The amino acid sequence of the tryptic peptides isolated from dogfish M4 lactate dehydrogenase.

The peptides which result from treatment of the S-[14C]carboxymethyl derivative of dogfish M4 lactate dehydrogenase (EC 1.1.1.27) with trypsin have been isolated and their sequences have been elucidated. Each identical subunit has a molecular weight of 36,000 and on the basis of the amino acid composition 40 unique tryptic peptides are anticipated. Thirty-seven of these peptides have been isolated and have been completely characterized. The amino acid sequences of these peptides are presented.


Action of bacteriophage T4 ultraviolet endonuclease on duplex DNA containing one ultraviolet-irradiated strand.

Previous studies have indicated that the ultraviolet endonuclease of bacteriophage T4 acts specifically at pyrimidine dimer sites in ultraviolet-irradiated DNA. At such sites the enzyme could conceivably catalyze endonucleolytic incision of the DNA either on the dimer-containing strand or on the strand directly opposite to the dimer. In the present work, a direct test of these alternatives was made. Substrate molecules containing one irradiated and one unirradiated strand were prepared from differentially isotopically labeled purified complementary strands of bacteriophage lambdaDNA. Following incubation with the enzyme, the sedimentation profiles of the DNA strands in alkaline sucrose density gradients were compared. The results show that the enzyme selectively nicks the irradiated strand.


Inhibition by 6-O-tosyl galactosides of beta-galactoside phosphorylation and transport by the lactose phosphotransferase system of Staphylococcus aureus.

The effect of various galactose derivatives, substituted at C-6, on the phosphoenolpyruvate:beta-galactoside phosphotransferase system of Staphylococcus aureus was studied. Cells were grown by an improved procedure, which resulted in a 5- to 10-fold increase in cell yield. The four protein components of the system were separated. A membrane fraction containing negligible levels of the soluble components was prepared by alternate cycles of sonic treatment and differential centrifugation. The in vitro system reconstituted from these fractions was used to test the ability of the galactose derivatives to inhibit the phosphorylation of lactose analogs, under conditions where the membrane-bound component, Enzyme IIlac, was rate limiting. Derivaites in which the hydroxyl group of C-6 was missing, or replaced by a fluoro, O-methyl, or carboxyl group had no affinity for Enzyme IIlac, as judged by their inability to inhibit phosphorylation. Surprisingly, derivatives containing arylsulfonyl groups at C-6 were potent inhibitors; the O-tosyl compound has an apparent affinity five times that of galactose. The arylsulfonyl substitution in an absolute requirement; neither O-benzyl or O-methanesulfonyl derivatives were inhibitory. The specificity of the inhibition by tosyl derivatives parallels that of unsubstituted substrates; tosyl galactosides of the beta configuration were inhibitory, but those of the alpha configuration were not. The tosyl derivatives also strongly inhibited the uptake of lactose analogs into whole cells; the requirement for the arylsulfonyl moiety was again observed. The chemical analogy between the tosyl galactosides and possible intermediates in the transport-phosphorylation step catalyzed by Enzyme IIlac provides a possible explanation for the unexpected properties of these derivatives.


Presence of displacement loops in the covalently closed circular chloroplast deoxyribonucleic acid from higher plants.

Chloroplast DNAs (ctDNA) from pea and corn plants were examined in the electron microscope for the presence of replicative intermediates. Pea and corn ctDNAs were each found to contain two displacement loops (D-loops). The D-loops were 820 (+/- 90) base pairs long in pea ctDNA and 860 (+/- 125) base pairs long in corn ctDNA. In each ctDNA, the two D-loops were located at positions that were 7100 +/- 240) base pairs apart. The displacing strands of the two D-loops were located on opposite strands of the parental DNA molecule and they were seen to expand toward each other. The D-loops in the ctDNA from pea and corn exhibited branch migration and thus were easily distinguished from the denatured regions that were also present in these closed circular ctDNAs. In addition, the positions of the D-loops were found to be distinct from the positions of the denaturation loops (Den-loops). The Den-loops were also shown to be located at AT-rich regions in these ctDNA molecules. D-loops and Den-loops were also found in the circular and catenated ctDNA oligomers from pea and corn plants. Mapping the positions of the D-loops relative to the positions of the Den-loops showed that the structure of the D-loop-containing region in the pea and corn ctDNAs has been conserved to a greater extent than the structure of the rest of the two ctDNA molecules.


Respiratory properties of rat liver mitochondria immobilized on an alkylsilylated glass surface.

Rat liver mitochondria are shown to adhere to the alkylsilylated glass beads in essentially a monolayer. The amount of mitochondria bound to the beads reaches a maximum where the length of the alkyl groups covalently linked to the beads exceeds eight carbons. Mitochondria immobilized on the beads and placed in a flow system exhibit normal: (a) respiratory control, (b) phosphate to oxygen ratio, (c) uncoupling by 2,4-dinitrophenol and carbonylcyanide p-trifluoromethoxyphenylhydrazone, and (d) inhibition by cyanide, azide, rotenone, oligomycin, and antimycin. Reversibility of the effects of 2,4-dinitrophenol, cyanide, and azide was rapid and complete. Inhibition by rotenone, oligomycin was essentially irreversible. Mitochondria have been maintained in a viable state on the beads at 27 degrees for periods up to 4 hours. The use of immobilized organelles appears to offer a new technique for the study of membrane-bound particles whereby substances can be rapidly added and removed while monitoring the composition of solution flowing over the particles.


Biological evaluation of polymers. I. Poly(methyl methacylate).

A series of poly(methyl methacrylate) formulations differing widely in chemical and physical properties was employed for the evaluation of primary screening methods for the assessment of acute toxicity. Materials and USP extracts of materials were tested in parallel. Tissue culture, hemolysis, intradermal irritation, systemic toxicity, muscle implant and histopathologic responses were determined for each of 27 formulations. A determination of the nonvolatile methanol extractable components was carried out on each formulation. The formulations varied with respect to percent, w/w, methyl methacrylate, N,N-dimethyl-p-toluidine, stannous octoate, 3, 4-diamino-toluene and, also, with respect to curing conditions. Volatile components, primarily methyl methacrylate, of three selected formulations were determined quantitatively by vacuum distillation and mass spectrographic analysis. Statistical analysis of the primary data indicated a significant correlation of residue weight (methanol extractable) with hemolytic activity (r = 0.93) and with the cumulative biological response (r = 0.9). Multiple linear regression analysis of residue weights with hemolysis and intradermal irriation responses gave the highest overall correlation (r = 0.96). Hemolytic activity and tissue culture responses were significantly correlated (r = 0.87). It was concluded that the observed variation of biological test results reflected significant differences in the toxicity of the test materials. The poly(methyl methacrylate) series examined was relatively low in toxicity and the biological tests examined, particularly the in vitro tests, were found to be responsive to formulation and curing conditions which indicated their suitability for primary toxicity screening.


