic hydroxyl group (acetanilide, aniline, and phenylbutazone) do not interfere. During the analytical procedure acetaminophen is consumed. One mole of acetaminophen leads to an apparent loss of four moles of glucose. The hexokinase/glucose-6-phosphate dehydrogenase method (Boehringer Hexokinase method) is not affected by these substances.


Reduced variation of tracer binding in digoxin radioimmunoassay by use of (125I)-labeled tyrosine-methyl-ester derivative: relation of thyroxine concentration to binding. Between-sample variation in tracer binding in the 125I-labeled digoxin radioimmunoassay was investigated with two tracers, 3-O-succinyl-digoxigenin-[125I]-labeled tyrosine and [125I]-labeled tyrosine-methyl-ester-digoxin. Digoxin-free serum samples having various concentrations of thyroxine were assayed with both tracers. The percentage of tracer bound when the samples were assayed with the first-mentioned tracer was increased significantly for the low thyroxine groups when compared to the normal (P less than 0.001) or the high thyroxine groups (P less than 0.05). Little difference existed when the latter tracer was used. There was variation in tracer binding when serum from dogs dosed with thyrotropin was assayed with the first tracer, but there was little variation with the second. Tracer binding may be influenced by thyroxine-binding proteins. Variation in tracer binding appears to be reduced when [125I]-labeled tyrosine-methyl-ester-digoxin is used.


Serum alkaline phosphatase measured by using thymol blue monophosphate and a bichromatic analyzer. Thymol blue monophosphate is evaluated as a substrate for use in kinetic bichromatic analysis of serum alkaline phosphatase activity. The effect of abnormally high concentrations of hemoglobin, bilirubin, or chylomicrons is negligible. The expected range for ambulatory adults established by assaying sera from 206 blood donors, was 12-44 U/lliter at 37 degrees C. Comparison of the method with a commonly used p-nitrophenyl phosphate method yielded a correlation coefficient, r, of 0.9949. Advantages of the method are the reduced cost per test and the exceptionally stable substrate.


Simple modification of a liquid scintillation counter to decrease cost of and time for radioimmunoassay. A simple, inexpensive modification in the elevator mechanism of a liquid scintillation counter allows for radioimmunoassay tubes (12 X 75 mm) containing the antibody-bound radioligand to be counted directly, thereby obviating the need for counting vials and considerably decreasing the amount of fluor used. Furthermore, because assay and counting are all done in the same tube, the modification shortens assay time and increases precision by avoiding what is often the most time consuming and inaccurate step, the transfer of antibody-bound radioligand to a counting vial. We find that use of this modification results in about a 75% decrease in the overall cost for materials and a substantial decrease in assay time.


Toward absolute methods in clinical chemistry: application of mass fragmentography to high-accuracy analyses. We review recently developed reference methods based on mass fragmentography (specific ion monitoring) with use of isotope-labeled internal standard, for determination of cholesterol, triglycerides, urea, glucose, cortisol, progesterone, and testosterone. With an optimal ratio between standard and material to be determined, the relative standard deviation of these methods, based on duplicate determinations, is between 1.3 and 2.7%. The very high specificity of the methods, in combination with the fact that the ratio between labeled and unlabeled molecules is determined with a high degree of accuracy, makes it likely that the most significant errors in the methods are related to errors in pipetting. The possibility is discussed that these methods might be developed into "absolute" or "definitive" methods by subjecting each individual step in the determination to a detailed analysis with respect to possible error. We also discuss some of the consequences of the different reference methods on the choice of routine methods.


Spectrophotometric and electrochemical determinations of L(+)-lactate in blood by use of lactate dehydrogenase from yeast. Lactate can be determined rapidly in blood by spectrophotometric and amperometric (enzyme electrode) procedures based on its oxidation by ferricyanide, the reaction being catalyzed with yeast L(+)-lactate dehydrogenase (cytochrome b2) (EC 1.1.2.3). In the photometric method lactate can be measured in a few minutes, but blood samples must first be deproteinized. In the amperometric procedure no treatment of blood is needed except ferricyanide addition. The enzyme electrode we used has a response time shorter than 1 min when its critical variables are optimized. Preliminary standardization is reduced to minimum operation, because electrode response is proportional to lactate concentration over a wide range (0.1 to 8.0 mol/liter) and many determinations can be done with little cost in enzyme. A simple electrical device ("two-electrode device") is described that is well suited for furture micro-cell construction. Lactate determinations on a series of normal blood samples show no deviation between results by these new methods and the usual ultraviolet spectrophotometric lactate tests.


Erythrocyte uroporphyrinogen I synthase activity in diagnosis of acute intermittent porphyria. Measurement of the activity of uroporphyrinogen I synthase provides an excellent laboratory aid in the diagnosis of acute intermittent porphyria, particularly in those patients who are asymptomatic or in whom the disease is not biochemically manifested by porphyrin precursor excretion. We describe here a simplified fluorometric method for measuring the activity of this enzyme in whole blood. The assay is based upon a coupled-enzyme procedure in which added delta-aminolevulinic acid and the dehydratase that is present in erythrocytes are used to generate porphobilinogen as substrate for uroporphyrinogen synthase. After appropriate incubation the protein is removed we sensitivity, specificity, and precision of the assay compare well with previously described procedures. Activity in nonporphyric male subjects was 31 (SD, 6.0) nmol of porphyrin formed per milliliter of erythrocytes per hour at 37 degrees C. Application of the method for identifying gene carriers of acute intermittent porphyria is demonstrated in three generations of an affected family.


