Ultrastructural studies on the vitellogenesis of Tetrodontophora bielanensis (Waga) (Collembola). Vitellogenesis in Tetrodontophora bielanensis (Waga) is of the "mixed" type. Part of the yolk material is produced inside the oocyte (autosynthesis), while part is absorbed by micropinocytosis. During antosynthesis polyribosomes, rough endoplasmic reticulum and dictyosomes take part. Regardless of their origin, mature yolk spheres are constructed identically and are composed of three elements: cortex layer, matrix and crystals. Histochemical tests show that polysaccharides are present in the yolk spheres. Lipid droplets have been observed in the ooplasm; they develop without visible contact with any of the organelles. Among the reserve materials the following have been found: rough endoplasmic reticulum, dictyosomes, polyribosomes, mitochondria and a few microtubules.


Sperm development in the teleost Oryzias latipes. In Oryzias latipes the processes of spermatogenesis and spermiogenesis occur within testicular or germinal cysts which are delimited by a single layer of lobule boundary cells. These cells, in addition to comprising the structural component of the cyst wall, ingest residual bodies cast off by developing spermatids. Therefore, they are deemed to be the homologue of mammalian Sertoli cells. The germ cells within a cyst develop synchronously owing to the presence of intercellular bridges connecting adjacent cells. Since bridges also connect spermatogonia, it seems probable that all of the germ cells within a cyst may form a single syncytium and do not exist as individual cells until the completion of spermiogenesis when the residual bodies are cast off. Significant differences between spermiogenesis in O. latipes and in the related poeciliid teleosts are discussed.


Fine structure of the interplatelet area in the gills of the macrophthalmia stage of the river lamprey, Lampetra fluviatilis (L.). The interplatelet epithelium of the gills of freshwater and seawater adapted macrophthalmia stages of the river lamprey, Lampetra fluviatilis, was studied by electron microscopy. Three main cell types: basal, ion-excretory and superficial cells were identified and described. Ion-excretory cells were exposed to the environment in sea water but covered by a layer of superficial cells in fresh water. Electron-dense secretory granules in the apex of the cell were more abundant in fresh water. In fresh water, a few of the superficial cells contained numerous mitochondria and were identified as ion-uptake cells. In view of the scarcity of ion-uptake cells, the possibility that the ion-excretory cells are also responsible for ion-uptake in fresh water is discussed. Phagocytic removal of degenerating ion-excretory cells may be an important mechanism for the conservation of materials in a starving animal.


Fine structural observations on the protonephridium of the terrestrial triclad Geoplana pasipha. The protonephridial system of the terrestrial triclad Geoplana pasipha was studied by electron microscopy. In general construction it conforms to that of the freshwater planarian Dugesia tigrina (McKanna, 1968, 1968a). Ultrastructural details permit the identification of proximal, collecting, osmoregulatory and nephridiopore regions. Intralumenal material is frequently observed within the tubules, whose cells are always joined by septate junctions. Cilia are numerous and possibly contribute to the flow of water and solutes along the system. Proximal tubules have a peculiar, particulate membrane coat, while a continuous coat, densely stainable with ruthenium red lines the more distal, unciliated tubules.


Further histophysiological observations on the lower esophagus of the rabbit. Previous neurohistological studies have been extended to include the structures contained solely or mainly within the junctional esophageal segment which may play an important role in the sphincter mechanism. The main findings were: 1) a progressive cranio-caudal thickening of the muscularis mucosae; 2) a conspicuous thickening of the circular muscle layer; 3) abundant and close interconnections between the esophageal striated fibres and gastric smooth muscle cells; 4) presence of annulo-spiral elastic fibres coiled around bundles of striated musculature; 5) increase of the intramural nerve component, particularly Auerbach's plexus, which consisted of a continuous nervous layer containing twice as many neurocytes as found in the upper esophageal segments; 6) presence of numerous interconnected motor endplates often possessing ultraexpansional fibres and secondary endplates. The findings are discussed with emphasis on functional correlations in order to attain a unitary morpho-functional view.


The discrimination of nine different types of synaptic boutons in the fundus striati (Nucleus accumbens septi). An attempt has been made to discriminate additional types of synapses than have been previously described in the nucleus accumbens septi of the cat, which can, according to Brockhaus (1942), justifiably be termed the fundus striati due to the fact that it possesses all of the morphological and some of the neurochemical features of the striatum. This was undertaken in order to correlate at least one type of synapse with each different afferent pathway. Nine distinct types of synapses could be differentiated electron microscopically: Type I: axo-spinous synapses with sparse, small, round vesicles which seemed to be the nigro-striatal endings (35%). Type II: axo-somatic or axo-dendritic en passant synapses containing small, round vesicles (3%). Type III: axo-spinous synapses filled with densely-packed, small, round vesicles displaying strong postsynaptic thickenings which seem to be cortico-striatal (17%). Type IV: large axo-spinous synapses with densely-arranged, small, round vesicles contacting larger spines branching off a pedicle (9%). Type V: axo-somatic or axo-dendritic synapses containing large pleomorphic vesicles, probably axon collaterals (1%). Type VI: axo-somatic or axo-dendritic synapses with elongated small vesicles (20 X 45 nm) (3%). Type VII: large axo-somatic or axo-dendritic synapses filled by densely-packed, small, round vesicles (11%). Type VIII: large axo-somatic or axo-dendritic synapses containing loosely-arranged, small, round vesicles (8%). Type IX: axo-somatic or axo-dendritic synapses containing large, round vesicles in a translucent axoplasm (13%).


Ultrastructure and permeability of lymph node microvasculature in the mouse. The microvasculature of lymph nodes and Peyer's patches consists of arterioles, capillaries and venules. The postcapillary segment comprises high-endothelial venules (HE venules) as well as ordinary venules. In order to study the ultrastructure of the microvasculature, particularly with respect to the nature of intercellular junctions, lanthanum and ruthenium red were used as tracers. Furthermore, to evaluate the permeability properties of the different segments of the microvasculature, intravenously injected horseradish peroxidase (HRP; MW: 40,000) was used. All segments of the microvasculature are permeable to HRP. However, the mechanism of transport across the vascular wall varies in the different segments, apparently correlated with a gradual decrease in number of transport vesicles and a gradual attenuation in the sealing of the endothelial cells. Tight junctions are present in arterioles, and it is assumed that HRP reach the basal lamina exclusively by vesicular transport. Incomplete or focal tight junctions are present in the capillaries, and both intercellular and vesicular pathways are observed. In the venules the intercellular pathway seems to be the dominant one, while vesicular transfer is negligible. However, some micropinocytic vesicles in the HE venule endothelial cells probably represent the initial stage of an intracellular digestion.


Effects of cystic fibrosis serum on the rat parotid gland. Injections of serum from human patients with cystic fibrosis into adult rats caused pronounced structural modifications and increased mitotic rate in the parotid gland. Mitotic rate was increased from a low level of 0.02/1,000 acinar cells in parotid glands of adult rats to 6.5/1,000 acinar cells after 2 or 3 days of serum injection. At the light and electron microscopic levels, significant acinar cell atrophy and degranulation were observed. Cellular necrosis, and increases in quantity of lysosome-like dense bodies, mast cells, and macrophages were also detected. These changes are suggestive of tissue response to injurious foreign protein. Furthermore, the fact that normal sera pronounced the same kind of effects (but greatly reduced in extent) strengthens the view that these effects result from the immunologic response of the host organ to foreign antigen. Since, however, the responses of the rat parotid to cystic fibrosis serum were considerably more marked than those elicited by normal serum, the rat parotid may thus have potential usefulness in assaying for the presence of human cystic fibrosis factor.


Changes in the fine structure of venous endothelium in the rabbit following adrenaline infusion. The fine structure of the endothelium lining the femoral and mesenteric veins in the rabbit was examined following the infusion of concentrations of adrenaline varying from 0.5 to 5.0 mug/kg. Control animals were infused with saline. Vesiculation of the endothelial cells was observed following each infusion of adrenaline, the effect being most marked with the highest dose used. It is suggested that this vesiculation may be associated with the release of antihaemophilic globulin and/or plasminogen activator from the endothelial cells.


Microtubule fascicles in the stem processes of cultured sensory ganglion cells. Microtubule fascicles, resembling those characterizing the initial segment of multipolar neurons, have been observed by electron microscopy within and close to the origin of the stem process of some unipolar ganglion cells in explant cultures of embryonic chick dorsal root ganglia. Each fascile comprised 2-6 closely spaced parallel microtubules linked by electron dense cross-bridges. Since similar observations have been made on stem processes in vivo, the possibility that linked microtubules occur commonly in this site is considered. The observations are discussed in relation to a possible correlation between the presence of microtubule fascicles and the initiation of action potentials.


A light and electron microscopic study of the ciliated urn of Phasolosoma agassizii (Sipunculida). The free swimming ciliated urn found in the coelomic fluid of Phascolosoma agassizii has been studied by electron microscopy. The urn is a multicellular structure composed of three cell types: (a) ciliated cells which possibly function in capturing cell debris and foreign particles; (b) cupola cells which are capable of phagocytozing latex particles; and (c) lobe cells which are capable of phagocytozing carbon particles. The lobes are separated from the ciliated cells by a semilunar area, with mucoprotein staining characteristics, containing fibrils which appear to be the structural support for the urn. Ciliated cells and lobes are attached to the semilunar area by hemidesmosomes.


The ultrastructure of the human fetal pineal gland. II. Innervation and cell junctions. The innervation of the pineal gland, the cell junctions in this organ and junctions between ependymal cells in the pineal recess were investigated in 27 human fetuses (crown-rump length 30-190 mm). Free nerve boutons containing clear and a few dense core vesicles were present in the pineal parenchyma and in the perivascular spaces. The boutons did not make "synaptic" contacts with the pinealocytes. No evidence for the presence of noradrenaline in the vesicles of nerve boutons was found. Gap junctions, intermediate-like junctions and desmosomes were frequently seen between the pinealocytes. Ruthenium red was used in three fetuses as an extracellular marker. The continuous endothelial cells surrounding the capillary lumen were connected by tight junctions. This indicates the presence of a blood-brain barrier. Tight junctions were present between the ependymal cells in the pineal recess. These junctions constitute an extracellular barrier between the pineal and the cerebrospinal fluid.