Corrosion of surface defects in fine wires.

Defects were observed on the surfaces of various fine diameter wires commonly used in biomedical applications. These surface irregularities were viewed at high magnifications using a scanning electron microscope which has a much greater depth of field than normal light microscopy. Defects include scratches, pits, and crevices, which are the result of commercial wire drawing practices. Corrosion test results show that imperfections can serve as sites for localized corrosion attack which could lead to premature failures.


The influences of electrical potential and surface finish on the fatigue life of surgical implant materials.

The influence of both imposed anodic potential in Ringer's solution and surface finish on the fatigue lives of annealed 316 type stainless steel and annealed pure titanium were measured and statistically compared to fatigue data run in air. The applied potentials in simulated extracellular fluid approximated conditions existing within the body while also producing the types of surface defects actually found on removed long time implants within the time interval of the accelerated R. R. Moor fatigue tests. Differentiating tests were run at single levels of applied cyclic stress well above the endurance limits. In Ringer's solution, the fatigue life of the 316 stainless steel decreased with increasing applied potential, and at +500 mV was significantly shorter than when run in air. At each condition, the 316 stainless steel was independent of initial surface finish. In contrast, the fatigue life of titanium improved rapidly with increasingly fine surface finishes. Furthermore, compared to air, the application of +500 mV in Ringer's solution improved the life of the rough surface finished material and markedly increased to number of cycles to failure for the electopolished specimens.


Ultrathin coating by plasma polymerization applied to corneal contact lens.

Plasma coating (deposition of polymer under the influence of plasma) is utilized to modify the surface properties of corneal contact lens. An ultrathin layer (thickness of roughly 200 A) of plasma polymer of acetylene/H20/N2 is applied to poly(methyl methacrylate) (PMMA) corneal contact lenses. The surface becomes highly wettable with water compared to uncoated lens. When coated and uncoated contact lenses are placed on rabbit eyes, a remarkable difference in accumulation of mucous matter is observed. With the control lenses, the accumulation of mucous matter in a week is sufficient to affect the optical clarity of the lenses, whereas the coated lenses show no change after three months continuous wearing. The comparative degree of adhesion of the corneal epithelium cells onto glass, modified glass, PMMA, and coated PMMA surfaces is studied using tissue cultures and phase contrast microscopy. The coated PMMA surface exhibits a degree of tissue adhesion lower than that of control PMMA and higher than that of glass surface, and no sign of toxicity of the coated surface is observed by the tissue cultures.


Reaction associated with a silicone rubber gel: an experimental study.

A blend of Silastic 382 (Room Temperature Vulcanizing, RTV) Medical Grade silicone oil and a catalyst was prepared in vitro, in both the catalyzed and noncatalyzed state, and injected subcutaneously in mice, rats, and rabbits. When properly blended, this catalyzed material, referred to as "silicone gel," formed a soft rubbery mass that remained at the site of injection. Properly catalyzed silicone rubber gel produces no macroscopic inflammatory reaction, attracts few polymorphonuclear leucocytes, and after 5-6 days a thin fibrous capsule begins to form aroung the gel. No degeneration of the silicone gel was observed during the 62 days of this experiment. Additional rats with this silicone gel have been under observation for 8 months and clinically have shown no changes in the local mass of silicone. If the catalyst is partly oxidized when added to the silicone fluid, the degree of gelling is much less. A local mass usually forms at the site of injection with some of the fluid diffusing into the tissue, forming minute cysts. The inflammatory reaciton is characterized by polymorphonuclear leucocytes, associated with many macrophages and giant cells phagocytizing oil droplets and particles of the diatomaceous earth. The pathogenesis of the inflammatory reaction is discussed, referring to the ionic change and the emigration of polymorphonuclear leucocytes to particles of plastics embedded in tissue.


Viscoelastic properties of human dentin.

Stress relaxation measurements were performed on thick-walled cylinders of radicular human dentin. The experimentally measured relaxation moduli were used to obtain an approximation to the logarithmic distribution function of relaxation times. This distribution function was used to predict the behavior of other viscoelastic properties. In particular, the prediction of the strain and strain rate dependence of the modulus was determined and compared with experimental results.


Long-term experience with poly(glycol monomethacrylate) gel in plastic operations of the nose.

Between 1964 and 1970 the authors implanted Hydron gel in 62 patients with nose malformations. The cosmetic defects most frequently operated on were the saddle, and scoliosis of the nasal dorsum. By the end of 1973, 39 of the total number of cases were checked again. In two cases, the gel had to be removed shortly after the operation because of local inflammatory changes. No signs of hypersensitivity could be detected. Calcifications were detected by x-ray examination in 54.2% of the cases. Thirty-four of the patients subjected to the checkup were satisfied with the results of their operations. The authors considered the results to be very good in 16 cases, and unsatisfactory in 6 cases. Calcifications detected by x-ray examination did not affect the final cosmetic effect. None of the examined patients had any complaints in connection with long-term application of the gel. Hydron gel seems to be a suitable implant material in plastic operations of the nose.


Pharmacological properties of the plasticizer, acetyl N-tributyl citrate, and its extraction from poly(vinyl chloride) tubing.

Aqueous extracts of a PVC transfusion tubing contracted isolated guinea-pig ilea. A pharmacological and chemical study (infrared spectroscopy, gas-liquid chromatography) showed that this was due to extraction of the plasticizer, acetyl N-tributyl citrate (ATBC). The concentrations of ATBC to produce contractions of the isolated guinea-pig ileum (from 1 mug/ml) can be compared with those achieved after recirculating tubing under conditions simulating use (up to 6 mug/ml). In assessing the toxicity of plastic tubing, there is a need to determine extraction of consituents under conditions simulating use and the effects of such extracts on a variety of tissues.


An ultrastructural study of the biocompatibility of poly(2-hydroxyethyl methacrylate) in bone.