Laboratory confirmation of the diagnosis of cystic fibrosis. The recent commercial introduction of a method for detecting albumin in meconium makes screening for cystic fibrosis feasible for many hospitals. If the tests is adopted, confirmatory tests should be available. Quantitative analyses of sweat for sodium by flame photometry and for chloride by silver titration and ion-sleective electrodes are now used as confirmatory tests. We compare results of these confirmatory methods applied to presons with cystic fibrosis, respiratory disorders, or digestive disorders, and to control subjects.


Radioimmunoassay for serum cortisol with 125I-labeled ligand: comparison of three methods. We evaluated a previously modified double-antibody radioimmunoassay for serum cortisol. It was compared with the conventional double-antibody method that includes the usual extraction step, and also with an antibody-coated tube method. In this modified method, cortisol was released from its binding globulin by enzymatic degradation rather than by extraction with ether, and a preincubated mixture of first and second antibody was used to separate antibody-bound cortisol from free. These two steps shortened total asssay time significantly. Results still correlated well (r = 0.87) with results by the conventional melthod, but the antibody-coated tube method gave lower results (r = 0.61). Because of its good correlation with the conventional method, this method was thought to be more accurate. In 52 normal subjects, mean cortisol concentrations at 0800 and 1700 hours were 161 +/- 52 (SD) mug/liter and 91 +/- 27 mug/liter, respectively. In 16 normal subjects, cortisol values before and after dexamethasone treatment (1 mg at midnight) were 134 +/- 53 mug/liter and less than 20 mug/liter. In the same subjects, cortisol concentrations before and 30 min and 60 min after Cortrosyn (synthetic corticotropin1-24, 0.25 mg) administration were 103 +/- 25, 205 +/- 45 and 223 +/- 51 mug/liter, respectively.


An 125I-labeled cortisol radioimmunoassay in which serum binding protein are enzymatically denatured. We report an iodine-125 radioimmunoassay for cortisol in biological fluids, in which interfering binding proteins are enzymatically denatured. An antiserum to cortisol-3-carboxymethyloxime-bovine serum albumin, extremely low cross-reacting with other corticosteroids, was raised in rabbits. A cortisol-3-carboxymethyloxime tyrosine methyl ester derivative was synthesized and labeled with iodine-125 by standard radioiodination techniques. To eliminate the need for extraction and recovery procedures, we digested interfering binding witha proteolytic enzyme, which then was heat-inactivated before adding the labeled derivative and the premixed, preincubated antiserum complex. There was quantitative analytical recovery of esogenous cortisol added to sera from a normal man, a normal woman, and a pregnant woman. Values for the same samples agreed after extraction and chromatographic purification and agreed well with values obtained by other techniques by independent reference laboratories. The five-step assay can be done in 6 h or less.


Correlation of selected serum constitutents: 2. Consistency of intra-individual correlation values, means, and variances during four months. We examined whether inter-individual differences in correlation coefficients previously found among subjects truly reflect consistent inter-individual differences or are time-related within an individual. The consitutents studied in this investigation were (a) the enzmes aspartate aminotransferase, alkaline phosphatase, and lactate dehydrogenase; and (b) the non=protein nitrogen-containing constituents urea, uric acid, and creatinine. Ten healthy women were each subjected to 15 venipunctures over a five-week period (Series I), and, after a two-month interval, were again subjected to 15 venipunctures over a second five-week period (Series II). Before statistical analysis, the data were corrected for the batch-to-batch (day-to-day) arnalytical variation. There was a signiificant (P less than .05) change in the covariance structure (variances or correlation coefficients, or both) between the two series in four of the 10 subjects for the combination of enzymes, and in three other subjects for the combination of nonprotein nitrogen constitutents. Although we found a significant (P lees than .05) average intra-individual variation in the mean values from series to series in the cases of the three enzymes and urea, the magnitude of the inter-series variation in means was relatively small. CV's were: alkaline phosphatase, 3.4%; lactate dehydrogenase, 2.3+; aspartate aminotransferase, 3.3%; urea, 5.0%; uric acid, 1.0%; and creatinine, 1.2%.


An evaluation of the Beckman chloride/carbon dioxide analyzer. We compared the Beckman Cl/CO2 Analyzer to the American Instrument Company chloride titrator for measurement of chloride in serum, sweat, and spinal fluid. We also compared it to the Natelson gasometer and a modified continuous-flow (AutoAnalyzer) technique for measurement of carbon dioxide in serum. Effects of bilirubin and hemoglobin on results for both the CO2, and Cl were negligible. The linearity, stability, and precision of the equipment was studied. We believe the Beckman Cl/CO2 Analyzer to be ideal for simultaneous measurement of chloride and carbon dioxide in a pediatric or emergency-determination setting.