Studies on silk secretion in the Trichoptera (F. Limmephilidae). II. Structure and amino acid composition of the silk. The ultrastructure and amino acid composition of the secreted silk of two species of trichopteran larvae, Pycnopsyche guttifer (Walk.) and Neophylax concinnus McL., were investigated. The spinnerets of these two animals were also examined by scanning electron microscopy. The silk consists of double-stranded, flat ribbons (1-4 mu wide), composed of bundles of 15-25 A filaments. There are two components of the silk: the fiber proper and a surrounding coat thought to be a silk "gum". Only the outer coat is positive to the EM PATP technique of Thiery (1967), which indicated the presence of neutral sugars. Amino acid analyses of Pycnopsyche silk show that, like other silks, two predominant amino acids are glycine and serine. Arginine, unexpectedly, is the third most abundant and there are a large number of basic and long side-chain amino acids. X-ray diffraction studies of the silk indicate that it has a less crystalline, more amorphous structure than that of other silks.


The ultrastructure of the cells of Leydig in the white-crowned sparrow (Zonotrichia leucophrys gambelii) in relation to plasma levels of luteinizing hormone and testosterone. In male White-crowned Sparrows subjected to 20 h daily photoperiods there is an approximately 3-fold increase in the plasma concentration of luteinizing hormone (LH) on the first long day after which a quasi-stable level is maintained for at least 42 days. This increase is followed by an increase in numbers of cells of Leydig and an enhancement of their steroidogenic features, a decrease in transitional interstitial cells, and an increase in plasma level of testosterone. With the decline in plasma LH, as photorefractoriness develops, the steroidogenic features of the cells of Leydig undergo disorganization. For as yet unexplainable reasons the plasma levels of testosterone decline before the decrease in plasma LH and before the degeneration of the steroidogenic features of the cells of Leydig.


Limitations of the double sucrose gap voltage clamp technique in tension-voltage determinations on frog atrial muscle. The purpose of this study was to evaluate the limitations of the double sucrose gap voltage clamp technique in the determination of tension-voltage relationships for frog atrial muscle. Tension-voltage relationships were determined under two conditions. In one case we determined both the tension response and slow inward current associated with an apparent step depolarization (step-clamp) as a function of the magnitude of the step depolarization. In the second case, an action potential was elicited, the voltage clamp was applied early during the plateau phase of the action potential, and the tension response was determined as a function of the clamp potential (action potential-clamp). Under both step-clamp and action potential-clamp conditions, the waveform of the tension response rose to a peak value (Tp) and then decayed with time to a tension that was maintained for the duration of the depolarization. The Tp-clamp potential relationships obtained under step-clamp and action potential-clamp conditions were similar. Microelectrode measurements of transmembrane potential of cells in the "voltage-clamped" region of the preparation demonstrated the lack of temporal and spatial voltage control under both step-clamp and action potential-clamp conditions, and also demonstrated that acquisition of spatial voltage control occurred at about the same time that the tension response reached its peak value. These data indicate that this voltage clamp technique does not allow an accurate determination of the so-called phasic tension-voltage relationship in frog atrial muscle because of a lack of temporal and spatial control of voltage during the rising phase of the tension response.


Measurements of disordered flows distal to subtotal vascular stenoses in the thoracic aortas of dogs. Instantaneous blood velocity measurements employing a constant temperature hot film anemometer were obtained in the region distal to externally enforced, subtotal vascular stenoses in the descending thoracic aortas of anesthetized dogs. Our objectives were to determine alterations in velocity waveforms and energy spectra as the degree of stenosis was increased. We paid particular attention to distinguishing features of the flow which were characteristic of turbulence. Our results indicate that, for the vessels studied, disturbances in the velocity waveforms occur for very minor localized constrictions. The energy spectra follow certain similarity parameters within a restricted region of the distal velocity field. For severe stenoses relatively high levels of energy exist in frequency ranges which previously have been found to produce poststenotic dilation. The measurements suggest that velocity waveforms and energy spectra provide a very early clue to the existence of localized occlusive vascular disease in larger vessels and that, within a limited region distal to a stenosis, the degree of constriction may be estimated by similarity analysis of the energy spectra.


Neurogenic sympathetic vasoconstriction of the rabbit basilar artery. When examined by fluorescence microscopy the rabbit basilar artery contains a rich adrenergic-like plexus at the adventitiomedial junction. The fluorescence disappears upon chronic reserpinization and bilateral superior cervical ganglionectomy. Transmural stimulation of intramural nerves a results in a response which is predominantly constrictor but also contains a small, inconstant dilator component. The constrictor response is abolished by chronic reserpinization, bilateral superior cervical ganglionectomy, and cold storage of the preparation. The constriction is prevented by the adrenergic neuron blocking agents guanethidine and bretylium but not by such alpha-adrenergic receptor blocking agents as phenoxybenzamine (PBZ), phentolamine, and tolazoline. Our results show that doses of the three latter agents sufficient to abolish contractions to norepinephrine (NE) in concentrations of up to 10(-2) M only potentiate and prolong the contractile response to nerve stimulation. The beta-adrenergic receptor blocking agent, propranolol, and inhibitors of NE neuronal uptake, such as desipramine (desmethylimipramine, DMI) and cocaine, do not influence the size of the neurogenic response. These results suggest that the vasoconstrictor component of the rabbit basilar artery response to transmural nerve stimulation (TNS) is mediated via sympathetic adrenergic-like neurons, but at the same time also raise the question whether the transmission process is typical of classic adrenergic neuroeffector mechanisms.


Effects of acutely induced hypertension in cats on pial arteriolar caliber, local cerebral blood flow, and the blood-brain barrier. Acute hypertension was induced in 19 anesthetized cats by the intravenous administration of angiotensin. The caliber of pial arteries was measured by a television image-splitting technique and local cerebral blood flow by the hydrogen clearance technique. As the blood pressure was increased, pail arterioles constricted and cerebral blood flow remained relatively constant, showing  that autoregulation of cerebral blood flow was intact. At mean arterial pressures of more than 170 mm Hg arteriolar dilation appeared. In smaller arterioles (initial diameter less than 100 mum) a segmental dilation (the "sausage'string" phenomenon) frequently preceded uniform dilation. This arteriolar dilation was associated with a marked increase in local cerebral blood flow indicating that the upper level of autoregulation had been breached. In no cat was vasospasm or a decrease in blood flow observed during induced hypertension. Hypertension also caused dysfunction of the bloodbrain barrier since, in 17 out of 19 of the cats examined, there was extravasation of protein-bound Evans blue into brain tissue. In only one of the 19 cats subjected to neuropathological analysis was  ischemic brain damage identified and this was restricted to minimal ischemic cell change. The results indicate that severe, induced hypertension in cats produces cerebral arteriolar dilation, an increase of cerebral blood flow, and dysfunction of the blood-brain barrier. These observations may be of importance in understanding the pathogenesis of hypertensive encephalopathy.


Physiological loading of isolated mammalian cardiac muscle. Cat papillary muscles were subjected to a complex loading function resulting from an analysis of the heart as a pump. The papillary muscle was assumed to be a hypothetical bundle of circumferential muscle fibers in the wall of a simplified cylindrical ventricle. The loading included inertial, resistive, and capacitive components of the cardiovascular system. Changes of ventricular dimensions were taken into account by application of a Laplace relationship. When this complex dynamic loading function was imposed on a shortening muscle by means of an electromagnetic feedback system, the developed force continuously changed with time. The time course of this changing force corresponded to the time course of calculated stress in the intact ejecting heart. Directly displayed force-velocity loops also were similar to loops obtained for the intact heart. Loads proportional to velocity of shortening (damping), acceleration of shortening (inertia), and to the square of shortening velocity (Bernoulli) were investigated separately. Cardiac muscle appeared rather insensitive to inertial loads, and the contribution of inertial loads in the early phase of a contraction under physiological pump loading was minimal. Moreover, during all these dynamic loadings, as long as loading was dynamically increasing or decreasing, velocity of shortening was respectively lower or higher at any muscle length and total load, when compared to velocity at the same length and load under static (constant preload and afterload only) loading.


Compression of the coronary arteries by the fibrillating canine heart. We performed experiments to test whether the subendocardial ischemia which reportedly accompanies elective ventricular fibrillation (VF) during cardiopulmonary bypass might be the result of mechanical compression of the coronary vessels. The left coronary artery of the open-chest, anesthetized dog was cannulated and perfused with arterial blood through an extracorporeal circuit. Coronary inflow rate was held constant with a pump and the coronary vessels were dilated maximally by infusing adenosine. Any change in perfusion pressure or the transmural distribution of flow in these hearts would have been due to changes in compression. When the hearts were stopped in diastole by vagal stimulation, infusion of microspheres 15 mum in diameter revealed a subendocardial to subepicardial (inner to outer) flow ratio (I/O) of 1.2. When the same hearts were caused to fibrillate spontaneously (not electrically maintained) the I/O fell to 0.9. Little change in coronary perfusion pressure occurred between arrest and VF. When the contractile activity during VF was attenuated by intracoronary sodium pentobarbital (120 mg) the I/O rose toward that seen during arrest. However, augmentation of muscle activity by infusion of isoproterenol during VF failed to change the I/O. Finally the I/O fell in proportion to the degree of distention in the fibrillating ventricle. The results that we observed indicate that muscular contraction during VF preferentially inhibits subendocardial flow through vascular compression.


Studies on the nature of a prostaglandin receptor in canine and rabbit vascular smooth muscle. The contractile response of rabbit renal arteries and canine tibial arteries to prostaglandins A2, B2, F2alpha, E1, E2, D2, and B1 was associated with a reduction in total sulfhydryl group content of smooth muscle. The total sulfhydryl content of rabbit renal and canine tibial arteries and was not affected by norepinephrine or potassium chloride. Reduction of disulfide groups with dithiothreitol (DTT) selectively inhibited contractile responses to angiotensin and prostaglandins; 5,5'-Dithiobisnitrobenzoic acid (DTNB), a sulfhydryl group-oxidizing agent, reversed the inhibitory effect of DTT on the contractile responses to prostaglandins. Alkylation of free sulfhydryl groups with ethacrynic acid did not affect the contractile response of isolated canine tibial or rabbit renal arteries to any agonist studied. Ethacrynic acid added to muscle strips exposed to DTT resulted in alkylation of sulfhydryl groups produced by reduction of disulfide bonds and irreversibly prevented DTNB-induced reversal of DTT inhibition of contractile responses to prostaglandins. However, addition of ethacrynic acid to muscle strips contracted by prostaglandins did not inhibit subsequent responses to these acidic lipids. These findings support the hypothesis that contractile responses of rabbit renal and canine tibial arteries to prostaglandins are dependent on interactions between prostaglandins and disulfide groups located in or on the vascular smooth muscle cell, and the concept that membrane disulfide groups may be integral components of vascular smooth muscle receptors for prostaglandins.