Because of the unique properties for swelling in a fluid medium and variable modulus of elasticity, hydrophilic methacrylates may be of potential usefulness for implanting prosthetic devices in bone. To investigate the biocompatibility, electron micrographic studies were done on specimens of poly(2-hydroxyethyl methacrylate) imbedded in bone for varying periods of time. Ultrastructural examination revealed progressive ossification of surrounding connective tissue up to the tissue polymer junction over a 6 month period. There was no evidence of inflammatory response. Based upon this study, it would appear that bone adjusts to an implant of hydrophilic methacrylate by ossification to the implant junction. This supports continued studies into the usefulness of this material as an implant.


The anatomy of orthopaedic malpractice. A study of two hundred and fifty cases.

Nine per cent of all of the Aetna Life and Casualty Company's closed malpractice cases during an eleven and a half-year period were orthopaedic. Two hundred and eleven orthopaedists (164 Board-certified), twelve general surgeons (eight Board-certified), and nineteen general practitioner-general surgeons were involved. Allegations of improper treatment (seventy cases), poor results (severty-four cases), postoperative infection including seven cases of gas gangrene (thirty-one cases), diagnostic error (twenty-six cases), back-surgery complications (twenty-six cases), miscellaneous complications (sixteen cases), and cardiac arrest (seven cases) were the grounds for these malpractice cases. When there was evidence of careful clinical appraisal, good clinical judgment, impeccable surgical technique, good doctor-patient rapport, and well documented records, the defense was successful.


Non-operative management of central fracture-dislocations of the hip.

Thirty-eight patients with central fracture-dislocations, seen over a seven-year period and all treated non-operatively, were evaluated. A follow-up study was done on twenty-four patients; the remainder had either died or could not be located. Several techniques were used to maintain the reduction obtained by closed manipulation done under anesthesia. The superiority of the method of traction using pins in the distal and proximal lateral parts of the femur and the importance of prolonged traction were demonstrated.


Arthroplasty of both hips and both knees in rheumatoid arthritis.

Eleven patients with rheumatoid arthritis underwent total joint replacement of both hips and both knees (four-joint arthroplasty). All patients had increased function and decreased pain. Severe rheumatoid arthritis in both hips and knees does not preclude a satisfactory result after replacement arthroplasty.


The Syme amputation in children.

Twenty-three children had proportionate growth of bone and soft tissue between two and eleven years after a Syme ankle disarticulation. All walked with permanent prostheses within three months after operation. Ischemic necrosis occurred twice after inadvertent ligation of the posterior tibial artery. Suture of the extensor tendons into the heel pad eliminated its posterior migration. Prosthetic realignment compensated for progressive genu valgum in ten of fourteen amputees with fibular hemimelia. On the basis of these gratifying results, the Syme ankle disarticulation is suggested as early treatment for certain congenital anomalies of the lower extremity.


Percutaneous pin fixation of open tibial fractures. Double-frame anchorage using the Vidal-Adrey method.

From 1970 to 1973, twenty-eight severe open tibial fractures in twenty-seven patients were treated with stable external transfixation by the Vidal-Adrey double-frame method. Excluding one patient who had an amputation because of vascular damage at the time of injury, wound healing was satisfactory in all patients with no primary soft-tissue or deep infection. There was one late infection following secondary intramedullary nailing three months after injury. Secondary skin and bone grafts were part of the original plan of treatment and were easily performed. All patients were able to start range-of-motion exercises in the early postoperative period. In the case of a severely comminuted tibial fracture combined with arterial injury, this method made it possible to observe the limb for a few days before amputation for vascular insufficiency. The average time in the frame was 4.9 months, and then a patellar-tendon bearing plaster case or splint was used. The mean time until full weight-bearing without external support was 7.9 months. In the twenty-two patients who could be assessed, the final result was excellent or good in seventeen and acceptable in five.


Rheumatoid wrist deformities and their relation to ulnar drift.

With a new technique of roentgenography to reveal more accurately the degree of ulnar drift and radial deviation in hands of patients with rheumatoid arthritis, it was shown that the radial deviation was less marked than would be ascertained with conventional roentgenograms. Therefore radial deviation probably does not constitute a cause for ulnar drift. The study led to clinical implications as regards the position of splinting (with the wrist in ulnar deviation) and as regards operations to correct deformities of the wrist due to rheumatoid arthritis.


High-pressure injection injuries of the hand.

In high-pressure injection injuries to the hand the most commonly injected materials are automotive grease, diesel oil, and paint. We treated twenty-six patients with high-pressure injection injuries of the hand and found that the injection of paint resulted in a poorer prognosis than did the injection of grease. Amputation is often necessary with paint injections into a digit, while injections into the palm have a somewhat better prognosis. The disability time of patients with paint-injection injuries appeared to be directly related to the time elapsed until amputation of the involved digit. Although we were unable to specifically relate the ultimate result to the time elapsed between injury and proper treatment, we continue to recommend early aggressive wide débridement of these injuries.


The effect of sensory denervation on rabbits' knee joints. A light and electron microscopic study.

Unilateral sensory denervation of the hind limb in a group of rabbits caused progressive atrophy of cells in all structures of the knee joint, whether or not the joint was protected by a plaster cast. Immobilization without denervation caused proliferative changes, first in the synovium and then in the articular cartilage. The initial changes in the articular cartilage following sensory denervation occurred in the middle layers, suggesting that nutritional deficiency was involved. In contrast, the first changes in the cartilage of intact immobilized limbs occurred in the superficial layers and were most likely of mechanical origin.


Contribution of collagen and mineral to the elastic-plastic properties of bone.

Tension testing of wet bovine haversian cortical bone demonstrated marked plastic behavior. Progressive surface decalcification of this bone with dilute hydrochloric acid resulted in progressive decreases in the tension yield point and the ultimate stress with no change in the yield strain or ultimate strain unless decalcification was complete. The slope of the plastic region remained identical throughout decalcification. These findings are consistent with an elastic-perfectly plastic model for the mineral phase of bone tissue in which the mineral contributes the major portion of the tension yield strength. The slope or stiffness of the plastic region of the stress-strain curve is a function only of the properties of collagen, which itself plays a minor role in the tension yield strength of bone.


Excessive thoracic lordosis and loss of pulmonary function in patients with idiopathic scoliosis.

