Beyond despair and disengagement: a transactional model of personality development in later life. It is argued that ego analytic, disengagement, and symbolic interaction theories of personality share an insufficient consideration of the dynamics of the interaction between developmental and social forces transpiring during the aging period. An alternative, transactional model is proposed, positing that the effects of the environment to which the elderly person is subject are the product of: (1) the people and settings comprising that environment and (2) the manner in which the individual acts upon and is acted upon by these people and settings. The model highlights the need for research in which present behavioral and environmental events assume as much importance as past ones.


Effect of prostaglandins on the blood-aqueous barrier of the perfused cat eye. With a technique of direct visualization of the arterially perfused cat eye, the pressure head may be chosen so that fluorescein added to the perfusate just barely stains the ciliary processes. After addition of PGE1, PGE2, PGF2alpha arachidonic acid, or indomethacin, with or without PG's, no more dye emerged from the processes. The addition of acetylcholine in the eserinized eye floods the processes with dye, apparently affecting the pore size of the blood-aqueous barrier; PG's may slightly inhibit, rather than facilitate, the emergence of fluorescein from the processes. It is speculated that in the cat PG's elevate pressure and protein content by backflow from the circle of Hovius, which is the equivalent of Schlemm's canal.


Collagen crosslinking in keratoconus.

Collagen crosslinking in keratoconus. The examination of reducible collagen crosslinks in keratoconus cornea revealed the presence of lysinonorleucine in amounts far greater than in normal age-matched corneas. There was no indication of decreased hydroxylysine levels in keratoconus, and there were no clinical indications of a generalized connective tissue disorder. The abnormal levels of dehydrolysinonorleucine in the tissue may represent a change in hydroxylation of selected lysyl residues of normal collagen or the synthesis of abnormal collagen, perhaps an unusual type.


Antimuscarinic effects of stereoisomers of tropicamide on rabbit iris sphincter. The antimuscarinic activity of optical isomers of tropicamide were compared on the isolated rabbit iris sphincter. The increasing concentrations of both the (--)- and (+)-isomer shifted the dose-response curve of carbachol to the right in a parallel fashion. The competitive reversible muscarinic blocking effects of both isomers were confirmed by pA2 plots. The pA2 values from the nonpigmented irides for (--)- and (+)-tropicamide were 7.88 and 6.18, respectively. Thus the (+)-isomer has only 1/50 the blocking activity of the (--)-isomer. Although both isomers are slightly less active in the pigmented iris, the activity difference between the isomers was high. From the nonpigmented iris, the blocking effect of the active (--)-tropicamide was readily reversed by washing, whereas reversal of this isomer's effect from the pigmented iris was relatively slow.


Intraocular pressure after optic nerve transection. Intraocular pressure response to systemically administered osmotic agents was studied in albino rabbits with one optic nerve transected and the fellow optic nerve left intact. There eas a significant increase in intraocular pressure of both eyes following water ingestion but no significant difference in the pressure rise of the two eyes. There was a significant decrease in intraocular pressure of both eyes following glycerol ingestion but no significant difference in the pressure fall of two eyes. These results do not support the hypothesis that the optic nerve carries fibers which are part of the control system for intraocular pressure.


Spatial context of residential moves by elderly persons. This is a descriptive analysis of the spatial context of residential moves by the over age forty-five population in the United States. It utilizes residential mobility and state of birth statistics calculated from a 15 per cent sample of the United States population drawn for the U.S. Census. It specifically focuses on the likelihood that the age sixty-five and over white and black mover will relocate within his same county or state of residence. The paper reveals that the preponderance of residential moves by the elderly (whether black or white) are within the same county. Only a relatively small percentage make interstate moves. The majority of elderly live in the state of their birth. However, there is no evidence that as a consequence of aging moves become more spatially restricted until after age seventy-five. It is argued that greater emphasis be placed on not why elderly persons move, but why when they do, their residential relocations occur within varying spatial contexts.


The transscleral VER: prediction of postoperative acuity. In 33 patients with corneal or lens opacities, the VER was recorded in response to a series of retinal stimuli delivered by the transscleral route (TVER). Based on the preoperative TVER characteristics, predictions of the postoperative acuities were made on a 1 to 5 scale. The predicted acuities were compared with those measured 6 months after surgery and showed that the TVER is a good predictor of postoperative acuity (r = 0.8). The criteria used for acuity estimates are presented.


The transscleral VER: normal responses.

The transscleral VER: normal responses. Normative data taken on a sample of 10 eyes indicate that transscleral visual evoked response (TVER) stimulation of the cortical evoked response with a range of lights and at lower adaptation levels produces replicable, low-variance responses which may be of sufficient quality for the clinical evaluation of retinal and visual pathway condition. Response amplitudes were related to the radiant peak-power of white stimuli but were greater for red stimuli. Attenuation of the subjective brightness of the stimulus delivered through the inferior lid and sclera was about 0.4 log units compared to corneal delivery. Typical signal:noise ratios were 5:1.


Mechanics of intraocular gas.

Mechanics of intraocular gas. The relationship between the volume of an intraocular gas bubble and the area of retina covered by the bubble was studied with the use of both a transparent model and a mathematical model of the vitreous cavity. The arc of contact of intraocular bubbles was calculated for vitreous cavities of various diameters. A 0.28 cm3 bubble will cover 90 degrees of retina and be of sufficient size to manage many of the problems for which an internal retinal tamponade would be useful. Larger retinal tears require disproportionately large increases in bubble volume to achieve modest increases in the area of retina covered. Estimating bubble size by observing the height of the bubble meniscus in the dilated pupil is subject to errors induced by small shifts in the angle of observation. A correct evaluation requires that the plane of observation be adjusted so that it coincides with the plane of the meniscus.


The fine structure of nuclear changes in superior limbic keratoconjunctivitis. Superior limbic keratoconjunctivitis (SLK) is a condition of unknown etiology. Histological signs include acanthosis, dyskeratosis, keratinization, and ballon degeneration of the bulbar conjunctival epithelium. Ultrastructural examination of biopsy material from five eyes of patients with SLK shows numerous nuclear changes in the conjunctival epithelial cells. These include abnormal distribution and aggregation of nuclear chromatin, the presence of filaments within the nucleus, and dense accumulations of cytoplasmic filaments which surround the nucleus, resulting in "strangulation" and the formation of multilobed nuclei or multinucleated cells. These changes do not appear to have been described previously in any cell type.


In situ assay of poxvirus-induced and virion-contained deoxyribonucleases in DNA polyacrylamide gels. Cowpox virus and vaccinia virus WR specific DNases were identified by an in situ assay using DNA-containing acrylamide gels. A DNase present in the isolated virions of strain cowpox and vaccinia WR was identified. Its properties as determined by the in situ assay resembled those of the well-characterized DNase V1. The assay was also used to follow the time course of 'acid' DNase induction by cowpox virus in primary chick embryo fibroblasts.


Sedimentation of multicomponent viruses: evaluation of sedimentation coefficient ratios. Ratios of the sedimentation coefficients for alfalfa mosaic virus components are shown to be independent of the virus concentration and the density of the solvent. Different numbers of components are observed in solvents of different density. This implies that in sedimentation velocity experiments an estimate of the number of components of a multicomponent virus should involve centrifugation in solvents of different density. For some viruses, estimates of the sedimentation coefficients of individual components can be obtained from the coefficient ratios observed in unfractionated solutions and the sedimentation coefficient of the most easily purified component.


Recovery of tobacco mosaic virus RNA Replication after incubation at 40 degrees. Incubation of tobacco mosaic virus (TMV)-infected tissue at 40 degrees for 12 h destroyed the capacity for TMV RNA replication. Upon shift of the tissue from 40 to 25 degrees, synthesis of TMV RNA did not resume. Incubation at 40 degrees for 1 h did not destory the TMV RNA replicase, because viral RNA synthesis resumed upon return to 25 degrees. However, upon further incubation at 25 degrees, the synthesis rate of TMV RNA gradually declined. Upon still further incubation at 25 degrees (16-20 h), the synthesis of TMV RNA in tissue incubated at 40 degrees for either 1 or 12 h recovered. This recovery was inhibited by cycloheximide but not by 2-thiouracil.


Time-course of actinomycin D inhibition of tobacco mosaic virus multiplication relative to the rate of spread of the infection. The time-course of inhibition of tobacco mosaic virus synthesis in mechanically inoculated leaves by actinomycin D was not correlated with cell-to-cell spread of the infection, as previously shown to be the case with 2-thiouracil and guanidine inhibition. The amount of inhibition by actinomycin D declined after inoculation much more rapidly than the infection spread. An actinomycin-D-sensitive step or its product occurred in cells before they became infected.


Estimation of the cell-to-cell spread of tobacco mosaic virus infection in mechanically inoculated leaves. Procedures to estimate cell-to-cell spread of tobacco mosaic virus (TMV) infection in mechanically inoculated leaves are presented mathematically and experimentally. Based upon the mathematical estimation, it is demonstrated that resistance of TMV infection to 2-thiouracil and guanidine in mechanically inoculated leaves is correlated to the fraction of cells infected. The addition of 2-thiouracil or guanidine prevents cell-to-cell spread of TMV infection.


Electron microscopic localization of 5'-nucleotidase in the stratum intermedium and ameloblasts. 5'-nucleotidase was demonstrated at the fine structural level in the stratum intermedium and ameloblasts of the first mandibular molars of CD-1 mice. The enzyme was localized with the Wachstein & Meisel (1957) method along the plasma membranes of the cells of the stratum intermedium and ameloblasts. While 5'- nucleotidase was present throughout the stratum intermedium, only the proximal region of the plasma membranes of ameloblasts was demonstrably active for this enzyme. 5'-Nucleotidase has been implicated in transport of metabolites across cell membranes, and its localization in the present study supports this implication as well as the transport functions of the stratum intermedium and the stratum intermedium--ameloblastic interface.


Alkaline phosphatase activity in human polymorphonuclear leukocytes. Alkaline phosphatase has been localized ultracytochemically in PMN of man with normal and elevated levels of this enzyme. Contrary to guinea-pig PMN, no activity appears to be present in the specific granules. Instead, the plasma membrane and the membrane of the endocytic vacuoles show a strong staining. However, the demonstration of this activity depends on the preparatory procedure employed for PMN isolation. The use of dextran and Ficoll-Hypaque in the isolation procedure induces a marked increase in alkaline phosphatase staining of the PMN plasma membrane. Strongly increased activity at this site has been found in PMN from cancer patients. In most of them, additional staining has been observed in atypical vesicles and sometimes in the Golgi apparatus. These findings are discussed in the light of some previously reported controversial biochemical and cytochemical data on the distribution of alkaline phosphatase in human PMN.


