The ultrastructure of the androgenic gland in Porcellio scaber Later. (terrestrial isopods). This paper presents the normal ultrastructural features of the androgenic gland in Porcellio scaber. It describes the changes in the organelles of its cells through the course of the secretory cycle, which terminates with the regression and destruction of these holocrine glandular cells. As products of this activity, the electron-opaque bodies (granules, lysosomes, agglomerates) and clear substances accumulate in the cytoplasm. With the destruction of the cell membrane, the entire cell content passes into the spaces of the haemocoele. The morphological modifications of the mitochondria and rough endoplasmic reticulum are the most striking changes.


Cytochemical demonstration of latency of lysosomal hydrolases in digestive cells of the common mussel, mytilus edulis, and changes induced by thermal stress. Latent beta-glucuronidase and glucosaminidase activities have been demonstrated in small cytoplasmic particles, which may possibly be primary lysosomes, as well as some larger granules of the digestive cells of the common mussel. Latency was indicated by increased staining of these structures following incubation in buffer at pH 4.5 at 37 degrees C. The exposure of mussels to temperatures of 25-28 degrees C over a period of four days induced a significant decrease in the latency of lysosomal glucosaminidase. Thermal death produced labilization of lysosomes although selective release of hydrolase activity was indicated by the differential latency of glucosaminidase and glucuronidase. The injection of hydrocortisone induced a significant increase in latency in stressed animals, indicating that the stress response involved changes in structure and function of membranes.


Freeze-etching studies on ultrastructural changes of endothelial cells in the thyroid of normal, TSH-treated and thyradin-treated mice. Freeze-etching images of the capillary endothelium in the thyroid of normal, TSH-treated and Thyradin (powdered thyroid)-treated mice were examined. Numerous pores represent vesicular stomata or fenestrations. The number of the pores and their population density are increased in TSH-treated mice, and decreased in Thyradin-treated animals. In addition, the width of the parajunctional zone and of the flat ray free from endothelial pores is smaller in TSH-treated mice and larger in Thyradin-treated animals. These facts indicate that the number of endothelial pores changes according to the functional activity of the gland.


Regional differences in mitotic activity due to injury in mouse skin. Mitotic activity adjacent to a wound inflicted at different sites in the mouse skin was measured 24 h after injury. A regional difference in the epidermal mitotic activity due to injury was noted. Mitotic activity was high in the anterior parts of the body including the head, lower in the middle to posterior regions of the body and lowest in the posterior-most parts of the body. Regional differences in epidermal mitotic activity due to injury were demonstrated in both female and male mice. The existence of a cranio-caudal gradient in epidermal response to injury is suggested.


Electron microscopic microprobe analysis of mineralized collagen fibrils and extracollagenous regions in turkey leg tendon. Bundles of tibia tendon from 19 week-old turkeys were deep frozen, freeze dried and embedded  in styrol methacrylate or Epon. In the distal mineralized region, bundles of unmineralized collagen fibrils as well as mineralized regions consisting of round microcompartments with low contrast surrounded by a mineral sheath with high contrast were found. The inner regions with low contrast corresponded to the mineralized collagen fibrils, while the contrast-rich peripheral zones corresponded to the mineralized collagen-free "ground substance". Using electron microscopic microprobe analysis, it was shown that the peripheral mineralized region, consisting mainly of closely packed needles, often contained 100% more mineral substance than the central, mineralized collagen zone, which consisted mainly of plate-like crystallites. Possible reasons for this difference in mineral content are discussed on the molecular level.


Quantitative fluorescence studies of the effects of catecholamines and hydrocortisone on endogenous amine levels in neurones and small intensely fluorescent cells of embryonic chick sympathetic ganglia in vivo and in vitro. Chick embryo lumbar sympathetic ganglia (11 day) cultured for three days and uncultured (in vivo) ganglia of comparable age were freeze-dried and processed by the formaldehyde-induced fluorescence technique for the demonstration of biogenic monoamines. The catecholamine levels within principal neurone cell bodies and small intensely fluorescent (SIF) cells were then examined in plastic sections of the in vivo and in vitro ganglia by a quantitative fluorescence method under various experimental conditions. Culture of ganglia for three days in the presence of hydrocortisone acetate (10 mug/ml) resulted in an increased SIF cells fluorescence (P less than 0.001 compared to control) and a green to yellow colour shift in the fluorophore of SIF cells. No detectable alteration in the fluorescence level of neurones was observed. When neurones after three days in culture were incubated for 1 h in exogenous catecholamines, a significant increase in fluorescence levels (interpreted as an increase in catecholamine content) occurred with noradrenaline (2 X 10(-6) M; 2 X 10(-5) M). SIF cells in ganglia removed directly from 14-day old chicks similarly took up noradrenaline and dopamine, and also adrenaline (2 X 10(-5) M). Morphological results are presented which indicate that the cellular appearances and architecture of cultured ganglion explants are very similar to those in comparable ganglia in vivo.


Seasonal changes in the fine structure of the accessory sex gland in the mole (Talpa europaea). The mole has a single pair of accessory sex organs with features of both the prostate and the seminal vesicle, for which the term prostate gland is not appropriate. Seasonal changes occuring in this gland were related to four periods: a) the quiescence period, b) the maturation period, c) the active period and d) the involution period. During the quiescence period the cuboidal epithelial cells display a quasi-embryonic fine structure and are sparse in cytoplasmic organelles, but rich in glycogen and lipopigment. With the onset of sexual activity glycogen and lipopigment disappear and the rough endoplasmic reticulum as well as the Golgi apparatus begin to proliferate. The fully active gland is lined by a low epithelium with parallel stacks of rough endoplasmic reticulum, a large Golgi apparatus and several lysosomes and secretory granules. In the involution period the gland collapses and the epithelial cells are eliminated by hetero- and autophagic processes. During this period a great number of presumably endocrine cells were observed. The results were compared with findings in experimental studies and those on postnatal development of accessory sex glands in laboratory animals.


ER-annulate lamellar associations of mucosal epithelial cells of the rat jejunum. Mucosal cells of the rat jejunum vary greatly in the relative abundance and kind of endoplasmic reticulum (ER). Many cells have few rough surfaced cisternal ER elements; these cells frequently are located in basal villous regions and generally are less differentiated than those near apices. Profiles of rough ER cisternae were often encountered in stacks of 3-4 parallel units in apical cells. A number of differentiating basal cells were found to contain ER-annulate lamellar associations arranged similarly to the ER stacks of apical cells. The annulate lamellae of this complex resemble those described for other rapidly differentiating or embryonic cells and may be derived from the nuclear envelope. They could be involved in the formation of the ER.


Occurrence of cilia in the fundus striati of the cat. In the fundus striati of the cat a few multiple ciliated astroglial cells and a single-ciliated small neuron were observed. In astrocytes cilia display a 9 + 2 subfiber configuration.


Coronary artery spasm: the effect of cardiovascular laboratory premedication practice. Eighty-four university cardiovascular laboratories responded to a questionnaire concerning premedication practice and observation of noncatheter-induced coronary artery spasm during routine coronary angiography. Half of the laboratories stated that they had not observed the phenomenon. The data suggest that premedication practice may account in part for the relative rarity of this phenomenon.


Determination of the origin of elevated plasma CPK after cardiac catheterization. Episodes of chest pain are not common in patients undergoing cardiac catheterization. The diagnostic implications of this symptom may be complicated by the occasional appearance of electrocardiographic changes mimicking those seen in acute myocardial infarction, and by the frequent elevation of conventionally measured serum enzymes. Exclusion of infarction is particularly important when coronary revascularization is contemplated. Since the MB CPK isoenzyme is relatively specific to myocardium, we assayed CPK isoenzymes in plasma samples from 184 patients undergoing cardiac catheterization to determine whether CPK elevations accompanying catheterization can be distinguished from those associated with myocardial infarction. Samples were obtained every 2 hr for 24 hr, and CPK isoenzymes quantified by a kinetic fluorometric method. Total plasma CPK increased in all patients (mean peak 0.238 +/- 0.042 (SD) IU/ml) but MB CPK remained normal in 181 patients (less than 0.005 IU/ml). In three remaining patients, MB CPK was elevated and myocardial infarction was confirmed by 99mTc (SN) pyrophosphate scan. Twelve patients after catheterization, in whom no intramuscular premedication was given, exhibited only minimal elevation of total plasma CPK. In contrast, 100 control patients with acute myocardial infarction exhibited peak total CPK activity averaging 0.833 +/- 0.037 (SD), and MB CPK was elevated in all cases (0.078 +/- 0.027 (SD) IU/ml). Thus, CPK elevations after catheterization reflect release of enzyme from noncardiac sources rather than from injured myocardium. Furthermore, increased plasma MB CPK activity may be considered a reliable index of myocardial infarction in patients undergoing cardiac catheterization.


The significance of early changes of positive and negative dP/dt following contrast ventriculography. The contrast agents used for cardiac angiography have pharmacologic effects on the myocardium and on peripheral and coronary circulations. These effects are a stress to the left ventricle. We have studied the sequential changes in heart rate, LVEDP, positive and negative dP/dt in 34 patients following left ventriculography. These patients were divided into three groups: nine normal patients, 14 patients with valvular or coronary artery disease and abnormal left ventricular function at rest, and 11 patients with significant coronary artery stenosis but normal left ventricular function at rest. Changes in dP/dt most clearly defined the normal and abnormal response. In the normal group the mean positive dP/dt rose abruptly at 30 sec after the ventriculogram, whereas the abnormal group showed a mean decrease. The extent of change varied, however, for patients within each group. Negative dP/dt decreased in both groups but more so in the abnormal group. As did the other groups, patients with coronary artery disease but with normal ventricular function showed individual variation in response. Their mean changes in positive and negative dP/dt were intermediate. Positive dP/dt decreased at 30 sec (similar to, but less than, the group with abnormal ventricular function) and negative dP/dt also diminished at 30 sec with a mean value between the normal and abnormal groups. The results of this study support the concept that left ventricular dysfunction may be elicited by the stress of contrast injection. More importantly, such dysfunction seen both with abnormal and normal left ventricles at rest, may be reflected by the relatively simple measurement of positive dP/dt after ventriculography.