Spontaneous action potentials of cells in the canine sinus node. Right atria were excised from the hearts of 20 young dogs (15 +/- 5 weeks old) and maintained in vitro in order to perform microelectrode impalements. The atria contracted spontaneously for at least 6 hours at a stable rate of 127/min when they were perfused with Krebs-Ringer bicarbonate solution through the sinus node artery. Cells within a small area near the sinus node artery and between the superior vena cava and right atrium consistently yielded transmembrane potentials typical of pacemaker cells. This area was verified histologically to be the midportion of the sinus node. Electrical characteristics of these sinus node pacemaker cells were: maximum diastolic potential, -56 +/- 7 mV (mean +/- 1 SD); action potential amplitude, 56 +/- 8 mV; overshoot, 0 +/- 2 mV. Smooth transitions from phase 4 to phase 0 were always present. From these experiments we conclude that the excised perfused canine right atrium provides a useful new experimental cardiac preparation because it maintains stable spontaneous activity for many hours in vitro and the sinus node within it is readily accessible for study by intracellular microelectrodes.


Evidence for an adenosine receptor on the surface of dog coronary myocytes. Adenosine and theophylline were linked covalently to oxidized stachyose to produce compounds too large to penetrate cell membranes. These compounds were used in two conscious and six open-chest anesthetized dogs to test the hypothesis that there is an adenosine receptor on the surface of the coronary myocyte. Intracoronary infusions of the adenosine derivative produced dose-dependent coronary vasodilation which was antagonized by theophylline; two types of theophylline derivative antagonized the coronary vasodilatory action of adenosine. Although these results show that both adenosine and theophylline exert their coronary effects at the surface of the smooth muscle cells, this evidence does not establish that they are competing for a common receptor.


Action potentials in chick atria. Ontogenetic changes in the dependence of tetrodotoxin-resistant action potentials on calcium, strontium, barium. Action potentials were recorded from chick embryo atrial muscle cells bathed in Tyrode's solution. Tetrodotoxin (TTX), 3.1 muM, was added to block the early, transient, Na+-dependent conductance system. Rectangular stimuli were used to evoke action potentials the peak amplitude (Ep) of which depend on the external concentration of divalent cations, [Me2+]0. The relationship between Ep and [Me2+]0 shifted to the right with increasing age. For example, the slope of Ep was 33 +/- 2,22 +/- 1 and 11 +/- 3 mV per 10-fold change in [Ca2+]0 on the 9th, 12th, and 18th incubation days, respectively. In solutions with reduced [Ca2+]0, Ep increased when Ba2+ or Sr2+ was added to the bath. The potency of Me2+ in generating action potentials was Ba2+ greater than Sr2+ greater than Ca2+ and this sequence did not change during development. Action potential amplitude, which was reduced in 18-day preparations, was increased by isoproterenol (increased Ca2+ conductance, gCa2+) and by tetraethylammonium (TEA) ion (decreased K+ conductance, gK). The results show that (1) Me2+-dependent action potentials support membrane excitation in chick atrial cells treated with TTX, and (2) the ability of Me2+ to support action potentials decreases during ontogenesis. We conclude from these experiments that the ontogenetically related decrease in Me2+-induced action potentials is the result of a reduction in gMe2+/gK+ during stimulation.


Esophageal echocardiography.

Esophageal echocardiography. Esophageal echocardiography has been developed for use in patients with chronic obstructive pulmonary disease and is a safe diagnostic procedure which provides high resolution mirror image echoes of many cardiac structures. Conventional anterior and esophageal echocardiograms were performed in 38 subjects. Esophageal echoes were of diagnostic quality in all 38 subjects, anterior echoes were of diagnostic quality in only 18. Measurements from anterior and esophageal echocardiograms correlated well for aortic valve diameter (r = 0.87), left atrium diameter (r = 0.96), mitral valve EF slope (r = 0.97) and less well for aortic root diameter (r = 0.69).


The natural history of truncus arteriosus. The cases of 23 patients whose condition was diagnosed as truncus arteriosus, type I or II, and who were seen at the Mayo Clinic during the decade preceding 1967, that is, before corrective operation became feasible, were reviewed. Ten were infants (through one year of age), and all ten have died. Eight ranged in age from more than one year through seven years of age, and all are living, except one, who diet 11 years after diagnosis. Five were older than seven years, and all had severe pulmonary vascular obstructive disease; three have died. Thus, 14 of the 23 have died, and all but one surviving patient have mild to moderate disability. The generally grave prognosis for patients with truncus arteriosus warrants continued use of corrective operation, but suggests that the greatest benefit can be realized by successful correction in the infant with congestive heart failure and in early childhood before the development of severe pulmonary vascular obstructive disease.


The surgical management of discrete and diffuse supravalvar aortic stenosis. Between 1956 and 1976, 18 patients underwent surgery for supravar aortic stenosis at the The Children's Hospital Medical Center, Boston. Discrete obstruction, present in 11, was treated by insertion of a prosthetic gusset placed across the area of narrowing and extending into the noncoronary sinus of Valsalva. There was one operative death. Residual gradients (measured in five patients) ranged from 4-55 mm Hg, one of which was supravalvar in location. Significant aortic regurgitation was not common preoperatively. The diffuse form of supravalvar obstruction, a more difficult surgical problem, was present in seven patients. There were three operative deaths. Complete relief of the pressure gradient was achieved only in one instance by insertion of a left ventricular-aortic bypass shunt diverting the majority of the cardiac output into the descending thoracic aorta. This patient is now asymptomatic 20 months following operation. On the basis of this experience, it is suggested that patients with the diffuse form of supravalvar obstruction, and perhaps even those with a hypoplastic annulus alone, would benefit from a left ventricular-aortic bypass shunt.


Transatrial resection of the obstructed right ventricular infundibulum. Obstructions of the right ventricular infundibulum were resected through the orifice of the tricuspid valve in 21 patients, 15 of whom had tetralogy of Fallot. At operation the systolic pressure difference between the right ventricle and pulmonary artery after repair averaged 18 mm Hg (range 0-40 mm Hg). In patients with tetralogy, cardiac index four hours after operation averaged 2.8 L/M2/min. One patient with tetralogy and severe pulmonary hypertension died. Twelve patients with tetralogy were recatheterized 10 to 186 days after operation. The mean systolic pressure difference between right ventricle and pulmonary artery was 23 mm Hg. Residual obstructions were in the pulmonary valvular annulus. Cineangiograms did not show paradoxical motion of the right ventricular wall. Transatrial resection of right ventricular infundibular obstructions carries with it none of the consequences that often follow right ventriculotomy and this surgical approach satisfactorily relieves infundibular obstructions.


Intracardiac repair of cardiac malformations with atrioventricular discordance. Twenty-six patients with atrioventricular discordance and a variety of ventriculo-arterial connections have had appropriate intracardiac repairs. Seven of 26 patients (26.9%) died early postoperatively, but only two (11.1%) of 18 patients operated upon since May 1972 have died. Ten patients (40%) developed permanent complete heart block. Two patients died late postoperatively; 16 (84.1% of those surviving the early postoperative period) were in NYHA Class I or II at late follow-up. Seventeen of the patients had "corrected transposition." Six developed tricuspid incompetence with regurgitation into the left atrium after repair. Two patients with atrioventricular discordance and double outlet right ventricle survived operation and are asymptomatic but have permanent pacemakers. One patient died after repair of double outlet left ventricle with atrioventricular discordance. Three of four patients with atrioventricular discordance and "pseudotruncus arteriosus" survived repair with valved external conduits and are well. One of two patients is well after repair of "isolated ventricular inversion" and ventricular septal defect.


Failure of demand pacing with small surface area electrodes. As pacemaker electrode myocardial contact area is reduced, the energy required to stimulate the heart decreases; but the effect of surface area on an electrode's ability to transmit R-wave potentials has not been well documented. Endocardial and intramyocardial R-wave potentials were measured in ten dogs with seven commercially available pacemaker electrodes of different surface area. With a load impedance of 1000 ohms, there was a direct correlation between surface area and the R-wave potentials measured. The amplitude of the R-wave that resulted from conduction from the heart to the sensor via the electrode system decreased with decreasing surface area. A similar direct correlation was seen between the R-wave potentials measured and the threshold current and energy requirements. With the present trend toward utilization of small surface area electrodes to reduce pacemaker cell drain, care must be taken to optimize pacemaker circuit impedance if sensing problems are to be avoided.


Effects of perhexiline maleate on coronary flow distribution in the ischemic canine myocardium. Intravenous perhexiline maleate in a canine preparation with fixed coronary flow increases coronary diastolic pressure. It also redistributes coronary flow so as to preserve endocardial flow. Myocardial oxygen consumption was reduced and lactate uptake enhanced by the drug. It had no effect upon the threshold for ischemic-induced left ventricular failure.


Valve ring abscess in active infective endocarditis. Frequency, location, and clues to clinical diagnosis from the study of 95 necropsy patients. Analysis of 95 necropsy patients with active infective endocarditis (AIE) involving 128 native cardiac valves (aortic = 59, mitral = 48, tricuspid = 20, and pulmomic = 1) disclosed 27 patients with ring abscesses involving 30 valves: the aortic valve ring was infected in 24 patients and only an atrioventricular valve ring (mitral in two, and tricuspid in one) in three patients. Comparison of the following parameters showed no significant differences between the 27 patients with and the 68 patients without ring abscess: age, sex, antibiotic treatment or length of treatment, status of the cardiac valve(s) before infection, and the kind of infecting organism. Comparison of the following parameters, however, showed significant (P less than 0.05) differences between the 27 patients with and the 68 patients without valve ring abscess: 1) infection of the aortic valve; 2) occurrence of valvular regurgitation of recent origin; 3) presence of pericarditis; 4) presence of high degree of atrioventricular block; and 5) short duration of symptoms leading to severe debility or death. These five features, therefore, serve as a clinical clues to the presence of valve ring abscess in patients with AIE.


