Influence of PGE1 on renal function in severe hemorrhagic shock.

The influence of intravenously administered PGE1 on renal function in standardized hemorrhagic shock in dogs was examined in this study. Infusion rates as high as 1.04 micrograms/min/kg were evaluated. Although arterial blood levels as high as 2.47 ng/ml of plasma compared to control values of less than 0.20 ng/ml were attained during postreinfusion treatment, no beneficial influences on renal functional parameters (hemodynamics, electrolyte and water handling) were observed. In fact, treated animals took up blood from the arterial reservoir more quickly and expired sooner following blood transfusion than an untreated series. A further deleterious change in renal function was a decrease in renal concentrating capability. It is concluded that with the severe grade of hemorrhagic shock employed in these studies, organ blood perfusion was restricted to the extent of limiting effective PGE action.


Correlation between skeletal muscle vascular decompensation and survival: roles of tissue ischemia and innervation.

A constant-flow, cross-perfused, vascularly isolated gracilis muscle preparation was used to examine the hypothesis that locally produced and released products of ischemic muscle metabolism are responsible for the vascular decompensation (vasodilation) reported to occur in late oligemic hypotension. Well-oxygenated donor arterial blood perfused recipient gacilis muscles at a constant flow rate of 5.2 +/- 0.5 ml/100 gm/min while the recipient animals were subjected to a modified Wigger's hemorrhage protocol. Arterial and venous blood gases taken across the gracilis muscle at regular intervals during the experiments verified adequate tissue perfusion. Of the thirteen studies reported, only ten shocked recipient dogs progressed to irreversible shock postreinfusion. This series was identified as the "recipient-irreversibly shocked group." The remaining three shocked animals recovered from the shock protocol and were labeled "recipient-reversibly shocked series." The initial response to blood loss in both groups was intense vasconstriction, with the greatest initial constriction occurring in the irreversibly shocked series. The three animals that survived the protocol were able to sustain this compensatory effort, but the ten that ultimately progressed into irreversible shock postreinfusion invariably demonstrated a significant loss of vascular tone during late oligemia (conductance rose from 43% to 63% of control). Thus evidence is presented which indicates decompensation during adequate tissue perfusion, or absence of ischemia. A strong correlation was also shown to exist between sustained compensatory vasoconstriction in the gracilis muscle, and survival. The suggestion is made that part of the loss of vascular tone may be related to prejunctional inhibition of adrenergic transmission or alpha-receptor fatigue, with a minor role being played by the vasodepressor products of local tissue ischemia.


On the protective role of the liver in the hypotensive state following intestinal ischemia.

Regional intestinal hypotension (arterial inflow pressure about 30 mmHg) was induced in cats by partially occluding the superior mesenteric artery with an adjustable clamp. The superior mesenteric vein was cannulated and the intestinal venous outflow recorded by a drop counter. The intestinal venous blood bypassed in one series the liver and was returned to the animal via the jugular vein. In two other series it was returned via the portal vein and in one of these also the hepatic arterial pressure was kept at 30 mmHg. Following two hours of regional intestinal shock a general cardiovascular derangement was evident in all series. This was not influenced by directing the intestinal venous blood flow through a normotensive or hypotensive liver. Characteristic intestinal mucosal lesions were found in all series. It is concluded that bypassing or directing the intestinal venous blood through a normotensive or hypotensive liver did not affect the local and general cardiovascular effects of regional intestinal hypotension.


Analysing parent-child interactions in clinical practice: the development of a procedure and method of reporting.

Research in child developmental psychology is concerning itself increasingly with the fine-grain study of infant-caregiver interactions and as a result awareness of the importance of early interaction patterns in the development of children has grown very rapidly. The research has been based on an observational approach made possible by the use of video-recording and the development of sophisticated methods of interaction analysis. The methods employed and the research findings have important implications for those working clinically with young handicapped children and their parents, but as yet the research methods have been too complex and time-consuming to be used directly in practice. This paper describes the development of a procedure which can be used to observe and analyse the interactions of pre-school handicapped children with their caregivers in a clinical setting, using modifications of existing research methods and based on current findings. Examples of the coding procedure, transcript sheet and reporting methods are given in the context of a pilot application. It is suggested that the approach is valuable as a conceptual framework which the observer can use to improve his observational skills, provides a sound base for assessment and remediation work and can incorporate new research findings as they become available.


Catecholamines and hemorrhagic shock in awake and anesthetized rats.

Catecholamines in plasma and tissue were determined during hemorrhagic shock in the rat. Two groups of rats were compared. 1. Awake rats bled to 70 mm Hg for 4 hours. 2. Anesthetized rats (pentobarbital sodium 60 mg/kg) bled to 35 mm Hg for 4 hours. The mortality rate was similar in both groups. The bled volume was also similar. The awake rats responded with tachycardia upon bleeding while the anesthetized rats responded with bradycardia. The basal plasma levels of noradrenaline (NA), adrenaline (A) and dopamine (DA) in the awake rats were 2.87, 4.09, and 0.51 nmol/l respectively and in the anesthetized rats 0.97, 0.54, and 0.56 nmol/l respectively. At the onset of bleeding there was a more rapid increase of plasma A and NA in the awake rats than in the anesthetized rats. In the awake rats plasma A reached its peak value (70 nmol/l) at 1 hour and then decreased, while NA showed a slow continuous rise to 17 nmol/l at 4 hours. In the anesthetized rats plasma A remained at a high level (about 60 nmol/l) between 1 and 4 hours, while there was a continuous rise of NA to 17 nmol/l at 4 hours. In these rats a very high DA level (17 nmol/l) was also found at 4 hours. The tissue content of NA was not significantly decreased in the heart while a significant decrease was seen in the skeletal muscle after bleeding for 4 hours. In the heart there was a substantial increase of A after bleeding. The A content of the adrenals decreased to about 25% of the initial value in the awake animals. The results show that barbiturate anesthesia considerably depresses the initial sympatho-adrenal response to bleeding.


Meldreth dribble-control project.

Seven subnormal spastic children aged 15--16 years, who had the additional problem of profuse dribbling, were trained to associate an auditory cue, from a small box pinned to their clothes, with swallowing. A significant reduction in dribble rate was obtained for all children within the first week. This method of dribble control has two advantages: it requires very little professional time, and the child's ability to control his dribbling is emphasized from the beginning.


Reversal by calcium of rat heart cell dysfunction induced by human sera in septic shock.

Twenty human sera obtained during the first 24 hours following clinical onset of different septic shocks were evaluated for their effects on electric an mechanical activities of rat heart cell cultures. Possible antagonistic action of calcium was tested on these effects. The results showed that septic shock sera, when compared to normal sera, increased action potential duration and depressed contractility of beating cardiac cells. Addition of calcium reversed these two actions, though markedly higher concentration was needed for depressed contractility. The in vitro deleterious effects of septic shock sera on cell cultures were related neither to decrease in extracellular calcium, since total calcium level was moderately below normal values, nor to inhibition of low calcium channel, since the action potential was not suppressed in presence of K+ excess. This study suggests the involvement of humoral factors mimicking the cardiac effects of low calcium concentration. If calcium is involved in in vitro cardiac dysfunction, it could result only from a decrease of available calcium in cytosol.


Rate of rise of myocardial PCO2 during early myocardial ischemia in the dog.

We have investigated the rate of rise of myocardial PCO2 (PmCO2) after coronary artery occlusion using a new method for this measurement. Previous studies of PmCO2 have been limited by the slow response of the only available method, and no increase in MmCO2 prior to 3 minutes after occlusion has been found. We have implanted a miniature PCO2 electrode, with a 63% response time of 14 seconds, into the left ventricle of 14 open-chest dogs. After abrupt coronary occlusion, PmCO2 began to rise in 13.6 +/- 1.1 seconds in heparinized dogs and in 7.5 +/- 0.7 seconds in unheparinized dogs. The subsequent magnitude of the increase in PmCO2 was 24, 88, 171, and 222 mm Hg at 2, 5, 10, and 15 minutes after occlusion. The rate of rise of PmCO2 was essentially linear from 1 minute to 10 minutes at a rate of 18.3 mm Hg/min. The rate of rise was slower during the first 30 seconds after occlusion (6.1 mm Hg/min) and also from 30 seconds to 1 minute (9.7 mm Hg/min). This rate of rise is much greater than that previously observed and reflects the severe myocardial acidosis developing during ischemia. A rise in PmCO2 is one of the earliest metabolic changes that has been observed during myocardial ischemia.


Decreased myocardial contractility in papillary muscles from atherosclerotic rabbits.

To determine the effect atherosclerosis has on myocardial contractility, we studied the contractile properties of right ventricular papillary muscles from 34 atherosclerotic and 17 control rabbits. We produced atherosclerosis by feeding for 2 to 8 months a diet of 5% lard, 5% peanut oil, 0.5% cholesterol, and 89.5% rabbit pellets. The controls received only rabbit pellets during the same time interval. Contracting isometrically 12 times per minute at 25 degrees C, muscles from the atherosclerotic rabbits developed tension at a lower maximum rate (max dT/dt), had a longer latency, and required longer to develop tension at the maximum rate and to develop peak tension. In isotonic contractions, they shortened with lower maximum velocities and required longer to accelerate to maximum velocity and to shorten maximally. We found no evidence that developed tension or distance shortened differed between the two groups of muscles. Raising the contraction frequency to 24 contractions per minute between the two groups of muscles. Raising the contraction frequency to 24 contractions per minute brought performance of the two groups of muscles closer in both types of contraction. Norepinephrine (1.5 x 10-5 M) nearly abolished differences between performance of the two groups. The loss of contractility correlates poorly with coronary and aortic atherosclerosis. It occurred early in the feeding of the atherogenic diet. We think it was due to a lipid-induced defect in the cardiac cell's handling of calcium.


Normal levels of fibrinopeptide A in patients with primary hyperlipidemia.

Fibrinopeptide A (FPA) levels were measured in a group of 130 controls and patients with various types of primary hyperlipidemia to investigate whether an increased steady state level of thrombin activity is present in hyperlipidemic patients. In a subset of 56 subjects, levels of clotting factors II, VII, and X were measured as well. FPA levels in hyperlipidemic patients were not significantly different from those of control subjects. Furthermore, on multiple regression analysis, no significant relationships were found between FPA levels and the concentrations of serum cholesterol or triglyceride, or log triglyceride levels. Statistically significant relationships were found between all three clotting factor levels and triglyceride concentration. The correlation coefficients for these relationships, however, were low, so that the correlations are of questionable pathophysiological significance. A weak relationship also was found between the plasma levels of cholesterol and of factor II. Thus, although small increases in various clotting factors may be found in patients with hyperlipidemia, plasma FPA levels are normal. These data indicate that hyperlipidemia is not associated with a steady state of increased thrombin activity in vivo in humans.