Idiopathic thoracic lordoscoliosis is more common and more productive of respiratory compromise than is kyphoscoliosis. In some patients with idiopathic scoliosis, thoracic lordosis is the predominant component of the disease. Five such patients, all of whom had idiopathic scoliosis with excessive thoracic lordosis, progressive deformity despite Milwaukee brace treatment, and significant compromise of pulmonary function, are presented. Harrington instrumentation (distraction rod only) and spine fusion improved the deformity and respiratory function. The recommended treatment for this type of idiopathic scoliosis is early recognition and prompt surgical correction. The Milwaukee brace should be avoided. Postoperative management must include early ambulation combined with vigorous breathing exercises.


Giant-cell tumor of the radius treated by massive resection and tibial bone graft.

Six cases of aggressive or malignant giant-cell tumor of the distal end of the radius were treated by massive resection and reconstruction using a corticocancellous autograft taken from the tibia. There was no local recurrence or metastasis noted on follow-up (average, eight years and three months). An excellent functional result was achieved in each case.


Gluteal fibrosis.

Five cases of gluteal fibrosis are presented. The anatomical extent of the gluteal involvement explains the clinical picture. Reports of gluteal fibrosis and fibrosis elsewhere suggest that the lesion was the same in all five patients. Intramuscular injections were a possible etiological factor. All the reported sites were those usually used for administration of intramuscular injections. Because the use of intramuscular injection is so common in Spain another factor is necessary to explain the involvement in only a small number of children.


Complications of silicone implant surgery in the metacarpophalangeal joint.

Of 162 metacarpophalangeal silicone prostheses inserted from 1967 to 1973, fifteen fractured, two were associated with an infection, and there was one case of skin necrosis. A recurrence of ulnar drift occurred in four hands. Three prostheses had to be removed and in one such case additional significant foreign-body reaction was evident. Although the complication rate was over 10 per cent, the failure rate was fairly low.


Immediate occlusions dependent on the type of reconstruction.

In 507 reconstructive procedures on patients with chronic occlusions, we saw 4.1% immediate occlusions. We performed 409 thrombendarteriectomies, 40 dacron grafts, and 32 venous bypasses. As cause of the immediate occlusion, we found in 23 cases a dissection of the intima in the distal segment of the restored artery, in one case a bleeding from a perforation was discovered. Intimal dissection was found mostly after orthograde thrombendarteriectomy of the femoral and popliteal artery. The results of orthograde TEA could be improved by intraoperative angiography or direct opening of the distal popliteal artery. Intimal dissection after bypass operations are generally the result of technical errors.


Intraoperative angiography.

The thromboendarterectomy can be improved with intraoperative angiography, sources of error may be considerably reduced. The control possibilities of intraoperative angiography offer advantages also for the various bypass operations as surgical errors can be found and corrected during the operation.


Anatomical concepts to be observed when performing a vascular junction.

An anatomical study shows that the vascular junctions are always very well rounded in accordance with the direction of the blood flow. When performing an anastomosis, the further one departs from this configuration, which we can define as perfect, the more flow disturbances are likely to arise. This may jeopardize the satisfactory outcome of surgery.


Polycythaemia and thrombocythaemia and their influence on reconstructive arterial surgery.

Relative polycythaemia and thrombocythaemia have a deleterious effect after artery grafting. Bleeding is more common than thrombosis, though both occur.


Competition between collateral vessels in femoro-popliteal by-pass grafting.

In patients with severe ischaemia and maximum collateral development there is no significant competition between the patent superficial femoral artery and common femoral to popliteal by-pass vein graft provided that there is a block in the main line arterial channel between the superficial femoral artery and the popliteal anastomosis of the vein graft.


Causes of rethrombosis following arterial reconstructions below the renal arteries.

Causes of rethrombosis after primary reconstructive surgery for arterial occlusive disease below the renal arteries are discussed, based on experience with 1,700 reconstructions since 1958, and data from literature. Important for reoperation is the fact that mortality of secondary procedures is much higher than for the primary ones (13% versus 0.9%). Longterm anticoagulant therapy is advocated. Results of reoperations are excellent.


Rethrombosis analysed in reference to anticoagulant therapy.

Of 1,103 re-examinations on femoro-popliteal operations, 485 in reference to anticoagulant therapy could be more closely analyzed. A very significant difference was found on relapse occlusions with or without Coumadin in relation to the disease stage. A significant difference could not be proven between the frequency of occlusion and the type of operation with or without Coumadin. After correction of chronic occlusions the indication for an anticoagulant therapy is poor distal outflow and stage III or IV disease.


Influence of the type of anastomosis: end-to-side anastomosis versus end-to-end.

The by-pass technique of arterial reconstruction has some theoretical disadvantages, particularly, unnecessary abandoning of useful main arteries, undesirable flow patterns and the possible descent of debris from the by-passed portion of artery. An oblique end-to-end technique can avoid these undesirable features and also avoid the creation of an over-sized vessel at the site of end-to-side anastomosis. It is suggested that the least procedure compatible with success should be used in arterial reconstruction in the lower limb and an ascending range of options open to the surgeon is given.


Interruption of the aortic arch associated with patent ductus arteriosus and ventricular septal defect. Proposal of a new surgical technique for total correction.

A four-year-old girl underwent complete correction of the association of interruption of the aortic arch (Type A, Celoria and Patton's classification), patent ductus arteriosus, and ventricular septal defect. The surgical procedure consists of establishment of a continuity between the ascending and descending aortas utilizing the patent ductus arteriosus and the anterior wall of the pulmonary arterial trunk, reconstruction of the rest of pulmonary arterial trunk with pericardium, and closure of the ventricular septal defect. Though the patient expired from cerebral complications, the hemodynamic result after repair was quite satisfactory. The procedure described makes total correction of this complex anomaly feasible at one operation through a median sternotomy and seems to be a method of choice for most patients with this association of anomalies, unless there is severe narrowing of the patent ductus arteriosus.


Effects of lumbar sympathectomy on thromboangiitis obliterans.

The clinical response to lumbar sympathectomy has been reported. The incidence of improvement was as follows: Coldness 52 percent, ulcer 58 percent, rest pain 64 percent, and claudication 31 percent, respectively. The blood flow in the anterior tibial muscles did not increase within 5 weeks postoperatively. There were correlations between the effects and the clinical stages, the interval from the onset to the operations, the abuse of tobacco, and the degree of reactive hyperemia, but not relation was observed between as to the site of obstruction. In the experiments, the influence of acetylcholine on blood flow increased after the operations and the duration of vasoconstriction following administration of noradrenalin became shorter. On the basis of the clinical and experimental facts, postoperative improvement seems to be due to increased blood flow to the peripheral skin and to relief of vasospasm.