Aspartate aminotransferase activity in human serum. Factors to be considered in supplementation with pyridoxal 5'-phosphate in vitro. The pyridoxal phosphate reactivation of the apo form of aspartate aminotransferase (EC 2.6.1.1) in human serum has been studied with "normal" and above-normal activity of this enzyme. The extent of the reactionation did not depend on the presence of the substrates, L-aspartate or 2-oxoglutarate. Reactivation was greatest with 110 mumol of added pyridoxal phsophate present per liter during a preinucation for 7 min in tris(hydroxymethyl)methylamine buffer wit;h serum volume fractions ranging from 0.017 to 0.267. In comparison with measurements prformed with no exogenous pyridoxal phosphate present, we found two potential sources of error when this cofactor was added: (a) reagent and sample blanks in the pyridoxal phosphate-supplemented system were two- to eightfold higher and (b) progress curves were nonlinear when L-aspartate rather than 2-oxoglutarate was used as the startin substrate. Aspartate aminotransferase measurement sith pyridoxal phosphate supplementation was slightly more precise than without.


Serum ionic fluoride: normal range and relationship to age and sex. We used the Orion fluoride electrode system to determine the normal range of serum ionic fluoride concentrations and to investigate its relationship to sex and age (A). 87 normal men, aged 18-92 years (mean, 46 years), and 49 normal women, age 19-64 years (mean, 38 years), participated in the study. At the 95% confidence limits, males less than 45 years old had a normal range of 0.29 to 1.52 mumol/litre and males greater than or equal to 45 years old 0.29 + 0.0101 (A-45) to 1.52 + 0.0101 (A-45) mumol/litre. Females, however, had a normal range of 0.022A - 0.32 to 0.022A + 1.07 mumol/litre. A group of 51 men 18-44 years old was compared with a group of 36 men 46-92 years old. The mean serum F- of the older group was shown to be significantly greater (P less than 0.01) than that of the younger group. Factors related to serum ionic fluoride values are (a) tea as an important source of dietary F-, (b) the lack of significant variation during daytime hours, and (c) the lack of significant difference in concentration between serum and plsma F-.


Low apparent creatine kinase activity and prolonged lag phases in serum of patients with metastatic disease: elimination by treatment of sera with sulfhydryl agents. We find lag phases exceeding 20 min in measuring creatine kinase activity, by using the kinetic creatin phosphate leads to creatine assay, in sera of some patients with carcinoma metastatic to the liver. Such long lag phases are accompanied by a decreased apparent enzyme activity. These problems are eliminated by adding sulfhydryl agents to the serum before assay, but not by adding more of such agents to the assay reagent. beta-Mercaptoethanol is supperior to Cleland's reagents, glutathione, and cysteine. The long lag phases could not be explained by inadequate activity of the coupling enzymes, interference with the coupling steps, high proportions of cardiacisoenzymes activity, simple oxidation of the enzyme, low concentrations of albumin, or increased concentrations of glutathione reductase, lactate dehydrogenase, or uric acid. We conclude that the prolonged lag phases reflect inadequate reactivation of the enzyme by sulfhydryl agents under the usual assay conditions. Reactivation before assay can prevent potentially serious negative errors in the assay of creatine kinase.


Evaluation of three commercially available menual "kit" methods for triglycerides in serum. We compared three commercially available kits for use in determining triglycerides (triacylglycerols) in serum, by different methods, for accuracy and precision, with use of pooled sera of low, normal, and high triglyceride concentrations. Accuracy varied from -17% to +16% of the independently determined value, depending on the method and triglyceride concentration. In general, the Calbiochem method was the most nearly accurate, and only it met Center for Disease Control criteria for precision at all three triglyceride concentrations. The relationships of mean values to the suggested normal range for each method varied, some significant differences being found. Our data demonstrate the importance of a correlation between the independently determined value and the normal range for the method used and stresses the need for normal-range values to accompany all patients' reports.


More sensitive high-pressure liquid-chromatographic determiantion of theophylline in serum. We present procedures for determining theophylline in 50 mul of serum. The drug is extracted into a small volume of solvent that contains an internal standard, 8-chlorotheophylline. The extract is Analyzed by isocratic reversedphase chromatography, with measurement of eluted theophylline at 273 nm. Day-to-day reproducibility within 5% is attainable for the concentration range 5--20 mg/liter. Other xanthines and related metabolites do not interfere. Sensitivity is 1 mg/liter. The correlation coefficient, when results by a spectrophotometric procedure were compared, was 0.989. Amobarbital, secobarbital, phenobarbital, and diphenylhydantoin do not interfere. Total analysis time for a single sample is 15 min.


Gas-chromatographic identification of drugs in gastric aspirate samples: a rapid screening procedure for emergency toxicology. Gastric aspirate has not been used frequently in the identification of ingested drugs. Because there is only qualitative significance in such a sample, a new method is described that facilitates identification. A 1-ml gastric aspirate sample is extracted with 100 mul of chloroform and analyzed directly by gas chromatography. Seventeen drugs commonly involved in overdose cases are included in this 25-min procedure. The gastric drug screen has been applied to more than 500 patients, and results are described.