Effects of ischemia on tissue metabolites in red (slow) and white (fast) skeletal muscle of the chicken.

Brief periods of ischemia have been shown to produce marked reactive hyperemia in both red (slow) and white (fast) skeletal muscle. However, evidence is lacking for specific vasodilator metabolites which are rapidly produced in ischemic skeletal muscle. The present study examined the effects of 1 and 3 minutes of ischemia on creatine phosphate (CrP), adenine nucleotide metabolism, and anaerobic glycolysis in red anterior (ALD) and white posterior latissimus dorsi (PLD) muscles of the chicken. Tissue metabolite concentrations were determined from perchloric acid or trichloroacetic acid extracts using enzymatic assay or high pressure liquid chromatography. CrP or adenine nucleotides were not significantly altered in either muscle following 1 or 3 minutes of ischemia. However, adenosine increased by 611% in the ALD at 1 minute. Following 3 minutes of ischemia, adenosine concentrations were elevated by 439% and 201% in the ALD and PLD, respectively. The PLD showed the greatest increases in inosine and IMP. Inorganic phosphate increased by 67% and lactate increased by 142% in the ALD at 3 minutes. The PLD, which is reported to have a high anaerobic glycolytic capacity, showed no increase in lactate. These results support the hypothesis that adenosine may be a mediator of akeletal muscle reactive hyperemia following short periods of ischemia.


Circus movement in canine right ventricle.

The present study reports on the epicardial spread of excitation during premature beats and during the initial stages of ventricular fibrillation, both of which were induced by single-test stimuli during regional ischemia or local hypothermia. Simultaneous recording of the activity at 48 epicardial sites on the right ventricle of dog hearts enabled us in some instances to demonstrate a circus movement.


The prolonged pressor response to renin in the nephrectomized rat.

Angiotension I dose-response curves and renin clearances were studied in nephrectomized and paired sham-nephrectomized control rats under pentobarbital anesthesia. Both threshold and slope of the angiotensin dose-response curves were decreased 22 hours after nephrectomy. In addition, the ratio of renin clearance (determined during renin infusions) in the 22-hour-nephrectomized rat to that in paired 22-hour sham-nephrectomized controls was 0.50 +/- 0.03 (mean +/- SEM, P less than 0.001, n = 12 pairs). The finding of reduced renin clearance was confirmed by an indirect assessment of "effective renin clearance" based on a comparison of the blood pressure decline after renin injections with angiotensin I dose-response curves in the same rat. Overall, approximately half of the 50% fall in renin clearance could be accounted for by an immediate effect of removal of the kidney on renin clearance. This role of the kidney in renin clearance was confirmed by the finding of a renal venous-arterial renin ratio of 0.9 +/- 0.03 (P less than 0.005) during renin infusion in normal rats. It is concluded that both changes in the angiotensin I dose-response curve and decrease in plasma renin clearance contribute to the postnephrectomy prolongation of the renin pressor response in the rat.


Platelet kinetic studies in patients with hyperlipoproteinemia: effects of clofibrate therapy.

Studies of platelet and fibrinogen kinetics in 27 patients with hyperlipoproteinemia and 28 control subjects demonstrated shortened platelet survival and increased platelet turnover in seven patients with type III and 10 patients with type IV-V hyperlipoproteinemia (p less than 0.01). There was no correlation between platelet survival time and specific lipid levels, vascular disease, sex or age. Platelet kinetics were not significantly altered from control values in eight patients with familial hypercholesterolemia. Platelet aggregation studies and fibrinogen kinetic measurements did not differ in any of the hyperlipoproteinemic groups of patients from those in control subjects. Despite significant changes in plasma lipids induced by clofibrate, platelet survival was significantly extended only in patients with type IV-V hyperlipoproteinemia (p less than 0.05). These results are consistent with the hypothesis that atherogenesis in patients with types III--V hyperlipoproteinemia may be associated with a process of endothelial desquamation, and type IIa hyperlipoproteinemia may involve nondesquamating endothelial injury.


Platelet function studies in heart disease. VI. Enhanced platelet aggregate formation activity in congestive heart failure: inhibition by sodium nitroprusside.

We studied 11 patients with congestive heart failure and 10 normal volunteers for in vivo platelet aggregate formation activity. The patients with heart failure had significantly (p less than 0.01) more circulating platelet aggregates than the normal volunteers. During sodium nitroprusside infusion, the number of circulating platelet aggregates declined to normal levels and in vitro platelet aggregation responses to epinephrine and adenosine diphosphate were also suppressed significantly (p less than 0.01). This was associated with a 30% decline in systemic vascular resistance and a 28% increase in cardiac output. In other in vitro experiments, sodium nitroprusside was found to have direct, dose-related platelet aggregation inhibitory actions. This study suggests that an increase in vascular resistance in certain heart failure patients may in part be related to an increase in circulating platelet aggregates. Direct inhibition of platelet aggregation by sodium nitroprusside may be a mechanism of its beneficial effects in heart failure.


Left ventricular volume from paired biplane two-dimensional echocardiography.

To evaluate the applicability of two-dimensional echocardiography to left ventricular volume determination, 30 consecutive patients undergoing biplane left ventricular cineangiography were studied with a wide-angle (84 degrees), phased-array, two-dimensional echocardiographic system. Two echographic projections were used to obtain paired, biplane, tomographic images of the left ventricle. We used the short-axis view (from the precordial window) as an anolog of the left anterior oblique angiogram, and the long-axis, two-chamber view (from the apex impulse window) as a right anterior oblique angiographic equivalent. A modified Simpson's rule formula was used to calculate systolic and diastolic left ventricular volumes from the biplane echogram and the biplane angiogram. These methods correlated well for ejection fraction (r = 0.87) and systolic volume (r = 0.90), but only modestly for diastolic volume (r = 0.80). These correlations are noteworthy because 65% of the patients had significant segmental wall motion abnormalities. The volumes determined from the minor-axis dimensions of M-mode echograms in 23 of the same patients correlated poorly with angiography.


Left ventricular volumes by gated equilibrium radionuclide angiography: a new method.

To compare radionuclide end-diastolic (EDV) and end-systolic (ESV) volumes with angiographic volume, we studied 52 patients with equilibrium radionuclide angiography using 99mTc-human serum albumin within 48 hours of contrast angiography. Each RR interval was divided into 20--28 equally timed frames and a time-activity curve generated. End-diastolic counts were taken at the early peak of the curve and end-systolic counts at its nadir. Counts were divided by the total number of processed heart beats and normalized for: 1) dose per body surface area; 2) plasma volume; and 3) counts/ml of plasma. A cardiac phantom was developed and serial volumes were studied using a normalization factor. Radionuclide values were expressed as dimensionless units and compared with either biplane angiographic volumes (in the patient studies) or known phantom volumes. Good correlations were obtained with methods 1 and 2 in 35 patients (r greater than 0.84), but the best correlation was obtained in 17 patients when normalization for counts/ml of plasma was used (r = 0.98; y = 0.255 x -0.121). The standard error of the estimate (SEE) was +/- 11.5 ml for EDV and +/- 7.3 ml for ESV. The phantom study also showed an excellent correlation (r = 0.99), with a SEE of +/- 6.5 ml. We conclude that a radionuclide method independent of geometric assumptions can be used to estimate left ventricular volume in man.


Early malfunction of transvenous pacemaker electrodes. A three-center study.

A 3-year study by three medical centers has revealed a 1-year electrode malfunction rate of 7.4%; most malfunctions occurred within the first 30 days. The incidence of unavoidable early malfunction (3.2%) fell within the 5% standards suggested by the committee report of the Inter-Society Committee on Heart Diseases. Incidences of obscure cause (3.2%) may be difficult to identify prospectively and may be, to a certain extent, unavoidable. The majority of the malfunctions (4.2%) showed specific clues that indicated that they were preventable. Successful repositioning was achieved on the first attempt in 80.6% of the cases with malfunction, and only 0.7% required ultimate myocardial electrode implantation. The principal clues to potentially unsatisfactory positioning included the presence of a large right ventricle with or without tricuspid insufficiency, current thresholds greater than 0.5 mA and ST-segment deviations on the intracardiac electrogram of less than 2 mV. Electrode malfunction may be more common with bipolar than with unipolar electrodes; but significant differences in the incidence of malfunction among different unipolar electrodes were observed. These data indicate that further developments in transvenous electrode design are warranted.


The estimation of sinoatrial conduction time in rabbit heart by the constant atrial pacing technique.

This study compared estimates of sinoatrial conduction time (SACT) obtained by constant atrial pacing (CAP) and premature atrial stimulation (PAS) with measured SACT in isolated rabbit right atrial preparations. Transmembrane potentials and surface electrograms were recorded from the sinus node and crista terminalis, respectively. The crista terminalis was paced 5, 10 and 15 beats/min faster than the spontaneous sinus rate with a train of eight pulses. Estimate of SACT by CAP was taken as the difference between the first atrial return cycle and the mean spontaneous cycle length. SACTs at 5, 10 and 15 beats/min faster were 76 +/- 10, 86 +/- 10 and 96 +/- 10 msec (mean +/- SEM; n = 12), respectively; correlation coefficients with the true SACT were 0.7, 0.54 and 0.4. Consecutive determinations of SACT by PAS and CAP in the same preparation (n = 6) at 10 beats/min faster gave SACTs of 86 +/- 13 and 79 +/- 14 msec, respectively, compared with true SACTs of 79 +/- 10 msec. Shortening of sinus node action potential, depression of automaticity and shifts in the site of the primary pacemaker contributed to the errors in both techniques. Estimation of SACT by CAP may be further complicated by failure of sinus node capture. Principles to minimize some of these errors are also presented.


Reentrant ventricular arrhythmias in the late myocardial infarction period. 7. Effect of verapamil and D-600 and the role of the "slow channel".