Thoracic aorta-to-femoral artery by-pass. For the treatment of complete obstruction of the abdominal aorta at the level of the renal arteries.

Complete obstruction of the abdominal aorta at the renal artery level is often a difficult surgical problem in case of long standing thrombosis, short suprarenal aortic space, and extensive periarterial inflammatory reaction. Six patients in whom revascularization of the lower extremities was performed through a prosthetic by-pass from the thoracic aorta to the femoral arteries, were reported here. One patient underwent also a left nephrectomy for renal hypertension, chronic renal artery obstruction, shrunken kidney. Both the short and long term results have been satisfactory in all cases.


Surgical treatment of giant left ventricular aneurysms. The intraventricular resection technique.

The intraventricular resection technique for giant aneurysm of the left ventricle decreases anoxic cardiac arrest time and controls the detachment of intramural thrombus. To perform this technique, it is necessary to expose only a longitudinal segment on the anterior aspect of the aneurysm to permit a ventriculotomy parallel to the anterior descending coronary artery 4-5 cm away. In the same way, to resect a giant aneurysm of the diaphragmatic aspect, only a segment parallel to the posterior descending coronary artery needs to be exposed. Then with the clear intraventricular vision of the limit between the fibrous sac and the contracting left ventricle, the surgeon rapidly detaches the aneurysm. In any case besides the relation of this limit, the transecting line must keep away at least 4 cm from the implantation of the papillary muscle of the mitral valve, in order to leave an adequate functional chamber for the left ventricle. The early visualization of the mitral apparatus during the resection of giant aneurysm is another basic advantage of the intraventricular approach. The ventriculotomy is closed with a running suture and coronary circulation is restored. Anoxic cardiac arrest averaged 15 minutes in the five out of six cases of giant aneurysm treated with this technique. In the period July 1972-December 1973, 28 aneurysms of the left ventricle with varied associated pathology have been treated in this surgical unit, with 14% (4 cases) mortality. By contrast, no death has been registered in this severely ill group of six patients with giant left ventricular aneurysm. In cases I and VI myocardial revascularization was added. Two important aspects contribute to the excellent long term result in this group. 1. Correction of the altered geometry and consequent dysfunction of the left ventricle. 2. Correction of the functional ischemia of the contracting myocardium. The presence of giant aneurysm increases the left ventricle wall tension, including the contracting mass, and consequently the myocardial oxygen consumption.


Separation of enantiomers on packed columns containing optically active diamide phases.

Chromatography of fourteen protein amino acids has been studied on 2-4- m long packed columns containing chiral diamide phases. Twelve of the thirteen optically active compounds examined could be resolved, with R greater than or equal to 1 in many instances. Two of the phases, N-docosanoyl-L-valine tert.-butylamide and N-lauroyl-L-valine 2-methyl-2-hepatadecylamide, can be employed at temperatures as high as 190 degrees and 180 degrees, respectively, without losing their efficiency, even after prolonged use. The problem of peak overlap in the analysis of mixtures of different amino acids was examined and partially solved.


Methionine dipeptide stationary phases for the resolution of enantiomers.

N-Trifluoroacetyl-L-methionyl-L-methionine cyclohexyl ester as well as the disulfide and disulfone derivatives of this phase have been synthesized. The methionine phase exhibits improved properties as a stationary phase for the separation of amino acid enantiomers as compared to other dipeptide stationary phases and can be used at temperatures ranging from 70 to 150 degrees. The sulfoxide phase, however, was incapable of resolving the enantiomeric derivatives. Physical properties of the sulfone compound limit its use as an effective resolving agent.


New considerations in detector-application relationships.

This report relates new ways of using refractometer and ultraviolet (UV) detectors in liquid chromatography (LC) which involve less sample preparation and handling. A new cell design is proposed which extends the sensitivity of UV detectors by correcting for refractive index effects as light passes through a cell. Finally, a technique called "trace enrichment" is proposed for concentrating non-polar organics, in very low concentration, from polar sources (river water) for analysis by LC.


Concurrent determination of alpha-tocopherol and free fatty acids in human plasma by glass open tubular capillary column gas chromatography.

A new gas chromatographic procedure was developed for the concurrent analysis of mixtures of long-chain acids (as methyl esters), cholesterol (as the trimethylsilyl ether) and the tocopherols (as trimethylsilyl ethers). This method was used in a study of free fatty acids and alpha-tocopherol in the plasma of normal subjects and stroke patients. The chromatographic separation was based upon the use of thermostable glass open tubular capillary columns prepared with Silanox (procedure of Lin, Pfaffenberger and Horning) and a new polar liquid phase (Schwartz-Mathews polyphenyl ether sulfone phase). The German-Horning injection system was used in a modified gas chromatograph.


Analysis of long-chain acids or human plasma phosphatidylcholines (lecithins) and cholesteryl esters by glass open tubular capillary column gas chromatography for stroke patients and for normal subjects.

Methods are described for long-chain acid compositional analyses of human plasma phosphatidylcholines and plasma cholesteryl esters. Conventional procedures were employed for the isolation of these lipids and for the preparation of methyl esters of the acids. The gas chromatographic analytical procedure was based upon the use of thermostable glass open tubular capillary columns with a new polar phase. The methods were used in a study of plasma lipids in stroke patients and in normal young adult subjects.


Cholesterol oxidase. Further studies of substrate specificity in relation to the analytical characterisation of steroids.

The substrate specificity of cholesterol oxidase has been further examined with respect to the size and shape of the steroid 17beta-side-chain and oxygenation of the nucleus. The relatively unreactive 17-keto-delta5-3beta-hydroxysteroids were converted to satisfactory substrates by formation of the isopentyl- or benzyloximes. The results obtained have been used to illustrate further uses of the enzyme in the gas-phase analytical characterisation and selective oxidation steroids. Oxidations were studied by kinetic experiments, gas-liquid chromatography-mass spectrometry.


Mass fragmentography of 5-hydroxytryptophol and 5-methoxytryptophol in human cerebrospinal fluid.