Error introduced by specimen handling before determination of inorganic phosphate concentrations in plasma and serum. Values for serum inorganic phosphate (Pi) concentrations in groups of healthy adults vary widely, the coefficient of variation ranging from 10 to 15%. We undertook to determine in 23 healthy adults whether part of this variation could be accounted for by (a) drawing blood in syringes vs. evacuated tubes (b) the time between blood sampling and separation of serum or plasma, and (c) the prevention of clotting. Values were unaffected by a, decreased significantly with time at room temperature between blood sampling and separation of cells in both serum and plasma, and were significantly lower in plasma than in serum. The group coefficient of variation for Pi averaged 13% and was uninfluenced by the blood-processing technique.


Improved thyroxine radioimmunoassay, for filter paper discs saturated with dried blood. I describe an improved procedure for estimating thyroxine in dried blood on 0.32-cm filter paper discs, by radioimmunoassay. A weak anion-exchange resin is used to separate free and antibody-bound radiothyroxine and, by measuring the resin-bound 125I-labeled thyroxine rather than the supernatant radioactivity, a tedious and cumbersome step is eliminated. The unique properties of the resin permit considerable latitude in the experimental conditions, in contrast with charcoal, without compromising sensitivity or precision. There is excellent correlation between thyroxine values obtained from discs and those of the corresponding plasmas. Application of this procedure for large-scale screening of newborns has proved to be highly successful in identifying thyroid insufficiency in several babies before clinical signs were apparent.


Radioimmunuassay compared to thin-layer and gas--liquid chromatography for detecting methadone in human urine. Radioimmunoassay was compared to thin-layer and gas--liquid chromatographic methods for detection of methadone in the urine of patients undergoing methadone maintenance therapy as treatment of heroin abuse. With urine samples known to contain methadone, 84% were positive by thin-layer chromatography as compared to 99% positives by the other two methods. This difference is attributed to the difference in sensitivity of the three methods. All three methods gave consistently positive results with urine samples from patients receiving 25 mg of methadone per day or more. With smaller daily doses the percentage of positive results obtained with thin-layer chromatography decreased. Analysis of urine samples not containing methadone showed no incidence of cross reaction of other drugs with the methadone radioimmunoassay. The methadone radioimmunoassay appears to be both sensitive and reliable; however, certain other factors limit its use as a primary screening method.


Relationship between erythrocyte 2,3-diphosphoglycerate and age in a normal population. The concentration of 2,3-diphosphoglycerate in erythrocytes was determined in 369 male and 376 female normal subjects, 1 to 86 years of age, derived from the Greek population. It was inversely correlated (P less than 0.01) with age. Persons over age 60 showed a decrease (P less than 0.05) in 2,3-diphosphoglycerate concentrations as compared those 1-20 years of age.


[Colorimetric determination of calcium in serum using methylthymol blue]. A simple, specific and rapid colorimetric method for the determination of calcium concentration and serum using methylthymol blue is described. The reagent given can be used both in a manual method and in an automatic method using an auto-analyser without dialysis. The results obtained by the automatic method are compared to those found by the manual method and those obtained by atomic absorption.


Accumulation of lactosyl ceramide in leukocytes of patients with adult Gaucher's disease. Glycosphingolipids were isolated from the leukocytes of 10 patients with Type I, chronic nonneuronopathic (adult) Gaucher's disease and 12 normal subjects, by silicic acid column chromatography and thin-layer chromatography. Quantitation of the individual glycosyl ceramides was achieved by the determination of hexose and sphingosine content, using colorimetric and fluorometric procedures. Lactosyl ceramide, which is the main glycolipid in leukocytes of normal subjects, was significantly increased in the leukocytes of patients with Gaucher's disease. On the other hand, the amount of glycosyl ceramide, which is the main glycolipid accumulating in the reticuloendothelial cells of patients with Gaucher's disease, was similar in Gaucher and in control leukocytes.


gamma-Carboxyglutamic acid in human urine. gamma-Carboxyglutamic acid, the amino acid responsible for the vitamin K dependent, Ca2+-binding structures of some of the blood coagulation proteins, has been identified in human urine. The amino acid wasisolated and its identity was proved by comparing it with synthetic gamma-carboxyglutamic acid by electrophoretic and chromatographic methods and by mass spectrometry. The isolated compound was also converted to glutamic acid by heat decarboxylation, a reaction consistent with its anticipated structure. A method for the quantitation of free gamma-carboxyglutamic acid in human urine was developed. The method consisted of an anion-exchange concentration step followed by automatic amino acid analysis using a pH 2.0 lithium citrate buffer. In three non-fasting adult males the urinary excretion of gamma-carboxyglutamic acid ranged between 27 and 42 mumol/24 h and in six non-fasting adult females it ranged between 19 and 32 mumol/24 h. One fasting adult male excreted 36 mumol/24 h.


A system of interpretation of infrared spectra of calculi for routine use in the clinical laboratory. We have devised systematic schemes for objectives qualitative analysis of the IR spectra of calculi. These schemes are presented as flow charts based on questions each requiring a yes or no answer before going on to the next step. The specific question asked in each step concerns the similarity of a limited region of the spectrum to reference spectra. For renal calculi, 5 regions are evaluated and a maximum of 10 decisions is necessary to complete the analysis. Biliary calculi are treated similarily, with 5 spectral regions evaluated and a maximum of 8 yes-no decisions required. The method requires less technician time and is much less susceptible to differences in subjective judgment than the wet chemical procedure. Three percent of stones contain unusual components and are quickly recognized as such and identified using a library of reference spectra.