Reentrant ventricular arrhythmias (RVA) were analyzed in dogs 3--7 days after ligation of the anterior descending coronary artery using averaged "composite" recordings of electrical activity of reentrant pathways (RP) from the epicardial surface of the infarction zone (IZ). Verapamil (V) and D-600 (D) (0.2--0.5 mg/kg i.v.) resulted in slight-to-moderate improvement of conduction in RP with abolition of spontaneous RVA and RVA initiated by premature depolarizations. The effect of V was not blocked by pretreatment with propranolol (0.5 mg/kg i.v.). Using a standard microelectrode technique and strips of epicardial muscle from the IZ, D (0.5--1 X 10(-6) g/ml) slightly improved the upstroke velocity and membrane responses of depressed ischemic cells. In contrast, tetrodotoxin (5 X 10(-7) g/ml) further depressed or abolished action potentials of ischemic cells. We conclude: 1) the moderate antiarrhythmic effect of V and D on RVA is the result of improved conduction in RP; 2) this action is partly explained by improvement of a depressed sodium channel and is not related to catecholamine release; 3) slow-response action potentials play no significant role in the genesis of ischemia-related RVA, which probably results from depression of the fast response.


Straddling and displaced atrioventricular orifices and valves.

This is an anatomic study of 96 hearts with straddling or displaced atrioventricular (AV) valves and orifices. In the complete form, both the annulus and the peripheral connections of either AV valve straddle a ventricular septal defect (VSD) and connect to both ventricles. In the annular form, only the annulus, and in the peripheral type only the peripheral connections of the valve are found in both ventricles. In displaced AV valve, the entire annulus and periphery of one AV valve are displaced into the opposite chamber. These anomalies are commonly seen in complete transposition with or without ventricular inversion, and in double outlet right or left ventricle. Straddling mitral valve is frequently seen in the Taussig-Bing heart. Any type of VSD may be associated with straddling tricuspid valve; however, they usually are of the AV canal type. Straddling and displaced AV valves should be differentiated from criss-cross hearts in which both AV valves are completely connected to oppositely placed ventricles.


Concealed nonparoxysmal junctional tachycardia.

Electrophysiologic studies were performed in a patient who had an apparently uncomplicated complete trifascicular block. His bundle recordings revealed atrioventricular dissociation with: 1) an atrial rate of 58 beats/min, 2) an idioventricular escape rate of 45 beats/min, and 3) nonparoxysmal junctional tachycardia (His bundle rhythm) at a rate of 65--85 beats/min. The latter arrhythmia was electrocardiographically silent, influencing neither atrial nor ventricular events. The arrhythmia probably reflected digitalis intoxication (digoxin level of 3.3 ng/ml). A repeat electrophysiologic study 4 days after digoxin was discontinued revealed complete trifascicular block (distal to H) with intact conduction between the atrium and the His bundle (AH of 150 msec). Thus, electrophysiologic study demonstrated an electrocardiographically silent but clinically relevant arrhythmia, suggesting that His bundle recording should be part of diagnostic study during temporary pacemaker implantation in patients with atrioventricular block.


Atrial flutter with exit block.

The mechanism of atrial flutter is controversial. A 76-year-old woman with rheumatic heart disease was referred to our clinic with an unusual rhythm disturbance which initially appeared to be classic atrial flutter at a rate of 300 beats/min. Later tracings, however, demonstrated a rate exactly one-half that of the earlier ECGs, with an identical p-wave morphology and vector. This latter rhythm also behaved in a manner expected for a flutter mechanism in that both spontaneously and with carotid pressure high-degree atrioventricular block occurred without alteration of the underlying atrial mechanism. Finally, the two rates interchanged spontaneously over several days without any significant interval changes in medical therapy. These findings were initially explained as probable digoxin toxicity. The underlying mechanism, however, was more likely atrial flutter with exit block and in this patient may have represented another facet of her sick sinus syndrome. This unusual phenomenon is discussed in terms of previous reports and possible implications for the mechanism of atrial flutter.


Determination of naproxen and its desmethyl metabolite in human plasma or serum by high performance liquid chromatography.

1. A liquid chromatographic method has been developed for the simultaneous determination of the anti-inflammatory agent naproxen and its metabolite, 6-0-desmethyl-naproxen, in human plasma or serum. 2. After addition of p-chlorowarfarin as the internal stardard, plasma is acidified and extracted with either chloroform (for assay of naproxen only) or ethyl acetate (for assay of naproxen and desmethyl-naproxen), the organic solvent is evaporated, the residue is dissolved in acetonitrile and injected onto a reverse phase column coupled with an ultra-violet absorbance detector. 3. Drug and metabolite can be detected readily in concentrations above 2 micrograms per ml in 0.5 ml samples. Salicylic acid and its metabolites and a number of other durgs were found not to interfere with the assay.


Use of Beckman BUN analysers to determine urine urea.

In this paper we report a method to enable the Beckman BUN analyser to be used for urine urea nitrogen determinations. Satisfactory urine samples could be obtained by diluting the urine with 10 volumes of 0.85% saline. We also report our experience with the use of reagent prepared in house and with a modified pipette, to reduce the cost of using the instrument.


Influence of two preparations of bovine albumin on the total bilirubin as determined by Thomson's method in its reference solution.

The influence of bovine albumin on the determined total bilirubin was examined by estimation of bilirubin in standard solutions prepared with albumin solutions of five different concentrations. The determinations were made by six different methods. The results show that when bilirubin was determined by Thompson's method the estimated bilirubin decreases by increasing the concentration of albumin. This discrepancy appears in preparations from two different industrial sources of albumin.


Biochemical parameters of normal rabbit serum.

An attempt was made to establish the normal range of 27 different parameters in the serum of 45 healthy, purebred, New Zealand White rabbits (3-5 kg). The reproducibility of the results was acceptable. Sensitive isoenzyme patterns (LDH, CK) could change, however, significantly if different methods were used in the bleeding procedure. Isoenzyme patterns (LDH and CK) of rabbit sera were different from that of human sera. All obtained results were compared with earlier data sporadically published in literature on rabbits.


Crossed immuno-electrodiffusion in the diagnosis of immunoglobulin fragment abnormalities.

The use of crossed immuno-electrodiffusion in the diagnosis of alpha-chain disease is described. The same basic technique is applicable in diagnosis of diseases in which immunoglobulin fragment abnormalities may occur. Crossed immuno-electrodiffusion is shown to be a superior method for demonstrating the abnormaility by providing more powerful resolution. Immunoselection (an adaptation of the classical method of immuno-electrophoresis) or rocket immuno-electrodiffusion both have limitations which are overcome by the use of crossed immuno-electrodiffusion. Mixture of antibodies is avoided by separating antibody-containing gels with interposition of intermediate gel layers free of antibody. Furthermore, precipitin rockets of some specific proteins can also be identified morphologically.


Gas-chromatographic analysis for chlorpromazine and some of its metabolites in human serum, with use of a nitrogen detector.

We describe a gas-chromatographic method for measuring chlorpromazine and its metabolites chlorpromazine sulfoxide, mono-N-desmethylchlorpromazine, and di-N-desmethylchlorpromazine at therapeutic concentrations in human serum, with use of a nitrogen detector. The compounds are extracted from serum at pH 10.5 into hexane/isoamyl alcohol, re-extracted into dilute HCl, and then extracted into hexane after alkalinization of the HCl. The N-desmethylated metabolites are measured as their respective N-trifuloracetyl derivatives; the parent drug and its sulfoxide are measured as the unchanged bases. Promazine is the internal standard. As little as 5 micrograms of chlorpromazine, 20 micrograms of chlorpromazine sulfoxide, 20 micrograms of mono-N-desmethylchlorpromazine, and 10 micrograms of di-N-desmethylchlorpromazine per liter can be measured in 2-mL samples of serum. The within-run coefficients of variation for assays of these drugs at 100 micrograms/L are 2.7%, 5.6%, 5.1%, and 5.3%, respectively. The procedure was applied to patients receiving therapeutic doses of chlorpromazine and to patients who had ingested an overdose of chlorpromazine.


Chemical assay, involving liquid chromatography, for aminoglycoside antibiotics in serum.

We describe a chemical assay involving "high-pressure" liquid chromatography for the quantitative determination of three aminoglycosides: netilmicin, tobramycin, and gentamicin. The drugs are separated from serum by means of precipitation of the serum proteins with acetonitrile after dilution with a buffer. The aminoglycosides are quantitatively extracted into the supernate, which is further purified by a two-step partition procedure involving derivatization of the drugs with o-phthalaldehyde. The drug derivatives are separated by reversed-phase chromatography and detected by on-line fluorometry. Sensitivity is 1 mg/L for tobramycin and 0.5 mg/L for netilmicin and gentamicin. Intra- and interassay variation was below 8%. Analytical recovery of each of the three drugs was 92 to 100%. Correlation with microbiological and radioimmunological assay methods was good. The assay is rapid (about 30 min), precise, and specific, and seems suitable for use in a routine clinical laboratory.


Non-chromatographic radioimmunoassay procedure for urinary aldosterone.

We describe a sensitive, specific, and simple procedure for measuring aldosterone in human urine, which requires no chromatographic purification before quantification by radioimmunoassay but does include hydrolysis and extraction steps. Rabbit anti-aldosterone serum is sued, generated against aldosterone-18,21-dihemisuccinate coupled to human serum albumin. The antibody cross reacted little with other structurally related steroids that are in human urine. Our procedure was validated by comparing values for urinary aldosterone in human urine, with and without preliminary purification by chromatography on either paper (y = 0.92x + 2.9; r = 0.99; p less than 0.01) or (Sephadex LH-20) column (y = 0.98x + 0.6; r = 0.99; p less than 0.01). Values by our procedure also correlated well (y = 1.03x - 0.8; r = 0.99; p less than 0.01) with those obtained with use of a validated commercial "kit" for urinary aldosterine. All reagents for the proposed method are available commercially.


Use of specific inhibitors to disciminate alkaline phosphatase isoenzymes originating from human liver, placenta and intestine: absence of meconial alkaline phosphatase in maternal serum.

We used the inhibitors bromotetramisole, L-phenylalanine amide, and L-phenylalanine in combination to measure intestinal phosphatase in maternal serum and amniotic fluid. By using high concentrations of these inhibitors, it was possible to measure the three isoenzymes separately. We found no evidence of the presence of meconial alkaline phosphatase in the serum of the mother (six cases) after meconial passage in utero.


Urinary 3-methoxy-4-hydroxymandelic acid as measured by liquid chromatography, with on-line post-column reaction.