Palatal shelf epithelium: a morphologic and histochemical study in X-irradiated and normal mice. The palatal shelf epithelium of normal and irradiated mice was examined morphologically and histochemically, utilizing the periodic acid-Schiff (PAS) technique for the demonstration of the basement membrane and the Nitro BT method for succinate dehydrogenase activity in order to demonstrate the metabolic competence of its cells. The 'programmed cell death theory' was not supported by the present investigation, since the cells of the medial ridge epithelium retained their structural and metabolic integrity even subsequent to the formation of cell nests. Additionally, the medial ridge epithelium of mice with radiation-induced cleft palates demonstrated normal structural and metabolic integrity long past the prospective time of fusion.


Electron cytochemistry of developing and mature eosinophils in the bone marrow of the fowl and the duck. Enzyme cytochemical studies have been carried out on eosinophils in the fowl and the duck. Peroxidase was found in all regions of the Golgi apparatus, the rough endoplasmic reticulum and the perinuclear cisternae of the early cells. In fowl eosinophil granules irregular deposits of peroxidase and arylsulphatase final reaction product were found, but the acid phosphatase deposits were even. In the duck in contrast, peroxidase was demonstrated in the external part of the granule only. Acid phosphatase and arylsulphatase were found in both the interna and the externa of the duck eosinophil granules. An ammoniacal silver nitrate reaction for the presence of the histone arginine was also studied. Silver deposits were found occupying all regions of the granules of eosinophils from both species of bird. The presence of the hydrolytic enzymes acid phosphatase and arylsulphatase in avian eosinophil granules supports the theory that these structures are lysosomal in nature and that they correspond with mammalian eosinophils in this respect.


Species variation in the distribution of cholinesterases in the ovary of the plains viscacha, cat, ferret, rabbit, rat, guinea-pig and roe deer. Sections of ovary from plains viscacha, cat, ferret, rabbit, rat, guinea-pig and roe deer have been histochemically processed to demonstrate acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) in nervous and non-nervous tissue. The effects of different reproductive states on enzyme activity were observed in some animals. AChE-containing nerves were sparse in rabbit and rat but plentiful in cat and roe deer. Nerves containing BuChE were not detectable in ferret or guinea-pig and were rare in cat. Species variations in the activity and type of enzyme were also found in non-neuronal tissues. Some blood vessels in the ovaries of guinea-pig and viscacha contained AChE. No other species showed a reaction for AChE in non-neuronal stromal tissue but BuChE was present at this site in all animals except rat. Granulosa cells reacted for AChE only in cat and rabbit while luteal cells were reactive in cat, rabbit and roe deer. Some BuChE activity was present in granulosa and or luteal cells in all species except roe deer. In rat, BuChE activity in luteal cells increased during pregnancy and the early phase of pseudopregnancy. The difficulty of assigning a function to ovarian cholinesterases is discussed.


Cyclic and pathologic changes of the mare endometrium as detected by biopsy, with a note on early embryonic death. Normal histologic changes that occurred in concert with the ovarian cycle were evaluated. When combined with behavioral and physical findings, this evaluation enabled determination when there was synchrony between ovarian steroids (estrogen and progesterone) and their target tissues of endometrium. A system for classification as well as definitions of patterns types and degrees of severity of inflammatory and fibrotic changes was developed to facilitate a uniform system for description and communication. An attempt was made to relate the pathologic  changes to their effect on fertility when fertility was defined as the ability to conceive, maintain, and deliver a live foal. This was done by dividing the types of endometrium into 3 categories, based on increasing degrees of extensiveness and severity of the various changes, and it was possible to show a high correlation of category to foaling rate. Embryonic and fetal loss from endometria with widespread periglandular fibrosis was observed. This was considered to be the major diagnosable cause of this type of reproductive failure.


Clinically important structures of the equine hock. A study method has been devised to review clinically important structures of the equine hock joint on a fresh specimen. The review can be done alone; dissection takes about 2 hours; special tools or materials are not required.


Repair of physeal fractures of the tuber olecranon in the horse, using a tension band method. Physeal fractures of the tuber olecranon of 4 horses were treated, using the tension band method of compression fracture repair. A cortical bone screw and Steinmann pin were placed through the proximal fragment into the distal parent bone parallel to the palmar border of the olecranon. The tension band was placed dorsal to the Steinmann pin and anchored in a hole through the shaft of the ulna 10 cm distal to the tuber olecranon. Of the 4 foals, 2 became pasture sound, 1 is now being worked under saddle, and the 4th showed no signs of lameness 5 months after surgery.


Navicular disease in the hindlimbs of the horse. Of 124 horses determined to have navicular disease during a 5-year period, 6 had clinical and radiographic signs of the disease in both hindlimbs. These 6 were all castrated males, ranging in age from 3 to 12 years, and were used for work of similar nature: roping, cutting, and pleasure. Treatment consisted of posterior digital neurectomy and corrective shoeing in 2 cases, posterior digital neurectomy alone in 2 cases, and corrective shoeing alone in 2 cases. Following treatment, lameness subsided, and all 6 horses were returned to full use.


Evaluation of inferior check ligament desmotomy for treatment of acquired flexor tendon contracture in the horse. The usefulness of inferior check ligament desmotomy as a treatment for acquired tendon contracture in horses was evaluated in 13 cases of deep digital flexor (DDF) contracture and in 3 cases of combined superficial digital flexor (SDF) and DDF contracture. In 8 of 9 cases of DDF contracture in which the dorsal surface of the hoof had not passed beyond being vertical to the ground, the surgical procedure was successful in returning the limb to a normal position and function. Compared with DDF tenotomy, inferior check ligament desmotomy was a superior treatment technique because of decreased postoperative pain, better postoperative appearance, and improved return to function. In 4 cases of DDF contracture in which the dorsal SDF contracture was also involved, inferior check ligament desmotomy was generally insufficient to allow return of the limb to normal alignment and function.


Pathophysiology of airway obstruction in horses: a review. Obstruction of the upper and lower airways is common in horses. In the upper airway, paresis of abductor muscles of the nares and larynx allows inspiratory collapse of soft tissues, which is accentuated by factors increasing upper airway resistance and by high inspiratory flow rates. Intrapulmonary airway obstruction occurs due to accumulation of secretions, release of chemical mediators in response to a variety of stimuli, and parasympathetic stimulation of airways. Obstruction of large airways increases the work of breathing, whereas obstruction of small airways may cause no measurable increase in resistance but does cause uneven distribution of ventilation and hypoxemia, especially during exercise. Poor collateral ventilation accentuates gas exchange problems in the horse with airway obstruction and may be a factor causing intrapulmonary hemorrhage during racing.


Internal abdominal abscesses in the horse: a study of 25 cases. internal abdominal abscesses in 25 horses, including 8 horses that died or were euthanatized, were found to be caused by Streptococcus equi, Streptococcus zooepidemicus, or Corynebacterium pseudotuberculosis. Breed or sex predilection was not found. Although horses of all ages were affected, those under 5 years of age were more commonly affected. Nearly all of the horses had a history of respiratory catarrh or lymphadenitis. Horses with internal abdominal abscesses also had intermittent, prolonged colic or chronic weight loss. Most horses had increased rectal temperature, increased heart and respiratory rates, and varying degrees of anorexia. Chronic depression anemia, increased plasma fibrinogen concentration, increased total plasma protein with hypergammaglobulinemia, and hypoalbuminemia were prominent clinicopathologic findings. Results of rectal examination or surgical exploration often revealed an abdominal mass. Peritoneal fluid from 14 horses was examined, and the protein content was greater than 2.5 g/dl, with increased numbers of neutrophils. Culture results were negative in all 14 cases, although bacteria were seen intracellularly in 5 cases. Long-term (2- to 6-month) antimicrobial therapy with procaine penicillin G resulted in clinical resolution in most of these animals.


Scanning electron microscopy of Gasterophilus intestinalis lesions of the equine stomach. The lesions caused by larvae of Gasterophilus intestinalis in the cardiac region of the equine stomach were funnel-shaped ulcers surrounded by a rim of hyperplastic epithelial cells. Bacteria were commonly seen on the rim of epithelial cells, at the base of some ulcers, and within the cavities produced by the hooks of the larvae. Cellular debris and mucus were within the ulcer.


Chronic enteritis associated with the malabsorption and protein-losing enteropathy in the horse. Chronic granulomatous enteritis associated with weight loss and hypoproteinemia was identified in 2 horses. Both horses continued to have normally formed feces. Malabsorption of carbohydrate and lipid, with concomitant gastrointestinal protein loss was demonstrated in 1 case. One horse was treated symptomatically and gained 108 kg. In both cases, principal gastrointestinal lesions were partial to total villus atrophy and transmural mononuclear leukocytosis, with lymphocytes and histiocytes predominating. The cause of the condition was not identified in either case.


Epiglottic entrapment by arytenoepiglottic folds in the horse. An abnormality of the epiglottis and arytenoepiglottic folds that caused epiglottic entrapment was diagnosed in 21 horses. Until recently, this entrapment was poorly understood. Definitive diagnosis of epiglottic entrapment can be made only by endoscopic examination of the epiglottis, arytenoepiglottic folds, and soft palate to differentiate the abnormality from dorsal displacement of the soft palate. Dorsal displacement of the soft palate is often associated with entrapped epiglottis. Epiglottic deformity, especially hypoplasia, is often associated with the entrapment. The abnormality was detected in horses 1 to 16 years old. Because of the relatively large number of young animals (11 being less than or equal to 2 years old), a congenital predisposition was suggested. This suggestion was strengthened by the fact that many of the horses had deformities of the epiglottis that were considered congenital. Because some of the horses had trained and raced satisfactorily before signs of upper airway obstruction developed, it was assumed that the abnormality may be a sequel to epiglottic injury.