A comparison of formulas used to estimate mixed venous saturations. True mixing of venous blood in the absence of shunt occurs in the pulmonary artery. In the presence of left to right shunt at a level proximal to the pulmonary artery, mixed venous blood for oxygen saturation (MVO2) is estimated by using an average of blood samples taken from the chamber proximal to the shunt. In atrial septal defect, the determination of MVO2 is calculated by using blood samples from the superior vena cava (SVC) and the inferior vena cava (IVC). Several formulas have been proposed, utilizing varying combinations of blood samples taken from the SVC and IVC. In the present investigation, 100 patients without evidence of shunt were studied during routine cardiac catheterization. Duplicate blood samples were taken from the pulmonary artery (PA), the SVC, and the IVC, and were analyzed for oxygen-saturation. If one assumes that the PA blood sample represents true venous blood mixing (TMVO2), the following formulas were used for comparison: 1)PA = SVC; 2) PA = IVC; 3) PA = (SVC + IVC)/2; 4) PA = (2SVC + IVC)/3; 5) PA = (3SVC + IVC)/4; and 6) PA = (2IVC + SVC)/3. When one uses the standard two variable regression equations, this study shows that the 90% confidence limits are wide. The correlation, however, is somewhat better if one uses the formulas 3)-6). Therefore, the error that may be introduced in calculating the TMVO2 may be substantial and can critically alter the estimation of the shunted blood volume.


Cineangiographic measurements with the help of a vertebral grid system. Cineangiographic measurements of cardiac structures require knowledge of the magnification factor due to nonparallelity of the X-ray beams. A new method is presented to determine the magnification factor from cineangiograms based on the patient's height and the height of three vertebral bodies and the adjacent intervertebral distances. The method enables measurement of cardiovascular structures from projected cineangiographic films where the appropriate magnification factor is missing. Furthermore, doubtful magnification factors can be checked out as to their accuracy.


Effects of Adalat (nifedipine) on left ventricular hemodynamics in angina pectoris: comparative study with propranolol. The hemodynamic effects of nifedipine and propranolol administered intravenously were studied in 17 patients with angina pectoris. Nine patients received nifedipine and eight received propranolol. The hemodynamic parameters were compared at rest and during supine bicycle exercise at work loads known to produce angina. Exercise-induced angina improved in four out of nine patients following nifedipine and in one out of eight patients following propranolol. Nifedipine significantly reduced the increment of left ventricular end-diastolic pressure and Vmax during exercise. Intravenous propranolol significantly suppressed the increment of heart rate, max dp/dt, tension time index, and Vmax during exercise. Although the exact mode of action of nifedipine remains uncertain, it is suggested that it decreases myocardial oxygen requirements primarily through a reduction of left ventricular volume, whereas propranolol suppresses the positive chronotropic and inotropic responses of the heart muscle to exercise.


Systemic vasodilatation following diazepam after combined sympathetic and parasympathetic blockade in patients with coronary heart disease. Clinical and hemodynamic benefits have been documented after giving diazepam to patients with coronary heart disease. The mechanisms by which this agent produces its hemodynamics effects are poorly understood. Partial autonomic blockade was induced in 10 patients by the intravenous administration of 5 mg propranolol and 0.8 mg atropine. Coronary and systemic hemodynamics were normal before and unchanged (p greater than 0.05) after propranolol and atropine administration. However, myocardial extraction of lactate improved in 9 out of 10 patients (from 11% to 24%, p less than 0.05). The patients then received 0.1 mg/kg diazepam intravenously. Diazepam had no effect on the coronary sinus blood flow and resistance while myocardial extraction of lactate further improved in all but one patient (from 24% to 35% at 5 min and 34% at 15 min, p less than 0.05). Heart rate increased by a mean of 4 beats at 5 min (p less than 0.01). Cardiac output was unchanged. Systolic arterial pressure (SAP) decreased significantly (p less than 0.001) at 5 and 15 min (6-10%) and left ventricular end-diastolic pressure (LVEDP) also diminished significantly (p less than 0.001) at 5 and 15 min (18-19%). These changes occurred despite autonomic blockade. These findings suggest, in the absence of change of myocardial blood flow and contractility, a direct peripheral action of the drug on arteries and/or veins.


First heart sound: a phono-echocardiographic correlation with mitral, tricuspid, and aortic valvular events. We evaluated the relationship of S1 recorded by phonocardiography at the mitral area with motion of the mitral, tricuspid, and aortic valves, recorded by simultaneous echocardiography in 20 cardiac patients with a normal PR interval. The first major component of S1 coincided with mitral valve closure in 20 of 20 patients (100%) and also with tricuspid valve closure in 14 of 20 patients (70%). The second major component of S1 coincided with aortic valve opening in 20 of 20 patients and also with tricuspid valve closure in six of 20 patients (30%). We conclude that the first major component of S1 coincides with mitral valve closure in all patients but may also coincide with tricuspid valve closure in many patients, and the second major component of S1 coincides with aortic valve opening in all patients but may also coincide with tricuspid valve closure in some patients.


Cavernous hemangioma of the right atrium: presumptive diagnosis by coronary angiography. The angiographic findings in a patient with an interesting vascular tumor of the right atrium are described. The tumor was supplied by vessels from both the right and left coronary arteries and consisted of dilated blood spaces in which contrast material persisted for a prolonged period of time.


Anomalous origin of the right coronary artery from the left sinus of Valsalva with associated chest pain: report of two cases. Ectopic origin of the right coronary artery from the left sinus of Valsalva is an infrequent coronary anomaly. The right coronary artery then passes between the aorta and pulmonary artery. We report two such cases with chest pain suggestive of angina pectoris in the absence of atherosclerosis, as demonstrated by selective coronary arteriography. A technique for selectively catheterizing the ectopic right coronary artery is described.


Patent ductus arteriosus in adult patients with aortic valvular disease: the importance of routine screening for a left-to-right shunt at cardiac catheterization. Two patients, catheterized primarily for the preoperative assessment of clinically apparent aortic stenosis and insufficiency, were found to have a patent ductus arteriosus as well at cardiac catheterization. The association of aortic valve disease and patent ductus arteriosus is reviewed, and the importance of making the diagnosis prior to aortic valve replacement is stressed, emphasizing the routine use of the hydrogenplatinum electrode system.


Design for obtaining the half-axial projection. A device for obtaining the half-axial projection complementing single plane cine equipment is described. The design enables a single technician to raise and lower the patient's upper half of the body between 0 degrees and 20 degrees of longitudinal deviation from the rotating cradle, set between 0 degrees and 70 degrees of horizontal deviation. Materials cost less than $100 and can be assembled by personnel in most hospitals.


Error in thermodilution cardiac output measurement caused by variation in syringe volume. It is important to measure the exact volume of 5% dextrose injected when thermodilution cardiac output determinations are made. The marked volume on plastic syringes and their variability may contribute to a small but significant error in the results.


Technique for transjugular placement of an umbrella filter in a left inferior vena cava. Transjugular placement of a Mobin-Uddin umbrella fillter in an anomalous left inferior vena cava to prevent recurrent pulmonary embolization may present technical difficulties. Preforming a slight curve in the applicator wire a short distance from its tip allowed catheterization and proper position of a fillter in two cases.


Toxic effect of alantolactone and dihydroalantolactone in in vitro cultures of leukocytes. Alantolactone, an allergenic sesquiterpene lactone, is toxic to leukocytes in in vitro cultures. Cell (1 X 10(6) cells/ml of culture) stimulation by pokeweed mitogen (PWM) decreases with increasing amounts of terpene. A concentration of 1 mug/ml of culture decreases stimulation by 50%. The reduced terpene, dihydro-11,13-alantolactone (DHA) is also toxic. A concentration of 1.7 mug/ml of culture of DHA brings about a 50% decrease in stimulation. Both compounds affect cell viability as measured by dye exclusion. It is suggested that the toxicity of alantolactone is not due to the presence of the alpha-methylene group conjugated to the carbonyl function of the gamma-lactone system.


Distribution along DNA of the bound carcinogen N-acetoxy-N-2-acetylaminofluorene in chromatin modified in vitro. Chromatin from duck erythrocytes was modified in vitro by the carcinogen N-acetoxy-N-2-acetylaminofluorene (N-Ac-O-AAF). The distribution of the carcinogen along the DNA molecule was studied using staphylococcal nuclease which allows the fractionation of chromatin DNA into two zones. It was shown that the carcinogen binds preferentially to the regions of chromatin sensitive to the enzyme; however, the regions of DNA tightly bound to histones and resistant to the enzyme react comparatively well. The single-strand specific nuclease S1 which digests DNA modified by the carcinogen in vitro did not digest chromatin under the conditions used. Some possible mechanisms for the interaction of the carcinogen with chromatin are discussed.


Prevention by copper of cadmium sequestration by metallothionein in liver. Following chronic CdCl2 administration to rats, more than 98% of the metal in liver supernatant is bound to the low molecular weight binding protein, metallothionein. Simultaneous administration of high doses of Cd and copper salts result in an increase in toxicity which is accompanied by a failure of Cd sequestration by metallothionein in vivo. This may be due to an aggregation of metallothionein which has been observed in the presence of copper in vitro.


Automatic activity in depolarized guinea pig ventricular myocardium. Characteristics and mechanisms. Membrane potential was changed uniformly in segments, 0.7-1.0 mm long, of guinea pig papillary muscles excised from the right ventricle by using extracellular polarizing current pulses applied across two electrically insulated cf preparations superfused with Tyrode's solution at maximum diastolic membrane potentials ranging from-35.2+/-7.5 (threshold) to +4.0+/-9.2 mV. The average maximum dV/dt of RAD ranged from 17.1 to 18.0 V/sec within a membrane potential range of -40 to +20 mV. Raising extracellular Ca2+ concentration [Ca2+]0 from 1.8 to 6.8 mM, or application of isoproterenol (10(-6)g/ml) enhanced the rate of RAD, but lowering [Ca2+]0 to 0.4 mM or exposure to MnCl2 (6 mM) abolished RAD. RAD were enhanced by lowering extracellular K+ concentration [K+]0 from 5.4 to 1.5 mM. RAD were suppressed in 40% of fibers by raising [K+]0 to 15.4 mM, and in all fibers by raising [K+]0 to 40.4 mM. This suppression was due to increased [K+]0 and not to K-induced depolarization because it persisted when membrane potential was held by means of a conditioning hyperpolarizing puled gradually after maximum repolarization. These observations suggest that the development of RAD in depolarized myocardium is associated with a time-dependent decrease in outward current (probably K current) and with increase in the background inward current, presumably flowing through the slow cha-nel carrying Ca or Na ions, or both.