A specific and very sensitive method for the determination of 5-hydroxy-tryptophol (5-HTOL) and 5-methoxytryptophol (5-MTOL) in extracts from human cerebrospinal fluid (CSF) involving the use of mass fragmentography and pentafluoropriopionyl derivatives is described. 5-HTOL and 5-MTOL were determined in human CSF of three patients with leukaemia and from nine patients with neurological disorders. The concentration of free 5-HOT in CSF was in the range of 0.1-33 ng/ml and that of 5-MTOL was 0.3-13.9 ng/ml. For the first time the presence of these compounds in human material has been shown. The concentration of these two alcohols in CSF is markedly lower than the concentration of 5-hydroxyindoleacetic acid. These results suggest that human cerebral 5-hydroxytryptamine is preferentially metabolized to 5-HTOL-hydroxyindoleacetic acid rather than to 5-HTOL and 5-MTOL.


A micro method for the determination of volatile metabolites in biological samples. A preliminary report.

A new method for analyzing volatile metabolites in biological fluids and tissues has been developed. The procedure involves extraction of the sample with diethyl ether, concentration on a short column of glass wool by elution of the diethyl ether, then thermal desorption of the trace constituents into a chromatographic system. Small samples of 100 mul are quite adequate. The method is applicable to serum, plasma, urine and other fluids. Small tissue samples can also be used with this technique.


Gas chromatographic-mass spectrometric study of volatile organic metabolites in urines of patients with diabetes mellitus.

Abnormally increased concentrations of the aliphatic alcohols ethanol, n-propanol, isobutanol, n-butanol and isopentanol and the ketones 4-heptanone and cyclohexanone in human urine reflect metabolic disorders related to diabetes mellitus. For the determination of these low-molecular-weight metabolites, the components are trapped on an absorbent, separated by gas chromatography and identified by mass spectrometry. After standardization of the adsorption and desorption techniques, the procedure is applicable for comparative studies and for screening.


Specific detection of volatile metabolites in urines of normal subjects and patients with diabetes mellitus using computerized mass fragmentography.

Volatile urinary components are analyzed by a combination of gas chromatography, mass spectrometry and a data-acquisition system. Mass fragmentograms using the mass spectrometric data on magnetic tape are recorded for the primary aliphatic alcohols ethanol, n-propanol, isobutanol and isopentanol and the ketones 4-hepatonone and cyclohexanone. The mass fragmentograms are used as selective profiles to facilitate recognition of abnormalities in the urinary components in cases of diabetes mellitus.


Automated high-resolution gas chromatographic system for recording and evaluation of metabolic profiles.

A fully automated high-resolution gas chromatographic system is described that incorporates repetitive sampling of volatile biological samples onto a glass capillary column, and a simultaneous flame ionization and nitrogen-sensitive detection prior to data acquisition and computer handling. Reliable headspace sampling procedure and reproducible column characteristics that are essential to automation have been studied. The application of this system to the reproducible chromatographic recording of human urinary volatile constituents is shown.


Identification of microorganisms by gas chromatographic-mass spectrometric analysis of cellular fatty acids.

The cellular fatty acid compositions of strains of Pseudomonas aeruginosa, P. cepacia, and P. maltophilia were determined by gas-liquid chromatography. A variety of acids was detected in these organisms, including branched and straight-chain acids, cyclopropane, and hydroxy acids. Comparison of the presence and relative amounts of these acids was useful in distinguishing the three species. Three branched-chain hydroxy acids not found in other bacteria were present in P. maltophilia and were identified by gas chromatography, mass spectrometry, and infrared spectroscopy.


The use of gas chromatographic-mass spectrometric-computer systems in pharmacokinetic studies.

Pharmacokinetic studies involving plasma, urine, breast milk, saliva and liver homogenates have been carried out by selective ion detection with a gas chromatographic-mass spectrometric-computer system operated in the chemical ionization mode. Stable isotope labeled drugs were used as internal standards for quantification. The half-lives, the concentration at zero time, the slope (regression coefficient), the maximum velocity of the reaction and the apparent Michaelis constant of the reaction were determined by regression analysis, and also by graphic means.


Gas chromatographic-mass fragmentographic determination of trazodone in rat plasma.

A sensitive and specific combined gas chromatographic-mass fragmentographic method for the determination of trazodone in rat plasma is described. After extraction with diethyl ether and washing of the extracts in order to prevent interference from endogenous materials, trazodone and the internal standard benperidol were separated on an OV-1 column. The minimum amount of trazodone detected was 20 ng by gas chromatography with the use of a flame ionization detector and 200 pg when using the mass fragmentographic technique. Plasma levels in rats treated with a single intravenous dose (10 mg/kg) of trazodone are also reported.


High-performance liquid chromatographic analysis of amino acids and peptide-hormone hydrolysates in the picomole range.

A high-pressure liquid chromatographic system for the separation of amino acids and peptide-hormone hydrolysates is described. A modified high-pressure liquid chromatography (Hewlett-Packard, model 1010B) equipped with an automatic gradient-generating system, and the applications of fluorescamine and ion-exchange resins of particle size 8 +/- 2 mum permit the detection of amino acids in the picomole range with good resolution. Compared with conventional amino acid analyzers, the times for separation are reduced by a factor of 3--4.


The performance of microparticle chemically-bonded anion-exchange resins in the analysis of nucleotides.

The performance of microparticles in the ion-exchange chromatographic separation of nucleotides was investigated. A microparticle chemically-bonded strong anion-exchange column packing was used for the analysis of the 5'-mono-, di- and -triphosphate nucleotides of adenine, guanine, hypoxanthine, xanthine, cytosine, uracil and thymine. It was found that excellent resolution of the majority of the nucleotides was obtained at ambient temperatures. The packings were stable and the sample capacity was greater than with pellicular resins. The retention times, peak shapes, heights and areas were highly reproducible and there was good linearity of response. Use of this column packing in biochemical studies is reported.


Liquid chromatographic separation and detection of nanogram quantities of biologically important dicarboxylic acids.

Phenacyl and benzyl derivatives of some biologically significant dicarboxylic acids have been prepared in a quantitative manner using a crown ether catalyst. They were separated using reversed-phase chromatography. The column used was packed with Corasil II to which a C9 phase (nonyl) was bonded. Water-methanol (68:32) was used as the mobile phase. Detection limits in the range of 5--15 ng have been obtained for a non-optimized system. The ease of preparation of these derivatives, coupled with their excellent chromatographic properties, makes this a very attractive procedure for the investigation of mixtures containing biologically significant acids. The implications and future of this technique are discussed.