The cortisol response during heart-lung bypass. The response patterns of plasma cortisol and plasma free cortisol have been studied in 20 consecutive patients undergoing cardiac surgery involving the use of heart-lung bypass. Sixteen consecutive patients undergoing closed mitral valvotomy have been used as controls. Total plasma cortisol levels fell at the onset of extracorporeal circulation (ECC) and did not rise significantly throughout the period of ECC. The peak cortisol level in the bypass patients occurred at 24 hours postoperatively with elevated levels still present at 48 hours. This pattern was significantly different from the control patients (P less than 0.001) who exhibited the typical cortisol stress response pattern. Synthetic ACTH stimulation of the adrenal cortex during heart-lung bypass produced a positive response (i.e., a rise in plasma cortisol greater than 7.5 mug/100 ml) in all six patients studied while the six mitral valvotomy patients had no response to synthetic ACTH administration during operation (P less than 0.01). Plasma free cortisol estimations indicate that a rapid and significant rise in % free cortisol occurred at the onset of ECC, but that despite the rise in percentage free, the overall plasma free cortisol concentration (i.e., total plasma cortisol X % free) was significantly lower during ECC in the bypass patients when compared with control (P less than 0.01). Twenty-four hours postoperatively these concentrations were significantly higher in the bypass patients (P less than 0.001).


Effect of hypertonic mannitol and isoproterenol on regional coronary flow following right ventriculotomy. We studied the changes which occurred in regional coronary blood flow after right ventriculotomy and the subsequent effects of hypertonic mannitol or isoproterenol infusion. Regional coronary flows were measured with radioactive microspheres (9 micron) in open-chest anesthetized dogs. Either hypertonic mannitol (25%) was infused at 3.2 ml/min or 7.6 ml/min for 30 minutes, or normal saline at 7.6 ml/min for 30 minutes followed by isoproterenol at 0.05-0.10 mug/kg/min. In the mannitol treated animals right ventricular, left artrial and aortic pressures, heart rate, and cardiac output did not change significantly following vertical ventriculotomy, whereas in the saline-isoproterenol treated animals aortic pressures fell significantly. Coronary flow to the peri-incisional area fell from 41 to 24 ml/min-100 g (P less than 0.05) following ventriculotomy, increased by 34% (P less than 0.05) when osmolality rose after mannitol by 37 mOsm, but was unchanged with isoproterenol. The data indicate that a vertical ventriculotomy reduces flow to adjacent myocardium and that subsequent infusion of hypertonic mannitol at moderate rates significantly increases coronary flow to this region.


The protective effect of glucose-insulin-potassium on the response to atrial pacing. The effects of glucose-insulin-potassium infusion (GIK) on atrial pacing-induced angina, ST depression, abnormal left ventricular end-diastolic pressure during pacing interruption (LVEDPi) and lactate metabolism (L), were studied in 18 patients: ten had angina during pacing = Ischemic group, and eight (5 normals and 3 with coronary artery disease) remained asymptomatic = Nonischemic group. The study consisted of 8-10 minute periods of control, pacing and recovery, before and after GIK. No untoward effects were observed. Comparison of the pacing responses (GIK vs pre-GIK states) showed that during GIK, angina occurred in only 4 patients, while significantly less severe changes were observed in ST depression (1.4 +/-0.5 vs 2.4 +/- 0.4 mm) and LVEDPi (16 +/- 3 vs 23 +/- 3 mm Hg). Lactate extraction was also higher (8.1 +/- 10.9 vs -5.2 +/- 11.1%), but not significantly so, although L became normal in 4 subjects and improved in another. These results indicate that GIK infusion was well tolerated and had a beneficial effect on pacing-induced myocardial ischemia.


A study of comparative blood pressure measures in predicting risk of coronary heart disease. The Western Collaborative Group Study is a prospective study of 3,154 employed men, aged 39-59 years. Coronary heart disease (CHD) occurred in 257 subjects during 8.5 years of follow-up. The multiple logistic risk model was used to assess the comparative strength of systolic, diastolic, mean arterial and pulse pressure for the prediction of CHD in two age decades after adjustment for age, serum cholesterol, cigarette smoking, behavior pattern and weight. The risk of CHD was more strongly associated with the systolic than the diastolic pressure. The general practice of assessing the importance of blood pressure based only on the diastolic component should be reassessed.


The early posthospital phase of myocardial infarction. Prognostic stratification. Prognostic stratification was carried out on 518 patients less than or equal to 65 years of age who were discharged from the hospital following a definite or probable acute myocardial infarction and followed for four months. The total population was made up of 272 patients hospitalzed in 1973 and 246 patients hospitalized in 1974; one hundred and forty-two variables were collected on each patient. The clinical characteristics of the 1973 and 1974 populations were remarkably similar, and both groups had a four-month posthospital cardiac mortality rate of 4%. Two prognostic stratification schemes were developed on the 1973 population which identified low and high risk groups with meaningfully different four-month cardiac death rates. Both stratification schemes were tested on the 1974 population, and one of the two schemes was validated as identifying a significantly increased cardiac mortality rate in the high as opposed to the low risk group. The four-month posthospital cardiac mortality rate was 3% in the low and 14% in the high risk group (Z = 2.70, P less than 0.003). The high risk group was characterized by two or more of the following characteristics: 1) history of angina at ordinary levels of activity or at rest; 2) CCU hypotension and/or congestive heart failure; 3) ventricular premature beat frequency greater than or equal to 20/hr on a six-hour electrocardiographic tape recording. The low risk group had none or only one of the above characteristcis. The prognostic power of this stratification scheme is such that sixteen percent of the posthospital population can be identified as high risk, and this subgroup contains forty-six percent of the patients who die of cardiac cause in the four-month posthospital interval.


Functional significance of regional ischemic contraction abnormalities. To evaluate the progression of segment function following induction of ischemia, the left anterior descending coronary artery was ligated (eight dogs) or cannulated and perfused at various pressures via a bypass-oxygenator (six dogs). Mercury-in-silastic length gauges were sutured to the anterior left ventricle, and pressure was recorded by a catheter-tipped transducer. Segment function was determined from the area of the pressure-length loop by plotting instantaneous left ventricular pressure against segment length and by evaluation of the degree of systolic shortening. Segment function decreased linearly as flow in the left anterior descending artery was decreased in a stepwise fashion by reduction in perfusion pressures from 100 to 20 mm Hg. With both left anterior descending coronary artery ligation and stepwise flow reduction, the pressure-length loop invariably showed four clearly identifiable morphologic patterns which relate conceptually to the specific left ventricular contraction patterns: dyssynchrony, hypokinesis, akinesis, and paradoxic systolic expansion. Re-oxygenation following occlusion invariably revealed return to a normal pattern in reverse order. This study demonstrates that a consistent and predictable progression of segmental contraction abnormalities occurs with ischemia.


Echocardiographic assessment of left-to-right shunt volume in children with ventricular septal defect. Echocardiograms were performed in 20 infants and children with isolated ventricular septal defects (VSD) undergoing cardiac catheterization. The magnitude of the left-to-right shunt was expressed as the pulmonary-to-systemic flow ratio (Qp/Os) and was compared to a ratio of the echographic left atrial (LA) diameter to the aortic root (Ao) diameter (LA/Ao). The Qp/Qs was also compared to LA/m2. Thirty-three normal children served as controls. The LA/m2 and Ao/m2 were significantly larger in normal infants under one year of age than in children above one year. The LA/Ao, however, was not influenced by age or size. A strong linear relationship was found between Qp/Qs and the LA/Ao (r = 0.96). The LA/Ao measurement appears to be helpful in the noninvasive assessment of the left-to-right shunt in patients with VSD.


Determinants of ventricular septal motion. Influence of relative right and left ventricular size. To test the hypothesis that the ventricular septum moves during systole toward the center of the ventricular mass (so that the end-diastolic position of the septum within the heart should determine both the direction and the magnitude of septal motion during systole), echocardiograms from patients with several different hemodynamic burdens were analyzed. A linear relation was noted between the end-diastolic intracardiac position of the ventricular septum and the direction and magnitude of systolic septal motion in 1) forty three patients with an atrial septal defect )regression coefficient r = 0.80), 2) fourteen patients with other causes of right ventricular volume overload (r = 0.82), 3) nineteen patients with left ventricular volume overload (r = 0.74), 4) ten patients with right ventricular pressure overload (r = 0.93), 5) ten patients with left ventricular pressure overload (r = 0.80), 6) twenty-eight normal subjects (r = 0.82). We conclude that, in the presence of normal ventricular activation and contraction, the direction and magnitude of sepatal motion during systole is determined by the intracardiac position of the septum at enddiastole.


Echocardiographic evaluation of the stent mounted aortic bioprosthetic valve in the mitral position. In vitro and in vivo studies. Echocardiograms were performed on 20 clinically stable patients following mitral valve replacement with glutaraldehyde-preserved porcine aortic heterografts and three patients with antibiotic sterilized aortic homografts mounted in the mitral position. Such valves were evaluated in a test chamber at varied flow rates resulting in improved understanding of movements seen with the echocardiogram in vivo. The technique for recording the valvular stent and leaflets is described and a method for measuring several parameters is demonstrated. Initial diastolic slope averaged 2.4 +/- 0.5 cm/sec (range 1.9 to 3.3 cm/sec). Left ventricular outflow tract measured from the anterior portion of the stent to the interventricular septum averaged 1.5 +/- 0.5 cm at end-diastole and 1.3 +/- 0.6 cm at end-systole. Leaflet excursion averaged 1.5 +/- 0.3 cm (with a range from 1.0 to 2.1 cm). The ratio of internal to external stent diameters averaged 0.66 +/- 0.05 (with a range from 0.56 to 0.74).


Asthma due to inhaled chemical agents--amprolium hydrochloride. A man with no previous history of allergy or chest disease developed asthma after exposure to a heavy atmospheric concentration of amprolium hydrochloride which is a constituent of a poultry-food additive. Subsequent experimental exposure produced an immediate asthmatic reaction which was inhibited by sodium cromoglycate.


Investigation of a respiratory disease associated with an air-conditioning system. This paper describes the investigation of respiratory disease presenting in a group of men employed on the printing floor of a factory. The air-conditioning units supplying this floor were found to be contaminated with a variety of micro-organisms. The specific cause of the disease was not identified but an alteration in the system of humidification led to the complete resolution of symptoms.