Phasic right coronary artery blood flow in conscious dogs with normal and elevated right ventricular pressures. We studied phasic right coronary blood flow in well trained normal dogs and dogs with pulmonic stenosis. We installed electromagnetic flow transducers and pressure tubes under anesthesia to monitor right coronary blood flow, cardiac output, central aortic blood pressure, and right ventribular pressure. In normotensive dogs, systolic flow amplitude equaled early diastolic flow levels. The ratio of systolic to diastolic flow at rest was substantially greater in the right coronary bed (36+/-1.3%) than in the left circumflex bed (13+/-3.6%). Right diastolid flow runoff, including the cove late in diastole, resembled left circumflex runoff. Blood flow to the normotensive right (37+/-1.1 ml/min 100(-1) g) and the left (35+/-1.0 ml/min(-1) g) ventricular myocardium indicated equal perfusion of both cardiac walls. Throttling of systolic flow was related directly to the right ventricular systolic pressure level in the dogs with pulmonic stenosis. Retrograde systolic flow occurred in severe right ventricular hypertension. The late diastolic runoff pattern in dogs with pulmonic stenosis appeared the same as for the normotensive dogs. We obtained systolic to diastolic flow ratios of 1/3 the value of normotensive hearts in high and severe pulmonic hypertension. Electrocardiograms and studies of pathology suggested restricted blood flow to the inner layers of the right myocardium in the dogs with severe and high right ventricular hypertension. Normotensive and hypertensive peak hyperemic flow responses were similar, except for an increased magnitude of diastolic flow, with proportionately less systolic flow in hypertensive states.


Reduction in baroreflex cardiovascular responses due to venous infusion in the rabbit. We studied reflex bradycardia and depression of mean arterial blood pressure (MAP) during left aortic nerve (LAN) stimulation before and after volume infusion in the anesthetized rabbit. Step increases in mean right atrial pressure (MRAP) to 10 mm Hg did not result in a significant change in heart rate or MAP. After volume loading, responses to LAN stimulation were not as great and the degree of attenuation was propoetional to the level of increased MRAP. A change in responsiveness was observed after elevation of MRAP by only 1 mm Hg, corresponding to less than a 10% increase in average calculated blood volume. after an increase in MRAP of 10 mm Hg, peak responses were attenuated by 44% (heart rate) and 52% (MAP), and the initial slopes (rate of change) were reduced by 46% (heart rate) and 66% (MAP). Comparison of the responses after infusion with blood and dextran solutions indicated that hemodilution was an unlikely explanation for the attenuation of the reflex responses. Total arterial baroreceptor denervation (ABD) abolished the volume-related attenuation was still present following bilateral aortic nerve section or vagotomy. It thus appears that the carotid sinus responds to changes inblood volume and influences the reflex cardiovascular responses to afferent stimulation of the LAN. On the other hand, cardiopulmonary receptors subserved by vagal afferents do not appear to be involved.


Ventricular pressure-volume curve indices change with end-diastolic pressure. Many indices have been proposed to describee the diastolic pressure-volume curve mathematically and permit quantification of the elastic properties of the myocardium itself in hopes that changes in the muscle caused by disease would b.e reflected in the diastolic pressure-volume curve. To date, none of the proposed indices has been shown convincingly to discriminate one group of patients from another. While this situation in part arises from the relatively large amount of noise introduced by the technical difficulties of measuring synchronous pressures and volumes during diastole in man, ther is a more fundamental difficulty. In practice, one can measure only a short segment of the entire pressure-volume curve, and the values of all diastolic pressure-volume curve parameters investigated change significantly when one uses different segments of the same pressure-volume curve to compute them. These results were derived from relatively noise-free pressure-volume curves obtained by filling nine excised dog left ventricles at a known rate and monitoring pressure-volume curve used to compute the parameter. Merely increasing measurement fidelity will not resolve this problem, because none of these parameters accurately characterizes the entire diastolic pressure-volume curbe from a segment like that which one can reasonably expect to obtain from humans.


Quantification of baroreceptor influence on arterial pressure changes seen in primary angiotension-induced hypertension in dogs. We studied the role of the sino-aortic baroreceptors in the gradual development of hypertension induced by prolonged administration of small amounts of angiotensin II (A II) in intact dogs and dogs with denervated sino-aortic baroreceptors. Short-term 1-hour infusions of A II(1.0-100 ng/kg per min) showed that conscious denervated dogs had twice the pressor sensitivity of intact dogs. Long-term infusions of A II at 5.0 ng/kg per min (2-3 weeks) with continuous 24-hour recordings of arterial pressure showed that intact dogs required 28 hours to reach the same level of pressure attained by denervated dogs during the 1st hour of infusion. At the 28th hour the pressure in both groups was 70% of the maximum value attained by the 7th day of infusion. Both intact and denervated dogs reached nearly the same plateau level of pressure, the magnitude being directly related both the the A II infusion rate and the daily sodium intake. Cardiac output in intact dogs initially decreased after the onset of A II infusion, but by the 5th day of infusion it was 38% above control, whereas blood volume was unchanged. Heart rate returned to normal after a reduction during the 1st day of infusion in intact dogs. Plasma renin activity could not be detected after 24 hours of A II infusion in either intact or denervated dogs. The data indicate that about 35% of the hypertensive effect of A II results from its acute pressor action, and an additional 35% of the gradual increase in arterial pressure is in large measure a result of baroreceptor resetting. We conclude that the final 30% increase in pressure seems to result from increased cardiac output, the cause of which may be decreased vascular compliance. since the blood volume remains unaltered.


Acute and chronic dose-response relationships for angiotensin, aldosterone, and arterial pressure at varying levels of sodium intake. We examined the acute and chronic dose-response relationships between intravenously infused angiotensin II (A II) and the resulting changes in arterial pressure and plasma aldosterone concentration at varying levels of sodium intake. Sequential analysis of plasma aldosterone at each A II infusion rate resulted in an acute dose-related increase in plasma aldosterone which was markedly attenuated after the first 24 hours of infusion, the final level being directly related to the dose of A II and inversely related to sodium intake. A II infused at 5,15, and 23 ng/kg per min was associated with an initial increase (2nd to 8th hour) in plasma aldosterone to 2,6, and 9 times control values, respectively, in dogs receiving 40 mEq Na+/day. But, after the 1st day, aldosterone averaged only 1, 1.7, and 3 times control values for the next 2 weeks at the same rates of A II infusion. Dogs receiving 120 mEq Na+/day during A II infusion exhibited only a transient increase in plasma aldosterone during the 1st day. Sustained hypertension developed over a period of a week at all doses of A II at normal and high sodium intake, but did not occur at any dose of A II in sodium-depleted dogs. Increasing sodium intake from 40 to 120 mEq/day resulted in higher levels of hypertension, 125% compared to 140% of ocntrol values for dogs infused with A II, 5.0 ng/kg per min. We conclude that primary angiotensin-induced hypertension need not be associated with increased levels of plasma aldosterone, which appears to remain elevated only with amounts of A II greater than those required to sustain a significant degree of hypertension.


The effect of cardiac contraction on collateral resistance in the canine heart. We determined whether the coronary collateral vessels develop an increased resistance to blood flow during systole as does the cognate vascular bed. Collateral resistance was estimated by measuring retrograde flow rate from a distal branch of the left anterior descending coronary artery while the main left coronary artery was perfused at a constant pressure. Retrograde flow rate was measured before and during vagal arrest. We found that in 10 dogs the prolonged diastole experienced when the heart was stopped caused no significant change in the retrograde flow rate, which indicated that systole has little effect on the collateral resistance. However, when left ventricular end-diastolic pressure was altered by changing afterload or contractility, a direct relationship between end-diastolic pressure and collateral resistance was noted.


Effect of electrotonic potentials on pacemaker activity of canine Purkinje fibers in relation to parasystole. Isolated false tendons excised form dog hearts were mounted in a three-chamber tissue bath. Isotonic sucrose solution was perfused in the central chamber to provide a region of depressed conductivity between the fiber segments in chambers 1 and 3, which were perfused with Tyrode's solution. The electrotonic influence of spontaneous or driven responses evoked in chamber 3 during the first half of the spontaneous cycle of a chamber 1 peacemaker delayed the next spontaneous discharge. This effect changed to acceleration when the chamber 3 segment fired during the second half of the spontaneous cycle. We found that subthreshold depolarizing current pulses 50-300 msec applied across the sucrose gap caused similar degrees of delay or acceleration. Furthermore, hyperpolarizing currents caused the reverse pattern. The results indicate that the discharge pattern of a parasystolic focus may be altered by the electrotonic influence of activity in the surrounding tissue. The significance of these findings is considered in relation to the mechanism of production of parasystolic rhythms.


The evolution of early fibromuscular lesions hemodynamically induced in the dog renal artery. I. Light and transmission electron microscopy. In view of the important roles of arterial intimal fibromuscular lesions as precursors of atherosclerotic plaque and occlusive lesions in arterial reconstructions, a model has been developed for the rapid hemodynamic induction of these lesions by anastomosis of the dog right renal artery to the inferior vena cava. Light and transmission electron microscopic observations were made on the arterial shunt after periods of rapid flow ranging form 10 minutes to 2 hours to identify initial factor(s) and evolutionary mechanisms in the etiology of the lesions. The sequence of events included aberrations in ruthenium red staining of the endothelial luminal membrane at 10 minutes, multilayered thickening of the subendothelial basement membrane (BM) at 15 minutes, and initial reorientation and migration of smooth muscle cells (SMC) into the intima along with the appearance of areas of degeneration of the internal elastic lamina (IEL) at 30 minutes. The endothelial cells were still intact in some areas overlying the SMC migration and IEL degeneration, but they were separating from the surface in other such areas. As subendothelium became exposed, some platelet adherence was noted. By 2 hours, the entire wall reaction was fully developed. Initial observations indicate that in the evolution of this hemodynamically induced lesion visible alteration in the endothelial cells is not prerequisite to degeneration of the underlying IEL and reorientation and migration of medial SMC.


Variations in 35SO4 incorporation into glycosaminoglycans along canine coronary arteries. A possible index of artery wall stress. Focal areas of accentuated wall stress along the course of canine coronary arteries may be revealed by the level of 35SO4 incorporation into glycosaminoglycans (GAG). In the anterior descending artery, 35SO4 incorporation in higher in the proximal than in the distal region and may be extraordinarily high as the vessel enters a proximally located muscle bridge and at the takeoff region of multidirectional branches. In the circumflex artery, the incorporation also is higher in the proximal than in the distal region and is high at the genu where the posterior descending artery forms. There are differences in uptake of 35SO4 in vessels even when the arteries arise from the same vascular bed.this was shown by the higher incorporation in the left coronary artery than in the right coronary artery. A general anatomical agreement exists between these sites of high 35SO4 incorporation and previously described locations of interval elastic disruption ans proliferation of intimal connective tissue in the dog.