Interruption of aortic arch in two foals. Two foals with a history of normalcy at birth developed a lack of exercise tolerance and weakness in the first few days of life. Weakness, inability to rise, and reluctance to suckle were common complaints. Physical examination of both foals revealed a cardiovascular abnormality, with a loud systolic murmur audible over both thoracic walls. Additional diagnostic techniques were blood gas analysis, radiography, cardiovascular catheterization, and necropsy. Necropsy findings were ventricular septal defect, atrial septal defect, patent ductus arteriosus, and congenital absence of the aortic arch. Additionally, 1 foal had anomalous drainage of the cranial vena cava into the left atrium.


Equine myositis and septicemia caused by Acinetobacter calcoaceticus infection. Myositis and septicemia caused by Acinetobacter calcoaceticus were diagnosed in a mare. The infection was characterized clinically by ventral swelling and edema, diarrhea, listlessness, and rectal temperature of 39.4 C. The mare was treated symptomatically for 2 days but died on the 3rd day. Conditions seen at necropsy were myositis, enteritis, typhlitis, colitis, and hepatitis. Lymph nodes were moderately enlarged throughout the body. Gross lesions in musculature were edema, scarring, petechiae, and an occasional exxhymosis. The enteritis was catarrhal, with excessive mucus and moderate hyperemia. The typhlitis and colitis were hemorrhagic. The swollen liver had a diffuse mottled pale and red pattern. Microscopic lesions in skeletal muscle consisted of petechiation, necrosis, scarring, and edema. Cardiac muscle was also scarred and necrotic, but edema was not prominent. Periacinal necrosis was found in the liver. Acinetobacter calcoaceticus was isolated from myocardium and liver.


Role of horse flies in transmission of wquine infectious anemia from carrier ponies. Equine infectious anemia virus was transmitted from an acutely ill and an inapparently infected pony to uninfected ponies by the interrupted feeding of horse flies (tabanids). Transmission from acutely ill ponies was not accomplished following: (1) the interrupted feeding of a single horse fly, (2) bites of horse flies that had fed on an acutely affected pony 24 hours earlier, (3) bites of horse flies that had oviposited after feeding on an acutely affected pony, or (4) the inoculation of larval material derived from horse flies that had fed to repletion. It was concluded that horse fly transmission of equine infectious anemia virus is mechanical only and that infected horses that are free of clinical signs can be a source of virus for insect transmission.


Gas-liquid chromatographic determination of chlordene epoxide in catfish. Chlordene epoxide was detected in the range of 0.04 to 0.27 ppm in the edible portion of 3 catfish samples during the analysis of 36 fish samples by electron capture gas-liquid chromatography (GLC) for pesticide and industrial chemical residues. Two of these catfish samples were from suppliers in Louisiana and the third was from a Texas supplier. Chlordene was also present; the amount of chlordene epoxide in the 3 samples averaged 46% of the amount of chlordene present. The epoxide was identified by comparison of the mass spectra and the GLC retention times of the samples with reference material prepared in the laboratory.


Determination of benzo(a)pyrene in foods. An analytical method was developed for determining benzo(a)pyrene in foods, suitable for routine use. The method consists of 4 cleanup steps: (1) alkali cleavage of sample, (2) preliminary silica gel column chromatography, (3) selective extraction with concentrated sulfuric acid, and (4) further silica gel column chromatography. Recoveries of benzo(a)pyrene added to 50 g (or 10 g) food at levels of 0.4 ppb (or 2 ppb) ranged from 70% for short-necked clam and mackeral to 85% for chicken meat. The sulfuric acid extraction step affords a simple method for isolating benzo(a)pyrene from various kinds of interfering substances which could not be separated by existing methods.


High pressure liquid chromatographic determination of methomyl and oxamyl on vegetable crops. A reverse phase high pressure liquid chromatographic method is presented for the separation and determination of residues of the carbamates oxamyl and methomyl on vegetables. A liquid-liquid extraction and cleanup procedure is applied to the vegetable extract. Samples are eluted from a muBondapak C18 column and quantitated by ultraviolet absorbance at 240 nm. Recovery data for vegetable samples spiked at 2 ppm are presented.


Gas-liquid chromatographic determination of chlorinated norbornene derivatives in fish. 1,2,3,4,7,7-Hexachloro-2,5-norbornadiene (I), 1,2,3,4,5-endo,7,7-heptachloro-2-norbornene (II), and 1,2,3,4,7,7-hexachloro-5,6-endo-epoxy-2-norbornene (III) have been identified as contaminants in Mississippi River fish by combined gas-liquid chromatography (GLC)-mass spectrometry. The method used to determine residues of these compounds, which are associated with production of the pesticide endrin, is similar to the AOAC multiresidue method for chlorinated pesticides. The residues are extracted from the sample with acetonitrile, transferred to petroleum ether, and chromatographed on a Florisil column. Compounds I and II are eluted from the Florisil with petroleum ether and compound III is eluted with ethyl ether-petroleum ether (6 + 94). The concentrated eluates are analyzed by using electron capture GLC. The method provides adequate recoveries of all 3 chlorinated norbornene derivatives and allows quantitation at or below the 0.05 ppm level in fish with a 20 mg sample equivalent injection.


Simple combustion-vapor trapping technique for determination of mercury in fish. A simple combustion train was constructed for analyzing mercury in fish samples. A nitric acid trap was used to capture the mercury vapors which were released later by adding a tin salt. The method is rapid, accurate, and reproducible and permits one person to analyze 40 samples daily. Sample matrix had no apparent effect on the recovery of mercury. Mean recoveries from fish samples spiked with 0.3 and 1.0 microgram mercuric chloride/g ranged from 97 to 105% with an average recovery of 101.5% and a standard deviation of 2.74%.


Quantitative determination of chlorpromazine. HCl in tablets, spansules, injectables, and bulk chemical by nuclear magnetic resonance spectroscopy. A nuclear magnetic resonance (NMR) procedure is described for the quantitative analysis of chlorpromazine. HCl in bulk chemical as well as in final dosage forms--tablets, spansules, and injectables. The method is based on measurement of a characteristic signal of chlorpromazine relative to an internal standard. Three different internal standards are specified: Cyclohexane was selected because of the convenience and rapidity with which samples could be prepared for assay. Piperonal was used to verify the method and to show that precision and accuracy were not affected by the volatility of the cyclohexane. Tetramethylammonium bromide was used as an internal standard for Thorazine injectable. No interferences were found from stearates and other tablet excipients. The NMR procedure provides a simple, direct, and specific assay with a precision of +/- 1-2%.


Proton magnetic resonance spectroscopic determination of disulfiram: collaborative study. A proton magnetic resonance spectroscopic method for determining disulfiram in the bulk drug product and in the formulated material was collaboratively studied. The method depends on the use of chloroform-d as a solvent and hexamethylcyclotrisiloxane as the internal standard. No interference from tablet excipients was observed. The method is rapid and specific. Eighteen laboratories analyzed duplicate samples of a bulk drug product, a 250 mg tablet composite, and a 500 mg tablet composite. The average per cent results and standard deviations were 99.7 +/- 1.4, 100.9 +/- 2.0, and 99.9 +/- 2.2, respectively.


Ion exchange method for determining mephentermine sulfate in drug formulations: collaborative study. Ion exchange chromatography is used to separate mephentermine sulfate from drug formulations. The drug is subsequently measured by ultraviolet spectrophotometry. Assays on 4 commercial samples of tablets and injectables gave recoveries from 97.6 to 104% of declared. A collaborative study involving 2 tablet and 2 liquid formulations gave mean recoveries ranging from 94.7 to 99.3% and coefficients of variation from 1.39 to 2.00%.


Gas-liquid chromatographic determination of p-chloroacetanilide in acetaminophen. Gas-liquid chromatography GLC) coupled with column chromatography was used to accurately determine as little as 25 ppm p-chloroacetanilide in acetaminophen. p-Chloroacetanilide was eluted from a pH 8 phosphate-buffered diatomite partition column by using purified tetrachloroethylene (acetaminophen was retained). This solution was concentrated, internal standard (docosane) was added, and p-chloroacetanilide was determined by using a 0.9 m X 2 mm glass column packed with 3% Poly A 103 on Supelcoport and a flame ionization detector with electronic integration. Standard curves were linear for 10-100 ppm p-chloroacetanilide. Various chromatographic materials were investigated for optimal retention characteristics. High pressure liquid chromatography (HPLC) was also evaluated as an alternative; however, lack of reproducibility of the HPLC column favored the GLC procedure.


Stimulation of glucagon of in vivo phosphorylation of rat hepatic pyruvate kinase. Rat hepatic pyruvate kinase (type L) has been purified to homogeneity by a simple, rapid procedure involving DEAE-cellulose chromatography and elution from a blue Sepharose column. The enzyme was homogeneous by the criteria of sodium dodecyl sulfate disc gel electrophoresis, had a subunit molecular weight of 57,000, and a specific activity of 558 units/mg of protein at 30 degrees. In order to test whether the enzyme is phosphorylated in vivo, rats were injected with radioactive inorganic phosphate. Incorporation into pyruvate kinase was determined after purification of the enzyme to homogeneity as well as after specific immunoprecipitation of the enzyme from partially purified preparations. Sodium dodecyl sulfate disc gel electrophoresis revealed that 32P was incorporated into the enzyme in both cases. Glucagon administration in vivo resulted in a 200 to 300% increase in the incorporation of 32P into the enzyme which was correlated with an inhibition of enzyme activity and an elevation of hepatic levels of cyclic AMP. These results represent the first demonstration of in vivo phosphorylation of a hepatic glycolytic enzyme and strongly support the hypothesis that glucagon regulates pyruvate kinase activity, at least in part, by a phosphorylation mechanism.


N epsilonB29-(+)-biotinylinsulin and its complexes with avidin. Synthesis and biological activity. Insulin was modified with d-biotin-N-hydroxysuccinimide ester in dimethylformamide. Mono-, di-, and triacylated insulins were separated by preparative isoelectric focusing. Monoacylated derivatives (isoelectric point 5.1) were fractionated twice on DEAE-cellulose to yield pure N epsilonB29-biotinylinsulin. The structure of the product was established by amino acid analysis before and after deamination. N epsilonB29-biotinylinsulin had biological activity indistinguishable from insulin on glucose oxidation and lipid synthesis assays using isolated rat epididymal fat cells. Complexes of N epsilonB29-biotinylinsulin with avidin, having essentially all but one binding site filled with biotin, were prepared in order to obtain a 1:1 insulin:avidin ration. The elicited identical maximal biological responses, but showed a potency decreased to 5% of that of insulin. Such complexes conjugated with ferritin will provide a useful tool in the development of electron microscopic stains of insulin receptors.