Hypersensitivity to acetylsalicylic acid (ASA) and tartrazine in patients with asthma. One-hundred and forty asthmatics were tested perorally with acetylsalicylic acid (ASA), and/or with the azo-colour tartrazine; a fall in PEF of more than 20% was accepted as a positive result. About one quarter of the patients displayed a positive reaction to one of the two tested agents. No significant correlation was found between the reactions of these, and the presence of atopy, nasal polyposis, sinusitis, rhinitis, sensitivity to cold air, the age at onset, duration of asthma, or history of sensitivity to alcoholic drinks. The history suggested sensitivity to ingested, possibly coloured, food and drink, in only about one third of the tartrazine-positive cases. The ASA provocation tests were mainly applied to patients with doubtful or negative histories of sensitivity to ASA-containing drugs. The frequency of cross-reactivity between the two tested agents was statistically significant; patients reacting to tartrazine were for the most part, also sensitive to ASA. Tests for sensitivity to analgesics and food additives should be conducted as a routine measure in asthmatics, and sensitive patients should be given information on suitable medication and dietary control.


IgE antibodies to house dust, mite, animal allergens and moulds in house dust hypersensitivity. Fifty-three patients were investigated by intradermal tests and Phadebas RAST and, in most cases, the serum IgE level was determined. They were tested for sensitivity to house dust, Dermatophagoides pteronyssinus, cat, dog, horse and mould allergens. All cases with IgE antibodies to house dust were found to have specific IgE to other tested allergens as well. Eighty-nine per cent of the patients being RAST positive for house dust were also positive for D. pteronyssinus and in the majority of those cases IgE antibodies to other allergens were detected. The results reaffirm previous findings that the mite is not the only allergen in house dust and that extract of house dust is a mixture of several allergenic substances, which make it less suitable for differential testing. It is concluded that serum IgE determination is of value as a screening procedure in the diagnosis and that the use of a number of specific allergens in RAST is valuable in pinpointing the offending allergen in house dust hypersensitivity.


Pulmonary responses to particulate materials capable of activating the alternative pathway of complement. The cellular sequence of events evoked by the intratracheal injection of both respirable mouldy hay dust and zymosan in unsensitized rats and rabbits was markedly similar and contained many features seen in lung biopsies from farmer's lung cases. It is considered that a combination of ability to activate the alternative pathway of complement and the particulate nature of mouldy hay dust and zymosan produced the pulmonary response seen and as such, considerable insight has been gained into mechanisms by which unsensitized individuals respond to dust inhalation. It is suggested that alternative pathway activation may produce an attack of farmer's lung in the absence of precipitins or other forms of hypersensitivity to the dust or its components and that cases designated as 'pulmonary mycotoxicosis' are more rationally explained by this mechanism.


Dependence of immediate hypersensitivity on the month of birth. This report describes a retrospective study of 1421 patients allergic to pollens and 728 patients allergic to animal epithelia. In males, but not in females, the month-of-birth distribution differed significantly from that in the total population of Finland (P less than 0-01 in pollen allergy and P less than 0-05 in epithelium allergy). The phenomenon was most clearly apparent in boys aged 10-14 born in 1960-4 (P less than 0-01 in pollen allergy and P less than 0-001 in epithelium allergy). When the relative risk of immediate hypersensitivity was plotted as a function of the month of birth, a two-phasic curve with maxima in March-May and September-November was obtained for both pollen allergy and animal epithelium allergy. The results indicate that boys, but perhaps not girls, have a sensitive period early in life during which one or more environmental factor and seasonal variations in this factor influence the development of subsequent immediate hypersensitivity.


The diagnosis of aspirin idiosyncrasy by analgesic challenge. Analgesic challenge was employed to identify fifty patients with aspirin idiosyncrasy. No serious reactions were observed using the method described. Patients with a history of the condition were highly sensitive and reacted to small doses of aspirin or to paracetamol. Patients with a positive challenge test, but previously unaware of aspirin idiosyncrasy, were less sensitive: they required larger challenge doses of aspirin and did not respond to paracetamol challenge. In the absence of an in vitro test, analgesic challenge is the only means of confirming the presence of aspirin idiosyncrasy.


Immediate (type I) allergic responses to platinum compounds. A series of platinum complexes has been used for allergy tests in a number of platinum refinery workers who are known to be sensitive to hexachloroplatinate ([PtCl6]2-) salts. The results show that the allergy-eliciting compounds are confined to a very small group of ionic complexes containing reactive halogen ligands. Neutral complexes and those containing more strongly bound ligands are totally inactive. These variations in activity are interpreted in terms of established kinetic and thermodynamic parameters.


Curve prediction in automated analyses in routine laboratories. 1. The hydraulics of first- and second-generation AutoAnalyzers introduce lag and exponential deformations of the square wave signal expected from the colorimeter. These factors limit sampling rates by causing sample interaction. Curve regeneration carried out on Technicon Flame IV modules, using a digital approach, with a Hewlett-Packard 2100A computer, has successfully compensated for exponential deformation of sodium, potassium, chloride and carbon dioxide channels in routine laboratory use for one year. A sampling rate of 138/hr has been used; faster rates are possible. 2. Reduced sample and reagent consumption are benefits in addition to the increased analysis rate.


Intestinal absorption of water and electrolytes. 1. Recent advances in knowledge of intestinal physiology have provided some insight into disturbed mechanisms and their clinical effects; for example, diarrhoea can now be defined biochemically as excessive fluid and electrolyte loss due to their malabsorption or excessive secretion. 2. Because of differences in structure and in absorptive and secretory mechanisms, the various parts of the gut perform different functions. In  the jejunum, transport activity is extensive and the rapid equilibration of its content provides the optimal absorptive mixture. Functionally, the ileum and colon are similar; compared with the jejunum, they have greater absorptive capacity for electrolytes and generate significantly higher transmural electrical potentials. In the colon, some transport mechanisms are potentiated by adrenocortical steroids. 3. Water and electrolyte absorption and secretion are the end-products of bidirectional fluxes across the intestinal wall that are several times greater than net movement in either direction. Secretion is the surplus of negative flux (into the lumen) and absorption the surplus of positive flux (out of it). 4. Many electrolyte transport mechanisms require the absorption of other electrolytes or non-electrolytes, and some are concerned with electrolyte exchange. Water transport is always passive, in the direction of solute flow, but its solvent drag can move solutes across the intestinal membrane.


Diagnosis of chronic pancreatic disease. The diagnosis of pancreatic disease is difficult. The first step is clinical suspicion, based on the symptoms and signs. If pancreatic disease is suspected, investigation is necessary to prove this diagnosis. Investigation aims to answer two questions: a) is there pancreatic disease and b) if so, what type? The first question may be answered by demonstrating abnormal pancreatic function, using pancreatic function tests, whereas the second is answered by using techniques to demonstrate structural (anatomical) abnormalities of the pancreas. a) The methods to establish abnormal pancreatic function consist of 1. tests to demonstrate abnormal digestive capability, 2. tests to study pancreatic exocrine secretion, and 3. tests to study endocrine secretion. The tests of group 1 are: chemical fat balance study before and during enzyme replacement therapy, faecal nitrogen balance study, and the demonstration of either the malabsorption of vitamins A, D and K or the sequelae of their malabsorption (low serum calcium, high alkaline phosphatase, prolonged prothrombin time, etc.). Abnormal vitamin B12 absorption also may be present. 2. The tests designed to study pancreatic exocrine secretion are determination of the presence or absence of proteolytic enzymes in the stool, the secretion test, the pancreozymin stimulation test and the Lundh test. The serum amylase and lipase values are of little help in assessment of pancreatic function. 3. The tests to study endocrine function are the glucose tolerances test (which frequently gives abnormal results in pancreatic disease), and radioimmunoassays for insulin and gastrointestinal hormones (which may be increased in patients with functioning tumours of the islet cells). b) The techniques used to establish structural abnormalities of the pancreas are: duodenal cytology (during secretin tests), radiological techniques (abdominal survey films, barium meal, hypotonic duodenography, roentgenography of the biliary tract, barium enema, and angiography,) gastroscopy, duodensocopy, endoscopy and retrograde pancreatography, echography, scan and laparotomy. The relative value of these tests is discussed.


Tests of intestinal function.

Tests of intestinal function. The most important tests for investigating the functions of the small bowel are faecal-fat analysis, the determination of xylose tolerance, vitamin-B12 absorption, and 14C-glycoholate transport, barium examination of the small bowel, and small bowel biopsy. With these tests, most disorders of digestion and of absorption can be adequately investigated and diagnosed.


Xylose absorption and its clinical significance. 1. Xylose absorption by the small intestine probably includes an active process. 2. For xylose testing, the 25-g dose appears to be preferable to 5 g. 3. Factors that influence the test result include intestinal bacterial overgrowth, reduced xylose metabolism in cases of liver disease, sequestration into ascites, age, and the state of renal function. 4. The test results differentiate patients with extensive disease of the upper small intestine from normal subjects, and from patients with steatorrhoea due to pancreatic insufficiency, in most but not all instances. Blood xylose levels combined with urinary xylose output aids discrimination.


Bile-acid metabolism and the liver.

Bile-acid metabolism and the liver. Primary bile acids are exclusively synthesize, and all bile acids are conjugated, in the liver. When hepatic function is altered by disease, not surprisingly the bile-acid metabolism reflects this change. 1. In chronic liver disorders the glycine: taurine ratio of serum and bile is reduced. 2. In cases of relapsing acute hepatitis and  relapsing chronic active hepatitis the serum bile acids are increased, providing an aid to early diagnosis. Their pattern often distinguishes chronic cholestatic conditions from Laennec's cirrhosis. 3. In chronic liver disease, the concentration of intraduodenal bile acids is reduced; when the reduction is severe, this probably accounts for co-existent steatorrhoea. 4. In Laennec's cirrhosis, both the synthesis and pool size of cholic acid are markedly depressed; in primary biliary cirrhosis, however, preliminary data indicate a decrease in the chenodeoxycholic acid component.


How an enzyme works.

How an enzyme works. The structure of enzyme active sites and the nature of the catalytic process are reviewed. The impressive efficiency of these protein catalysts appears to stem from such factors as proximity and orientation of enzyme and substrate moieties, strain, and the occurrence of distinctive microenvironments within catalytic centres. Carboxypeptidase, lysozyme, and aspartate transcarbamylase, which have been extensively investigated by many techniques, serve to illustrate the application of these concepts.


Errors in measuring enzyme activity by reaction-rate methods. 1. The two types of assay used in clinical enzymology -- reaction-rate and two-point assays -- are usually performed with different types of analyzers. A third type, the multi-point analyzer, has been introduced recently. 2. Reaction-rate and two-point enzyme assays have sources of error inherent in their methodologies. There are also major errors due to the analytical system itself; these are considered systematically.