Evaluation of the force-frequency relationship as a descriptor of the inotropic state of canine left ventricular myocardium. The short-term force-frequency characteristics of canine left ventricular myocardium were examined in both isolated and intact preparations by briefly pertubing the frequency of contraction with early extrasystoles. The maximum rate of rise of isometric tension (Fmas) of the isolated trabeculae carneae was potentiated by the introduction of extrasystoles. The ratio of Fmas of potentiated to control beats (force-frequency ratio) was not altered significantly by a change in muscle length. However, exposure of the trabeculae to isoproterenol (10(-7)M) significantly changed the force-frequency ratio obtained in response to a constant frequency perturbation. Similar experiments were performed on chronically instrumented conscious dogs. Left ventricular minor axis diameter was measured with implanted pulse-transit ultrasonic dimension transducers, and intracavitary pressure was measured with a high fidelity micromanometer. Atrial pacing was performed so that the end-diastolic diameters of the beats preceding and following the extrasystole could be made identical. Large increases in the maximum rate of rise of pressure (Pmas) were seen in the contraction after the extrasystole. The ratio of Pmax of the potentiated beat to that of the control beat was not changed by a 9% increase in the end-diastolic diameter, produced by saline infusion. Conversely, isoproterenol significantly altered this relationship in the same manner as in the isolated muscle. Thus, either in vitro or in situ, left ventricular myocardium exhibits large functional changes in response to brief perturbations in rate. The isoproterenol and length data indicate that the force-frequency ratio reflects frequency-dependent changes in the inotropic state, independent of changes in length.


Site of myocardial infarction. A determinant of the cardiovascular changes induced in the cat by coronary occlusion. The influence of site of acute myocardial infarction on heart rate, blood pressure, cardiac output, total peripheral resistance (TPR), cardiac rhythm, and mortality was determined in 58 anesthetized cats by occlusion of either the left anterior descending (LAD), left circumflex or right coronary artery. LAD occlusion resulted in immediate decrease in cardiac output, heart rate, and blood pressure, an increase in TPR, and cardiac rhythm changes including premature ventricular beats, ventricular tachycardia, and occasionally ventricular fibrillation. The decrease in cardiac output and increase in TPR persisted in the cats surviving a ventricular arrhythmia. In contrast, right coronary occlusion resulted in a considerably smaller decrease in cardiac output. TPR did not increase, atrioventricular condition disturbances were common, and sinus bradycardia and hypotension persisted in the cats recovering from an arrhythmia. Left circumflex ligation resulted in cardiovascular changes intermediate between those produced by occlusion of the LAD or the right coronary artery. Mortality was similar in each of the three groups. We studied the coronary artery anatomy in 12 cats. In 10, the blood supply to the sinus node was from the right coronary artery and in 2, from the left circumflex coronary artery. The atrioventricular node artery arose from the right in 9 cats, and from the left circumflex in 3. The right coronary artery was dominant in 9 cats and the left in 3. In conclusion, the site of experimental coronary occlusion in cats is a major determinant of the hemodynamic and cardiac rhythm changes occurring after acute myocardial infarction. The cardiovascular responses evoked by ligation are related in part to the anatomical distribution of the occluded artery.


Intrarenal site of action of calcium on renin secretion in dogs. We studied the effects of intrarenal calcium infusion on renin secretion in sodium-depleted dogs in an attempt to elucidate the major site of calcium-induced inhibition of renin release. Both calcium chloride and calcium gluconate reduced renal blood flow and renin secretion while renal perfusion pressure was unchanged. These data indicate that calcium inhibition of renin secretion did not occur primarily at the renal vascular receptor; decreased renal blood flow is usually associated with increased renin secretion. Calcium chloride infusion increased urinary chloride excretion without affecting sodium excretion, and calcium gluconate failed to increase either sodium or chloride excretion. Also, the filtered loads of sodium and chloride were unchanged during the calcium infusions. These results give no indication that calcium inhibited renin secretion by increasing the sodium or chloride load at the macula densa. The effects of intrarenal calcium infusion on renin release were also assessed in dogs with a nonfiltering kidney in which renal tubular mechanisms could not influence renin secretion. The observation that calcium still suppressed renin release in these dogs provides additional evidence that the the major effect of calcium involved nontubular mechanisms. Thus, it appears likely that calcium acted directly on the juxtaglomerular cells to inhibit renin secretion.


Uptake of infarct-imaging agents in reversibly and irreversibly injured myocardium in cultured fetal mouse heart. We studied the specificity of uptake of infarct-imaging agents for reversibly or irreversibly injured myocardium independently of blood flow by using intact beating fetal mouse hearts in organ culture. Reversible injury resulted from deprivation of oxygen and glucose for 4 hours at 37 degrees C; irreversible injury, from similar deprivation at 42 degrees C. At the end of the insult, uptake of 99mTc(Sc)-labeled pyrophosphate, glucoheptonate, or tetracycline was markedly increased in irreversibly damaged and, to a lesser degree, in reversibly injured hearts. After 24 hours of recovery, necrotic hearts accumulated even more pyrophosphate and tatracycline but less glucoheptonate. Uptake of radioiodinated tetracycline increased only in irreversibly injured hearts. Pyrophosphate uptake was not reduced in hearts cultured in calcium-free medium. These finding suggest that 99mTc(Sn)-labeled pyrophosphate, tetracycline, and glucoheptonate preferentially localize in irreversibly damaged myocardium; the 99mTc(Sn) complex modifies the specificity of uptake; and the uptake of 99mTc(Sn)-pyrophosphate appears unrelated to calcium uptake.


Stimulation of renin release from rabbit renal cortex by arachidonic acid and prostaglandin endoperoxides. The mechanism by which renal prostaglandins stimulate renin secretion in vivo is unknown. In this in vitro study we measured the effects of activation of the prostaglandin (PG) system on renin release from slices of rabbit renal cortex. The PG precursor arachidonic acid (C20:4), a natural PG endoperoxide (PGG2), two stable synthetic PG endoperoxide analogues (EPA I and II), PGE2, PGF2alpha, and two different PG synthesis inhibitors [indomethacin and 5,8,11,14-eicosatetraynoic acid (ETA)] were used to evaluate the possibility of a direct action of the cortical PG system on renin secretion. Renin release increased significantly with time after addition of C20:4, PGG2, EPA I, and EPA II to the incubation medium. Stimulation of renin release was se-related for C20:4 in concentrations of 0.6 to 4.5 X 10(-6) M, for EPA I in concentrations of 0.7 to 2.8 X 10(-6) M, and for EPA II in concentrations of 1.4 to 14.0 X 10(-6) M. Indomethacin (10(-4) M) and ETA (10(-4) M) significantly decreased basal renin release as well as the renin release stimulated by C20:4 and EPA I. PGE2(10(-12) to 10(-6) M) had no effect on renin release, whereas PGF2alpha (10(-12) to 10(-6) M) decreased renin release in a dose-dependent manner. These data raise the possibility of a direct action of the renal cortical PG system on renin secretion. The results further indicate that stimulation of renin release by C20:4 may depend more specifically on the action of PG endoperoxides than on the primary prostaglandins.


Investigation of the theory and mechanism of the origin of the second heart sound. To investigate further the origin of the second heart sound we studied human subjects, dogs, and a model in vitro of the cardiovascular system. Intra-arterial sound, pressure, and, where possible, flow and high speed cine (2,000 frames/sec) were utilized. The closure sound of the semilunar valves was of higher amplitude in be ventricles than in their respective arterial cavities. The direction of inscription of the main components of intra-arterial sound were opposite in direction to the components of intraventricular sound. Notches, representative of pressure increments, were noted on the ventricular pressure tracings and were coincident with the components of sound. The amplitude of the closure sound varied with diastolic pressure, but remained unchanged with augmentation of forward and retrograde aortic flow. Cines showed second sound to begin after complete valvular closure, and average leaflet closure rate was constant regardless of pressure. Hence, the semilunar valves, when closed, act as an elastic membrane and, when set into motion, generate compression and expansion of the blood, producing transient pressure changes indicative of sound. The magnitude of the initial stretch is related to the differential pressure between the arterial and ventricular chambers. Sound transients which follow the major components of the second sound appear to be caused by the continuing stretch and recoil of the leaflets. Clinically unexplained findings such as the reduced or absent second sound in calcific aortic stenosis and its paradoxical presence in congenital aortic stenosis may be explained by those observations.


Hierarchy of ventricular pacemakers.

Hierarchy of ventricular pacemakers. To characterize the pattern of pacemaker dominance in the ventricular specialized conduction system (VSCS), escape ventricular pacemakers were localized and quantified in vivo and in virto, in normal hearts and in hearts 24 hours after myocardial infarction. Excape pacemaker foci were localized in vivo during vagally induced atrial arrest by means of electrograms recorded from the His bundle and proximal bundle branches and standard electrocardiographic limb leads. The VSCS was isolated using a modified Elizari preparation or preparations of each bundle branch. Peacemakers were located by extra- and intracellular recordings. Escape pacemaker foci in vivo were always in the proximal conduction system, usually the left bundle branch. The rate was 43+/-11 (mean+/-SD) beats/min. After beta-adrenergic blockade, the mean rate fell to 31+/-10 beats/min, but there were no shifts in pacemaker location. In the infarcted hearts, pacemakers were located in the peripheral left bundle branch. The mean rate was 146+/-20 beats/min. In isolated normal preparations, the dominant pacemakers usually were in the His bundle, firing at a mean rate of 43+/-10 beats/min. The rates of pacemakers diminished with distal progression. In infarcted hearts, the pacemakers invariably were in the infarct zone. The mean firing rates were not influenced by beta-adrenergic blockade. The results indicate that the dominant pacemakers are normally in the very proximal VSCS, but after myocardial infarction pacemaker dominance is shifted into the infarct. Distribution of pacemaker dominance is independent of sympathetic influence.


Methods for the estimation of three vitamin dependent red cell enzymes. The estimation of vitamin dependent red cell enzymes before and after in vitro vitamin supplement offers a new approach to the assessment of vitamin nutritional state. To facilitate the clinical application of the principle involved, enzyme assay and aspartate aminotransferase have been made suitable for routine use. Reference ranges have been obtained for enzyme activity and vitamin effect, and the coefficients of variation for the techniques under normal use have been calculated. The assays are useful for hospital patients suspected of poor vitamin B nutrition i.e. the elderly, and chronic alcoholics.