Optimization of T assay: a model study.

Optimization of T assay: a model study. Factors involved in the optimization of competitive binding radioassays have been analyzed using a thyroxine (T4) radioassay as a model system. The effects of kinetic errors are minimized by the use of the lowest possible incubation temperature for the separation step and by the use of labeled T4 of high purity (which is not consistently available from commerical sources). The use of labeled T4 containing significant quantities of labeled triiodothyronine (T3) can lead to a marked increase in bias and a decrease in precision with relatively small errors in the separation step due to the relatively short half-life of the T3-binder complex. The determination of the dissociation rate constant (not to be confused with the affinity constant) allows one to make useful estimates of the tolerances that can be allowed in the separation step in order to achieve a  desired level of accuracy and precison.


An isolation system for aldosterone and tetrahydroaldosterone prior to estimation by gas liquid chromatography or radioimmunoassay. We report a method of isolation of aldosterone and tetrahydroaldosterone from urine and undiluted plasma which is based on extraction with Amberlite XAD-2 resin and purification by low pressure liquid chromatography using microbore LH-20 columns. Thin-layer chromatography on polyamide plates is introduced as the final step of a procedure designed to reduce destruction of steroids to a minimum, and to provide, in addition, a facility for multisample processing. Using a synthetic substrate, it has been shown that enzyme hydrolysis of steroid conjugates is possible whilst still adsorbed to the resin after extraction although the yield is lower than when hydrolysis is performed in solution.


Solid-phase radioimmunoassay of plasma triiodothyronine. A direct radioimmunoassay method for circulating triiodothyronine was set up, using an insolubilized (cellulose-coupled) antiserum and 8-anilino-1-naphthalene-sulfonic acid (ANS) as a protein inhibitor. Particular attention was paid to the evaluation of changes in the physico-chemical properties of the antibody sites under the experimental conditions adopted: no substantial decrease of immunoreactivity was found to be associated with the insolubilization process, while complex effects following the use of protein inhibitors were observed. The standardized procedure was defined in terms of analytical reliability both by a comparison with a reference (charcoal-dextran) method and by evaluation of sensitivity, precision and accuracy.


Rapid and effective separation of thyroid hormones and iodide by thin-layer chromatography. The use of 1,3-dichlorotetrafluoroacetone allows the conversion of the iodoamino acids into a chemical form convenient for following chromatographic separation on silica as an adsorbent and benzene or chloroform as a solvent. The time required for derivatization and TLC separation is less than one hour. The outstanding resolution of the substance of interest, as well as the sensitivity of the radiochemical analysis, makes the method convenient for the rapid determination of minute quantities of thyroid hormones.


Comparison of a simplified radioimmunoassay with a gas-liquid chromatographic method for urinary aldosterone. A simplified radioimmunoassay for the measurement of aldosterone in urine, requiring only an extraction without any further purification is described. The results obtained on 47 urine specimens were compared with those obtained by a gas-liquid chromatographic method. An excellent correlation was found (r = 0.932) and values obtained by both methods were similar so that this RIA has been satisfactorily adopted in our laboratory for one year. Some discrepancies, however, led us to consider the problem of preservation and storage of biological fluids.


The effect of a short burst of exercise on activity values of enzymes in sera of healthy young men. We report the effect of exercise on the activity values of five enzymes in sera as studied in four healthy male volunteers. The underlying purpose of this present study was to produce an increase in the activity values in the sera of selected enzymes found in muscle. Then by observing the decay rate of these enzymes, we computed the inter-individual differences in clearance rates serum half-life) of these enzymes. Blood specimens were collected just prior to exercise, 1 h after excerise, and on eight additional times up to 93 h after exercise. All specimens were assayed on one occasion for activity values of creatine kinase, asparate aminotransferase, alanine aminotransferase, alkaline phosphatase, and lactate dehydrogenase. We found increases in the three muscle enzymes with average increases being: creatine kinase, +116%; asparate aminotransferase, +41%; and lactate dehydrogenase, +32%; all of which remained above baseline values for 53 h or longer. In the case of creatine kinase, a monoexponential decay curve depicted the data (from the 19-h specimen to the 67-h specimen). The calculated "apparent serum half-life" for creatine kinase varied from 38 h to 118 h in the subjects tested.


An assessment of the clinical usefulness of plasma ribonuclease assays. The usefulness of plasma ribonuclease assays was studied in (i) patients with possible protein deficiency, (ii) patients with myelomatosis, (iii) patients with carcinoma of the breast. In each group, the major factor associated with elevation of plasma ribonuclease was impairment of renal function. The assay was therefore of little value in the assessment of patients with myelomatosis or carcinoma of the breast. However, in the patients with possible protein deficiency and normal renal function, an elevation of plasma ribonuclease is, in general, associated with a decrease in serum albumin, transferrin and cholinesterase. Plasma ribonuclease may therefore be a useful parameter in the assessment of protein nutritional status.