Demographic and analytic factors affecting the normal range of serum enzyme activities. 1. The effect of demographic variables such as age, sex, body weight, social class and blood pressure upon the activity of serum enzymes in healthy humans is reviewed. A system developed by the author and his colleagues, which automatically adjusts the results of enzyme activity measurements to allow for demographic influences, is described. This allows derivation of a "demographically corrected" normal range; for most enzymes this range is much narrower than that provided by the conventional mean +/- 2 SD approach. 2. The influence of precision and analytical factors upon the normal range for serum enzymes is discussed. Data from a British national quality-control survey reveal a depressing picture of interlaboratory precision for commonly determined enzyme estimations. Examples are given (serum aspartate aminotransferase and guanase) which illustrate the influence that precision of a method may have on the normal range for that enzyme.


Analytical toxicology:  Applications of element-selective electrolytic conductivity detection for gas chromatography. We applied a commercially available microelectrolytic conductivity detector to toxicological problems of qualitative and quantitative analysis by gas chromatography. The detector can be used for the sensitive and selective detection of halogen-, nitrogen-, and sulfur-containing compounds. Relative response in different element-selective detector variables such as reaction gas, reaction catalyst, and furnace temperature can be used to further improve qualitative identification by gas chromatography.


Radioimmunoassay, enzyme immunoassay, spectrophotometry, and gas-liquid chromatography compared for determination of phenobarbital and diphenylhydantoin. Sera from epileptic patients were assayed for phenobarbital and diphenylhydantoin by four different analytical procedures. Quantitative results obtained by radioimmunoassay (I) and enzyme immunoassay (II) were compared to each other and to the results obtained on aliquots of the same sample by gas-liquid chromatography (III) and ultraviolet spectrophotometry (IV). For phenobarbital the correlation coefficients were I vs. II, 0.909; I vs. III, 0.947; II vs. III, 0.917; I vs. IV, 0.950; II vs. IV, 0.953. For diphenylhydantoin the correlation coefficients were I vs. II, 0.953; I vs. III, 0.951; II vs. III, 0.957; I vs. IV, 0.862; II vs. IV, 0.898. The immunoassays can be substituted for liquid chromatography or ultraviolet spectrophotometry without changing the resulting clinical interpretations.


Efficacy of activated charcoal hemoperfusion in removing lethal doses of barbiturates and salicylate from the blood of rats and dogs. Rats were injected intraperitoneally with lethal doses of sodium pentobarbital (115 mg/kg) or a lethal mixture of sodium salicylate (500 mg/kg) and sodium acetazolamide (25 mg/kg). Within about 20 min, part of each group was connected to an extracorporeal circuit containing uncoated activated charcoal and part to an empty control circuit. After a 90-min hemoperfusion, the treated groups showed a significantly decreased mortality (58% to 14% for pentobarbital; 100% to 0% for salicylate). Dogs were injected intravenously with lethal doses of sodium phenobarbital (175 mg/kg). One group was treated by hemoperfusion through an empty device in a control extracorporeal circuit, a second group was treated with loose-bed activated charcoal devices, and a third group with fixed-bed activated charcoal devices. For both the fixed and loose-bed devices, a 5-h hemoperfusion markedly decreased mortality (100% to less than or equal to 15%). The lethal combination of salicylate and closed-circuit methoxyflurane anesthesia was also successfully treated in dogs. This study clearly demonstrates the lifesaving potential of uncoated activated charcoal hemoperfusion.


Rapid radioisotopic procedure for determination of nortriptyline in plasma. With the widespread use of tricyclic antidepressant drugs, the relationship between the concentration of the drug in the plasma and the therapeutic response is of considerable interest. We describe a double-isotope derivative dilution procedure for measuring plasma nortriptyline. In the method, [14C]nortriptyline is used for estimating procedural losses and [3H]acetic anhydride for derivative formation. The assay is rapid and adequately specific, sensitive, precies, and reproducible for routine clinical use. We used it to investigate the variation in steady-state drug concentrations in plasma of persons who were on a 150 mg/day dose of nortriptyline. Intra-individual variation from day to day was 10-14%. This variation was not significantly affected by the dosage schedule, the time of sampling after an oral dose, or the storage of the plasma samples. For 19 patients on 150 mg of nortriptyline per day, the mean concentration in plasma was 181 +/- 22 (SE) mug/liter, a value that compares well with our previous findings and those of other groups.


Gas-chromatographic micro-scale procedure for theophylline, with use of a nitrogen-sensitive detector. We report a micro-scale procedure for determination of theophylline by use of gas chromatography with nitrogen-sensitive detection. The procedure requires only 10 mul of serum, plasma, or saliva. This sample size makes the determination especially appropriate for monitoring in a pediatric population. With the sample volume used, background interference is equivalent to about 0.1 mg/liter and 0.5 mg of theophylline per liter can easily be measured. The new method correlates well with our earlier flame ionization gas-chromatographic procedure that required 1 ml of sample [Clin. Chem. 21, 1038 (1975)]. The selectiviey of the nitrogen detector allows a simplified extraction procedure. Between-run precision (CV) is 2.8% at a theophylline concentration of 14.8 mg/liter.


Simultaneous gas-chromatographic analysis for phenobarbital, diphenylhydantoin, carbamazepine, and primidone in serum. We describe a simple, sensitive determination of phenobarbital, diphenylhydantoin, carbamazepine, and primidone in serum, by use of gas-liquid chromatography with temperature programming. The methylated derivatives of these anticonvulsants are well resolved, as was 5-(p-methyl-phenyl)-5-phenylhydantoin, the internal standard. The proposed procedure requires only 0.20 ml of serum and can be done in less than 30 min. The lower limit of detection for each of the drugs is 0.5 mg/liter. Analytical recoveries of drug from serum were excellent and peak height and concentration were linearly related up to twice the toxic concentration for serum.


Gas-chromatographic determination of 5-fluorocytosine in human serum. We describe a sensitive and precise gas-chromatographic method, in which cytosine is used as the internal standard, for determination of an antifungal agent, 5-fluorocytosine, in serum. The trimethylsilyl derivative of this drug is well separated from the internal standard and from normal serum constituents. Amphotericin B does not interfere with the determination of 5-fluorocytosine. The lower limit of detection for 5-fluorocytosine is 1 mg/liter when 200 mul of serum is analyzed. Within-run precision (CV), established by analysis of 10 replicates, was 4.5% at a concentration of 19.9 mg/liter. Twenty-five serum samples were analyzed for 5-fluorocytosine by a microbiological assay and by the gas-chromatographic method. Mean value observed with the bioassay was 78.5 mg/liter and with our procedure was 69.4 mg/liter. When values for our assay were regressed against values for the bioassay, slope of the least-squares line was 0.85, intercept was 2.7 mg/liter, and r was 0.93.


Gas-chromatographic analysis for therapeutic concentrations of amitriptyline and nortriptyline in plasma, with use of a nitrogen detector. We describe a gas-chromatographic procedure for the simultaneous determination of amitriptyline and its active metabolite, nortriptyline, in therapeutic concentrations in human plasma, with use of a nitrogen detector. Both drugs are extracted at pH 10.5 into hexane/isoamyl alcohol, back-extracted into dilute HCl, and re-extracted into hexane/isoamyl alcohol after alkalinization of the HCl. The solvent is evaporated and the residue gas-chromatographed. Protriptyline is used as the internal standard. As little as 5 mug of amitriptyline or nortriptyline can be detected per liter of plasma. The coefficients of variation, for a concentration of 200 mug/liter, are 4.6% and 4.3% within-day and 8.6% and 3.4% day-to-day for amitriptyline and nortriptyline, respectively. The procedure was applied to patients receiving therapeutic doses of both drugs and also to patients who had taken overdoses of amitriptyline.


Liquid chromatographic method for monitoring therapeutic concentrations of L-dopa and dopamine in serum. We describe a new method for the simultaneous assay of 3,4-dihydroxyphenylalanine (L-dopa) and dopamine in serum. Both compounds have been determined quantitatively at concentrations as low as 10 mug/liter with a coefficient of variation of less than 4%. Peak heights were linearly related to concentration up to 10 mg/liter for each compound. Assay of human sera gave within-run and day-to-day coefficients of variation of 2.8% and 3.1% for L-dopa, and 2.9% and 3.7% for dopamine. The linear relationship between readings nA, y-axis) and concentration (mug/liter) is described by the following equations: y = (21.0 +/- 0.082)x + (0.46 +/- 0.31 SD) for L-dopa and y = (17.0 +/- 0.10)x + (0.08 +/- 0.04 SD) for dopamine. The procedure combines liquid/solid extraction, liquid chromatography, and controlled-potential electrochemistry. The simplicity, sensitivity, and accuracy of this method make it suitable for routine clinical analysis of serum samples to optimize bioavailability for individual patients. After the extraction procedure is completed, samples can be analyzed at the rate of 10/h.


Relative merits of some methods for amphetamine assay in biological fluids. We describe principles and details that we use for amphetamine determination in blood and urine by photometric, thin-layer chromatographic, gas-chromatographic, and immunologic (EMIT and radioimmunoassay) procedures. Results, interferences, stabilities, and economic considerations are intercompared and recommendations are made.


Automated enzymic determination of ethanol in blood, serum, and urine with a miniature centrifugal analyzer. We describe an automated spectrophotometric reaction-rate method for determination of ethanol in serum and urine with a miniature centrifugal analyzer. The theanol is selectively oxidized in the presence of alcochol dehydrogenase and NAD+ to form NADH, which is measured by the rate of change of its absorbance. Reaction rates are determined automatically, and unknown concentrations are calculated from a computer-generated working curve based on aqueous ethanol standards. Blood, serum, or urine specimens need not be deproteinized. The method permits duplicate analysis of at least 30 samples per hour. Coefficients of variation and relative errors are about 2-3% for ethanol concentrations of 0.3-3.0 mug per 2 mul of sample. Analytical recovery of ethanol added to serum is 92-103% (average, 98.5%). Comparisons with distillation-oxidation, gas-chromatographic, and conventional enzymic procedures give satisfactory agreement.


Gas-chromatographic determination of an antifibrinolytic drug, epsilon-aminocaproic acid. We describe a modified method for assay of epsilon-aminocaproic acid. Serum or cerebrospinal fluid is deproteinized, followed by cation-exchange column-chromatography, and N-trifluoroacetyl-n-butyl derivatives of amino acids are formed and separated by gas chromatography. Tranexamic acid, a nonprotein amino acid, was used as an internal standard. The assay is sensitive and precise, and results correlate adequately with those obtained with an automated amino acid analyzer (ion-exchange chromatography).