The accuracy of commercial calibration sera and assayed control sera. Commercial calibration sera and assayed control sera have been extensively studied in parallel runs. An attempt has been made to assess the accuracy of the stated values of these sera, using mehtods that in two major quality control schemes have been shown to be reliably accurate. The Technicon product has proven to be generally the most reliable of the calibration sera. Many discrepancies were found in the assayed control sera values, particularly for calcium, phosphate and bilirubin, and our results indicate that the products of some manufacturers need to be improved.


Kinetic determination of serum haptoblobin with a centrifugal analyzer. We describe a method for determining haptoglobin with a centrifugal analyzer that is based on haptoglobin combining stoichiometrically with hemoglobin to form a complex that has peroxidase-like activity proportional to the quantity of haptoglobin present. Under assay conditions, unbound hemoglobin exhibits only a small fraction of the total peroxidase activity. Activity is measured colorimetrically at 405 nm after reaction with o-dianisdine and ethyl hydrogen peroxide. The procedure is standardized by saturating aknown amount of hemoglobin with a serum whose hemoglobin binding capacity exceeds the amount of hemoglobin in the assay system. The mean and mean within-run precision of our method, determined by performing 17 replicate assays of both a pooled normal serum and a 10-fold dilution of the serum, was 1.13 g/liter (CV, 2.9%), and 106 mg/liter (cv, 5.8%), respectively. The 95 percentile estimate of the normal range by our method is 0.45-1.85 g/liter hemoglobin binding capacity. When results by our automated method were compared to those by a manual method [Scand. J. Clin. Lab. 2nvest. 18, 80 (1965)], the slope of the unweighted linear least-squares regression line was .970 the y-intercept 26 mg/liter, and the correlation coefficient .995.


Separation and quantitation of serum constituents associated with calcium by gel filtration. Gel filtration of serum by use of polyacrylamide beads (Biogel P-2) separates total colcium into four distinct peaks: an initial peak, corresponding to protein-bound calcium; a second peak containing the calcium complexes of citrate, phosphate, lactate, and sulfate; a third peak containing calcium bicarbonate; and a fourth peak or trough corresponding to the serum ionic calcium. An eluent containing (per liter) 140 mmol of sodium, 1.1o mmol of calcium, 0.50 mmol of magnesium, and 10 mmol of 2-([tris(hydroxymethyl)methyl]amino)ethanesulfonic acid(pH 7.40 at 37 degrees C) provides physiological conditions that determine the equilibria between these calcium components. Association constants determined under these conditions permit calculation of the expected concentration of the calcium complexes in each tube of eluent, and these concentrations closely correspond to the amount of bound calcium measured experimentally. The mean distribution of calcium in healthy individuals, as determined by this method, is (per liter): calcium protein, 1.00 mmol; calcium complexes, 0.31 mmol; and ionic calcium, 1.07 mmol.


Serum phospholipids determined without acid digestion. A method for determination of phospholipids in serum is described that involves complexing intact phospholipids with chromogenic reagent, thus eliminating the need for acid digestion, which is a mandatory step in all the current procedures for phospholipid analysis. Inorganic phosphorus and other serum components do not interfere in the determination. Excellent agreement between results of this new procedure and the conventional acid digestion method (r=0.986, y intercept=2.053, and slope=0.986) makes it an attractive alternative clinical method for determination of phospholipids in serum.


Microscale modification of a cation-exchange column procedure for plasma ammonia. A column cation-exchange resin procedure for plasma ammonia was modified to require only 100 mul of plasma per determination. Mean analytical recovery of standard from resin (six samples) was 98% (range, 94-100%) as compared to a mean of 63% (range, 58-70) for nine samples when a batch cation-exchange procedure was used. Absorbance was proportional to sample concentration up to 800 mumol/liter. Analytical recovery of standard from plsma (six samples) was complete (mean, 103%; range, 90-113). Thirteen aliquots of a specimen of fresh plasma from a single adult individual gave a mean value of 20 mumol/liter (range, 11-26). The mean plasma venous ammonia concentration for 27 adults was 16 mumol/liter (range, 0-39), and for 15 newborns it was 60 (range, 34-102). Values for capillary plasma measured at the same time were higher, and we discuss possible explanations for this. Values are given for infants and children from one month to 14 years of age. Effects of storage time and temperature on plasma ammonia concentration are discussed.


A spot test for uroporphyrinogen I synthase, the enzyme that is deficient in intermittent acute porphyria. We describe a spot test for detecting deficiency of uroporphyrinogen I synthase (EC 4.3.1.8), which is characteristic of intermittent acute porphyria. The specimens used for enzyme assay are 6.5-mm filter paper discs saturated with dried blood (less than 15 mul) that was collected by direct application from a fingerstick or from venipuncture, with or without anticoagulant. The enzyme in such specimens is stable for at least nine days at -20 or c degrees C or for two days at room temperature. The discs are incubated with porphobilinogen (0.11 mmol/liter) in tris(hydroxymethyl)aminomethane HCl buffer, pH 8.2, in the dark at 37 degrees C for 3.5 h. Trichloroacetic acid is added and, after centrifugation, the supernate is examined visually with a long-wavelength ultraviolet lamp. Samples from normal and porphyric subjects are readily differentiated, both by color and intensity of the resulting porphyrin fluorescence. Anemia is a potential source of falsely positive tests, but one may accurately determine the concentration of hemoglobin in the whole blood on the filter paper discs. Moreover, the fluorescence of normal but anemic samples clearly differs qualitatively from that of porphyric specimens. Another source of falsely positive tests, variation in enzyme activity creating an overlap zone of normal and porphyric results, has not been a confounding problem. The method thus seems to offer promise for screening populations for this disorder.


How accurate are lactate dehydrogenase isoenzyme estimations by the thin-layer agarose fluorescent technique? We offer an assessment of the accuracy of the thin-layer agarose fluorescent technique of Elevitch et al. [Am. J. Clin. Pathol. 46, 692 (1966)]. We used semi-purified human lactate dehydrogenase isoenzymes 1 and 5. Both the lactate lead to pyruvate and pyruvate lead to lactate assays [Clin. Chem. 20, 1462 (1974)] appear to give, within the errors of the techniques used, a substantially unbiased estimate of both LD-1 and LD-5, although this must remain a provisional conclusion until a definitive method of assay for the total and isoenzymic LD activities is created. Introducing a buffer into the substrate mixture (lactate lead to pyruvate assay) had no effect on these findings except at extremes of pH, when marked inaccuracies occurred.


Interferences with the starch-iodine assay for serum amylase activity, and effects of hyperlipemia. Recent reports imply or claim that amylase activity is inhibited in hyperlipemic sera, because diluted samples showed greater activities when assayed by a starch-iodine method. We find that dilutions of both clear and lipemic samples give higher-than-expected activities when assayed by a starch-iodine method, an effect attributable to the variable effects of protein, turbidity, and triglycerides in the hyperlipemic samples. Thus the starch-iodine method is unsuitable for assessing the effects of hyperlipemic samples on amylase activity. To do so, we used an alternative method, in which soluble dyed amylopectin is used as the substrate. This method exhibits apparent zero-order kinetics, and we detected no interfering factors. Plots of sample volume (x) vs. activity (y) for clear and hyperlipemic (triglycerides up to 80 g/liter) sera gave straight lines with y-intercepts of zero. Evidently hypertriglyceridemia does not inhibit amylase activity.


Screening for erros in galactose metabolism with the erythrocyte. We propose determination of the ratio of the rate of galactose metabolism to glucose metabolism by erythrocytes as a screening test for abnormalities in glucose and galactose metabolism. Packed erythrocytes (20 mul) are incubated for 1 h at 37 degrees C in 0.42 ml of a solution comprising phosphate buffer (pH 7.4), 0.4 mg of glucose, 60 mg of methylene blue, and 50 nCi of either [1-14C]glucose or [1-14C]galactose. Metabolism is then stopped by injecting dilute H2SO4 through a rubber septum sealing the flasks. On incubating the acidified solution for 1 h, the evolved CO2 is trapped in a well containing ethanolamine, which is suspended from the septum. The radioactivity of the well and its contents is measured in a scintillator, and from these data CO2 is calculated. (The scintillation medium is preheated with ethanolamine to eliminate chemiluminescence.) For normal adults mean values for CO2 are 0.468 mumol/liter of erythrocytes per minute for galactose and 37.8 mumol/liter of erythrocytes per minute for glucose. Homozygous galactosemics exhibit no galactose metabolism but the rate for flucose metabolism is normal. Results for parents of homozymotes are described. We review various causes for galactosemia and point out that transfer of galactose through the cell wall and into the erythrocyte is markedly reduced in certain cats and, although unreported, may possibly be a cause for galactosemia in humans.


Energy-dispersive x-ray fluorescence spectroscopy and inductively coupled plasma emission spectrometry evaluated for multielement analysis in complex biological matrices. Energy-dispersive x-ray spectroscopy and inductively coupled plasma emission spectrometry were evaluated as methods for routine multielement analysis of biological material. Standard samples included Standard Reference Materials (National Bureau of Standards), compounded mixtures, and supplements that provided a wide range of elemental concentrations for analysis. Elements included in this study were Zn, Pb, Ni, Mn, Fe, Mg, Cu, Ca, As, Se, Br, Rb, and Sr. Standards were analyzed as unknowns by participating laboratories. The two methods were evaluated for sensitivity, precision, and accuracy, and the results compared to those obtained for atomic absorption spectrometric analysis of identical standard unknowns. Both methods compared favorably and both were determined to be highly reliable for such an application. Advantages and disadvantages of each method are compared and discussed.


Stabilization of blood glucose by cooling with ice: an effective procedure for preservation of samples from adults and newborns. Glycolysis causes a considerable decrease in blood glucose when whole blood is kept at room temperature without preservative. The most commonly used preservative, NaF, makes analysis of other serum constituents such as sodium and calcium and urea difficult or impossible, an especially serious limitation when sample size must be restricted. In samples at room temperature without preservative, plasma glucose decreased 36 mg/liter per hour in blood from adults and 60 mg/liter per hour in blood from newborns. Cooling on ice slowed these rates to 3.9 and 11, respectively. Plasma potassium increased 0.3 mmol/liter per hour in cooled specimens from both adults and newborns. Sodium, calcium, chloride and urea values were unaffected. We conclude that cooling effectively stabilizes plasma glucose for 4 h in samples from both adults and newborns and that potassium may be measured with negligible change for as long as 1 h and other constituents for the entire period.