We describe a method for measurement of 3-methoxy-4-hydroxymandelic acid (vanillylmandelic acid, VMA) in urine. After the pH of the urine is adjusted to 2.7 and the sample is filtered, exactly 15 microL is injected onto a C-18 reversed-phase column. VMA is eluted from the column with 10 mmol/L phosphate buffer, pH 2.7, containing 30 mL of acetonitrile per liter. The eluate stream is combined with alkaline periodate and then passed through a 60 degrees C water bath. The VMA is completely oxidized to vanillin, which is detected and quantitiated by its absorbance at 360 nm. No deterioration of the column was noted after 167 such injections of urine samples. Long-term control data indicate a CV of 12 and 10% at VMA concentrations of 1.5 and 5.8 mg/L, respectively. Although results correlate well (r = 0.976) with those by the method of Pisano et al. [Clin. Chim. Acta 7, 285 (1962)], they average 10% lower. Of 20 compounds tested, only methyl dopa interfered with the procedure as described.


X-ray fluorescence and potentiometry compared for determining iodine content of thyroid glands.

We compare results for iodine quantitation by x-ray fluorescence of excised thyroid glands with results of the quantitation by means of an original, newly developed chemical method, a Schöniger combustion technique, in which iodie is quantitated potentiometrically. Subsequently, we establish the accuracy and clinical suitability of the former technique in quantifying the intrathyroidal iodine.


Performance assessment of the GammafloTM automated radioimmunoassay system by assaying for digoxin.

We report an evaluation of the GammafloTM automated continuous-flow radioimmunoassay instrument in which we used a digoxin assay to assess system performance. System operation was based on combined continuous-flow and column-chromatographic techniques. No drift or carryover was detectable in 180 within-assay consecutive determinations performed at a rate of 42 determinations per hour (5 h of continuous operation). Within-assay and between-assay precision were less than 6% (coefficient of variation). The automated method correlated well (r = 0.960 and 0.952, respectively) with two established manual digoxin radioimmunoassay procedures. The data suggest this automated system offers a valid alternative to manual radioimmunoassay procedures in terms of overall precision, simplicity of operation, and sample throughout capacity.


Decreased aminotransferase activity of serum and various tissues in the rat after cefazolin treatment.

Treatment of rats with cefazolin in vivo significantly suppressed activity of alanine and aspartate aminotransferases in serum and in the liver, brain, kidney, and heart. Simultaneous administration of pyridoxal further reduced enzyme activity except in the liver, where there was no change. Pyridoxal 5'-phosphate partly reversed the decreased enzyme activity in the serum, liver, and kidney, but did not return it to the amount observed in the control animals; enzyme activity remained suppressed in the brain and heart. The effect of cefazolin was dose related, but there was no sex-related difference. In contrast to its action on am-notransferase activity, cefazolin elicited no effect on alkaline phosphatase (pyridoxal-5'-phosphate hydrolase) in serum or on pyruvate carboxylase in the liver, heart, and kidney. Cefazolin exposed to the hepatic microsomal mixed-function oxidase system in vitro was partly converted into metabolites that inhibited serum alanine aminotransferase activity in vitro. The latter inhibition was reversed by the addition of pyridoxal 5'-phosphate.


Creatine kinase B-subunit activity in serum after immunoinhibition of M-subunit activity.

Creatine kinase (EC 2.7.3.2) B-subunit activity in serum may be routinely measured as residual activity after specific immunoinhibition of the M-subunit. We assessed the inhibition kinetics, specificity, completeness of inhibition, and inhibitory capacity of three different anti-M preparations, with use of isolated human BB, MM, and MB isoenzymes. The Scandinavian-recommended reaction system was used. We suggest a set of tentative quality requirements for anti-M for use in diagnosing acute myocardial infarction. The need to measure and subtract sample residual adenylate kinase activity was demosntrated. We describe a routine photometric method for determining B-subunit activity in serum. With the Scandinavian CK method the upper reference value for total creatine kinase in serum was found to be 150 U/L for women, 270 U/L for men. By bioluminescence, we found the upper reference value for B-subunit activity to be 6 U/L for both sexes. We discuss three different modes for applying B-subunit determinations to the diagnosis of acute myocardial infarction.


Urinary excretion of conjugated homovanillic acid, 3,4-dihydroxyphenylacetic acid, p-hydroxyphenylacetic acid, and vanillic acid by persons on their usual diet and patients with neuroblastoma.

We report quantitative data on beta-glucuronidase- and sulfatase-hydrolyzable conjugates of homovanillic acid, 3,4-dihydroxyphenylacetic acid, p-hydroxyphenylacetic acid, and vanillic acid in the urine of 20 apparently normal and healthy control persons and of three patients with neuroblastoma. We used organic solvent extraction and capillary gas chromatography. There was considerable person-to-person variation in the conjugation percentages calculated. Mean conjugated percentages of the four compounds for 16 normal healthy persons 2.5--40 years of age were, respectively, 12%, 33%, 14%, and 35%. For newborns and patients with neuroblastoma, these percentages were somewhat different. Increased amounts of vanillic acid were found in the urine of the patients with neuroblastoma, but results of a small metabolic study in rats suggest that this increase most probably is of dietary origin.


Comparison of acid phosphatase isoenzymes of human seminal fluid, prostate, and leukocytes.

We used ion-exchange column chromatography and electrophoresis on polyacrylamide gel to compare the acid phosphatase isoenzymes of prostate and leukocytes. The major isoenzyme of the prostate is band 2A; only a trace of band 2B was observed. However, the major isoenzymes of leukocytes are band 4 and band 2B, and only a small amount of band 2A was observed. The three isoenzymes isolated from leukocytes or prostate gland react to the antiserum prepared against the aicd phosphatase isoenzyme of seminal fluid. Acid phosphatases of leukocytes other than the three isoenzymes mentioned above did not interact with the antiserum.


Improved liquid-chromatographic method for determination of serum cortisol.

We describe a specific and precise method for measuring concentrations of cortisol in serum or plasma by liquid chromatography. Cortisol, together with an internal standard, equilenin, is extracted from 1 mL of serum or plasma and analyzed isocratically on a reversed-phase column with a mobile phase of acetonitrile/phosphate buffer (30/70, by vol.), at a flow rate of 2.0 mL/min. The eluted cortisol is detected by its absorption at 254 nm and quantitated by peak height measurements. Each analysis requires no longer than 15 min at the optimum column temperature of 50 degrees C. The lower limit of detection for cortisol is about 2 ng/sample for a standard solution; sensitivity is routinely 5 micrograms/L of serum. Analytical recoveries exceeded 95%, with good day-to-day precision (coefficients of variation between 4 and 7%). Of more than 50 drugs and steroids tested for possible interference, only the steroids cortisone, prednisone, and prednisolone may interfere with the analysis of cortisol.


Micro-scale method for liquid-chromatographic determination of chloramphenicol in serum.

We describe the use of "high-performance" liquid chromatography to measure chloramphenicol in as little as 25 microL of serum. Serum is treated to precipitate proteins with acetonitrile containing p-nitroacetanilide as an internal standard. Chloramphenicol is eluted with a mobile phase of methanol in pH 7.0 phosphate buffer (35/65 by vol). The drug is measured at 278 nm and simultaneously monitored at 254 nm; interfering substances are detected by examining the 278 nm/254 absorbance ratios. This method is sensitive to less than 0.5 mg/L and the standard curve is linear to at least 50 mg/L. Inter-day precision ranged between 3--6%. We encountered no interference from endogenous compounds or from other drugs we tested.


Measurement of Erythrocyte 2,3-diphosphoglycerate with a centrifugal analyzer.

Intra-erythrocytic concentration of 2,3-diphosphoglycerate is a major determinant of the oxygen affinity of hemoglobin. We report here the adaptation of its assay to a centrifugal analyzer, with use of a commercially available reagent. Results are calculated by using the reagent-blank-corrected absorbance change at 340 nm between 120 and 300 s for the samples and a 2.5 mmol/L standard. Under these conditions the standard curve is linear to 5 mmol/L. The compound in a 101-fold aqueous hemolysate is stable for several weeks at either -4 or -70 degrees C. Assay sensitivity and precision are excellent and results agree well with those by the corresponding manual method.


Diabetic ketoalkalosis.

The usual metabolic derangement in uncontrolled diabetes mellitus is metabolic acidosis, with an increase in the anion gap because of increased ketoacids and lactate. However, diabetic ketoalkalosis may occasionally be encountered, the prominent clinical feature of which is vomiting, with depletion of potassium, chloride, and hydrogen ions. Self-medication with absorbabe alkali may also contribute to the alkalosis. It would be dangerous to treat hyperlgycemic patients with alkali if their condition is ketoalkalosis instead of ketoacidosis.


Liquid-chromatographic measurement of taurine in cerebrospinal fluid of normal individuals and patients with meningitis.

Taurine was measured in cerebrospinal fluid by reacting it with fluorescamine to form a fluorescent derivative, followed by separation on a reversed-phase column and fluorometric detection and evaluation. The assay is rapid (17 min) and sensitive to as little as 1 mumol/L. The mean value for 27 cerebrospinal fluid samples collected from patients free from meningitis and aneurysm was 5.7 +/- 1.8 mumol/L. Twenty-two patients with bacterial meningitis showed a 0- to 20-fold increase in cerebrospinal fluid taurine, with a return to normal values after antibiotic treatment.


Improved determination of phenobarbital, primidone, and phenytoin by use of a preparative instrument for extraction, followed by gas chromatography.

A microprocessor-controlled automatic extractor, (PREMTM I) is described with which lipophilic components may be extracted from physiological fluids by means of a selective, solid-phase extraction cartridge, and the extracts presented in dry form for subsequent analysis. This instrument has been successfully evaluated for the extraction of phenobarbital, primidone, and phenytoin from human serum before chromatographic analysis. We describe a specific method for determination of these anticonvulsants, involving the PREP I and analysis, after flash-heater methylation, in a gas chromatograph equipped with a nitrogen-sensitive detector. Absolute recoveries ranged from 90 to 102%. Within-day and day-to-day precision (CV) ranged from 4 to 7% for all three drugs in therapeutic concentrations. Comparison of this method with results obtained with a liquid--liquid micro-scale extraction and a similar gas-chromatographic method resulted in correlation coefficients of 0.988 (phenobarbital), 0.978 (primidone), and 0.982 (phenytoin). These drugs in therapeutic concentrations can be simultaneously extracted and measured with confidence.


Development of a stable lipoprotein diluent for use in reconstituting lyophilized human serum for the preparation of clear, hyperlipidemic quality-control materials.

We describe a simple system for the preparation of a hyperlipidemic control serum. Beta- and pre-beta-lipoproteins are removed from the serum and prepared as a stable diluent; the extracted serum is then lyophilized. Upon addition of the lipoprotein diluent, which was observed to contain only liproprotein and sodium bicarbonate, the serum reconstitutes rapidly, usually within 5 min. By a suitable adjustment of the lipoprotein concentration in the diluent, a hyperlipidemic control serum may be produced with desired concentrations of lipids. Because the lipoproteins are not included in the destructive lyophilization step, the resulting product has remarkable clarity, precision, and stability.