Serum quinidine concentrations: comparison of fluorescence, gas-chromatographic, and gas-chromatographic/mass-spectrometric methods. Serum quinidine concentrations were determined in patients on chronic therapeutic doses. Although results were higher by a protein precipitate-fluorescence method as compared to a specific extraction fluorescence method, there was substantial correlation between results by the two methods (r = 0.945, P less than 0.001). We established the specificity of the extraction method by a methylation gas-chromatographic method in which the base peak in the mass spectra of the methylated products of both quinidine and cinchonidine, the internal standard, was monitored. We conclude that the protein precipitate method should be discarded.


Simultaneous measurement of phenobarbital, diphenylhydantoin, and primidone in blood by high-pressure liquid chromatography. We describe a sensitive, precise high-pressure liquid chromatographic method in which 5-(p-methylphenyl)-5-phenylhydantoin is used as the internal standard for simultaneous determination of diphenylhydantoin, phenobarbital, and primidone in whole blood and plasma. These anticonvulsant drugs are well separated from each other and from normal blood constituents in less than 10 min. The lower limit of detection for each drug is 100 ng for primidone, 200 ng for dilantin, and 300 ng for phenobarbital. The eluted drugs were detected by their absorption at 254 nm, and evaluated from their peak heights as compared to internal standard. The method was successfully adapted for pediatric samples (100 to 500 mul of whole blood or plasma). Fifty specimens were analyzed for phenobarbital and diphenylhydantoin and 25 specimens for primidone by a standard gas-chromatographic method and by our liquid-chromatographic method; the resulting correlation coefficient was greater than 0.98.


Radioimmunoassay for clindamycin.

Radioimmunoassay for clindamycin. We describe a radioimmunoassay for measuring clindamycin in serum extracts that is more accurate than the microbiological assay because of the minimal response of the matabolite, N-demethylclindamycin. The assay is not affected by the presence of other antiobiotics. As described it is as sensitive as the microbiological assay, but can be made more sensitive.


Assessment of interference by aspirin with some assays commonly done in the clinical laboratory. We have assessed the interference of acetylsalicylic acid (aspirin) with some common clinical laboratory assays, because of its widespread use and reported interference. The therapeutic regimens involved ingestion of 10 325-mg tablets daily for three days or eight tablets for five days or two weeks. Twenty-one commonly done determinations were run on control sera, and sera were collected during and after drug therapy. Significant changes in t-test values, indicating the significance of the standard deviation of the difference between controls and specimens drawn after drug therapy, were observed for chloride (increased), and for total protein, calcium, cholesterol, uric acid, bilirubin, and thyroxine (decreased). Aspirin therapy depressed the apparent concentrations of these constituents progressively in the three-day regimen and initially in the long-term regimens, followed by recovery toward zero t-values at the end of the longer regimens.


Results of a nine-laboratory survey of forensic toxicology proficiency. Toxicological determinations are crucial to coroners' or medical examiners' judgments that drugs are significantly involved in a death. However, differences in laboratory procedures, thoroughness of screening, and limits of detection may result in artifactual differences in the toxicological results and the subsequent interpretations of them. To test this possibility, we conducted a toxicology proficiency-testing survey of nine collaborating laboratories. The results for the proficiency samples point out starting interlaboratory differences in accuracy and precision of detection of drugs. These observed variations in toxicological proficiency may introduce a significant source of error in drug-death statistics and in epidemiological deductions based on these statistics.


Analysis of results of toxicological examinations performed by coroners' or medical examiners' laboratories in 2000 drug-involved deaths in nine major U. S. cities. Data were collected on 2000 deaths in which psychoactive drugs were involved. The data were submitted by medical examiners or coroners in nine U. S. cities from their case files. The 2000 cases comprise a representative sample from each of these cities of deaths from psychoactive drugs between 1972 through 1974. This report details inter-city differences in methods and practices of the toxicological examination as well as the type and numbers of drugs reported. Even when the same analytical method was used in various cities, there were differences in extraction solvent and extraction pH. Of the 3909 drugs detected, 2945 were quantitated; the number of drugs quantitatively measured per case studied ranged from a low of 0.82 for New York to a high of 2.20 for Washington, D.C. The number of different drugs quantitatively measured varied from 16 for New York to 30 for San Francisco; however, New York qualitatively assayed for the presence of a total of 25 drugs. The number and type of drugs found per case varied. Methadone, for example, was found in 60% of the cases reported by New York and in 49% of the cases from Washington, D.C., but in only about 10% of cases reported by Philadelphia, Dallas, Miami, and San Francisco, and in less than 1% of Los Angeles and Cleveland cases; it was not reported by Chicago. Phenmetrazine-caused deaths were reported only by Dallas (one case) and Washington, D.C. (29 cases). From the data as a whole, information is presented for 33 drugs as to the concentration in physiological tissues and fluids. Analysis of single psychoactive drug cases and single-drug-plus-ethanol cases shows that, in the presence of ethanol, the toxic blood concentration of imipramine, amytriptyline, meprobamate, thioridazine, morphine, propoxyphene, methaqualone, and all barbiturates was decreased by an average of 50%.


Serum theophylline analysis by high-pressure liquid chromatography. We have developed and evaluated a rapid, high-pressure liquid-chromatographic method for theophylline in serum. Only 0.2 ml of serum is required for each determination, and the sensitivity of this method is 0.5 mg/liter. This method, involving liquid extraction and silica adsorption chromatography, provides adequate selectivity, accuracy, and precision for routine or research applications. Little sample preparation is required before chromatography. We found no endogenous or exogenous interferences. Use of beta-hydroxypropyl theophylline as the internal standard provides reproducible results for this micro-scale method.


Rapid micromethod for measuring anticonvulsant drugs in serum by high-performance liquid chromatography. We describe an assay system for measuring phenobarbital, diphenylhydantoin, primidone, ethosuximide, and carbamazepine in 25 mul of serum. The procedure involves precipitation of proteins with an acetonitrile solution containing cyheptamide as an internal standard, and reverse-phase chromatography on a 4 mm X 30 cm column containing "muBondapak C18." The anticonvulsants are eluted with an equivolume mixture of potassium phosphate buffer (10 mmol/liter, pH 8.0) and acetonitrile at a flow rate of 0.8 ml/min, detected by their absorbance at 200 nm, and quantitated by measuring peak areas. When measurement of primidone is not required, a 254-nm detector may be used. Each analysis requires 10 min. Optimum column temperature has been found to decrease with use. Analytical recoveries for the five drugs varied from 92% to 102% with good precision (coefficients of variation between 2.8% and 9.2% for therapeutic and toxic concentrations). The results obtained with this method compare favorably with results obtained by gas-liquid chromatography (correlation coefficients between 0.77 and 0.98). In over 1300 patients' samples analyzed to date, the only drugs known to have interfered with the assay are gentamicin, diazoxide, and mephobarbital.


Rapid gas-chromatographic measurement of anticonvulsant drugs in serum. We describe a rapid method for precisely measuring concentrations of anticonvulsant drugs in serum. The simple procedure, adaptable to the clinical laboratory, can be used for the routine simultaneous measurement in sera of phenobarbital, primidone, dephenylhydantoin, and (or) ethosuximide. The extraction procedure requires no solvent evaporation and gives a good yield for primidone and quantitative extraction for the other drugs. The relatively small amount of "early phenobarbital" formed during on-column methylation permits a precise analysis of phenobarbital based on a single peak. Results can be made available directly, without manual measurements or calculations.


Method for alcohol determination in biological liquids by sensing with a solid-state detector. I describe a new method for quantitative alcohol determination in equilibrated headspace gas of biological liquids by means of a solid-state metal-oxide semiconductor (MOS) detector. After equilibration of the NaCl-saturated specimen at 40 degrees C or other convenient temperature in a closed vessel, a Taguchi MOS sensor is momentarily exposed to the headspace vapor. The resulting dc voltage change from the baseline conditions accurately reflects the alcohol concentration of the liquid specimen, and can be read in direct alcohol-concentration units. Calibration is linear to 3.00 g/liter (300 mg/dl). Values for analytical recovery ranged from 98 to 102%. Correlation of results for blood alcohol concentration by this method (yi) and an automated gas-chromatographic reference method (xi) was y, g/liter = 0.991x + 0.014, with r = 0.997 for 103 blood specimens having concentrations ranging from 0 to 2.89 g/liter (0-289 mg/dl), with a mean difference (xi - yi) - -6 mg/liter (0.6 mg/dl). The method is sensitive to 0.01 g/liter (1 mg/dl) and can be readily automated. An internal standard is not required.


Gas-chromatographic method for acetaminophen (N-acetyl-p-aminophenol) based on sequential alkylation. A gas-chromatographic procedure for acetaminophen is described in which the drug is chromatographed as the O-heptyl-N-methyl derivative. This derivative is prepared by a sequential alkylation procedure in which the phenolic hydroxyl group of the parent compound is alkylated off-column with heptyl iodide and the amide group is derivatized on-column by reaction with trimethylanilinium hydroxide. The internal standard, N-propionyl-p-aminophenol, is subjected to the same derivatization procedure. This gas-chromatographic procedure correlates well with conventional colorimetric and spectrophotometric procedures for acetaminophen and is more sensitive. Within-run precision (CV) was 2.0% at a serum concentration of 10.0 mg/liter (n = 10) and between-run precision was 4.0% over a period of eight months. This method is particularly applicable to studies of the pharmacokinetics of acetaminophen.


Quantitative gas-chromatographic flame-ionization method for p-chlorophenoxyisobutyric acid in human serum and saliva. We describe a gas-chromatographic method for p-chlorophenoxyisobutyric acid (I) the active metabolite of clofibrate. The drug and internal standards are separated from either serum or saliva by a double extraction procedure and converted to the corresponding butyl esters by reaction with iodobutane in a mixture of methanol and N,N-di-methylacetamide containing tetramethylammonium hydroxide. Within-run CV of this assay at a serum I concentration of 79.2 mg/liter was 2.3% and at a salivary I concentration of 2.5 mg/liter was 2.1%. Precision during four months of the serum and salivary assays at these concentrations was 4.1% and 6.2%, respectively. The mean serum concentration of I (12 h after dose) in patients receiving the drug at an average dose of 28.0 mg/kg per day was 109.6 mg/liter. Serum and salivary concentrations of I as determined by our procedure were used to calculate the unbound fraction of drug in human serum. Such measurements can be used to monitor therapy in patients with renal disease, where drug toxicity may arise from high concentrations of unbound I.