Evaluation of a new blood-collecting device ("microtainer") that is suited for pediatric use. We evaluated the prototype of a "Microtainer" (800-mul) tube (Becton-Dickinson Co., Rutherford, N.J. 07070), In which a semi-solid silicone material is used to separate serum from erythrocytes on centrifugation. We compared results for 18 serum analytes with those obtained for specimens collected by the "dripping" method of capillary blood collection. The serum was examined microscopically for platelets and leukocytes from both normal and leukemic specimens. The cellular elements were all contained in the erythrocyte layer. Hemolysis was negligible. Means for paired samples did not differ significantly, except for Na+, for which the difference was not clinically significant. Values for potassium and lactate dehydrogenase were unchanged 24 h after collection, even though the serum was left in the collecting device.


Improved batch and column separation in the assay of hemoglobin A2. Hemoglobin A2 is a batch fractionated on a column containing diethylaminoethyl-cellulose (DE52, Whatman) with a discontinuous buffer system at pH 8.9 and 7.3. A short micro-column method is also presented, which allows separation in less than 30 min with no interference from hemoglobin S. Both methods clearly distinguish normal from thalassemia specimens, and are simpler and more rapid than previously published methods.


Automated enzymatic determination of L-lactate in serum, with use of a miniature centrifugal analyzer. We describe an automated enzymatic reaction-rate method for spectrophotometric determination of lactate in serum with a miniature centrifugal analyzer. The L(+) -lactate is selectively oxidized in the presence of lactate dehydrogenase (EC 1.1.1.27) and NAD+ to form NADH, which is measured from its absorption. Reaction rates are determined automatically, and unknown concentrations are calculated from a computer-generated calibration curve with aqueous lithium lactate standards. Lactate concentrations in the range 0.32-1.6 mug/4 mul (80-400 mg/liter) of sample were determined with relative errors and coefficient of variation of 4.8%. Analytical recovery of lactate added to pooled serum was 89-112% (average, 101%). Comparison with a kit ("Rapid Lactate") method gave a correlation coefficient squared of 0.979 over a concentration range of 39-779 mg/liter.


Properties of the amylase produced in carcinoma of the lung. The properties of the amylase produced by carcinoma of the lung were studied. The abnormal amylase recognized in the serum of a patient with carcinoma of the lung had a mobility with beta-position and showed reduced migration to the cathodic side after neuraminidase digestion. This abnormal amylase had a close affinity for Concanavalin A and this affinity was not retarded by the neuraminidase digestion. However, the purified, tumor-extracted, amylase from the same patient had the same electrophoretic migration as normal human salivary amylase and was not affected by neuraminidase treatment. The abnormal affinity for Concanavalin A was not observed in this purified tumor-extracted amylase. It is suggested that some transglycosidation steps are needed for the appearance of the abnormal amylase in the patient's serum, and that the terminal sialic acid is independent of the affinity for Concanavalin A. The dissociation constants of the tumor amylase for several substrates were smaller than those of normal pancreatic or salivary amylases. Moreover, maltotriose had no affinity for the tumor-extracted amylase and it was not digested to maltose and glucose by the purified tumor extracted amylase. These differences in the kinetic properties and in the mode of digestion were of interest in the study of tumor-produced amylases.


The effect of uraemic metabolites on parathyroid extract-induced bone resorption in vitro. A study is reported of the effects of both pre- and post-dialysis serum and some of the known uraemic metabolites on parathyroid extract-induced bone resorption using an in vitro organ culture system. Serum from uraemic patients prior to haemodialysis was shown to inhibit the action of parathyroid hormone on bone and this inhibitory effect was not present after dialysis. Some of the uraemic metabolites studied and also phosphate exerted an inhibitory effect but the metabolites caused their inhibition at higher concentrations than those which are known to occur in uraemic patients. It is concluded that if uraemic metabolites play a role in vivo then it is probably due to a cumulative phenomenon.


A transient variant of serum lactate dehydrogenase isoenzymes. An unusual variant of the serum lactate dehydrogenase (LDH) isoenzyme pattern is described in an apparently healthy young woman. The abnormal pattern consisted of a single zone of LDH activity, having the same mobility as, but more diffuse than, normal LDH-4. The molecular weight of the abnormal LDH complex is approximately 280 000, but the nature of the additional component remains unknown, as the isoenzyme pattern spontaneously reverted to normal six weeks after it was first noticed.


Simultaneous analysis of catecholamine acidic metabolites by single ion monitoring and manual control of the magnetic field. A method for multicomponent analysis by single ion monitoring is presented. This method involves monitoring of one ion per selected compound in a GC run. To focus sequentially on each ion, the magnetic field strength of the mass spectrometer is manually reset to the proper value during the intervals between eluting peaks. Stationary phase bleeding provides a suitable reference background for rapid location of selected ions. This technique has been applied to the simultaneous analysis of 3,4-dihydroxyphenylacetic acid (DOPAC), vanillylmandelic acid (VMA) and homovanillic acid (HVA) in urine samples. Quantitative data for DOPAC and HVA are presented using 3,4-dihydroxyphenylpropionic acid (DHPPA) as an internal standard.


Long-term effects of aminoglutethimide on steroid metabolism in congenital adrenal hyperplasia. Five cases of congenital adrenal hyperplasia due to C21-hydroxylase defect were treated with a combination of aminoglutethimide and prednisolone. In the third year of treatment the urinary levels of 17-oxosteroids increased above normal values while the total 17-hydroxy-corticosteroids were normally low. Specifically, urinary pregnanetriol was normal in 3 cases. To determine the reasons for this disparity the adrenal metabolism of cholesterol, as judged by the urinary steroid metabolites, was studied. Fractionation of urinary steroid metabolites was by thin-layer chromatography (TLC) followed by gas-liquid chromatography (GLC). The results indicate that aminoglutethimide inhibits steroidogenesis less than prednisolone; that a pathway from cholesterol via 17 alpha, 20 alpha-dihydroxycholesterol to dehydroepiandrosterone is likely to operate after long-term aminoglutethimide therapy; that 11 beta-hydroxylase, at least for pregnenes may be inhibited by aminoglutethimide and that the metabolic breakdown of testosterone may be delayed by this drug.


Studies on the use of skin fibroblasts for the measurement of cystathionine synthase activity with respect to homocystinuria. The levels of cystathionine synthase have been examined in cultured skin fibroblasts obtained from a patient suffering from pyridoxine-non-responsive homocystinuria and compared with the normal and heterozygous states. Levels of synthase activity were found to vary with time in culture and composition of culture media. The physical properties of normal and abnormal synthase activities were markedly different. It has been concluded that caution has to be exercised when using cell culture methods for the purpose of defining normal, heterozygous and homozygous states with reference to homocystinuria.


High-speed liquid chromatographic determination of antiepileptic drugs in human plasma. A high-speed liquid chromatographic method for the simultaneous determinations of diphenylhydantoin, phenobarbital and carbamazepine in human blood plasma is presented. This method involves two step extraction procedures with chloroform and uses 2 X 50 cm long stainless steel columns packed with a anion exchange resin, with a mobile phase of 4 mM ammonium phosphate buffer solution, pH 6.2 at a flow rate of 0.40 ml/min. The results presented show linear calibration curves and quantitative determinations as low as 1.0 mug of each drug added to 0.5 ml plasma. This method has a sensitivity sufficient to detect human plasma levels after therapeutic clinical doses.


Urinary formiminoglutamate in man. Normal values related to sex and age. Effects of low calorie intake and alcohol consumption. A modification of the enzymic method of Tabor and Wyngarden for formiminoglutamate (FIGLU) estimation in urine is described. Alkaline degradation of FIGLU in blanks enhanced the sensitivity of the method. FIGLU excretion in 94 healthy persons indicated a sex and age dependence. Short term calorie restriction or fasting led to a decrease of FIGLU excretion while acute alcohol intoxication or chronic alcohol administration were accompanied by enhanced FIGLU excretion. It is suggested that decreased FIGLU excretion was due to histidine deficiency.


Uraemia and hyperuricaemia in acute myocardial infarction. Plasma urate, urea and creatinine were measured at daily intervals for up to six days in 22 patients suffering from acute myocardial infarction. All except 3 patients showed some increase in plasma urea concentration and a tendency to increases in plasma urate was also observed. The increases in plasma urate correlated significantly with the increases in urea. This leads to the questioning of earlier reports linking hyperuricaemia with myocardial infarction when the possibility of uraemia has been ignored. Urine studies in a small number of patients showed no marked reduction in urea excretion, implying that increased production may partly account for the rise in plasma levels.


Cellular glycosaminoglycans in lymphocytes from patients with cystic fibrosis. Cellular glycosaminoglycans were isolated from lymphocytes from patients with cystic fibrosis and controls. The isolated glycosaminoglycans were fractionated by cellulose acetate electrophoresis, analyzed for glucosamine and galactosamine content, and subjected to hydrolysis with bovine testicular hyaluronidase. The total glycosaminoglycan content, the per cent glucosamine and galactosamine, and the distribution of cellular glycosaminoglycans in circulating lymphocytes in cystic fibrosis were no different from controls.


The effect of proteolytic activity on plasma renin activity assay. Carrier proteins are used in the radioimmunoassay of angiotensin I. It has been shown that the proteolytic activity found in commercial lysozyme will falsely elevate the observed plasma renin activity. This effect is probably due to the fact that human plasma contains proteinase inhibitors which inhibit this activity during radioimmunoassay of angiotensin I. This proteolytic activity can be inhibited by diisopropyl fluorophosphate or by heating lysozyme.


Polyamine distribution in cellular compartments of blood and in aging erythrocytes. Human blood was separated into pure preparations of erythrocytes, mononuclear leukocytes, polymorphonuclear leukocytes, platelets, and platelet free plasma. The mean concentrations of putrescine, spermidine, and spermine per 10(9) cells were found to be several orders of magnitude higher for leukocytes than erythrocytes. There was no significant difference between leukocyte types. Platelets and plasma had relatively low levels in proportion to the amounts contributed by erythrocytes and leukocytes to whole blood. Human erythrocytes were age-separated by density and the changes in polyamine concentrations in maturing erythrocytes were documented. There were highly significant statistical differences between young and old red blood cells for putrescine, spermidine and spermine. The clinical use of red blood cell polyamines as an indicator of the activity of the bone marrow in anemic states is suggested.