Quantitative determination of the binding of epsilon-aminocaproic acid to native plasminogen. The binding of epsilon-amino[14C]caproic acid (6-aminohexanoic acid, EACA) to native human plasminogen was determined using the ultrafiltration technique of Paulus (Paulus, H. (1969) Anal. Biochem. 32, 91-100) at free ligand concentrations ranging from 2 micrometer to 16 mM. One strong binding site (Kd = 0.009 mM) and approximately five weaker ones (Kd = 5 mM) were found. The constants were obtained by fitting the experimental points to the simple assumption of two sets of noninteracting sites. The distinct separation of the two kinds of sites allowed the correlation of the well known epsilon-aminocaproic acid-induced conformational transition in plasminogen with the saturation of the weaker group of binding sites by this ligand. The conformational transition was monitored by measurements of the sedimentation coefficient, as was done by others earlier. The midpoint of the transition occurred at approximately 3.3 mM free ligand. A dissociation constant of 0.32 mM was also obtained for L-lysine by measurements of the competition between this compound and labeled epsilon-aminocaproic acid for the strong binding site. The correlation between epsilon-aminocaproic acid binding and effects of the compound on various physical and functional properties is discussed. A discussion of the possible sources of error encountered in the technique used is also included.


Biosynthesis of beta nerve growth factor in mouse submaxillary glands. The biosynthesis of beta nerve growth factor (betaNGF) was studied in isolated mouse submaxillary glands incubated with L-[35S]cystine. Sodium dodecyl sulfate gels of anti-betaNGF immunoprecipitates from labeled gland homogenates showed a single major peak of radioactivity, which comigrated with purified betaNGF. This species was nearly completely precipitated by the addition of equivalent amounts of anti-betaNGF, but was absent from immunoprecipitates obtained by the addition of ferritin plus anti-ferritin. The cystine-containing tryptic peptides of the labeled species appeared identical with those of purified betaNGF. In submaxillary glands from adult male mice, labeling of betaNGF represented approximately 0.2% of the trichloroacetic acid-precipitable radioactivity. Castration reduced this value to one-third, while testosterone treatment of castrated animals restored the relative betaNGF synthesis to normal or more. No betaNGF synthesis could be detected in glands from female animals. Several tissues were examined for their ability to synthesize betaNGF in culture. Only submaxillary gland incorporated detectable amounts of radioactivity into betaNGF. Labeling of betaNGF could also be obtained by direct injection of isotope into the submaxillary gland in vivo. The results are discussed in terms of the integration of betaNGF synthesis into neuronal development and maintenance.


Purification and properties of the endonuclease specific for apurinic sites of Bacillus stearothermophilus. An endonuclease specific for apurinic sites when double-stranded DNA is used as substrate has been isolated from the thermophilic bacterium, Bacillus stearothermophilus; it is a monomeric protein of about 28,000 daltons, without action on normal DNA strands or on alkylated sites. The enzyme is quite thermoresistant in the presence of other proteins, has an optimal temperature of 60 degrees, needs monovalent cations for optimal activity, is insensitive to EDTA, and is inhibited by divalent cations; it has no associated exonuclease activity. These latter properties are closer to those of Escherichia coli thermoresistant endonuclease IV, which is also insensitive to EDTA and has no exonuclease activity, and very different from those of the main endonuclease for apurinic sites of the same bacterium. The B. stearothermophilus enzyme is more resistant to urea and detergents than the main E. coli endonuclease for apurinic sites and has a higher content of hydrophobic amino acids.


An approach to the isolation of biological particles using sedimentation analysis. A systematic, general approach for the design of an initial purification procedure for any biological particle is described in this communication. A series of centrifugations in fixed angle rotors has been used to obtain information on the sedimentation behavior of particles of interest (Anderson, N.G. (1967) Anal. Biochem. 23, 72-83). Refinements of this technique have facilitated the determination of sedimentation profiles of subcellular organelle markers in suspensions of murine spleen and brain. The degree of homogeneity of several particles with respect to size can be ascertained from the sedimentation profiles. Alterations in these profiles after mechanical disruption and treatment with detergents are readily measurable and have been found to be useful in both the characterization and isolation of subcellular particles. Because fixed angle rotors are used in these studies, the data obtained can be directly applied to the development of a preparatory scheme for purification of a desired particle. These methods for sedimentation analysis are readily applicable to subcellular organelles, macromolecular complexes, viruses, viral-like agents, and a variety of macromolecules.


Some properties of chromatin synthesized by mouse-L-cells temperature-sensitive in DNA replication. When ts A1S9 mouse L-cells are incubated at the nonpermissive temperature (38.5 degrees) DNA synthesis proceeds at the normal rate for 6 to 8 h; it then declines to attain 1 to 5% of this rate after 24 h. General protein synthesis from precursor leucine is relatively unaffected by the high temperature. In contrast, protein formation from lysine (and arginine) remains unchanged for 12 to 15 h after temperature upshift. It then drops and plateaus at about 25% of the initial rate after 32 h. The chromatin protein and DNA are fully conserved in ts A1S9 cells incubated at 38.5 degrees for at least 24 h after full expression of the ts defect. Temperature inactivation of the ts A1S9 gene product results in inhibition of de novo formation of chromatin. This is evidenced by coordinate suppression of incorporation of dThd and of lysine and arginine into chromatin-bound DNA and histone, respectively.


Isolation of eukaryotic ribosomal proteins. Purification and characterization of the 60 S ribosomal subunit proteins La, Lb, Lf, P1, P2, L13', L14, L18', L20, and L38. The proteins of the large subunit of rat liver ribosomes were separated into seven groups by stepwise elution from carboxymethylcellulose with LiCl at pH 6.5. Ten proteins (La, Lb, Lf, P1, P2, L13', L14, L18', L20, and L38) were isolated from three groups (A60, B60, and D60) by ion exchange chromatography on carboxymethylcellulose and DEAE-cellulose, and by filtration through Sephadex. The amount of protein obtained varied from 0.3 to 3.8 mg. Two of the proteins (La and L18') had no detectable contamination; the impurities in the others were not greater than 8%. The molecular weight of the proteins was estimated by polyacrylamide gel electrophoresis in sodium dodecyl sulfate; the amino acid composition was determined. Several additional acidic proteins were identified: P1a and P1b are phosphorylated derivatives of P1; P2a, P2b, and P2c are phosphorylated derivatives of P2. P1 and P2 are distinct proteins but both have large amounts of alanine (20.4 and 17.5 mol %).


Interrelationships between Water and cellular metabolism in Artemia cysts. VIII Sorption isotherms and derived thermodynamic quantities. An analysis of water vapor sorption by cysts of the brine shrimp, Artemia salina, has shown that at environmental water activities (aw) of 0.95 or less, the cysts equilibrate with the aw of their environment. Above this aw the metabolic activity of the cysts participates directly in their water content, and equilibration does not occur. In contrast, dried cysts killed by heat treatment or exposure to ammonia fumes equilibrated with all values of aw examined. Analysis  of the temperature dependence of sorption isotherms revealed that below cyst hydrations of about 0.3 g H2O/g dried weight the temperature coefficient for water sorption was negative, but became positive at hydrations appreciably in excess of this value. Estimates for the differential and integral net enthalpy and entropy changes accompanying the sorption of water have been calculated from isotherms. These results have been interpreted and integrated with those from previous work on the hydration-dependence of metabolic activity. All of the examined hydration properties of the cysts have been shown to be due chiefly to the cellular component, and not the acellular shell. Analysis of the data by the Bradley equation has shown that the hydration behavior of the shell obeys this relationship, whereas that of the cellular component does not.


Protein turnover in senescent cultured chick embryo fibroblasts. The over-all rates of protein synthesis, degradation and net accumulation were estimated in rapidly growing young and slowly doubling old cultures of chick fibroblasts. We find that not only the rate of protein synthesis is reduced in senescent cultures, but the average rate of protein degradation is also slowed down considerably. This decrease in the rate of protein breakdown in aging cells stands in contrast with the previously observed acceleration of this process by other conditions (such as serum deprivation or overcrowding) that lead to the cessation of cellular growth. Though the retarded protein degradation may contribute to the acculation of abnormal proteins in senescent cells we find that the breakdown of grossly abnormal puromycin peptides proceeds equally rapidly in young and old cultures. The protein content of senescent cells increases by 1.8-fold as compared to young cells, while the average cell volume is increased even more (almost 5-fold). By contrast, consideration of the over-all balance of protein metabolism in these cells indicates that the average concentration of metabolically turning-over proteins is somewhat higher in senescent than in young fibroblasts.


Age-related and carcinogen-induced alterations of the extracellular growth factor requirements for cell proliferation in vitro. Cells from thigh muscles of fetal rats proliferate readily in vitro in medium containing homologous adult rat "plasma". As the donor animals mature, these cells become unable to make DNA and proliferate in "plasma" medium, but retain an ability to proliferate in medium containing fetal bovine serum (FBS). This age-related loss of the ability to proliferate in "plasma"-medium is due to an increasing need by the cells for an exogenous prereplicative promoter which is found in FBS and a crude preparation of bovine luteinizing hormone. Adult cells (and possibly fetal cells) also require a "cycle-completion" factor which is found in FBS and adult rat "plasma". The requirements for such external proliferative promoters is completely eliminated by neoplastic transformation in vivo, and neoplastic adult cells isolated from a nickel sulfide-induced mixed rhabdomyo-fibro-sarcoma can make DNA and proliferate in vitro in the complete absence of exogenous growth factors.


Effects of an acridine half-mustard (ICR 191) on growth and ploidy of frog cells in culture. The effects of an acridine half-mustard, ICR 191, on the growth rate and ploidy of four haploid and two diploid lines of Rana pipiens cells in culture were studied. Growth curves indicate that the haploid and diploid cell lines were equally resistant to a 4-hour exposure of this drug (0.1 micrometer to 10 micrometer. ICR 191 treatment induced the haploid cell cultures to become diploid. The proportion of diploid cells increased progressively with respect to time after the 4-hour exposure period. The greater the concentration of ICR 191 applied, the more rapid the rate of conversion. Autoradiographic determinations of percent labelled nuclei indicate that DNA synthesis was not inhibited in haploid or in diploid cells. Therefore, the increased proportion of diploid cells did not originate from the small percentage of diploid cells in the initial population. Instead the haploid cells were converted to diploid cells. Time lapse cinematography indicated that the conversion mechanism was other than cell fusion. Conversion to higher ploidy did not occur when diploid cell cultures were exposed to ICR 191.