D-Mannose in human serum, measured as its aldononitrile acetate derivative.

We report a procedure for determining D-mannose in serum. After removal of proteins and lipids, the carbohydrate-containing fraction is treated to form the aldononitrile acetate derivative and analyzed by gas--liquid chromatography with a flame-ionization detector. D-Mannose and D-glucose had retention times of 31 and 34 min, respectively. Day-to-day reproducibilities (CV) of between 2 and 12% for mannose were attainable for quantities of 100--900 ng. Related hexoses and derivatives do not interfere. The sensitivity was such that 10 mg/L could be detected in a 0.1-mL sample of serum. This method may be of use in diagnosing invasive Candida infection.


Spectrophotometric measurement of carboxyhemoglobin and methemoglobin in blood.

This paper describes separate spectrophotometric procedures for rapidly measuring carboxyhemoglobin (COHb) and methemoglobin (MetHb) in blood. Absorbance measurements are made in the Soret region at a blood dilution near 1000-fold. For COHb estimation, the diluent contains sodium hydrosulfite, providing the two-component system COHb--Hb for absorbance measurements at 420 and 432 nm. The NA2S2O4 effectively prevents dissociation of COHb by oxygen. For MetHb estimation, the diluent contains KCN and carbon monoxide, providing the two-component system COHb--CNMetHb. Absorbance measurements are made at 420 nm, before and after addiely analyzed for the total of all MetHb forms present. Each procedure requires about 3 muL of blood and can be applied to human or animal blood. Results obtained by the present methods are in satisfactory agreement with currently accepted procedures, which require much larger samples or more elaborate equipment.


Inter- and intra-individual variations in the saliva/blood alcohol ratio during ethanol metabolism in man.

The inter- and intra-individual components of variation in the saliva/blood alcohol ratio have been calculated from experiments with 48 male subjects after they drank 0.72 g of ethanol per kilogram of body weight as neat whisky after a short fast. Saliva and blood ethanol profiles were monitored at 30--60 min intervals for up to 7 h after intake. The analytical component of variation inherent in an estimate of the saliva/blood alcohol ratio, expressed as coefficient of variation, was 1.75%. I calculated saliva/blood ethanol ratios for each subject at each sampling time by taking the antilogarithm of the difference (log saliva alcohol--log blood alcohol). The mean ratio between 60 and 360 min after drinking was 1.077 (n = 336) with 95% confidence limits of 1.065 and 1.088. Moreover, the individual ratios showed no systematic variation throughout the absorption, distribution, and elimination phases of ethanol metabolism. Using a two-way analysis of variance and allowing for analytical sources of variation, I determined that the inter- and intra-subject variance components were 53 and 47% of the total biological variation. The saliva/blood alcohol ratio during ethanol metabolism, determined once in a single individual, had a biologically derived coefficient of variation of 10%.


Micro-scale enzymic determination of ethanol in plasma with a discrete analyzer, the ABA-100.

An enzymic method for estimating ethanol has been adapted to the Abbott Bichromatic Analyzer-100. Fully automated, the method can accommodate up to 192 assays per hour. It requires only 2.5 muL of plasma and therefore is applicable to analysis for ethanol in blood sampled by finger-prick.


Evaluation and application of magnetizable charcoal for separation in radioimmunoassays.

We compared conventional charcoal separation of antibody-bound and free antigen with a novel method involving magnetizable particles containing charcoal. The magnetizable charcoal separation method was as effective as the conventional method for a wide range of radioimmunoassays, including those for several steroids and small polypeptide hormones. In a detailed comparison of conventional vs. magnetizable charcoal for radioimmunoassay of aldosterone, the magnetizable charcoal had several advantages: it did not require prior coating with dextran or protein, varied less with time and temperature, required no centrifugation, and took less time for separation. The correlation of results obtained with magnetizable and conventional charcoal separation was excellent (n = 89, r = 0.98, p less than 0.001), and the coefficient of variation for the separation with magnetizable charcoal was 0.5%.


Gas-chromatographic analysis of patterns of fatty acids of cholesteryl esters and phosphatidylcholine.

We have developed a quick, reliable method for compositional analysis of the C14 to C20 fatty acids of cholesteryl esters, based on the use of a double extraction-solvent system and whisker-walled capillary columns with a new polar phase. Fatty acids of phosphatidylcholine, after separation by thin-layer chromatography, were determined on the same column. Our normal values are in good agreement with those published previously. We found marked changes in the fatty acid composition of both cholesteryl esters and phosphatidylcholine in cord blood serum, in various disease states, and in post-mortem specimens. The previously reported correlation between oleic (18:1) and linoleic (18:2) acids in serum cholesteryl esters was confirmed for normal adults, but no such correlation exists in cord blood specimens. We also found a significant correlation between the relative amounts of fatty acids in cholesteryl esters and phosphatidylcholine for all unsaturated fatty acids and for palmitate in all groups of subjects.


Creatine kinase BB: a new tumor-associated marker.

Creatine kinase isoenzyme BB (CK-BB) is found in the serum of patients with various types of cancer. Using both radioimmunoassay and agarose electrophoresis, we found abnormal amounts of this isoenzyme in the serum of 15 of 17 patients with untreated prostatic carcinoma. Among patients with other types of adenocarcinomas and metastatic disease, serum CK-BB was increased in most of those who were unresponsive to therapy. In benign or malignant prostate tissue and in malignant pleural effusions, cytoplasmic CK-BB as determined by immunoperoxidase staining was found in epithelial cells. Prostatic fluid had high concentrations of CK-BB, as did malignant, but not benign, pleural fluid supernates. We conclude that glandular epithelial cells contain CK-BB, which is released in benign and malignant states and may appear in higher concentrations in the circulation in malignant states. These conclusions are consistent with predictions we have made from a model experimental system concerning characteristics of possible tumor markers. The observations indicate a role for CK-BB as a tumor marker, particularly for adenocarcinoma of the prostate.


Routine glucose determination in serum by use of an immobilized glucose dehydrogenase nylon-tube reactor.

We report a method for immobilizing glucose dehydrogenase on the inside surface of nylon tubes to produce an immobilized-enzyme nylon-tube reactor. The glucose dehydrogenase reactor is integrated into the flow system of a continuous-flow analyzer to facilitate routine analysis of serum glucose at 50 samples/h. We compared results with those by the reference hexokinase/glucose-6-phosphate dehydrogenase solution method. The coefficient of correlation was r = 0.996. A glucose dehydrogenase reactor made starting with 1 mg (250 U) of enzyme was stable during eight weeks of continuous use, that is, for nearly 3500 tests. This reduced the cost of the assay by at least 50-fold, compared with that for a commercial glucose dehydrogenase test pack method.


A simplified, colorimetric micromethod for xylose in serum or urine, with phloroglucinol.

We have developed a simplified xylose assay procedure that requires only 10 min and requires 50 microL of serum or 5 microL of urine. The reaction with phloroglucinol is more sensitive than the classic p-bromaniline color reaction, and requires only 4 min of heating for color development. A single reagent is mixed with the specimen directly, without prior protein precipitation. Analytical recovery of xylose added to serum was quantitative; precision studies resulted in a between-day coefficient of variation of 5.2%. Glucose, which has significant potential for interference in most other xylose procedures, reacts under the test conditions only to the extent of 70 mumol of apparent xylose per liter for a 5.5 mmol/L solution of glucose. The new procedure has been valuable in the assessment of malabsorption, especially in children and infants, where serum xylose is the preferred measurement.


Mechanized determination of the apparent unbound unconjugated bilirubin concentration in serum.

The peroxidase method for determining the apparent unbound bilirubin concentration in serum has been automated by use of a programmable, computer-directed spectrophotometer. This mechanized assay determines the total bilirubin concentration and apparent unbound bilirubin concentration in serum samples and titrates the serum with bilirubin to estimate the effect of increasing total bilirubin concentrations on the apparent unbound bilirubin concentration. The entire analysis requires 0.1 mL of serum and 4 min operation time, as compared with about 30 min for the manual method. The coefficients of variation for determination of the apparent unbound bilirubin concentration in bilirubin-enriched commercial control serum were 2.8% within-day and 5.6% between-day. Bilirubin--albumin binding in serum samples from infants with severe hyperbilirubinemia was analyzed by the manual peroxidase method, the automated peroxidase method, and Sephadex gel filtration. Good correlation was found among all three methods.


Separation of five isoenzymes of serum lactate dehydrogenase by discontinuous gradient elution from a miniature ion-exchange column.

We describe a simple, fast chromatographic technique for quantitatively separating the five isoenzymes of lactate dehydrogenase (LD; EC 1.1.1.27) in serum. A 250-microL serum sample is applied to a 6.0 x 0.7 cm column of QAE-Sephadex A-50 and eluted stepwise with five different buffers. The isoenzyme fractions, assayed by the method of Wroblewski and LaDue [Proc. Soc. Exp. Biol, Med. 90, 210 (1955)], are stable at room temperature for 24 h. For all five isoenzymes the average within-day coefficient of variation is 4.3%; the day-to-day CV for 20 days is 6.3%. Of some common potentially interfering substances tested, only sodium fluoride (238 mmol/L) was found to do so, by slowing the elution from the column and making the fractions turbid. The expected range in international (IUB) united and percent for each isoenzyme was determined from data on 73 men and 70 women. From these data we calculate, by a percentile estimate of a nongaussian distribution, a normal range of LD-1/LD-2 ratio of 0.55--0.87 for men and 0.52--0.91 for women. The LD isoenzyme patterns in both normal and above-normal samples of 147 sera, as evaluated by the present column method and by electrophoresis, correlated well.


Heparin interferes with albumin determination by dye-binding methods.

Heparin, when added in vitro at rather high concentrations (400--1000 kilounits/L) as an anticoagulant, interferes with albumin determination by bromcresol green by forming an insoluble precipitate with this dye. The interference is much less with bromcresol purple. It can be abolished by addition of hexadimethrine bromide.


An improved micromethod for theophylline determination by reversed-phase liquid chromatography.

We describe an improved micro-scale method for determining serum theophylline by reversed-phase liquid chromatography. Samples are deproteinized with two volumes of methanol containing beta-hydroxyethyltheophylline as an internal standard. The supernate is chromatographed with a methanol/sodium acetate buffer (15/85 by vol) mobile phase, and the amount of theophylline is calculated from the ratio between peak heights for theophylline and the internal standard at 273 nm. We also monitor for chemical interferences at 254 nm. We found no interferences from ampicillin, methicillin, cephalosporin, and cephalothin. None of the drugs tested or metabolites and compounds related to theophylline have been found to interfere. The method is rapid, accurate, sensitive, and specific for theophylline.