Quantitative determination of underivatized anti-convulsant drugs by high resolution gas chromatography with support-coated open tubular columns. A method is described to prepare routinely high resolution support-coated open tubular (SCOT) columns. A siliceous support material (Cab-O-Sil) is deactivated with benzyltriphenylphosphonium chloride and deposited on the inside wall of a glass capillary column. After additional coating with a polar stationary phase (OV-225) a number of anticonvulsant drugs can be analyzed without prior derivatization. The columns described show high plate numbers and do not deteriorate in use. The repeatability of the gas chromatographic analysis is better than 1.2% (Orel). The minimum detectable quantity is of the order of 10(-10) g. An improved procedure for isolating the drugs from serum is given, which results in an extraction recovery of better than 90% for the drugs of interest.


A new method for the determination of serum creatinine based on reaction with 3.5-dinitrobenzoyl chloride in an organic medium. A new method for the determination of serum creatine, based on its reaction with 3,5-dinitrobenzoyl chloride in an organic medium is described. Various analytical parameters are studied and comparisons are made with the routine picrate procedures, as well as the dinitrobenzoic acid methods reported in the literature. On the basis of the data submitted, the proposed method is recommended for routine clinical use.


The enzymatic spectrofluorimetric determination of uric acid in microsamples of plasma by using p-hydroxyphenylacetic acid as a fluorophor. A sensitive spectrofluorimetric micromethod for the determination of uric acid is presented together with its application to human and rat plasma. The method is based on the reactions or uricase and peroxidase coupled with p-hydroxyphenylacetic acid as a fluorophor. There is a very wide range of proportionality between the concentration of uric acid and the increase of fluorescence intensity. 10 ng of uric acid is still determinable. At most 25 mul of human or 50 mul of rat plasma is sufficient to obtain the accurate valve of endogenous plasma uric acid concentration. The uric acid levels of normal human and rat plasma measured by present method were within the ranges of concentrations previously reported. A comparative study of this method with a standard assay method is also presented.


An improved assay of thyrotropin in dried blood samples on filter paper as a screening test for neonatal hypothyroidism. A method for measuring thyrotropin (TSH) in eluates of dried blood samples on filter paper was evaluated and improved as a screening test for neonatal hypothyroidism. A linear relationship between the volume of eluate and the TSH value and good recoveries of endogenous TSH (104%) and added TSH (89%) were obtained, indicating that TSH in dried blood was extracted well by overnight elution and determined accurately by radioimmunoassay. The TSH content in the central portion of a spot was less (71%) than that in the peripheral portion. The TSH in dried blood samples on filter paper was stable at 4 degrees C, room temperature (about 25 degrees C) or 37 degrees C for 1 month. The TSH values of eluates were correlated with those of whole blood (r = 0.90) and serum (r = 0.81). Cases of primary hypothyroidism cound be readily differentiated from normal subjects by this method, even when eluates of their blood were combined with those of normal blood for assay of TSH.


Effect of incubation in human plasma on electrophoretic mobility of brain-type creatine phosphokinase. 1. Electrophoretic mobility of brain-type creatine phosphokinase (CPK) from rabbit, rat and human decreased after incubation in human plasma for 3 h or 17 h but did not change after incubation in isotonic saline solution. The presence of the reducing agent mercaptoethanol during incubation in human plasma tended to reduce the rate of change of electrophoretic mobility of BB-CPK. 2. The activity of brain CPK was almost completely lost after incubation for 17 h in human plasma. The addition of mercaptoethanol after incubation of brain CPK in human plasma could only partially restore the enzyme activity. 3. The decrease in electrophoretic mobility of rat brain CPK occurred after incubation in an ultra-filtrate of human plasma which contained molecules whose molecular weight did not exceed 1000. 4. Gel chromatography indicated that incubation of brain CPK in human plasma did not markedly change the molecular size of the enzyme.


Solid phase radioimmunoassay for determination of conjugated cholic acid in serum. A modified radioimmunoassay for conjugated cholates is presented. In this modification the antibody is chemically bound to Sepharose which enablproducibility (the coefficient of variation between samples is 4%). The whole procedure is carried out at room temperature and with a short incubation time (45 min). Serum can by analysed directly (no extractions or modifications needed). The assay is a suitable tool for liver function testing. A rough indication of total bile acid concentration in serum or bile can also be obtained with this assay.


Effect of agarose variability on the measurement of lysozyme activity. Lysozyme assays are often performed by a diffusion technique utilizing agarose gels impregnated with substrate organisms (lysoplates), but the results differ greatly from those obtained with spectrophotometric or immunologic techniques. We have investigated the effect of agarose composition on the lysoplate assay utilizing 10 different gels varying in ionic parameters. Standard curves generated with purified human lysozyme solutions were parallel, but the diameters of the zones of lysis varied inversely with gel sulfate content. The different agaroses had variable effects on determinations of normal serum lysozyme, and the results obtained on any given gel agreed with neither those found on other gels nor with independent assay in another system. The lysoplate assay should be utilized only in those laboratories that can obtain uniform agarose preparations and extensively calibrate normal ranges for their gels.


Diagnosis of disorders in amino acid methabolism by chemical ionization mass spectrometry. A semi-quantitative method has been developed for rapid determination of amino acids and other nitrogenous compounds in human urine. The method uses direct chemical ionization mass spectrometry with isobutane as reagent. The nitrogenous compounds are separated from the urine samples by ion exchange. Disorders in amino acid metabolism have been determined by this method in patients suffering from phenylketonuria, cystinuria and maple-syrup urihe disease.


Urinary levels of vanilmandelic acid and homovanillic acid determined by high-speed liquid chromatography. High-speed liquid chromatographic determinations of vanilmandelic acid and homovanillic acid in human urine have been made. The effects of taking refreshments on the assay were examined. HVA conjugates in urine also were hydrolyzed and determined. Urinary excretion of vanilmandelic acid, free and conjugated homovanillic acid in normal subjects and patients with neuroblastoma, phaeochromosytoma and Sipple's syndrome were determined and discussed. Urinary excretion values (mean +/- 1 S.D.) of vanilmandelic acid and homovanillic acid in 14 normal subjects were 2.0 +/- 0.7 mg/day and 4.0 +/- 1.0 mg/day, respectively.


Daily variation of lipids and hormones in sera of healthy subjects. The physiological day-to-day variation of selected hormone and lipid concentration values in sera of healthy subjects was studied. We drew blood specimens from 14 healthy volunteers, aged 22 to 40 years, (8 male and 6 female) at 0800 h on six separate days over a ten day interval. On one occasion all the twelve specimens from each subject (6 days X 2 replicates) were assayed for the hormones: thyroxine and cortisol; and the lipips: cholesterol and triglyceride which were analyzed by enzymatic methods. The assays were performed on one occasion in order to eliminate the batch-to-batch analytical variation which would tend to blur the physiological day-to-day variation. Using an analysis of variance technique, the total variation was separated into the physiological intraindividual day-to-day variation, the biological inter-individual variation, and the within-batch analytical variation. The mean physiological day-to-day variations in terms of percent coefficient of variation were 7.5% for thyroxine, 26.6% for cortisol, 4.8% for cholesterol, , and 25.0% for triglycerides.


A potential source of error in the enzymatic diagnosis of the neurolipidoses when radiolabelled sphinogolipids are used as substrates. High specific activity 3H-labelled glucocerebroside, galactocerebroside and sphingomyelin but not 3H-labelled ceramide, have been found to bind to the wall of glass scintillation vials thereby greatly decreasing the counting efficiency. The binding of the lipids was largely eliminated by counting samples in scintillation fluid containing methanol (6-10%, v/v). To avoid possible errors in the enzymatic diagnosis of the neurolipidoses, it is suggested that the latter solvent should be routinely incorporated into scintillation fluid used for counting labelled sphingolipids.


The binding of a glycopeptide component of wheat gluten to intestinal mucosa of normal and coeliac human subjects. A carbohydrate-containing component of wheat gluten (glyc-gli) has been prepared which is toxic to coeliac intestinal mucosa. Its amino acid composition is uniquely different from that of the parent gluten and from alpha-gliadin. This glyc-gli material has been shown to bind to membrane components of the coeliac intestinal cell but to bind only poorly to normal intestinal cell membranes. Furthermore this binding can be interfered with by exogenous free carbohydrate suggesting that the toxic moiety of gluten acts in an analagous way to plant lectins. These data suggest that the underlying defect in coeliac disease is related to the composition of the surface membrane of the enterocyte.


Prolactin in amniotic fluid: its correlation with maternal plasma prolactin. Prolactin concentrations in amniotic fluid from 319 women with normal pregnancies and 29 women with complicated pregnancies were determined by radioimmunoassay. Prolactin levels varied from 36 ng/ml to 1800 ng/ml mean +/- S.D. = 408 +/- 297) in the normal pregnancy group but showed no definite pattern of rise or fall during pregnancy. No difference in levels was found in complicated pregnancies. Prolactin concentrations in the plasma from 203 of these women were also assayed. The levels in the amniotic fluid were about 9 fold higher than those in the plasma. There was no significant correlation between amniotic fluid and plasma levels of prolactin.


Biphasic reaction kinetics in an anomalous isozyme of erythrocyte pyruvate kinase. A mutant erythrocyte isozyme of pyruvate kinase (PK) (ATP: pyruvate phosphotransferase, EC 2.7.1.40) has been found in associatin with chronic hemolytic anemia in two siblings who were doubly heterozygous for the isozyme and for quantitative PK deficiency of the usual form. The isozyme was characterized by approximately normal maximum reaction velocities but had markedly decreased affinity for the substrate, phosphoenolpyruvate (PEP), with 5-fold to 10-fold increases in half-saturation constants and decreased interaction between substrate binding sites. Two distinctly separable kinetic patterns for PEP were observed with multiple specimens. Concentrations of fructose 1,6-diphosphate (FDP) required for half-maximal activation were two orders of magnitude greater than controls, and optimal pH was lowered to 6.5. stability at 4 degrees C was markedly decreased, but the lost enzyme could be reactivated by fdp for periods even longer than normal controls.