Regulation of protein synthesis in human diploid fibroblasts: reduced initiation efficiency in resting cultures. The level of poly A+ RNA in growing cultures of human diploid fibroblasts is 1.8-fold times greater than in resting cultures. The level of functional ribosomes in growing cultures is 2.8 times that in resting cultures. Since transit times are similar in both types of cells, it can be concluded that the rate of protein synthesis in growing cultures is 2.8 times that in resting cultures. a reduced efficiency of mRNA translation at the level of initiation in resting cultures is proposed as a probable explanation for the fact that the decrease in protein synthesis rates is greater than the decrease in mRNA levels. This hypothesis is supported by the observations that: (a) poly A+ RNA is associated with smaller polysomes in resting than in growing cells, and (b) cycloheximide treatment of resting cells results in recruitment of nonpolysomal poly A+ RNA into polysomes and a shift of polysomal poly A+ RNA into larger polysomes.


Nature of cells forming erythroid colonies in agar after stimulation by spleen conditioned medium. Erythroid colony formation in agar cultures of CBA cells was stimulated by the addition of pokeweed mitogen-stimulated C57BL spleen conditioned medium. Both 48-hour colonies ("48-hour benzidine-positive aggregates") and day 7 large burst or unicentric erythroid colonies ("erythroid colonies") developed, together with many neutrophil and/or macrophage colonies. In CBA mice, the cells forming erythroid colonies occurred with maximum frequency (650/10(5) cells) in 10- to 11-day-old yolk sac and fetal liver but were present also in fetal blood, spleen and bone marrow. The frequency of these cells fell sharply with increasing age and only occasional cells (2/10(5) cells) were present in adult marrow. A marked strain variation was noted, CBA mice having the highest levels of erythroid colony-forming cells. The erythroid colony-forming cells in 12-day CBA fetal liver were radiosensitive (DO 110-125 rads), mainly in cycle and were non-adherent, light density, cells sedimenting with a peak velocity of 6-9 mm/hr. These properties are similar to those of other hemopoietic progenitor cells in fetal tissues. The relationship of these apparently erythropoietin-independent erythroid colony-forming cells to those forming similar colonies after stimulation by erythropoietin remains to be determined.


Changes in membrane dynamics associated with myogenic cell fusion. Changes in membrane dynamic properties associated with membrane fusion are studied employing in vitro myoblast fusion as a model system. We utilize a microscopic fluorescence relaxation approach which makes feasible the study of local variations in membrane dynamics within surface subdomains of single intact cells. Studies of the average rotational mobility of the fluorescent probe-1-anilino-naphthalene-8-sulfonate by this technique indicate that myoblast fusion activity is preceded by a generalized increased in membrane fluidity and that areas of cell contact between fusing cells exhibit higher fluidity and polarity, locally, than non-fusion regions.


Early transport changes during erythroid differentiation of Friend leukemic cells. Early transport changes occurring during Friend erythroleukemic cell differentiation are reported. A decrease in the rate of 86Rb transport was observed beginning approximately five hours after stimulation with 1.5% dimethylsulfoxide (DMSO), a potent inducer of Friend cell differentiation. By 12 to 14 hours after DMSO addition, the transport rate had stabilized at close to 60% of control level. This decrease in the rate of 86Rb transport preceded a previously reported decrease in cell volume. Other chemical inducers of Friend cells, such as hypoxanthine and ouabain, also caused early decreases in 86Rb influx. In contrast, xanthine, which does not induce Friend cell differentiation, also did not affect 86Rb influx. The transport of two amino acid analogues, alpha-aminoisobutyric acid and 2-aminobicyclo [2,2,1]-heptane-2-carboxylic acid, which differ in their mode of uptake, was also measured following induction by DMSO. The transport rates of both compounds decreased after a 12-hour exposure to DMSO. In contrast, the uptake of 3H-colchicine, a drug which diffuses passively across the cell membrane, was not significantly affected. Studies with several variant cell lines which do not synthesize hemoglobin in response to DMSO indicate that these non-inducible cells can be divided into two classes--those that demonstrate early changes in transport very similar to the changes observed in inducible cell lines and those which exhibit only small changes in transport. Results obtained using a revertant clone have helped to distinguish between those transport changes which are associated with the induction of hemoglobin synthesis and those which are not. In addition, these early transport changes may be useful in defining the stage in the differentiation process at which a particular variant line is blocked.


Assay of ribonucleotide reduction in nucleotide-permeable hamster cells. Ribonucleotide reduction was measured in Chinese hamster ovary cells made permeable to nucleotides by treatment with the detergent Tween-80. When compared to the respective ribonucleotide reductase activity in partially purified cell extracts, CDP and GDP reductase activities in permeabilized cells responded in a similar fashion to dithiothreitol, pH, MgCl2, FeCl3, substrate concentration and the presence of positive or negative allosteric effectors. At low protein concentrations both CDP and GDP reduction with whole cells increased linearly with cell number and was greater than the activity in corresponding cell extracts. Permeabilized cells were used to measure the level of CDP and GDP reductase in a hamster cell line resistant to the cytotoxic effects of hydroxyurea. The hydroxyurea-resistant cell line contained four to ten times more CDP and GDP reductase activity compared to parental or revertant cell lines. The permeabilized cell assay was also used to measure CDP and GDP reductase activities in Chinese hamster ovary cells synchronized by isoleucine starvation. CDP reductase activity was low in G1 arrested cells but increased 10-fold by 16 hours after the readdition of isoleucine to the growth medium. GDP reductase, which is present at much higher levels, is similarly induced after isoleucine addition, but only by 2-fold. The maximum activity of both CDP and GDP reductase occurred from 14 to 16 hours after isoleucine addition, which corresponded to the period of maximum DNA synthesis.


Mass fragmentographic determination of vanilmandelic acid, homovanillic acid and isohomovanillic acid in human body fluids. Vanilmandelic, homovanillic and isohomovanillic acids in body fluids were efficiently isolated by liquid chromatography on an Amberlite XAD-4 column and by organic extraction in a special apparatus. The purified metabolites were converted into their trifluoroacetylhexafluroisopropanol esters and analyzed by mass fragmentography. The working curves of the metabolites were linear from 0.5 to 5 ng injected. The minimum detectable concentrations of all the metabolites were 2 ng/ml for plasma and cerebrospinal fluid, and 120 ng/ml for urine. The metabolite concentrations in plasma, cerebrospinal fluid and urine of normal persons and patients were determined.


Assay of underivatized intrazepam and clonazepam in plasma by capillary gas chromatography applied to pharmacokinetic and bioavailability studies in humans. The assay procedure of underivatized, intact nitrazepam and clonazepam in human plasma is described, using gas chromatography with a support-coated open tubular column (OV-17), a solid injection system and electron-capture detection. Clonazepam is used as a internal standard in the assay of nitrazepam and vice versa. Linear calibration curves after a single extraction step were obtained in the concentration range 10--100 ng/ml plasma, with standard deviations less than 4.9%. The sensitivity limit of the method is about 1 ng/ml plasma for both drugs. The method was applied to pharmacokinetic and bioavailability studies of nitrazepam in humans. Seven healthy volunteers received two nitrazepam-containing tablet preparations (5 mg) and plasma concentrations were determined regularly from 15 min to 80 h following drug administration. The mean elimination half-life of nitrazepam was 27 h (range 13-34 h). Considerable intra-individual differences in peak level times between the two preparations were observed, whereas the extent of bioavailability was rather similar.


Rapid procedure for chromatographic isolation of DOPA, DOPAC, epinephrine, norepinephrine and dopamine from a single urinary sample at endogenous levels. A three-step procedure has been investigated to extract 3,4-dihydroxyphenylalanine (DOPA), 3,4-dihydroxyphenylacetic acid (DOPAC), epinephrine (E), norepinephrine (NE) and dopamine (DA) from a single urinary sample with the object of obtaining extracts pure enough for specific fluorimetric assay. The procedure described in this paper results from the combination of urine purification on an aluminum oxide column, separation by ion-exchange chromatography of the DOPA-DOPAC fraction from catecholamines, and ether isolation of DOPAC from DOPA. The whole procedure is rapid and easily performed in one work day. Extraction recoveries were 72.4 +- 3.5%, 76 +- 2%, 85.7 +- 3.3%, 85.6 +- 1.4% and 92.4 +- 5.5% for DOPA, DOPAC, E, NE and DA respectively (n=6). The lowest amounts of the five catechols that could be detected in urinary samples by a combination of this extraction procedure and the methods of assay used in our laboratory were 15 ng for DOPA, 40 ng for NE, 20 ng for E, 152 ng for DA and 2.95 micrograms for DOPAC. Urinary volumes convenient for accurate estimation of each compound were 20 ml for healthy human subjects. For pathological or pharmacological purposes, 5 ml of human urine were sufficient. The daily excretion of DOPA, DOPAC, E, NE and DA found by this procedure agrees with data obtained by other authors in healthy subjects. In pathological samples, our three-step procedure led to lower amounts than methods using alumina purification only. The discrepancies between the two methods are discussed in terms of development of internal standards, relative specificity of fluorimetric assays, values of blank eluates, and the possibility of interference from unknown abnormal body metabolites or pharmacological drugs not eliminated by a single-step alumina purification.


Rapid direct determination of trace amounts of salicylic acid in deproteinized serum by means of high-pressure liquid--liquid chromatography. A simple method for the quantitative analysis of salicylic acid in blood serum is described. A liquid--liquid chromatographic system, consisting of a long-chain aliphatic amine as the stationary phase and dilute aqueous perchloric acid as the mobile phase, enables the direct injection of deproteinized serum into the system. No change in the chromatographic properties of the system was noticed after 2000 injections of deproteinized serum. Quantitative analysis is possible using peak area or peak height measurements. The method has a high precision: relative standard deviations of 0.4% and 5% are found for samples containing 10 micrograms and 10 ng injected salicylic acid respectively. The detection limit is found to be about 1 ng slicylic acid, corresponding to 40 ppb salicylic acid in serum. Simultaneously administered drugs such as indomethacin, acetylsalicylic acid, caffeine and phenacetin, and metabolites of salicylic acid do not interfere with the analysis. The time course of the concentration of salicylic acid in serum is demonstrated after oral administration of 1 g sodium-salicylate. The phase system was also found to be suitable for the analysis of salicylic acid in urine.