Computerized gas chromatographic-mass spectrometric analysis of polycyclic aromatic hydrocarbons in environmental samples.

Substantial progress has been made in the last few years in the development of a rapid method for determining polycyclic aromatic hydrocarbons (PAH) in environmental samples. The three-step method consists of (i) a preliminary separation of PAH by solvent and/or column chromatography, (ii) identification by a combination of gas chromatography with quadrupole mass spectrometry and computer, and (iii) measurement by computerized gas chromatography using internal standards. Samples of industrial effluents, coke oven emissions, coal tar and airborne particulates have been investigated. The efficiencies of different gas chromatographic columns were evaluated during these investigations.


Trace analysis of organic volatiles in water by gas chromatography-mass spectrometry with glass capillary columns.

Traces of volatile organic materials in water have been concentrated by gas phase stripping and adsorption onto a porous polymer. A simple all-glass sampling device is proposed which allows efficient concentration at elevated temperatures. Sample transfer from absorbent into a gas chromatographic column is effected by a simple one-step procedure involving heat desorption. The capacity of the absorbent has been determined for a number of model substances which are found in water. Under the sampling conditions used, compounds being less volatile than benzene are usually quantitatively retained, with some exceptions. Separations were effected with highly efficient glass capillary columns. Water samples, collected from a small number of locations, included both tap water and untreated waters. A number of volatiles have been determined by gas chromatography-mass spectrometry in drinking water and in a river which flows  through an industrialized area. The drinking water examined contains a large number of chlorinated and brominated compounds whereas the river water is largely free from this class of substances. Derivatives of camphor and terpenes have been identified in this particular river.


Analysis for aqueous nitrates and nitrites and gaseous oxides of nitrogen by electron capture gas chromatography.

A highly sensitive gas chromatographic method is presented for the determination of aqueous nitrates. By suitable chemical pretreatment, this method is also applicable to the analysis of aqueous nitrites and gaseous oxides of nitrogen. The method has been applied to the analysis of drinking water, urine, and nitric acid. The technique involves the conversion of the nitrate ion to nitrobenzene with subsequent analysis by electron capture gas chromatography. The detection limit for nitrobenzene is ca. 10(-12) g, making possible the analysis of environmentally significant concentrations of aqueous nitrates and nitrites and gaseous oxides of nitrogen.


A comparison of the chromatographic properties of various polyadenylate binding materials.

The suitability of various materials for messenger RNA isolation and possible fractionation was examined by comparing the chromatographic behaviour of synthetic poly(riboadenylic acid) [poly(A)] and ribosomal RNA on each suport. The ratios of poly(A) to ribosomal RNA capacities of the various materials under different chromatographic conditions were used to estimate the potential selectivity of the columns for messenger RNA isolation. Using this criterion, benzoylated cellulose and oligo(deoxythymidylate)-cellulose should be the most selective materials of those studied. The elution conditions (temperature or salt gradient) used to obtain bound poly(A) were investigated to determine the optimum conditions for possible messenger RNA fractionation. It is concluded that oligo(deoxythymidylate)-cellulose appears to be the most suitable support of those investigated for messenger RNA isolation and possible fractionation.


Analysis of 1-(2-phenyladamant-1-yl)-2-methylaminopropane as its chlorodifluoroacetyl derivative.

The diastereoisomeric content of 1-(2-phenyladamant-1-yl)-2-methylaminopropane can be determined by gas chromatography following treatment with chlorodifluoroacetic anhydride. Racemisation occurs in the synthesis of 2-phenyladamantane from the corresponding 2-chloroadamantane in the Friedel-Crafts reaction with aluminium chloride, but fractionation of the diastereoisomers can occur in the purification steps. A method for determining 2-phenyladamantane in plasma and urine extracts at the nanogram level using an electron-capture detector is described and compared with a previously described radio-assay procedure.


Quantitative assay of maprotiline in biological fluids by gas-liquid chromatography.

A specific and sensitive gas-liquid chromatographic method has been developed for the assay of maprotiline in biological fluids. Maprotiline is isolated from the biological sample by base-specific extraction followed by conversion into the heptafluorobutyramide. The derivative is determined quantitatively by gas-liquid chromatography with an electron capture detector, nortriptyline being used as the internal standard. Amounts below 10 ng per biological sample can be measured.


Polychlorinated dibenzo-p-dioxins. Separation and identification of isomers by gas chromatography-mas spectrometry.

Attempts were made to synthesize all polychlorinated dibenzo-p-dioxin isomers containing six to eight chlorine atoms by micro-scale pyrolysis of different polychlorophenates. Eight of the ten possible hexachlorodibenzo-p-dioxins, the two hepta- and the octachlorodibenzo-p-dioxin were observed and separated by gas chromatography using glass capillary columns. Without actual isolation of these toxic materials, isomers were characterized by gas chromatography and mass spectrometry. Commercial chlorinate phenols were analyzed for the presence of these isomers. The major hexachlorodibenzo-p-dioxin observed in two commerical products was the unexpected 1,2,3,7,8,9-substituted isomer, which was not formed as the main dioxin component in any of the pyrolysis experiments. The same isomer was reported to be isolated from toxic fat and identified by X-ray crystallography.


Applications of moving-wire detectors for the liquid chromatography of fats and fatty acid derived oleochemicals.

Published applications of moving-wire detectors and other transport flame ionization detectors for the liquid chromatography of fats and oleochemical are briefly reviewed. The usefulness of this detector is illustrated by a number of examples taken from our own work at the Unilever Forschungsgesellschaft Hamburg. The chromatograms shown were chosen in which a way as to give an idea of how broad a range of applications may be covered by this approach.


Qualitative and quantitative gas liquid chromatography of triglycerides.

This article reviews the methodology required for triglyceride analysis by gas liquid chromatography. Compositions of the animal and vegetable triglyceride mixtures to which this technique has been applied are summarized.


Application of open-tubular column/gas-liquid chromatography to the analysis of complex mixtures of branched-chain fatty acids.