Measurement of clomipramine, N-desmethyl-clomipramine, imipramine, and dehydroimipramine in biological fluids by selective ion monitoring, and pharmacokinetics of clomipramine. To quantitatively determine tricyclic antidepressant agents, we used a combined gas chromatograph/mass spectrometer system, and deuterium-labeled internal standards. Recovery exceeds 95% and the coefficient of variation is less than 4% for human whole-blood samples supplemented with 5 to 15 ng of clomipramine hydrochloride or 20 to 60 ng of dehydroimipramine hydrogen fumarate per milliliter. For both amines, the detection limit is 0.3 mug/liter; Six healthy volunteers who received a single oral dose of 50 mg of clomipramine hydrochloride showed peak drug concentrations in the blood 3 to 5 h after administration, ranging between 14.4 and 30.1 mug/liter. Plasma/whole blood concentration ratios varied from 0.70 to 1.20, and the cumulative renal elimination from 0 to 72 h is less than 0.2% of the dose. This method is suitable for in vivo bioavailability studies of unchanged clomipramine, dehydroimipramine, and imipramine after a single oral dose of as little as 25 mg.


Gas-chromatographic quantitation of theophylline in small volumes of plasma. We describe a rapid procedure for quantitating theophylline in 100-mul plasma samples by use of a gas-liquid chromatograph equipped with a flame ionization detector. This methos is especially useful for monitoring theophylline concentrations in serum or plasma of infants, because sufficiently large blood samples can be readily obtained from a heel prick. The method is specific for theophylline in the presence of caffeine, theobromine and phenobarbital. For plasma concentrations equal to or greater than 5 mg/liter the average daily coefficient of variation was less than 7% while the coefficient of variation from day to day was less than 11%. The same approach can also be used to measure concentrations of phenobarbital in small volumes of plasma or serum, and is readily adapted to determination of theophylline and phenobarbital in larger samples.


Primidone analyses: correlation of gas-chromatographic assay with enzyme immunoassay. Serum specimens from patients on primidone therapy were assayed by two currently available procedures: a commercially available enzyme immunoassay (EMIT) and a published gas-chromatographic procedure. Both procedures were done with commercially available materials and equipment. Results by the two procedures agreed well, which suggests that the two methods could be used interchangeably. For the 94 specimens studied, the correlation coefficient was 0.98, and the least-squares values of slope and intercept were, respectively, 0.97 and 0.51 mg/liter.


Improved micro-radioimmunoassay of digoxin in serum, with use of 125I-labeled digoxin. A micro-radioimmunoassay of 125I-labeled digoxin is described in which 10-mul rather than 50-mul aliquots of sera are required. The method is a modification of a simplified, rapid, and accurate procedure, which is commercially available as a kit. There is excellent correlation between results by these two methods, in which lyophilized digoxin standards and insoluble antibody polymer are used. The digoxin concentration in capillary sera was not significantly different from that in the corresponding venous blood. It is clinically useful to monitor digoxin therapy in capillary sera, particularly in patients in whom blood sampling by venipuncture is inconvenient or difficult.


Adaptation of "EMIT" technique for serum phenobarbital and diphenylhydantoin assays to the miniature centrifugal analyzer. We used a miniature centrifugal analyzer in a spectrophotometric rate-measurement mode to determine the anticonvulsant drugs phenobarbital and diphenylhydantoin in serum, by use of a modified enzyme immunoassay ("EMIT", Syva Corp.) We decreased reagent cost per determination by at least sixfold by means of microscale techniques. Also, the analysis rate is increased by measuring multiple samples simultaneously. Our method requires only 3 mul of serum for duplicate determinations. Replicate analyses of sera containing phenobarbital and diphenylhydantoin gave reaction rates with a CV of 1.5%. Run-to-run CV was 15%. Analytical recovery for drug-supplemented serum samples was 98%, and results for a series of samples compared well with results obtained by gas chromatography (for phenobarbital, r = 0.95; for diphenylhydantoin, r = 0.91).


Simplified fluorometric assay for diphenylhydantoin in plasma. The fluorometric benzophenone procedure for diphenylhydantoin has been simplified by eliminating the need for preliminary extraction of plasma with an organic solvent. Assays are done directly on 0.2 ml of plasma by treating it with alkaline permanganate to form benzophenone, extracting the benzophenone with heptane, and then shaking the heptane layer with sulfuric acid and measuring the fluorescence of the acid layer. The assay is highly reproducible, and adequately sensitive to detect 1 mug of the drug per milliliter of plasma. Fluorometric and gas-liquid chromatographic assays of 154 plasmas gave results that were not significantly different, even in the presence of phenobarbital, other barbiturates and anticonvulsant drugs, or of various tranquilizers and other commonly used drugs. The assay is rapid, the unit cost per assay low.


Beta-carotene content of certain organs from two patients receiving high doses of beta-carotene. We measured carotene in the livers of two patients receiving beta-carotene therapy for photosensitivity. In both patients, the amount of carotene was within reported ranges for patients on normal diets; no abnormal amounts of beta-carotene had accumulated in the livers after oral administration of large amounts of beta-carotene. Light-microscopic examination of the liver (specimen obtained at cholecystectomy) of one patient with erythropoietic protoporphyria and cholesterosis showed findings characteristic of these conditions. Electron-microscopically, semicrystalline inclusions were seen in the mitochondria of the hepatocytes; such inclusions have been reported in several conditions, including erythropoietic protoporphyria and porphyria cutanea tarda, and in control subjects. Microscopic examination of the liver of the other patient, who had Rothmund-Rhomson syndrome and died of acute bacterial meningitis, revealed no abnormalities. The cerebrum of the latter patient contained 13 mug of carotene per kilogram (wet weight) of brain. No previous report of analysis of human brain for carotenoids could be found.


Anomalous behavior of glutathione reductase on dilution. Serum glutathione reductase activity decreases on standing at room temperature. Dilution of the sample slows this decay in sera from patients with a variety of clinical disorders, and reverses the inactivation in sera from patients with liver or biliary tract disease. Prolonged contact with dithioeythritol or acetate buffer, pH 6.4, at 4 degrees C prevents spontaneous inactivation of the native serum and abolishes the effect of dilution. These hitherto unreported phenomena are a source of potential inaccuracy in assays for this enzyme. The inactivation may be the result of conformational folding with "burying" of active sites, or of molecular aggregation brought about by hydrogen bonding or disulfide bond formation.


Evaluation of a new system ("Corvac") for separating serum from blood for routine laboratory procedures. We evaluated the suitability of a new system ("Corvac," Corning) for rapid, complete separation of cell-free serum from whole blood. Twenty-one different clinical chemical procedures were performed on about 1150 paired blood samples, several by more than one method, to afford a more comprehensive evaluation. The tube of clotted blood collected in the conventional manner ("Vacutainer") underwent a double centrifugation and served as the control sample, while its paired specimen collected in the new system was processed by a single centrifugation. We evaluated significant differences between the means of the paired samples, the standard deviations of the differences between the means, and the bias of the means for the values of each constituent for each collection method. For several tests we observed differences that were statistically but not clinically significant. Only in the case of lactate dehydrogenase were large differences consistently demonstrated.


Immobilized glucose oxidase used in the continuous-flow determination of serum glucose. We used a tubular glucose-oxidase wall reactor in the "AutoAnalyzer II" continuous-flow analytical system to determine glucose in blood serum. Sensitivity was high and wash characteristics were satisfactory with use of a 30-cm tube containing immobilized glucose oxidase. Results compared favorably with those of the conventional free-enzyme method. More than 25000 such assays can be performed with a single enzyme tube, which also shows long-term storage stability. Because of the steady-state chemistry 60 samples can be analyzed per hour. The linearity of the method is excellent and sample interaction from 5.0 to 1.0 g/liter is less than 5%. Results correlate well (greater than 0.993) with those obtained with both the neocuproine method used in the "SMA 12/60" multichannel analytical system and the free glucose oxidase method used in the AutoAnalyzer.


Lactate dehydrogenase activities in serum and plasma. We investigated lactate dehydrogenase activity in serum and plasma because of the conflicting data found in the literature. We assayed serum, platelet-rich, and platelet-poor plasma by two colorimetric endpoint methods and by an ultraviolet kinetic procedure. Platelet-poor plasma and serum had essentially the same activities by all three methods, whereas the activity in platelet-rich plasma plasma averaged fourfold that in platelet-poor plasma or serum when the assay was performed under conditions that result in lysis of platelets and release of their lactate dehydrogenase. When measurements were performed in platelet-rich plasma under conditions that prevented lysis of platelets, all three types of specimens gave the same results. This occurred when the osmolality of the reaction mixture was about 240 mOsm/kg of water. At an osmolality of about 120 mOsm, the activity of platelet-rich plasma was substantially lower than that of platelet-poor plasma or serum. If plasma must be used, the sample must first be centrifuged (3000 X g, 15 min) to provide a platelet-free plasma.


Studies of the reliability of constituent target values established in a large inter-laboratory survey. Lyophilized serum specimens were manufactured in such a way that fixed quantitative interspecimen relationships were established. The specimens were analyzed for about twenty constituents by several thousand laboratories in 1971, 1972, and 1973. Most of the consensus mean values derived from these analyses were found to relect accurately the relationships established in the manufacture of the specimens. In addition, analyses of some of the specimens for calcium by the National Bureau of Standards confirmed the relationships. I conclude that the consensus mean values derived for most constituents are reproducible and any biases that may be present are consistent from survey to survey.


Derivatives of 1-naphthyl ethylenediamine dihydrochloride for standardization of direct-bilirubin assays done with the Technicon "SMAC". The Jendrassik--Groff assay for direct bilirubin was adapted for analysis rates of 150/h on the Technicon "SMAC" continuous-flow analyzer. This requires development of a standard that is compatible with the other 19 channels on this analyzer. N-1-Naphthyl ethylenediamine dihydrochloride has been used for standardization of direct bilirubin assays, but we found it to be unsuitable because values for potassium are falsely elevated when potassium is determined with a valinomycin ion-selective electrode. This interference can be eliminated by alkylating the aliphatic amine group in the standard. The resulting compounds undergo the coupling reaction in the same way as the original compound and function equally well as standards for the direct bilirubin reaction. The only limitation of these analogs is their decreased solubility at physiological pH in some cases. Thus only certain alkyl groups are suitable.