Determination of di-and polyamines by high-performance liquid chromatographic separation of their 5-dimethylaminonaphthalene-1-sulfonyl derivatives. Using a Lichrosorb RP-8 reversed-phase column and a methanol--water gradient elution program, it is possible to separate within 40 min and to determine routinely in picomole quantities the natural di- and polyamines. The precision of the method is comparable to the thin-layer chromatographic procedures, the separations are most efficient, and the method can be fully automated. A modified gradient enables the repeated assay of spermidine and spermine within 20 min. The method is suited for polyamine analyses in tissues and body fluids.


Towards a definitive assay of creatinine in serum and in urine: separation by high-performance liquid chromatography. A fast and sensitive method for the separation of serum and urinary creatinine is described. For the preliminary purification of serum and urine, a cation-exchange column is used to remove protein, anions and neutral compounds prior to isolation of creatinine by high-performance liquid chromatography. A reversed-phase system with 0.01 M ammonium acetate solution as the mobile phase can separate creatinine in 7.5 min at a flow-rate of 1 ml/min. The purity of the separated creatinine is proved by derivatization using trifluoracetic anhydride, followed by gas chromatography and mass spectrometry. Although this method of purification was designed for incorporation into a definitive assay, the ease and speed of analysis makes is very attractive for routine clinical use.


Separation of urinary ultraviolet-absorbing metabolites by high-pressure liquid chromatography using a commercially available analytical unit. A high-pressure liquid chromatographic system for the separation of UV-absorbing and/or fluorescent urinary metabolites is presented. Thirty to forty UV-absorbing chromatographic peaks are obtained from a 200-microliters urine sample using a 100 cm x 2 mm column filled with a total porous ion-exchanger. The reproductibility of the separation has been studied and eighteen important substances have been located.


Polymorphism of A, B and H substances in human urine. The gel-chromatographic behavior of A, B and H substances from urine was examined and compared with that of previously described AHB salivary fractions. Urinary fractions 1 and 2 exhibited a molecular size smaller than those of salivary fractions 1 and 2. In the urine of 52 subjects a polymorphism of A, B and H substances, with four main types, was observed which is independent of the salivary one and of the so-called secretor and nonsecretor status.


Simplified micro-method for the quantitative analysis of putrescine, spermidine and spermine in urine. A simplified micro-method for the quantitative analysis of urinary polyamines is described. After acid hydrolysis of urine, the polyamines are converted to fluorescent 1-dimethylaminonaphthalene-5-sulfonyl (Dns; dansyl) derivatives and separated by means of thin-layer chromatography. Dns-NH2, which has been reported to interfere with the determination of putrescine, is well separated from di-Dns-putrescine. Putrescine, spermidine and spermine are quantitated by in situ scanning of their fluorescent spots on the chromatogram. The present method is both sensitive and reproducible. It eliminates a number of time-consuming steps and thus reduces preparative losses. Yet an adequate chromatographic resolution is obtained. Representative polyamine analyses of urine from normal volunteers and from cancer patients are reported. Elevated levels occur in the urines of pregnant women and of patients with various types of cancer.


Gas--liquid chromatographic determination of carbamazepine and phenylethylmalonamide in plasma after reaction with dimethylformamide dimethylacetal. A previously published procedure for the gas chromatographic analysis of carbamazepine has been modified and expanded to allow simultaneous determination of phenylethylmalonamide, a metabolite of primidone. Internal standards that closely resemble each compound are used, and derivatives are made by reaction with dimethylformamide dimethylacetal. This change of internal standard for carbamazepine and the use of a commercial, pretested column-packing material eliminate the major pitfalls of the original method.


Rapid and sensitive gas chromatographic quantitation of morphine codeine and O6-acetylmorphine in illicit heroin using an electron capture detector. Morphine, codeine and O6-acetylmorphine are reacted with heptafluorobutyric anhydride, rendering them suitable for electron capture detection and quantitation. The fluorinated derivatives are extracted from an acetonitrile-sodium bicarbonate solution into light petroleum in a rapid one-step extraction procedure. The derivatives are chromatographed on a stationary phase of 3% OV-17 on Gas-Chrom Q. Morphine, codeine and O6-acetylmorphine can be readily quantitated in heroin at levels as low as 0.001%, 0.01% and 0.01% respectively. Reproducibility, linearity and recovery studies are described.


Mass fragmentographic determination of pyrazinamide and its metabolites in serum and urine. A combined gas chromatographic-mass spectrometric technique is described for the simultaneous determination of pyrazinamide and its two main metabolites, pyrazinoic acid and 5-hydroxypyrazinoic acid. Serum (200 microliter) is deproteinized and evaporated to dryness; urine (20 microliter) is evaporated. The crude residues are silylated and selected ions are monitored in the chemical-ionization mode with isobutane as both chromatographic carrier and reagent gas. The sensitivity is 10 ng/ml for pyrazinamide and pyrazinoic acid and 20 ng/ml for the 5-hydroxy metabolite in a single analysis. Nicotinic acid and nicotinamide are internal standards. Both unchanged drug and metabolites were identified and quantified in the serum and urine of human subjects.


High-performance liquid chromatographic determination of erythromycin. An isocratic, reversed-phase high-performance liquid chromatographic (HPLC) method has been developed for the determination of erythromycin. The method uses a muBondapak C18 reversed-phase column with a mobile phase composed of acetonitrile-methanol-0.2 M ammonium acetate-water (45:10:10:35). The relative standard deviation of the method for quantification of erythromycin is about 0.6%, and the method is capable of selectively quantifying erythromycins A, B, C and at least nine other erythromycin epimers and degradation compounds in 15 min of chromatographic time. The bio-equivalent potencies of erythromycin powders calculated from the HPLC data agreed well with those of the microbiological assay method. The HPLC method developed is also applicable for the analysis of various derivatives of erythromycin.


Ion-pair high-performance liquid chromatographic separation of a multicomponent anticholinergic drug formulation. N,N'-Trimethylene-bis-(pyridinium-4-aldoxime)dibromide, 4-pyridine aldoxime, atropine sulfate, benactyzine hydrochloride, methyl paraben and propyl paraben are separated by ion-pair high-performance liquid chromatography. The method is specific for detecting and quantifying each compound in a complex mixture without solvent extraction or pretreatment. Levels as low as 1 ng on column are quantifiable by the procedure. All components are eluted within 9 min subsequent to the initial injection. Because of the simplicity of the method, the procedure is suitable for routinely monitoring the stability of the various compounds in the formulation during storage.


[High-performance liquid chromatographic separation and quantitative determination of valepotriates in valeriana drugs and preparations (author's transl)]. It is possible directly to separate and analyse, quantitatively and qualitatively, the valepotriates from Valeriana crude extracts or from commercial Valeriana preparations by high-performance liquid chromatography. The separations are achieved on 4 or 8 mm I.D. columns packed with silica gel (particle size 10 micron) with n-hexane-ethyl acetate mixtures as eluent. A refractive index detection system is necessary for determining all of the valepotriates. If the concentration differences between didrovaltratum and valtratum are very great, an ultraviolet (UV) detector must be used and the determination must be conducted in two steps. For valtratum drugs UV detection alone will suffice. As internal standards p-dimethylaminobenzaldehyde should be used for extracts and preparations from valtratum races, and benzaldehyde in the presence of didrovaltratum races. This determination is superior to the combined thin-layer chromatographic-hydroxamic acid method used hitherto with respect to time consumption, precision, and sensitivity.


Determination of individual hydroxyethyl rutosides in various animal body fluids by thin-layer chromatography and scanning densitometry. A method is described for the accurate determination in plasma of the beta-hydroxyethylrutosides by measurement of the fluorescence of their borocitrate complexes by scanning densitometry following separation by thin-layer chromatography. A modification is also described for estimation of individual hydroxyethylrutosides and their glucuronide conjugates in samples of bile and urine.


Separation of radioactive histamine and some of its metabolites by one-dimensional paper chromatography. A simple one-dimensional paper chromatograph technique is described for the separation of radioactive histamine and some of its metabolites in blood and tissues. After removal of protein, the supernatant fluid (200 microliter) is subjected to two-stage one-dimensional paper chromatography. This enables large numbers of samples to be chromatographed simultaneously. Using this technique in conjunction with liquid scintillation counting, it was possible to monitor the small amounts (0.02 nCi) of radioactive histamine and its metabolites in serial blood samples.


Determination of sodium phenobarbital in animal chow by high-pressure liquid chromatography. An analytical procedure is described for determining residues of sodium phenobarbital in animal chow at levels as low as 0.14 ppm. The methanol extract is subjected to a liquid-liquid cleanup at pH 13 and 1, further cleaned up on a silica gel column and assayed by high-pressure liquid chromatography by using an ultraviolet absorption detector at 210 nm. Data concerning extraction efficiency, partition values and stability of the chemical in animal chow are also presented.


Quantitative methods for the gas chromatographic characterization of acidic fermentation by-products of anaerobic bacteria. The present report describes improved chromatographic procedures which are capable of separating and quantitating complex mixtures of acidic fermentation by-products produced by anaerobic bacteria grown in two glucose-containing media. These methods are reliable and are sensitive, being able to detect as low as 0.5 mumoles of each by-product. Sample preparation has been simplified, and the methylation conditions have been optimized. It is also indicated in this investigation that each culture produced different patterns of by-products in each medium, indicating that the types and quantities of by-products produced in one medium cannot be used as a basis for characterization of these same cultures when grown in a different medium. Finally, it is shown that cultures can be characterized by the distinctive molar proportions of by-products they produce within each medium.