Enzymatic measurement of free and esterified cholesterol levels in plasma and other biological preparations using the oxygen electrode in a modified glucose analyzer. A method is described for assaying free and esterified cholesterol using the oxygen electrode in a modified glucose analyzer to measure the relative amount of oxygen utilization taking place during oxydation of free cholesterol by the enzyme, cholesterol oxidase. A second enzyme, cholesterol ester hydrolase, is utilized to generate free cholesterol from cholesterol esters. This assay procedure is rapid, specific, reproducible and applicable to the measurement of free and esterified cholesterol carried in the major plasma lipoprotein fractions of man and the rat and, in addition, it can be utilized for the assay of sterols in subcellular fractions of cells.


alpha1-Microglobulin, a new low molecular weight plasma protein. A new low molecular weight protein was purified from the urine of uraemic patients. The protein was found to be glycoprotein with a molecular weight of 31 500, determined by SDS-polyacrylamide gel electrophoresis, and a carbohydrate content of 19%. It was electrophoretically heterogenous and migrated in the slow alpha1-region. The mean serum level in 20 apparently healthy individuals was 32+/-10 mg/1. The serum level was normal in 5 patients with severe reduction of plasma protein synthesis caused by cirrhosis of the liver but elevated in patients with a decreased glomerular filtration rate. The mean urinary excretion in 5 healthy individuals was 1.3 mg/24 h. Increased excretion was seen in 10 patients with varying degrees of uraemia.


The effect of various stains on quantitative agarose-gel electrophoresis: evaluation of the results with the aid of an analog computer. The effect of dyes of varying purity and with different binding mechanisms on quantitative electrophoresis was examined with the aid of agarose-gel electrophoresis, the phoretogram curves being evaluated with an analog computer. Concurrent quantitative results were obtained with the three dyes tested, viz. Amido Black 10 B, Ponceau Red S and Procion Brilliant Blue. It is concluded that no falsification of electrophoresis results will be caused by dyes with colour-producing secondary components.


Determination of deuterium-labeled phenylalanine and tyrosine in human plasma with high pressure liquid chromatography and mass spectrometry. A method is presented for the recovery of deuterated phenylalanine and tyrosine from human plasma. Phenylthiohydantoine derivatives are formed (Edman reaction) which are separated and isolated by high pressure liquid chromatography. The relative concentration of the deuterated amino acid is determined by mass spectrometry. The results obtained from a healthy person after oral loading with 40% monodeuterated L-phenylalanine are presented. The method appears to be suitable for in vivo studies of phenylalanine metabolism in humans.


A reliable method for generating antibodies against pancreozymin, secretin and gastrin. High specific antibodies against the gastrointestinal hormones pancreozymin, secretin, and gastrin were generated by coupling these peptides with N,N'-carbonyldiimidazole to bovine serum albumin. None of the antisera showed any cross reaction with gut and pancreatic hormones tested for cross reactivity. The dilution of antisera which were useful for the detection of 5-250 pg of hormone in human serum were 1:150 000 for secretin, 1:2000 for gastrin, and 1:2000 for pancreozymin. The N,N'-carbonyldiimidazole reagent is therefore highly effective for binding labile peptides to protein carriers without destroying the immunogenic features.


Determination of creatine kinase isoenzyme MB activity in serum using immunological inhibition of creatine kinase M subunit activity. Activity kinetics and diagnostic significance in myocardial infarction. This is a new method for the determination of creatine kinase isoenzyme MB activity in serum. The method uses direct activity measurement of creatine kinase B subunit activity after blocking of CK-M subunit activity by inhibiting antibodies. The test takes no longer than 15 min. The method yields an intra-serial C.V. of 2.0-12.9%, and a C.V. from day to day of 5.5%. The detection limit is 3.4 U/l creatine kinase MB. In the 95 cases with proven myocardial infarction several types of creatine kinase MB activity kinetics could be determined. The percentage of creatine kinase MB of peak CK-total is 6-25%, with a mean of 11.1%. The amount of creatine kinase MB with respect to total CK activity after reinfarction is higher than the amount after initial infarction.


Improved method for measurement of serum levels of phenobarbital, carbamazepine, primidone and diphenylhydantoin by gas-liquid chromatography. A gas-liquid chromatographic method for the simultaneous determination of phenobarbital, primidone, carbamazepine and diphenylhydantoin in human serum following therapeutic doses has been developed. After extraction with chloroform, the anticonvulsant drugs were methylated with phenyltrimethylammonium hydroxide in dimethylformamide at 85 degrees C for gas-liquid chromatography. Linear temperature programming of a 1% OV-17 column was used to achieve separation and quantitation. The procedure described in the present paper is relatively simple, highly specific and sufficiently sensitive for use in routine clinical assays.


Simultaneous measurement of renin and renin substrate concentration in human plasma by a simple kinetic method. A simple kinetic method is described for measurement of plasma renin concentration (PRC) and plasma renin substrate concentration (PRS) in human plasma. It is based on the radioimmunological determination of angiotensin I generated when a plasma sample is incubated at 37 degrees C for 2, 4, 6 and 8 h. The use of exogenous renin and renin substrate preparations are not required. The specific velocity constant of reaction (K) was used to deduce the PRC. PRS determination was based on the stoichiometric conversion of renin substrate to angiotensin I. PRC and PRS have been determined in plasma from healthy subjects, plasma from health subjects 3 h after oral furosemide administration and in plasma from women, 12-20 weeks normally pregnant.


Determination of manganese in blood and urine by flameless atomic absorption spectrophotometry. A method for the determination of manganese in blood and urine is described. A chelate fo manganese with cupferron is extracted with methylisobutylketone and determined by flameless atomic absorption spectrophotometry. The method is directly applicable to urine but the determination of manganese in blood required a preliminary digestion step. With the use of internal standards, this technique allows the determination of manganese concentrations of the order of 1 mug/1 of urine or 1 mug/100 ml whole blood.


Determination of quinidine in human serum in the presence of diuretics. A new method for the determination of quinidine in human serum by fluorometry is described. The interference of triamterene encountered with other methods is greatly reduced, whereas a number of other diuretics do not interfere at all. The specificity is based on quenching of the fluorescence of cinchona alkaloids by chloride ions. The method has good reproducibility and linearity, and gives a recovery of 98%. A serum sample of 0.1 ml is sufficient for the assay. Comparison with two other methods showed that this procedure yields the total amount of cinchona alkaloids present in serum, which makes it extremely useful for routine clinical analysis.


Massive excretion of 2-oxoglutaric acid and 3-hydroxyisovaleric acid in a patient with a deficiency of 3-methylcrotonyl-CoA carboxylase. A three-month old child, presenting with a history of feeding problems, suspected respiratory infection and failure to thrive, later developed fits and a profound irreversible metabolic acidosis. Chromatographic investigation of the urine revealed a gross excretion of 2-oxoglutaric and 3-hydroxyisovaleric acids. The identity of these two acids was confirmed by mass spectrometry. Enzyme studies on liver obtained at post-mortem demonstrated a deficiency of 3-methylcrotonyl-CoA:carbon dioxide ligase (ADP) (EC 6.4.1.4).


The preparation of trimethylsilyl derivatives of sodium salts of organic acids. Evidence is presented to confirm that N,O-bis(trimethylsilyl)acetamide does not form trimethylsilyl derivatives of sodium salts of many organic acids. Further evidence suggests that such derivatives are formed when either hydroxylamine hydrochloride or trimethylchlorosilane are also present. This presents a useful technique of reducing losses of organic acids due to evaporation during lyophilisation by converting them to sodium salts and preparing volatile derivatives for gas chromatography directly from the salt residue.


Infantile polymyoclonus: Its occurrence in second cousins. Two second cousins with infantile polymyoclonus are described. All other published cases in the literature are sporadic,and the occurrence of two cases of this exceedingly rare disorder in one family raises the possibility of genetic transmission. Careful examination of the family pedigree is indicated in this disorder.


Lymphocyte proliferation inhibitory factor (PIF) in alcoholic liver disease. Lymphocyte proliferation inhibitory factor (PIF) was determined in the supernatants of PHA-stimulated lymphocytes from patients with alcoholic liver disease. PIF was assayed by determining inhibition of DNA synthesis in WI-38 human lung fibroblasts. A two-fold greater inhibition in thymidine incorporation into DNA by lung fibroblasts was observed in supernatants of PHA stimulated lymphocytes from patients with alcoholic hepatitis or active Laennec's cirrhosis as compared with that found in control subjects or patients with fatty liver. It is suggested that decreased liver cell regeneration seen in some patients with alcoholic hepatitis may be due to increased elaboration of PIF.


Features of the immune response to DNA in mice. I. Genetic control. The genetic control of the immune response to DNA was studied in various strains of mice F1 hybrids and corresponding back-crosses immunized with single stranded DNA complexed to methylated bovine serum albumin. Anti-DNA antibody response was measured by radioimmuno-logical technique. High responder, low responder, and intermediate responder strains were found and the ability to respond to DNA was characterized as a dominant genetic trait which is not linked to the major locus of histocompatibility. Studies in back-crosses suggested that this immune response is under multigenic control. High responder mice produce both anti-double stranded DNA and anti-single stranded DNA 7S and 19S antibodies, while low responder mice produce mainly anti-single stranded DNA 19S antibodies.


Anti-heart autoantibodies in ischaemic heart disease patients. One hundred and ninety-nine ischaemic heart disease (IHD) patients were studied with regard to the prevalence of anti-heart autoantibodies (AHA). The incidence of AHA in IHD patients was 1%: one out of 102 patients who suffered acute myocardial infarction (AMI), one out of seventy-two patients who suffered from acute coronary insufficiency (ACI), and none out of twenty-five patients with other signs and symptoms of IHD, had AHA in their sera. An additional 2% of patients who suffered from AMI developed detectable antibody levels during a follow-up period of 15 days. In comparison,, 53% of patients (eight out of fifteen) who underwent heart surgery and who had no AHA prior to operation, developed these antibodies in their sera during 1-2 weeks following operation.


Computerised axial tomography and the diagnosis of glioma: a study of 100 consecutive histologically proven cases. The reliability of computerised axial tomography (CAT) in the recognition of intracranial lesions is now accepted. Apart from ventricular displacement, compression or distension, abnormal patterns in the area of a lesion may be shown on the oscilloscope display and the corresponding polaroid film, and these may show additional enhancement patterns after the intravenous injection of a contrast medium. In an attempt to assess the changes shown with gliomas, both before and after contrast enhancement, and the possible relationship to the type of glioma present, and analysis has been made of 100 consecutive histologically proven cases. Other lesions occurring during the same period as the study, and giving a similar display, are illustrated and the differential diagnosis discussed.