Assay of plant sterols by use of cholesterol oxidase.

The cholesterol esterase/cholesterol oxidase enzyme kit can be used for analysis of plant sterols. The results are comparable to those obtained by conventional colorimetric analysis.


Enzyme-labeled immunosorbent assay for serum ferritin: method evaluation and comparison with two radioassays.

We report our evaluation of a commercially available procedure and reagents for determination of ferritin in serum by enzyme-labeled immunosorbent assay (ELISA). Results by our procedure and the "Fer Iron" (Ramco Labs) procedure shows a degree of association (r) of 0.95 and a regression equation of y = 1.03x - 33. Similarly, our procedure compared to the "Gamma Dab" (Clinical Assays) ferritin procedure shows a degree of association of 0.98 and a regression equation of y = 0.93x - 11. Between-day standard deviations were 6 and 22 micrograms/L (n = 24 and 20) for ferritin concentrations of 20 and 300 micrograms/L, respectively. Ferritin values showed no correlation with total iron concentration, but show a broad inverse relationship with iron-binding capacities. The favorable correlation with existing procedures and the speed of the analysis commend the use of ELISA for measurement of ferritin in serum.


A bichromatic method for total bilirubin with a CentrifiChem 400.

We describe a method for the bichromatic determination of total bilirubin with a CentrifiChem 400 centrifugal analyzer with the Boehringer DPD kit. After reading the blank at 620 nm, we read the test at 550 nm. Because only one rotor is required, the analytical time for bilirubin estimations is halved. The proposed method is no more prone to interference by hemoglobin or turbidity than a conventional approach or a reference continuous-flow method and has performed well over several months of routine use.


Low proportions of glycosylated hemoglobin associated with hemoglobin S and hemoglobin C.

Using a cation-exchange chromatographic method, we found normal or subnormal values for glycosylated hemoglobin in a few diabetic patients with persistent hyperglycemia. Subsequent investigations revealed that these unexpected results had originated from black patients with diabetes. In view of common occurrence of abnormal hemoglobins in the Negro population, we subjected blood preparations to electrophoresis on cellulose acetate and acrylamide gel. The results have shown the presence of hemoglobin S or hemoglobin C in each patient. When allowance was made for the percentage of the abnormal hemoglobin, the "corrected values" of glycosylated hemoglobin increased to the diabetic range. Furthermore, the corrected values agreed well with the "expected values" calculated from a regression line correlating fasting blood glucose concentrations and proportions of glycosylated hemoglobin in more than 300 diabetics with no evidence of hemoglobinopathy. We conclude that in diabetic patients presenting with hemoglobin S or hemoglobin C, there is a considerable decrease in the values for glycosylated hemoglobin as measured by cation-exchange chromatographic methods, and that this decrease is proportional to the percentage of the abnormal hemoglobin.


Hyperxanthinemia interferes with serum uric acid determinations by the uricase method.

Serum xanthine concentrations as high as 148 mg/L were noted after treatment of a patient with Burkitt's lymphoma who was receiving allopurinol. These markedly above-normal values for xanthine led to spuriously low values for serum uric acid as measured by the uricase method. Rapid tumor lysis in patients who are receiving allopurinol may lead to marked hyperxanthinemia, which in turn may obscure hyperuricemia in such patients when the uricase method is used for uric acid analysis. In such situations, uric acid concentrations should be measured by the phosphotungstate colorimetric assay.


An evaluation of the use of electrophoresis and carbon furnace atomic absorption spectrometry to determine the copper level in separated serum protein fractions.

The results of an evaluation of the use of atomic absorption spectrometry with carbon furnace atomisation to analyse the copper content of individual protein fractions separated by electrophoresis is reported. Albumin copper levels in patients with rheumatoid arthritis were found to be about 1 to 2% of the total serum copper. Both albumin and alpha 2 levels are affected by contamination from residual copper present on the strip. An investigation of this contamination indicated very low blank copper levels in the areas of the strip containing alpha 1 and beta proteins. These results, and the results of analysis of copper in the ultrafiltrate from the same serum samples, are combined to define the copper distribution in a group of patients with rheumatoid arthritis. The determinations that can most readily be carried out by this technique are discussed.


Dicarboxylic aciduria: the response to fasting.

The urine of a child who presented with an episode of a disease resembling Reye's syndrome was found to contain large quantities of the dicarboxylic acids adipic and suberic acids, as well as the glycine conjugate of suberic acid, suberyl glycine. A variety of other dicarboxylic acids, both saturated and unsaturated, were also found in the urine at the time of the attack. It was found that the excretion of these unusual metabolites could be markedly increased by fasting for periods of greater than 10 h. These results indicate that the patient may have a defect in fatty acid oxidation which becomes clinically significant during periods of prolonged fasting.


Immunochemical determination of two trypsins in human duodenal juice.

An application of electroimmunoassay to the separate determination of anionic and cationic trypsin in human duodenal juice is presented. The proportions of anionic to cationic immunoactive trypsin in duodenal juice from a group of children averaged 20 : 80. The ratio of immunoactive to esterolytically (BAPNA) active trypsin averaged 1.6 : 1, indicating the presence of inactive forms of trypsin in duodenal juice.


An assay for human erythrocyte catechol-O-methyltransferase activity using a catechol estrogen as the substrate.

A radiometric assay for catechol-O-methyltransferase (COMT) activity in human erythrocytes is described that employs 2-hydroxy[3H]estrone, and non-radiolabeled S-adenosylmethionine (SAM) as the cosubstrates. The ease of separation of the product of the reaction, 2-methoxy[3H]estrone from 2-hydroxy[3H]estrone makes it possible to achieve low reaction blanks. The assay is very sensitive, and only 200 microliter of whole blood are used per determination. The assay is highly reproducible. The interassay variability (coefficient of variation) was 6.5% for 24 assays of COMT activity in red blood cells in blood obtained daily for 24 days from one person. In incubations conducted at 37 degrees C for 30 min, the catechol-O-methyltransferase activity was a linear function of enzyme concentration (equivalent to 11 to 180 microliter of packed red blood cells). Employing this assay, we evaluated the catalytic conversion of 2-hydroxyestrone to 2-methoxyestrone by catechol-O-methyltransferase from human red blood cells and found that the apparent Michaelis constant and the apparent maximal rate of reaction were 3 x 10(-7) M and 6.7 x 10(-9) mol . ml-1 erythrocytes . h-1, respectively. The catechol-O-methyltransferase activity measured in erythrocytes obtained from 100 healthy subjects (men and nonpregnant women) was 8.2 +/- 0.17 (mean +/- S.E.) nmol 2-methoxyestrone . ml-1 erythrocytes . h-1.


Urinary excretion of 3-methylhistidine and creatine in carriers of Duchenne muscular dystrophy.

The mean molar ratio (0.0171) of 3-methylhistidine to creatinine in 24-h urine samples from carriers of Duchenne muscular dystrophy did not differ significantly from that of controls. The creatine/creatinine ratio was also normal.


Articular cartilage, blood serum glycosaminoglycans and glycoproteins in osteoarthritis deformans.

Glycosaminoglycans in articular cartilage from human femoral heads have been isolated and fractionated by means of a cellulose microscale technique. The glucosaminoglycans have been identified by different procedures as keratan sulphate, hyaluronic acid, heparitin sulphate, chondroitin 4-sulphate, chondroitin 6-sulphate and dermatan sulphate. Osteoarthritic cartilage showed a significant reduction of dermatan sulphate, chondroitin 6-sulphate, keratan sulphate and heparitin sulphate. The content of glycoprotein carbohydrate components in the cartilage (neuraminic acid derivatives, hexoses, 6-deoxyhexoses) also appeared to be reduced. It has been shown that chondroitin 4-sulphate, keratan sulphate and dermatan sulphate content was considerably increased in osteoarthritic serum. Among serum glycoprotein carbohydrate constituents only the content of 6-deoxyhexoses was slightly increased.


Fetal tissue amino acid concentrations in argininosuccinic aciduria and in "maternal homocystinuria".

Free amino acid concentrations have been measured in the tissues of a fetus at risk for argininosuccinic aciduria and of an obligate heterozygous fetus in a mother homozygous for homocystinuria. Argininosuccinic acid was detected in all tissues studied of the homozygous affected fetus from the heterozygous mother. Abnormal concentrations of methionine and cystathionine were observed in the tissues of the fetus who was an obligate heterozygote for homocystinuria. These abnormal free amino acid concentrations occur early in fetal development and may be related to later brain dysfunction.


Identification of a non-specific carboxylesterase in human pancreas using vinyl 8-phenyloctanoate as a substrate.

Human pancreatic tissue, pancreatic juice and sera of patients suffering from acute pancreatitis contain a vinyl 8-phenyloctanoate hydrolysing activity which was separated from true pancreatic lipase (EC 3.1.1.3). The enzyme, preliminary called "non-specific pancreatic carboxylesterase, was partially purified from human pancreatic tissue by DEAE-cellulose chromatography. Its molecular weight was found to be 54 000 by gel filtration on Sephadex G-100. The isoelectric point was estimated as 4.65 by isoelectric focusing. The results explain the poor correlation obtained when determinations of "serum lipase activity" using triolein and vinyl 8-phenyloctanoate as substrates are compared. However, since non-specific pancreatic carboxylesterase is liberated into the serum, determination of this new enzyme provides additional information in the diagnosis of pancreatic diseases.


Preservation of whole blood samples for creatine kinase isoenzyme MB estimations in the routine laboratory.

Activity of creatine kinase isoenzyme MB was maintained for 5 days at 4 degrees C in 5 ml whole blood collected in specimen bottles containing 9.5 mg EGTA and 2 microliters beta-mercaptoethanol for glutathione-activated CK-MB assays and 7.5 mg EDTA for N-acetyl-L-cysteine-activated CK-MB assays.


An ultra-micro method for the determination of total nitrogen in biological fluids based on Kjeldahl digestion and enzymatic estimation of ammonia.

An ultra-micro method for the determination of the total nitrogen-content of biological fluids and suspensions is described, based on a digestion in sulphuric acid and a enzymatic determination of the ammonia formed with glutamate dehydrogenase (EC 1.4.1.3). The proposed method yields the same results as the classical Kjeldahl procedure, but is less time-consuming. The detection-limit of the nitrogen, without loss of precision and accuracy, is much lower than in the original Kjeldahl procedure, and is in the order of 35 ng N per sample.