Control of the acute phase response. Serum C-reactive protein kinetics after acute myocardial infarction. In order to investigate the magnitude and kinetics of the C-reactive protein (CRP) response after differing degrees of tissue injury, we studied changes in serum concentration of this acute phase protein in 19 patients after mild or extensive acute myocardial infarction. An increase in serum CRP concentration was seen in all patients. The rate of increase in concentration was found to be exponential, with a mean hourly rate constant for the entire group of patients of 0.085 (doubling time, 8.2 h). Patients with extensive infarction attained mean serum CRP levels about 4 times as great as did patients with mild infarction. No difference could be shown in the mean rate constant between these groups, the greater CRP response in the former group resulting principally from a more protracted period of rise in serum CRP concentration. A lag period before serum CRP levels began to rise was noted in only 4 of the 13 patients in whom this could be assessed. 7 of 10 patients with presumed unstable angina (coronary insufficiency) showed no rise in CRP concentration, while a small increase as noted in 3 patients. The data suggest that acute tissue injury, such as myocardial infarction, rapidly leads to acceleration in synthesis of CRP, and that the duration of this period of acceleration is related to the extent of tissue injury.


The role of endotoxin in protection of adult rats from oxygen-induced lung toxicity. Adult rats show evidence of severe lung damage after 72h of continuous exposure to hyperoxia (96-98% O2). Treatment of adult rats with a solution of Plasmanate, inadvertently contaminated with endotoxin-producing organisms, or with purified endotoxin itself markedly altered the lung toxicity associated with hyperoxic exposure (survival in treated animals = 110/113 [97%] versus survival in untreated animals = 56/172 [33%]). After 72h of hyperoxic exposure, the endotoxin-treated rats demonstrated significant increases in lung superoxide dismutase, catalase, and glutathione peroxidase activity, a protectant enzyme response not seen in untreated adult rats. The basis for endotoxin's protective effect from hyperoxic lung damage is believed to be related to the stimulated increase in activity of the pulmonary antioxidant enzyme defense system. Some previously known actions of endotoxin are speculated to also serve a protective function by opposing some of the usual detrimental effects of high concentrations of O2 on the lung.


Role of gastrin heptadecapeptide in the acid secretory response to amino acids in man. Amino acids and peptides release gastrin and stimulate gastric acid secretion. However, the relation between gastrin release and acid secretory response is unclear. An isotonic mixed amino acid solution (casein hydrolysate) was continuously infused into the stomach of eight healthy human subjects. Acid secretion, measured by in vivo intragastric titration, increased 12.8 meq/h over the response to intragastric infusion of isotonic saline. Plasma gastrin heptadecapeptide (G-17) concentration, measured by specific radioimmunoassay, increased 13 pmol/liter during intragastric amino acid infusion. To determine whether this rise in plasma G-17 concentration could account for some or all of the acid secretory response, several doses of synthetic human G-17-I were infused intravenously into the same subjects. During i.v. G-17-I infusion, the stomach was continuously infused with isotonic saline. By graphically relating plasma G-17 concentration during i.v. G-17 infusion to concomitant acid secretion, it was determined that a 13-pmol/liter rise in plasma G-17 concentration could increase acid secretion 14.8 meq/h. Therefore, the rise in plasma G-17 concentration during intragastric amino acid infusion could have produced all of the observed acid secretory response. This suggests that gastrin heptadecapeptide is the major physiologic mediator of the human acid secretory response to meals containing mixed amino acids.


Mechanisms of fasting-induced increase in insulin binding to rat adipocytes. Fasting leads to an increase in the ability of adipocytes to bind insulin, and this was accounted for by an increase in the affinity of the receptors for insulin without any change in the number of receptors per cell. Binding affinity can increase because of a decrease in the dissociation rate constant (k(d)), an increase in the association rate constant (k(a)), or both. Kinetic studies demonstrated that fasting leads to a striking decrease in the rate at which insulin dissociates from its receptor, and the near two-fold prolongation of the time at which 50% of the bound (125)I-insulin dissociates (28+/-4 vs. 50+/-5 min) correlated quite well with the two-fold increase in binding affinity. On the other hand, the rate at which insulin associates with its receptor was essentially unchanged. Negatively cooperative interactions between receptors were readily demonstrated in cells from control and fasting animals, and the magnitude and sensitivity of this effect was the same in both groups of cells. It seemed likely that during fasting a change in the concentration of some substrate or hormone could lead to these effects on insulin binding. However, in vitro attempts to recreate the substrate and hormonal changes which occur in fasting produced no evidence to support this idea. (a) fasting leads to an increase in the ability of adipocytes to bind insulin because of an increase in binding affinity; (b) this increase in the affinity of the receptor for insulin was primarily accounted for by a decrease in the rate at which insulin dissociates from its receptors; and (c) fasting did not appreciably alter the negatively cooperative interactions displayed by adipocyte insulin receptors.


The actions of secretagogues on oxygen uptake by isolated mammalian parietal cells. The action of histamine, carbamylcholine, and gastrin on oxygen uptake by cells isolated from canine fundic mucosa was studied in vitro. Viable mucosal cells were prepared by exposure of separated mucosa sequentially to collagenase and EDTA. Oxygen consumption, determined by polarography, was chosen as an index of physiological response of mucosal cells to secretagogues. Isobutyl methyl xanthine (IMX), carbamylcholine, histamine, and gastrín each independently stimulated oxygen uptake by the unfractionated mucosal cells. The response to histamine was greatly enhanced when IMX was present. In fractions of varying parietal cell content obtained with the Beckman elutriator rotor, basal and stimulated oxygen uptake correlated with the parietal cell content of the fractions. The percentage increases in oxygen uptake in response to histamine, gastrin, carbamylcholine, and IMX were similar in enriched fractions with from 50 to 85% parietal cells and in unenriched starting fractions. The normalized dose-response relations for histamine with an IMX background and for carbamylcholine were also similar in these two fractions.The specificity of these responses was tested by use of an H(2)-histamine receptor antagonist, metiamide, and an anticholinergic agent, atropine. At the doses used, neither metiamide (0.1 mM) nor atropine (10 muM) inhibited basal oxygen uptake. Histamine, studied with an IMX background, was inhibited by metiamide but not by atropine, while carbamylcholine was inhibited by atropine but not by metiamide. Neither metiamide nor atropine inhibited gastrin-stimulated oxygen uptake. These data indicate that in this in vitro system parietal cells account for most of the increase in oxygen uptake produced by exposure to gastric secretagogues and that histamine, gastrin, and carbamylcholine each independently stimulate oxygen uptake by the parietal cell. The specificity displayed by atropine and metiamide in this in vitro system suggests that the parietal cell has specific receptors for each of these secretagogues.


The interaction of histamine with gastrin and carbamylcholine on oxygen uptake by isolated mammalian parietal cells. Using oxygen uptake as an index of the physiological response of isolated parietal cells, the interactions between histamine and gastrin and between histamine and carbamylcholine and the effects of atropine and metiamide on these interactions have been studied. Parietal cells were isolated from canine fundic mucosa by sequential exposure of separated mucosa to collagenase and EDTA. In previous studies carbamylcholine, isobutyl methyl xanthine, gastrin, and histamine have each been shown to increase oxygen uptake by these cells. Isobutyl methyl xanthine greatly enhanced the histamine effect. Carbamylcholine was inhibited by atropine but not by metiamide, histamine was inhibited by metiamide but not by atropine, and gastrin was inhibited by neither, suggesting that each of these agents has a direct action on the parietal cell. In the present studies, potentiating interactions between histamine and carbamylcholine and between histamine and gastrin have been demonstrated. Against a histamine (0.1 and 1 muM) plus isobutyl methyl xanthine (0.1 mM) background, the dose for 50% response for gastrin was approximately 1 nM, and the maximal response was obtained at 0.1 muM. When added to these combinations of stimulants, metiamide and atropine retained their respective specificities against stimulation by histamine and carbamylcholine, in that responses were inhibited to the level that was seen when the component of the pair that was not inhibited was given alone. The observation that histamine plus gastrin and histamine plus carbamylcholine produced maximal responses that were greater than the maximal response to histamine alone further supports the hypothesis that these agents each have direct actions on parietal cells. These observations are not consistent with the hypothesis that histamine is the sole mediator for the effects of other secretagogues. Furthermore, the inhibitory effects of atropine and metiamide on the specific cholinergic and histaminic components of the interactions that occur between secretagogues provide a possible explanation for the apparent lack of specificity of these agents on in vivo acid secretion.


Changes in human serum amyloid A and C-reactive protein after etiocholanolone-induced inflammation. Secondary amyloidosis is a complication of diseases characterized by recurrent acute inflammation. In this study, a standardized stimulus which induced fever and inflammation was given to six normal subjects (19-24 yr old) to follow the fluctuation in concentration of serum amyloid A (SAA), the precursor of the secondary amyloid fibril protein. After a single intramuscular injection of etiocholanolone (0.3 mg/kg), blood samples were drawn twice a day for 12 days for determination of SAA by solid phase radioimmunoassay. From a base line of <100 mug/ml, the SAA concentration began rising within 12 h to a maximum value at about 48 h of 1,350-1,800 mug/ml in three males and 380-900 mug/ml in three females and returned to base line by 4-5 days. The SAA response showed a similar time response to C-reactive protein (CRP), a well-documented acute phase protein which was assayed semiquantitatively by capillary tube precipitin reaction. CRP, but not SAA, showed a quantitative correlation with the amount of fever induced by etiocholanolone. One subject exhibited a second rise in SAA and CRP concentrations after acute over-indulgence with alcohol, suggesting that acute liver damage may have caused an acute phase reaction. Thus, a controlled episode of fever and inflammation produced a prompt and prolonged elevation of SAA and CRP concentrations. Unlike SAA, CRP has not been implicated in the pathogenesis of amyloidosis, although its relationship to the P component of amyloid has recently been established.


Prevention of chronic experimental pyelonephritis by suppression of acute suppuration. In order to evaluate the importance of suppuration, persistent infection, and scar formation in the evolution of Escherichia coli chronic pyelonephritis, we treated rats with different antibiotic regimens at different stages of the disease. The results show that (a) if acute suppurative pyelonephritis is aborted with early antibiotic therapy, chronic pyelonephritis is prevented; (b) chronic pyelonephritis can develop even after eradication of infection if acute suppuration persists beyond 3 days; (c) persistent infection does not lead to chronic pyelonephritis, if the acute suppuration is suppressed; and (d) residual infection, antigen-load, antibody, and(or) cell-dependent autoimmune processes did not play a significant role. We interpret these results as evidence that the pathologic entity recognized as chronic pyelonephritis results from kidney damage, scarring and shrinkage secondary to acute suppuration.