Pregnancy associated alpha2-glycoprotein in post-menopausal women receiving hormone replacement therapy. Serum pregnancy associated alpha2-glycoprotein (P.A.G.) concentrations were measured in a group of post-menopausal women receiving hormone replacement therapy. The P.A.G. concentrations increased from a mean basal level of 48.1 mug/ml (range 20.6--104.4 mug/ml) to a maximum after six months therapy of 359 mug/ml (range 82--1290 mug/ml). This represents a mean increase of 647%. Two months after cessation of therapy the P.A.G. concentrations decreased but the mean level (60.3 mug/ml) was still significantly higher than the pre-treatment level. There was a significant correlation between the pre-treatment P.A.G. concentrations and the levels after six months of therapy. These findings may be of relevance to the use of P.A.G. determinations in the monitoring of malignant disease.


Radioimmunoassay for aldosterone and deoxycorticosterone in human plasma. Comparison of various antisera and determination of normal values. This paper describes a study of the physico-chemical and radioimmunological properties of three antialdosterone antisera which permitted practical conclusions to be drawn. By its high degree of specificity, anti-aldo-3-oxime-BSA constitutes the most useful antiserum for the clinical assay of aldosterone. The principal advantage of this antiserum is that it allows both urinary and blood aldosterone radioimmunoassay without the necessity of including a chromatographic step. No problems arise with the blanks. This work also includes the study of two anti-deoxycorticosterone antisera. The aldosterone and deoxycorticosterone values, obtained from normal subjects under various physiological conditions, are in agreement with the values given in the literature.


Effect of fatty acids on thyroid function tests in vitro and in vivo. Addition of long-chain fatty acids to serum increased thyroxine (T4), measured by a competitive protein binding assay, and triiodothyronine (T3) uptake by Sephadex or resin (T3U tests). This is compatible with the assumption that fatty acids compete with thyroxine for binding sites on T4-binding proteins. When equimolar concentrations of various saturated and unsaturated fatty acids were added to serum it was observed that the effectiveness in raising tests based on protein binding of thyroid hormones incrreased serum T3 determined by radioimmunoassay (RIA). T4(RIA) was not significantly influenced by either saturated or unsaturated fatty acids. Serum T4(CPB) rose during storage at 22degreesC and 37degreesC but was stable at 4degreesC and --20degreesC for periods up to two weeks. The proportional increase in T4(CPB) and free fatty acids (FFA) indicated that this phenomenon was due, at least partly, to the interference from FFA formed during storage of the serum. There was also a small, significant increase in T3U, T3(RIA) and CT4I (a free thyroxine estimate) after storage of serum at room temperature or higher for one to two weeks. Serum T4(RIA) did not alter during two weeks of storage. In five subjects with raised serum FFA after eating a fat meal followed by a heparin injection an increase in T4(CPB), T3U, T3(RIA) and CT4I that was proportional to the increase in FFA was observed. This effect on the thyroid tests was small until the increase in FFA concentration exceeded 2 mmol/l. T4(RIA) did not respond to the increase in FFA. In ten patients with raised levels of FFA due to uncontrolled diabetes T4(CPB), T4(RIA) and T3(RIA) decreased while T3U increased. These unexpected alterations were probably related to the severe, chronic illness in these patients. Increased FFA in vivo seem to be of little importance for the interpretation of thyroid tests in clinical practice.


A simple quantitative method to determine short chain fatty acid levels in biological fluids. A simple method has been developed for quantitative acetic, propionic, isobutyric, butyric, isovaleric, valeric, isocaproic, and caproic acids in intestinal fluids, feces and blood. The method utilizes extraction with ether and gas chromatography. It is accurate over a wide range of SCFA concentrations and appears to be applicable to any biological fluid.


The quantitation of plasma phytanic acid by mass fragmentography. Plasma phytanic acid has been quantitated by mass fragmentography after its successive reduction and derivatization to the corresponding t-butyldimethylsilyl ether. The latter was estimated by selective ion monitoring of its characteristic (M-57) ion (M/z 355). The technique has the advantage of being more rapid, specific and sensitive than existing methods, permitting the determination of phytanic acid at levels greater than 5 mug/ml plasma.


Pharmacological influences on T4 to T3 conversion in rat liver. Five groups of 8 rats were treated with normal diet, diphenylhydantoin, dexamethasone, thyroxine (T4) and phenobarbital respectively for 14 days. After this period plasma triiodothyronine (T3), T4 and reverse T3 (rT3) concentrations as well as the T4 to T3 converting activity in the liver homogenates were determined. Plasma T3 was low after dexamethasone, T4 and phenobarbital. Plasma T4 was low after dexamethasone and elevated after T4. rT3 plasma concentrations were at the lower limit of detection in all groups. The converting activity in the liver homogenate was significantly increased after diphenylhydantoin and very much depressed after dexamethasone. These results support the concept that different drugs may influence the peripheral thyroid hormone metabolism, especially the T4 to T3 conversion. However other pathways might be affected also because plasma T3 levels did not always reflect the converting activity in the liver.