Dynamic deviation control in clinical chemistry laboratories based on the time variant behaviour of analytical processes.

Based on the time variant behaviour of analytical processes, a dynamic system of deviation control is discussed. Originating from industrial process control, measurability and controllability rules as measures of the optimum reconstructing and controlling efficiency of quality control systems are introduced. It is seen that when trying to reconstruct and control and analytical process, properties of the process and characteristics of the control system need to be taken into consideration. Application of these rules offers the possibility of investigating the effect of actions aimed at improving the performance of a controlling system, but also indicates if a certain precision requirement can be achieved with less effort and expense. An example illustrates the time variant behaviour of an analytical process used for urea determination in a clinical chemistry laboratory. It shows the applicability of measurability and controllability rules, with the emphasis on the measuring or reconstructing efficiency of the controlling system used in that laboratory for the surveillance of the urea determination.


Determination of D-penicillamine in serum and urine of patients with rheumatoid arthritis.

A chromatographic auto-analyser method is described for the determination of total D-penicillamine in biological fluids. After oxidation with performic acid, D-penicillaminic acid is separated from other ninhydrin-positive acidic amino acids by anion-exchange chromatography. Total cysteine/cystine is simultaneously determined as cysteic acid, which makes the method equally suitable for patients with cystinuria. The detection limit in serum and urine is 2 microM D-penicillamine, which is more than sufficient for clinical application. The metabolite S-methyl-D-penicillamine is oxidized by performic acid to a sulphone, which can be determined after separation by cation-exchange chromatography.


Water movement in red cells from burned patients. Its relationship to sodium retention and red cell filtrability.

Nine burned patients were investigated. Urinary sodium retention was associated with low red cell water and reduced red cell filtrability. The red cells from patients who recovered lost less water than normal cells when suspended in hypertonic NaCl solution but the proportionality of water loss or gain found for normal cells was maintained. The red cells from fatally burned patients lost proportional behaviour in hypertonic or hypotonic media. The abnormalities are not related to the area of the burn and may be the result of varying therapy. The monitoring of red cell water and water movement under tonicity stress is a convenient measure of the metabolic state of the cell.


Change in bile secretion in patients with calcium carbonate in their gallstones.

Common duct bile in patients with gallstones contains virtually no amylase activity. Therefore the raised levels of total CO2 concentrations found in common duct bile from patients with some calcium carbonate on the surface of their gallstones do not result from a reflux of pancreatic juice. A change in bile secretion is the likely explanation.


The recordings and cerebral properties of unaveraged electroretinograms in routine EEG's.

The occurrence of single unaveraged ERG activity in routine EEG's is documented in this report. The ERG activity is focal at Fp1 or Fp2 and often restricted to these electrodes. Mean values of latency and amplitude are 51.5 mSec and 31.8 uv. Significant differences with age, sex and light intensity are shown identical to those of corneally-recorded ERG's.


EEG's in an alcohol detoxification and treatment center.

EEG's were carried out on two hundred and one (201) alcoholics admitted to a four week treatment program. The majority were grossly intoxicated on admission and presented the usual differential problems during the first few days of hospitalization. In spite of therapeutic doses of medication before testing, 85% had totally normal EEG's and only 5.5% revealed clearly abnormal tracings. Of those for whom repeat records were later obtained, four of six with an abnormality showed a persistance of abnormality suggesting underlying organic dysfunction and not just "intoxication". EEG has a place in identification of the alcoholic patient at greater risk for neurological complications and may be useful in alerting the psychiatrist to obtain additional personal and family history, neurological examination, and when appropriate, further tests for potential structural abnormality. Routine use of structural tests such as computerized scan will aid in the identification of serious structural abnormalities but will not identify equally serious disorganizing functional abnormalities.


Pituitary-gonadal dysfunction in low-output cardiac failure.

Diminished cardiac output and digoxin therapy appear to have opposing effects on testosterone, oestradiol and luteinizing hormone concentrations. Depression of the levels of these hormones was significantly correlated with a decrease in cardiac index and elevation was noted by digoxin therapy of long duration. The effect of cardiac output is most marked on testosterone and that of digoxin on oestradiol.


Effect of the angiotensin II antagonist saralasin on plasma aldosterone concentration and on blood pressure before and during sodium depletion in normal subjects.

The effect of the angiotensin II antagonist saralasin on plasma aldosterone, plasma angiotensin II and blood pressure was studied in six normal supine subjects both before and during sodium depletion. Before sodium depletion, infusion of saralasin produced no consistent changes; during sodium depletion, infusion of the angiotensin antagonist caused a fall in plasma aldosterone and an increase in plasma angiotensin II in each subject. It is concluded that angiotensin II plays a major part in stimulating aldosterone secretion during sodium depletion in man.


Presence of positive feedback between oestrogen and LH in patients with Klinefelter's syndrome, and Sertoli-cell-only syndrome.

In five adult patients with Klinefelter's syndrome and two with Sertoli-cell-only syndrome, exogenous administration of 17 beta-oestradiol, resulting in a sustained and prolonged elevation of circulating oestrogen levels, had a biphasic effect on LH release. After an initial decrease, a dramatic rise in peripheral levels of LH (positive feedback) was observed in all patients. The timing and magnitude of the induced LH surge was similar to that observed in postmenopausal women after administration of large doses of Ethinyloestradiol. A similar positive feedback effect on the secretion of FSH could not be demonstrated. In two patients with the Sertoli-cell-only syndrome, oestrogen treatment induced marked variations in serum testosterone levels, which appeared to be related to the LH changes. Similar changes were not present in patients with Klinefelter's syndrome, indicating a decrease in the sensitivity of the testicular Leydig cell. These experiments demonstrate that positive feedback between oestrogen and luteinizing hormone is present in the adult human male.


Individual neurophysin concentrations in the pituitary and circulation of humans.

Specific, homologous human neurophysin I and II radioimmunoassays were established and used to measure the individual, immunoreactive neurophysin concentrations in human plasma. Circulating levels of human neurophysin I in normal individuals were less than 1 ng/ml and neurophysin II levels were 1-2 ng/ml. During dehydration, there was a significant rise in plasma neurophysin I, together with an increase in neurophysin II. Haemorrhage also was associated with a rise in plasma neurophysin I and II, but the percent increase was greater for I than II. In two subjects in whom nicotine inhalation caused a rise in plasma neurophysin I, there was no detectable increase in plasma neurophysin II. These stimuli which have been reported to release vasopressin from the posterior pituitary also are associated with the differential release of neurophysin I. Plasma neurophysin II levels could more clearly be shown to rise independently of plasma neurophysin I during events thought to be related to oxytocin release. Plasma neurophysin II levels were significantly elevated in women taking oral contraceptives. Similarly during pregnancy there was a progressive rise in plasma neurophysin II concentration which was proportional to the period of gestation. Plasma neurophysin II concentrations in seven of fifteen nursing women rose significantly during suckling. There was no  detectable change in plasma neurophysin I concentrations during any of these events. Plasma neurophysin I and II levels were both significantly elevated in fourteen patients with chronic renal failure and rose over haemodialysis, suggesting that the kidney may be the major route of clearance of the neurophysins. In humans the independent release of neurophysin II was associated with stimuli thought to release oxytocin, but neurophysin I showed only a differential release compared to neurophysin II in vasopressin stimulated events.


The relationship between the 'idiopathic oedema syndrome' and subclinical hypothyroidism.

Thyroid function has been studied in eleven patients with the 'idiopathic oedema syndrome'. Six patients have been shown to have subclinical hypothyroidism associated with the presence of thyroid antibodies. All responded to treatment with sodium thyroxine. Five patients had normal thyroid function, absence of thyroid antibodies and failed to respond to treatment with sodium thyroxine.


Hormonal regulation of the menstrual cycle in women with breast cancer: effect of adjuvant chemotherapy.

The hormonal features of the menstrual cycle determined by measurement of serum LH, FSH, oestradiol, progesterone and prolactin were normal in thirteen breast cancer patients clinically disease free and not taking anti-tumour therapy. Results of similar studies during seven menstrual cycles in four patients on an adjuvant therapy programme of L-phenylalanine mustard (L-PAM) + 5-flurouracil (5-FU) also had normal hormonal characteristics after three to fourteen courses of chemotherapy. Amenorrhoea with elevated gonadotrophins occurred after seven courses of chemotherapy in one 48-year-old woman. Adjuvant chemotherapy does not appear to result in major alterations in hormone secretion during menstrual cycles of women who continue to menstruate while on therapy.


Exchange of triiodothyronine derived from thyroxine with circulating triiodothyronine as studied in the rat.

At present it is widely assumed that T3 derived from T4 is rapidly and totally exchangeable within the volume of distribution of T3 secreted by the thyroid into the bloodstream. This concept is implied when conclusions are drawn from comparisons between a biological effect in a responsive tissue and circulating T3 and T4 levels. Such conclusions are often in conflict with those derived by comparing the biological effect with the concentrations of T3 and T4 in the responsive tissue itself. Thus, it appeared important to test the above assumption directly. Thyroidectomized rats have been treated for 4-4 1/2 days with a mixture of 131I labelled T4 (131T4) and 125I labelled T3 (125T3), which was either injected twice daily or administered by continuous i.v. infusion. The rats were bled, perfused, and their plasma and tissues submitted to extraction and paper chromatography. If the tested assumption were correct, the ratio between the T3 derived from T4 and the T3 injected as such (namely, the 131T3/125T3 ratio) should be the same in plasma, liver, kidney, heart, muscle, etc. It was evident that the 131T3/125T3 ratio was not the same for different tissues. The differences were not merely due to artefactual deiodinations. The presence of small amounts of 131I and 125I containing compounds in the T3 spot was considered as highly unlikely, though not totally excluded. The data thus suggest that T3 derived from T4 and the injected (or thyroidally secreted) T3 might not be totally exchangeable within an observation period which is considerably longer than the one for which complete equilibrium was previously assumed. If so, changes in the size of the T4 pool, or in the rate of T4 conversion to T3, might affect the concentration of T3 in a given tissue to an extent not disclosed from the circulating T3 levels alone. Several possible consequences of the present findings are discussed.


Experiences of patients who undergo laryngectomy.

Fifty patients undergoing laryngectomy are described. The sociological and psychological consequences of the operation for the patient and his relatives are described, and the factors which govern the development of oesophageal speech.


Longterm results of ultrasonic irradiation in Meniere's disease.