Multicompartmental analysis of cholesterol metabolism in man. Characterization of the hepatic bile acid and biliary cholesterol precursor sites. The present report has presented the first clear evidence in man for the existence of specific hepatic cholesterol precursor sites associated with the formation and secretion of bile acids and biliary cholesterol. These hepatic compartments derive virtually all their cholesterol from newly synthesized and lipoprotein free cholesterol. The model which is presented was formulated on current concepts of cholesterol metabolism in man and is concerned, at this initial stage, with the elucidation of the bile acid and biliary cholesterol compartments. The complexity of cholesterol metabolism in man necessitated an initial approach that would minimize the number of inputs of cholesterol into the system, allow for the sampling of several cholesterol compartments, and permit the simultaneous labeling of newly synthesized cholesterol and preformed cholesterol. To achieve these objectives, we studied the patient with a total bile fistula. Six patients were administered simultaneously pulse injections of labeled mevalonic acid and [(14)C]cholesterol. The qualitative features of the specific activity time course curves after labeled mevalonic acid revealed no precursor-product relationship between bile acid, biliary cholesterol, and plasma free cholesterol. The peak specific activity of the bile acids was reached in approximately 100 min and was higher than the biliary cholesterol, which was higher than the plasma free cholesterol. The plasma free cholesterol specific activity became higher than the other lipids after 12 h and remained higher throughout the period of study. Similar related observations were made with [(14)C]cholesterol. The data were then subjected to simulation analysis and modeling using the SAAM-27 computer program. Computer least-square fits of the data were obtained after the model was evolved. During the model development, the least number of compartments and transport pathways were introduced consistent with a good fit of the data. Of particular importance was the constraint that the model fit the data obtained from both [(14)C]cholesterol and labeled mevalonic acid. The same parameter values were used to fit the data from both tracers. The fluxes arrived at in the model indicate that 31% and 20%, respectively, of the cholesterol input into the bile acid and biliary cholesterol precursor sites were derived directly from the newly synthesized hepatic cholesterol. The remainder had its origin predominantly from lipoprotein free cholesterol. Plasma esterified cholesterol (as free) made a small contribution (11%) to the bile acid compartment. Similarly, 10% of the biliary cholesterol arose from an unknown hepatic site. The present report has provided the basis for a new procedure for studying in vivo cholesterol metabolism in man. Examination of the derived cholesterol flux rates between the compartments suggests the presence of an important mechanism regulating the partitioning of lipoprotein free cholesterol between the bile acid and biliary cholesterol precursor sites. Aberrations in the proportioning of precursor cholesterol between these sites could be a causative factor precipitating the excessive secretion of biliary cholesterol and the production of lithogenic bile.


Influence of heat and humidity on the airway obstruction induced by exercise in asthma. We examined the degree of airway obstruction that developed in eight asthmatics who exercised while breathing air under four conditions: (a) ambient room temperature and water content; (b) body temperature and ambient water content; (c) ambient room temperature fully saturated; and (d) body temperature fully saturated. These test conditions were performed in random order. Multiple aspects of pulmonary mechanics were measured before and 5 min after exercise. When air at ambient conditions was inhaled, the expected airway obstruction developed after exercise, and all variables changes significantly from their pre-challenge values. Heating the air to body temperature did not influence this response. Increasing the humidity at ambient temperatures significantly blunted the response, and by inhaling body temperature, fully saturated air completely prevented it from occurring. Thus, the water content of inspired air is an important variable in the development of exercise induced asthma.


Characterization of the immunochemical forms of calcitonin released by a medullary thyroid carcinoma in tissue culture. Immunoreactive calcitonin released by a medullary thyroid carcinoma in tissue culture has been found to exhibit heterogeneity when analyzed by gel chromatography and radioimmunoassay, in a pattern analogous to that seen in the circulation of the patient from whom the neoplasm was removed. To examine the cause of the heterogeneity, the immunoreactive material released by the tumor into tissue culture medium was further analyzed by gel electrophoresis in the presence of the protein denaturant 8 M urea, by gel chromatography after reduction and alkylation, by affinity chromatography on concanavalin A-agarose, and by bioassay in a renal adenylyl cyclase system of enhanced sensitivity. The results suggest that the larger immunochemical forms of calcitonin described in the circulation of patients with medullary thyroid carcinoma may be released directly from the neoplasm and need not derive from peripheral metabolism of the monomer. It could be demonstrated that a major proportion of the immunochemical enlargement is dependent upon intermolecular disulfide bridge formation whereas aggregation or non-convalent protein binding account for a smaller component of the heterogeneity. In view of the absence of binding of the immunoreactive material to the lectin agarose, carbohydrate side chains, at least of the alpha-d glucosyl variety, do not seem to contribute significantly to calcitonin enlargement. Additionally, the studies indicate that, at least by in vitro assay, the larger immunochemical forms of calcitonin, representing the majority of the immunoreactivity released by a medullary thyroid carcinoma, are biologically inactive.


Influence of basal insulin and glucagon secretion on potassium and sodium metabolism. Studies with somatostatin in normal dogs and in normal and diabetic human beings. To examine the role of basal insulin and glucagon secretion in potassium and sodium homeostasis, somatostatin, a potent inhibitor of insulin and glucagon secretion, was infused for 5 h into healthy human subjects, maturity-onset diabetes, juvenile-onset diabetics, and normal dogs. Infusion of somatostatin resulted in an increase in serum potassium (0.5-0.6 meq/liter) in normal subjects and maturity-onset diabetics, but not in juvenile-onset diabetics despite equivalent reductions in plasma glucagon in all three groups. A similar rise in serum potassium was observed in normal conscious dogs given somatostatin and was reversed by insulin replacement. Urinary excretion of potassium was unaffected by somatostatin. In dogs given intravenous potassium chloride in doses (0.375 meq/kg per h) which do not alter basal insulin levels, the rise in serum potassium (0.6 meq/liter in controls) increased 100% when somatostatin was administered together with the KCl infusion. Addition of replacement doses of insulin to the somatostatin infusion resulted in increments in serum potassium which were comparable to infusion of KCl alone. Urinary potassium excretion rose after KCl administration and was unchanged by the addition of somatostatin. Serum sodium concentration was unaffected by somatostatin administration in both the human and dog studies. However, urinary sodium excretion displayed a biphasic response falling by 20-60% within the first 2 h of somatostatin administration and then rising to values 50-80% above basal levels at 3-4 h. Inulin and p-aminohippurate clearances were unaffected by somatostatin. It is concluded that (a) potassium homeostasis is influenced by basal insulin levels in the absence of which serum potassium concentration rises and potassium tolerance declines; (b) this effect of insulin is mediated via extrarenal mechanisms of potassium disposal; (c) somatostatin has a biphasic effect on urinary sodium secretion, the mechanism of which remains to be established.


Erythroid precursors in congenital hypoplastic (Diamond-Blackfan) anemia. To explore the etiology of congenital hypoplastic anemia (CHA) or the Diamond-Blackfan anemia, erythropoietin responsive committed erythroid precursors were enumerated by the plasma clot method. These included blood and marrow erythroid burst-forming units (BFU-E) and marrow erythroid colony-forming units (CFU-E). The peripheral blood nucleated cells of 11 patients and the marrow cells of seven of these patients were examined. Studies were repeated in several patients during relapse and after induction of remission. BFU-E were undetectable in the marrow and blood of all but one relapsed patient, and the numbers of marrow CFU-E were depressed in all relapsed patients. Blood BFU-E remained low in all of the patients in remission. No evidence was obtained for suppression of normal CFU-E or BFU-E by CHA lymphocytes. Erythropoietin dose-response curves performed in two patients revealed a 10-fold increase in erythropoietin requirement for marrow CFU-E colony growth. This marked unresponsiveness to erythropoietin was strikingly improved by steroid therapy in one patient. We suggest that CHA is the result of a qualitative and/or quantitative deficiency of BFU-E. If BFU-E are produced, they must be relatively unresponsive to erythropoietin. The abnormal BFU-E give rise to erythropoietin unresponsive CFU-E and, thence, to proerythroblasts that are, in turn, trapped in that early stage of development because of their poor erythropoietic response. Hence, red cell production is deficient. Steroids appear to improve the erythropoietin response of CHA erythroid precursors.


Studies in porphyria. VII. Induction of uroporphyrinogen-I synthase and expression of the gene defect of acute intermittent porphyria in mitogen-stimulated human lymphocytes. A 50% reduction in the activity of uroporphyrinogen-I (URO) synthase in liver, erythrocytes, and cultured skin fibroblasts characterizes all patients with clinically active acute intermittent porphyria (AIP). The same enzyme defect has also been demonstrated in the erythrocytes and skin fibroblasts of completely latent gene carriers of this disorder and presumably exists in the liver as well. In this study, we examined whether or not the formation of URO-synthase is impaired in AIP cells using lymphocytes treated with mitogens or infected with Epstein-Barr virus. Both mitogens (phytohemagglutinin and pokeweed mitogen) and Epstein-Barr virus induced the synthesis of URO-synthase in lymphocytes, but the induction of URO-synthase in AIP lymphocytes was only 50% as compared with that in normal lymphocytes. The impaired induction of URO-synthase in AIP lymphocytes reflects a specific gene defect because AIP lymphocytes showed normal [(3)H] thymidine uptake into DNA, [(3)H] uridine uptake into RNA, and normal delta-aminolevulinic acid (ALA) synthase, ALA-dehydratase, catalase activities, and heme content. Utilizing the same methodology, the ferrochelatase deficiency of hereditary erythropoietic protoporphyria could also be identified. The K(m) of the induced URO-synthase in AIP cells was identical to that of the enzyme in normal cells. The induced URO-synthase of mitogen-treated AIP lymphocytes was not accompanied by a concurrent enhanced level of ALA-synthase. Moreover, the URO-synthase deficiency in lymphocytes from actively ill AIP patients was not different from the level of enzyme activity when they were in clinical remission, or when compared with the enzyme activity of cells from completely latent AIP gene carriers. The results of this study indicate that the URO-synthase deficiency in AIP may be the result of a gene mutation regulating the rate of synthesis of a normal enzyme rather than a mutation causing a structural abnormality of this enzyme protein.