The use of mass fragmentography in the evaluation of routine methods for glucose determination. A highly specific and accurate mass fragmentographic reference method for determination of serum glucose is described. A fixed amount of hepta-deuterated glucose is added to a fixed amount of serum. The mixture is lyophilized, converted into the penta-trimethylsilyl-methyloxime derivative and subjected to analysis with a combined gas chromatograph - mass spectrometer equipped with a MID-unit (multiple ion detector). The amount of unlabeled glucose was determined from the ratio between recordings at m/e 319 and 323. The two ions used correspond to the base peak in the mass spectrum of derivative of unlabeled and hepta-deuterium labeled glucose, respectively. The relative standard deviation of the method was 1.9%. The method was compared with different enzymatic methods based on the hexokinase reaction and the glucose oxidase reaction. Of the different methods tested, a glucose oxidase method with determination of maximal rate of consumption of oxygen gave results most close to the results of the reference method.


A diagnostic triad for portal cirrhosis. Abnormal serum aminotransferase activities with dominance of aspartate aminotransferase over alanine aminotransferase activity, and elevated serum adenosine deaminase activity and immunoglobulin. A concentration, were commonly encountered among patients with portal cirrhosis. The full triad was present in 31 of 49 cases (63%). As isolated abnormalities, these features were not uncommon in patients with other diseases of the liver and biliary tree, but the full triad was found only in 11 of 163 such cases (6.8%). The presence of this triad in a patient with unexplained hepatomegaly is indicative of portal cirrhosis.


Assay of plasma 25-hydroxy vitamin D.

Assay of plasma 25-hydroxy vitamin D. A radio-competitive protein-binding assay for 25-hydroxy vitamin D3 (25-OHD3) is described using diluted plasma from patients with vitamin D deficient osteomalacia as the binding protein. The 25-OHD3 is extracted from plasma with ethanol and assayed directly without further purification. An incubation of two hours is used to give suitable binding. The lower detection limit of the assay is approximately 23 pg, the intra-assay coefficient of variation is 5.4% and the inter-assay variation 15%. Tth a mean of 26.9 mug/l. Levels of plasma 25-OHD3 are undetectable in patients with vitamin D deficient osteomalacia and greater than 110 mug/l in patients taking large doses of vitamin D2.


The level and composition of seromucoid and nondialysable serum sugar-peptide fraction compared with the activity of galactosyl transferase (EC 2.4.1.38) of liver Golgi membranes in insulin-treated rats. Long-term treatment of rats with insulin does not influence the yield of the Golgi membrane-rich fraction obtained by Fleischers' method or the activity of galactosyl transferase (EC 2.1.1.38) in present in this fraction. Exogenous insulin increases the total levels of the seromucoid and serum middle-weight molecular glycopeptide fraction, but does not alter the proportions of neutral sugars bound with the peptide residues of both fractions.


Clinical studies in benign (Becker type) X-linked muscular dystrophy. Ten extensive families with benign (Becker type) X-linked muscular dystrophy have been studied, in which there was a total of 67 affected males. Reliable information was available on 41 of these males, 29 of whom were alive at the time of the study. The disorder was characterised by a predominantly proximal myopathy, associated with pseudohypertrophy of the calf muscles, particularly in the early stages of the disease. The early development of contractures was not a feature of the disease in these families and cardiac involvement, when present, was a late manifestation. The clinical findings in these patients are discussed in detail and compared with those in a large unselected group of patients with Duchenne muscular dystrophy. The results indicate that the best criterion for distinguishing between these two disorders is the age of becoming chair-bound. Electrocardiographic studies revealed no consistent abnormality and no evidence of an abnormal algebraic sum of the R and S waves in lead V1, as is found in patients with Duchenne muscular dystrophy. Serum creatine kinase levels are significantly elevated, particularly in the early stages of the disease, and in this way preclinical cases may be identified.


X-short arm deletion gonadal dysgenesis in two siblings due to unique translocation (Xp-;16p+). A family demonstrating short arm deletion of the X chromosome as a consequence of X-16 balanced translocation in the mother is reported. The two Xp- sisters exhibit clinical signs of gonadal dysgenesis, while the balanced carriers are phenotypically normal. To our knowledge this represents the only example of both the balanced carrier state for an X translocation and its genetic consequence occurring in the offspring, as well as the involvement of X-16 interchange. Literature data of 37 additional cases of verified X translocations are discussed.


Infantile XX male: a case report.

Infantile XX male: a case report. A case of infantile XX male syndrome with bilateral scrotal testes and penoscrotal hypospadias is presented. No evidence of XX/XY mosaicism of Y chromatin was obtained in preparations from cultures of the peripheral blood, skin fibroblast, or other tissues. Although true hermaphroditism was suspected, exploration of the bilateral gonadal structures failed to detect the presence of ovarian structures, either grossly or microscopically. Furthermore, exploratory laparotomy revealed no Mullerian structures. The difficulty of early diagnosis of XX males in infancy is emphasized.


Multiple none-erupting teeth, maxillo-zygomatical hypoplasia and other congenital defects: An autosomal recessive disorder. A new syndrome is described, affecting four persons in one family; it includes multiple non-erupting teeth with cementum defects, hypoplasia of the alveolar process and the maxillo-zygomatical region, genua valga and ear deformities. An autosomal recessive trait could be ascertained.


A familial syndrome of cranial, facial, oral and limb anomalies. A family is described in which two male infants have microcephaly, abnormal ears, anti-mongoloid slant, small mouth, cleft palate, flexed overlapping fingers with syndactyly of digits three and four, syndactyly of the second to the fifth toes, and normal karyotype. This seems to be a new syndrome.