Thirty-seven patients with Ménière's disease were treated with ultrasonic irradiation to the lateral and superior semicircular canals. In a longterm follow-up study, lasting alleviation of the vertiginous attacks was found in 24 out of 33 patients (72%). In most of the cases, hearing remained unchanged. It is suggested that the therapeutic effect is associated with a change in the endolymphatic drainage dur to histological alterations in the labyrinth produced by ultrasound.


Unilateral grommet insertion and adenoidectomy in bilateral secretory otitis media: preliminary report of the results in 91 children.

This study evaluates whether the use of grommets in the primary treatment really changes the course of secretory otitis media. The patient acts as his own control. Bilateral cases are selected and one ear is treated by a grommet, the other left intact, and all have their adenoids removed. Until now 91 children have been entered. A repeated operation was done in 13, and 6 of them had a contralateral grommet because of persisting middle ear effusion. A discharge occured from 25% of the operated ears. After the operation the intact ear showed a great improvement in the function of the Eustachian tube according to the tympanograms. The hearing level was normalized within 3 months in both intact and operated ears. In 28 children the grommets had been extruded for more than one month. By comparing the intact and the operated ears no significant statistical difference was found.


Rehabilitation of the hearing impaired adult in Denmark.

An outline is given of the rehabilitation of the hearing impaired adult in Denmark. Emphasis is put on the comprehensiveness and the professionalism of the services provided.


The development of audiological services in the Cardiff area.

An account of the development of audiological services in the Cardiff area is presented so that otolaryngologists may be encouraged to extend and improve the facilities present in their locality.


The mediation of the localization of polymorphonuclear leucocytes in glomeruli during the autologous phase of nephrotoxic nephritis.

A passive model of the autologous phase of nephrotoxic nephritis (NTN) in rabbits was developed to study the events at the initiation of this stage of the disease. Intravenous injection of sheep anti-rabbit glomerular basement membrane antiserum was followed 48 hr later by 125I trace labelled rabbit anti-sheep IgG. Animals were killed 3 hr after the second antibody injection. Experiments were undertaken to investigate whether or not a reaction between the passive antibody Fc piece and polymorphonuclear leucocyte (PMN) Fc receptor occurred, causing the localization of PMN in glomeruli in the autologous phase of NTN. The results indicate that such a reaction is an important, but not exclusive, factor in glomerular PMN localization. The complement independence of the localization was confirmed. In addition, it appeared that PMN-dependent intraglomerular fibrin deposition was mediated exclusively by a reaction between antibody Fc piece and PMN Fc receptor.


DNA binding activity of serum from patients with systemic lupus erythematosus.

The reaction conditions essential for reproducible use of the cellulose ester membrane filter assay for anti-DNA antibody levels in patients with systemic lupus erythematosus are described. A dependence of DNA-binding capacity on serum concentration was observed in the assay, requiring that serum concentrations be comparable in determinations of DNA-binding activity of different sera and when comparing different published studies. The DNA-binding capacity of serum from lupus patients was found to be significantly different from that of healthy controls. However, the binding capacities were not significantly different between lupus patients with and without nephritis. The relative avidity of the anti-DNA antibodies were estimated from plots of 1/DNA bound vs 1/DNA free and these data indicate that the avidities of the antibodies from the two groups of lupus patients are not significantly different. This observation suggests that the tightness of binding between the DNA and the serum anti-DNA antibodies cannot be used to predict immune complex-induced nephritis in lupus patients.


Effect of chronic developmental lead exposure on cell-mediated immune functions.

Studies were performed to investigate the effects of chronic, low level pre- and post-natal lead exposure on cell-mediated immune function in rats. Weanling female rats were exposed to lead (as lead acetate) in their drinking water at 0, 25, and 50 ppm for 7 weeks. At the end of 7 weeks they were mated with untreated males and continued on the same dosage throughout gestation and lactation. The offspring of these females were weaned at 21 days of age and continued on the same lead exposure regimen as their mothers. These offspring were used in immune surveillance procedures between 35 and 45 days of age. Lead exposure at the levels employed had no statistically significant effect on growth and did not result in overt signs of toxicity. Thymic weights were significantly decreased in both males and females of the two lead dosage groups. Furthermore, lead exposure resulted in suppression of responsiveness of lymphocytes to mitogen stimulation and in reduced delayed hypersensitivity responsiveness. Results indicate that chronic low-level lead exposure causes suppression of cell-mediated immune function.


Tissue distribution of brain-thymus shared antigens recognized by anti-brain xenosera in the rat, dog and man.

A comparative and quantitative study of the tissue distribution of brain-thymus shared antigens was carried out using rabbit antisera to rat, dog and human brain homogenates, assayed on rat, dog and human thymus cells, respectively. Quantitative absorption analyses with eleven different tissues showed that the tissue distribution of the brain-thymus antigens was strikingly different in the three species. In the rat, the antigens were present in large amounts on both brain and thymus, to a lesser extent on bone marrow cells, but not at all, or only slightly, on the other tissues studied. In the dog, the shared antigens were present in large amounts only on brain. They were present in smaller amounts (approximately 5% compared with brain) on thymus, spleen, lymph node and, unexpectedly, kidney, and to a slight extent on bone marrow cells. In both rat and dog, absorption to a plateau with liver indicated the presence of brain-thymus shared antigens of restricted tissue distribution. No brain-thymus antigens of restricted tissue distribution could be detected in man, since liver, heart, kidney, brain and all other tissue studied, excepting erythrocytes and platelets, could absorb out all the antibody.


Deficiency in kwashiorkor serum of factors required for optimal lymphocyte transformation in vitro.

Blastogenic responses of normal human peripheral blood lymphocytes cultured in media supplemented with serum from children with kwashiorkor were, on average, 47.7% of those observed when the same cells were cultured in the presence of normal AB serum. Incorporation of radioactive uridine was also diminished in the presence of normal AB serum. Incorporation of radioactive uridine was also diminished in the presence of kwashiorkor serum indicating that lectin-induced RNA synthesis was also affected. The kwashiorkor serum effect was not due to a cytotoxic action nor could it be attributed to the presence of saccharides or other inhibitors of the inducing lectins. Mixing experiments showed that kwashiorkor serum was not inhibitory, but that it lacked factors present in normal serum that are required for optimal lymphocyte blastogenesis. The deficiency of these factors could largely be rectified by supplementing kwashiorkor serum with an ultrafiltrate of normal serum containing components with molecular weights of less than 500 Daltons. We conclude that nutritional deprivation of severity sufficient to cause kwashiorkor leads to a deficiency of low molecular weight lymphocyte growth factors. This lack may contribute to the immunodeficiency associated with the disease.


Inhibition of mitogen-induced human lymphocyte proliferative responses by tetracycline analogues.

The effect of tetracyclines on mitogen-induced proliferative responses of human lymphocytes was examined. The results showed that of the three tetracycline analogues studied, doxycycline possessed the most potent inhibiting effects. This occurred at drug concentrations (1--10 micrograms/ml) easily attainable in serum during conventional dosage schedules. Other investigations have shown that tetracyclines also interfere with neutrophil function. Taken together, these findings may have clinical significance. Recovery from serious infections generally requires some minimal host immune responses, and the immunosuppressive side-effects of tetracyclines may have detrimental effects on patients with debilitating illnesses or impaired immunological defence mechanisms. Furthermore, tetracyclines may share some common properties of conventional immunosuppressive drugs, such as cytotoxicity, teratogenicity and cancerogenicity. The long-term use of tetracyclines for conditions such as chronic bronchitis, bronchiectasis and acne vulgaris needs to be re-examined.


ADCC against human erythrocyte target cells: role of the anti-target cell antibodies in determining lymphocyte killer activity.

Studies were undertaken to investigate the role of anti-target cell antibodies in determining whether lymphocytes can mediate antibody-dependent cellular cytotoxicity (ADCC) in vitro. Trinitrophenyl (TNP) modified Chang liver cells and human erythrocytes were employed as target cells and were coated with xenogeneic and allogeneic antibodies against TNP and natural cell surface antigens. Two cytotoxic effector cell populations were used: human peripheral blood mononuclear cells (PBMC) containing both lymphocytes and monocytes, and monocyte-depleted peripheral blood lymphocytes (PBL). With Chang targets, both PBMC and PBL mediated ADCC with xenogeneic anti-Chang and xenogeneic anti-TNP sera. With human erythrocyte targets, PBMC but not PBL mediated ADCC with human anti-blood group B serum, while both PBMC and PBL mediated ADCC with xenogeneic anti-TNP sera and also with a human anti-CD serum. These results demonstrate that the source of anti-target cell antibodies employed in ADCC reactions may determine whether or not lymphocytes are capable of mediating cytotoxicity.


Anaphylactic sensitivity of guinea-pigs drinking different preparations of cows' milk and infant formulae.

Guinea-pigs have been given various preparations of cows' milk or infant formulae to drink in an investigation of the capacity of these milk preparations to stimulate per os anaphylactic sensitivity. The treatment, presumably heat, used to concentrate the 'Evaporated' whole cows' milks (three brands were tested) almost abolished their sensitizing capacity to beta lactoglobulin. The brand presumed to be most heated as judged by the degree of caramelization had also lost most of its sensitizing capacity to casein. Injected parenterally, the 'Evaporated' milk adequately sensitized to anaphylaxis. An infant formula, which in its spray dried form was only moderately sensitizing to both beta lactoglobulin and casein, lost most of this sensitizing capacity when processed to a liquid concentrate state. The manufacturing process for the liquid concentrate involved greater and more prolonged heating than required for the spray-dried form. The relevance of these findings in the guinea-pig to cows' milk protein intolerance in the infant and possibly also to cot death is discussed.


Histamine 2 receptor-mediated immunomodulation in the mouse. I. Immunomodulation by the H2 agonist tolazoline.

Tolazoline, a drug used in the treatment of hypertension, has been described as a typical H2 agonist. In this study possible immunomodulating properties of tolazoline were investigated. A single injection of tolazoline 1 day before immunization caused an effect on delayed hypersensitivity that depended on the antigen dose. The response to 10(5) and 10(6) sheep red blood cells (SRBC) was decreased, whereas the response to 10(9) SRBC was enhanced. Administration of tolazoline 4 days after immunization predominantly affected the humoral response. The IgM response was inhibited in favour of the IgG response. Low doses of tolazoline, given to animals simultaneously with the elicitation for delayed hypersensitivity, lead to a more severe inflammation. The possible involvement of suppressor cells and vessels in tolazoline action is discussed. The application of tolazoline in the immunotherapy of human cancer is suggested.