Benzo-pyrones: their selective injury to rabbit vascular endothelium. 1. Three benzo-pyrones were tested in two types of high protein oedema. 2. In general, they prevented increases in permeability in superficial venules. 3. In the absence of an injurious stimulus they caused injury to the superficial capillaries and deep venules. 4. The injurious results may be due to a direct effect on the vessel walls or to an increase in intravascular pressure and flow. This would enhance leakage from already existing defects. 5. Despite the minor injuries the effectiveness of these drugs is so great that a net removal of protein by proteolysis from the tissues occurs, thus reducing the oedema. 6. The study contributes further to an understanding of the mode of action of the benzo-pyrones.


The inhibitory response of the rabbit anococcygeus muscle. 1. Strips of rabbit anococcygeus muscle were submaximally contracted by the addition of phenylephrine to the bath fluid. 2. Acetylcholine, dimethylphenylpiperazinium and electrical field stimulation caused relaxation. Relaxation induced by acetylcholine and field stimulation was blocked by tetrodotoxin but only the acetylcholine-induced response was inhibited by lignocaine, tetraethylammonium and hexamethonium. The responses to both acetylcholine and field stimulation were resistant to atropine, practolol, and sotalol. 3. Relatively high concentrations of ATP caused relaxation. 4. Histological investigation did not show any cells resembling autonomic ganglion cells. 5. It is suggested that both acetylcholine and field stimulation release an inhibitory transmitter from nerve endings and that the release by acetylcholine is mediated through nicotinic receptor stimulation.


Effects of calcitonin on the secretion of pancreatic juice induced by dopamine, secretin and pancreozymin. 1. Effects of calcitonin on dopamine-, secretin- and pancreozymin-induced pancreatic secretion were investigated in the isolated blood-perfused canine pancreas. 2. The volume of pancreatic secretion induced by pancreozymin given intra-arterially (i.a.) was decreased by an i.a. infusion of 1 u/min of calcitonin, but that induced by dopamine or secretin given i.a. was not affected by calcitonin treatment. 3. Amylase concentration in pancreatic juice either in spontaneous secretion in the resting state or in that of stimulated secretion by pancreozymin was decreased approximately 30% by calcitonin treatment, but amylase concentration in pancreatic juice induced by dopamine or secretin was not affected by calcitonin treatment. 4. Calcitonin had no effect on bicarbonate concentration in pancreatic juice stimulated by these secretagogues. 5. Calcium concentration in pancreatic juice in the resting state was reduced about 36% by calcitonin treatment. Calcitonin caused a decrease in a calcium concentration in the pancreozymin-induced secretion, but did not cause any change in the dopamine- or secretin-induced one. 6. These results suggest that calcitonin may affect the secretory mechanism of the acinar cells but not that of the ductular cells, and that the acinar cells are active even in the resting state.


Effect of secretin on pacemaker activity and contractility in the isolated blood-perfused atrium of the dog. 1. The effects of secretin on inotropic and chronotropic activity were investigated in nine isolated canine atrium preparations which were suspended in a bath and perfused with arterial blood from a carotid artery of a heparinized donor dog. 2. Secretin administered into the cannulated sinus node artery in a dose range of 0-1-10 units produced a dose-related positive inotropic and a biphasic chronotropic effect. 3. The positive chronotropic and inotropic responses to secretin were not suppressed by treatment with alprenolol in doses which blocked responses to noradrenaline. 4. The negative chronotropic response to secretin was not blocked by atropine in doses which blocked the response to acetylcholine. 5. After treatment with glucagon, secretin produced dose-related negative chronotropic and a positive inotropic effects. Thus glucagon may antagonize the positive chronotropic effect of secretin. 6. From these results, it is concluded that secretin has a direct effect on atrial rate and contractility.


Differing effects of the vasodilator drugs, prazosin and diazoxide on plasma renin activity in the dog. 1. The effects of intravenous (i.v.) administration of the vasodilator drugs prazosin or diazoxide on blood pressure and plasma renin activity were evaluated in the anaesthetized dog. 2. Prazosin and diazoxide both induced a rapid reduction in the mean arterial pressure to 73% and 75% of control values respectively. 3. Prazosin lowered plasma renin activity to 62% (P less than 0-025) of the control value whereas diazoxide raised plasma renin activity to 178% (P less than 0.05) of the control value. 4. The combination of vasodilatation and low renin activity observed following the administration of prazosin is unique, and may have clinical significance if these factors reduce the vascular complications of hypertension.


Renal vasodilation in response to coronary artery ligation in the dog. 1. The effects of coronary artery ligation on renal vascular resistance were determined with and without pretreatment with methylprednisolone succinate or atropine in anaesthetized dogs. 2. With coronary artery ligation alone renal blood flow is maintained constant in spite of a reduction in arterial blood pressure, indicating renal vasodilation. 3. Pretreatment with methylprednisolone succinate prevents the fall in arterial blood pressure. 4. After pretreatment with atropine, renal blood flow falls in response to the reduction in arterial blood pressure, suggesting inhibition of cholinergic renal vasodilatation.