Methyl and butyl esters of branched-chain fatty acids of subcutaneous triacylgycerols from barley-fed lambs were subjected to urea-adduction and the resulting fractions were analyzed as methyl esters by gas-liquid chromatography on wall-coated open-tubular columns. The chromatogram of each fraction was less complex than that of the original and there was evidence to suggest that, in the original mixture of branched-chain acids, components resistant to urea-adduction were present, but their occurrence was obscured in chromatograms by the presence of monomethyl-substituted acids.


GLC identification techniques for long-chain unsaturated fatty acids.

Relationships between GLC behavior and structures of long-chain fatty acids are discussed. These relationships have been developed for mono-olefinic and myethylene-interrupted polyolefinic acids and have been made possible by the extensive published retention data on polyester phases and by a rationalization of ideas of stationary phase polarity. The relationships have been extended to nonmethylene-interrupted polyolefinic acids and the concept of fractional chain-length (FCL) values is explored for polyolefinic acids.


Technology for the identification of unusual cis, cis octadecadienoic fatty acids.

In this article methods are presented for the separation and identification of unusual cis, cis dienoic and polygnoic long chain fatty acids. Special emphasis has been laid on the identification of cis, cis octadecadienoic acids. The steps followed are: after transesterification the fatty acid methyl esters are separated by preparative gas chromatography according to chain length followed by argentation chromatography on thin-layer plates. After hydroxylation of the double bonds with osmium tetroxide the polyhydroxy compounds are derivatized to the per-O-trimethylsilyl-ethers. Separation and identification of individual compounds are achieved by combined gas chromatography-mass spectrometry using SCOT columns and low ionization energy.


Effect of clonidine on growth hormone, prolactin, luteinizing hormone, follicle-stimulating hormone, and thyroid-stimulating hormone in the serum of normal men.

Clonidine (0.15 mg iv), a selective noradrenergic receptor agonist, increased serum growth hormone (GH) levels (greater than 6 ng/ml) on 8 out of 12 administrations to 6 normal men. This increase was independent of the hypotensive effects of the drug and unrelated to changes in serum cortisol. Clonidine induced a hyperglycemic effect in all subjects which was greatest 15 min after commencint the injection. No changes in blood sugar or GH occurred after placebo injection. Apomorphine, a selective dopamine receptor agonist, elevated GH in each of these 6 subjects (greater than 10 ng/ml). Clonidine had no effect on serum prolactin (PRL), luteinizing hormone (LH), follicle-stimulating hormone (FSH), or thyroid-stimulating hormone (TSH). These data are compatible with a dual dopaminergic and noradrenergic mechanism modulating GH secretion in normal men and with the absence of a noradrenergic mechanism in the regulation of PRL, LH, FSH, or TSH.


Hypothalamic-pituitary-gonadal function in patients with myotonic dystrophy.

Gonadal function was studied in three post-pubertal siblings (two male and one female) and one unrelated male patient with myotonic dystrophy. The diagnosis was confirmed in all cases by electromyography and muscle biopsy. Basal levels of plasma immunoreactive LH, FSH, testosterone, and estradiol were measured. Hypothalamic, pituitary, and gonadal reserve and responsiveness were evaluated by clomiphene, LHRH, and HCG tests. Histologic examination of gonadal biopsies was also performed. The results showed that gonadal failure present in the four patients had different characteristics. In the same family, hypothalamic amenorrhea was observed in the female patient, and hypothalamic eunuchoidism and hypergonadotropic hypogonadism with marked tubular and leydig cells failure in the male patients. The non-related male patient had hypergonadotropic hypogonadism with tubular failure but with a compensatory leydig-cell hyperplasia. These data are interpreted as demonstrating different expressivity of the hypogonadism associated with the same inherited muscle disease.


Allergic response to glucagon injection as a result of insulin contamination.

A 25-year-old obese woman with adult onset diabetes, with known insulin allergy, was evaluated for her insulin response to glucagon. Intravenous injection of glucagon produced severe generlaized allergic reaction. Skin tests with various insulin and glucagon preparations showed allergic reaction to be most pronounced with beef regular single peak and single component insulin, pork regular single peak, beef lente single peak, and neutral regular (beef-pork) single peak insulin. Allergic reactions to numerous glucagon preparations were found to be directly proportional to the amount of insulin contamination in those preparations. Purification of one glucagon lot by column chromatography verified the presence of proinsulin and insulin contaminants in the preparation.


Radioimmunoassay for cortisol using antibodies against prednisolone conjugated at the 3-position.

A radioimmunoassay for the measurement of cortisol in plasma was developed. The procedure requires a one-step precipitation of the proteins in plasma or a one-step extraction with methylene chloride in urine. The method is precise, accurate, sensitive, and very specific. The primary antibody was prepared against prednisolone-3- carboxymethoxime-bovine serum albumin. The method is sensitive to 12.5 pg/ml of plasma. The antiserum has little interference from other endogenous steroids and metabolites of cortisol. 0830 hour plasma levels of cortisol in twenty normal human adults averaged 150 +/- 33 ng/ml.


Effects of diphenylhydantoin, phenobarbital, and diazepam on the metabolism of methylprednisolone and its sodium succinate.

The metabolic clearance rates (MCR) of methylprednisolone (MP) (no. = 13) and methylprednisolone-21- Na-hemisuccinate (MPHS) (no. = 6) were studied in normal humans using tritium-labeled steroids. The cumulative appearance of the labeled steroid was examined for the whole urine and for three major urinary fractions. The MCR, half-life, and volume of distribution were, respectively, 383 +/- 72 (SD) liters/day, 165 +/- 49 minutes, and 61 +/- 12 liters for MP, and 234 +/- 37, 160 +/- 19, and 41 +/- 6 for MPHS. Diphenylhydantoin (DPH) administered to 4 subjects increased the MCR of MP from 424 +/- 71 to 977 +/- 132 (P less than 0.01), and decreased the half-life from 149 +/- 44 to 69 +/- 7 (P less than 0.001). Similar effects were found with phenobarbital (PB). Diazepam (DZP) had no effect. Major increases in urinary metabolites after DPH and PB were in the unconjugated ethyl acetate fraction, and this suggests that MP metabolism is significantly altered by hepatic microsomal hydroxylation enzyme induction by DHP and PB, but not DZP. This could occur with the formation of a 6beta-hydroxy derivative which could be readily cleared by the kidney. The urinary pattern of excretion for MPHS was similar to that of MP. The MCR of MPHS was affected to a lesser extent by DPH and PB than was the MCR of MP(P less than 0.01). Therefore, the use of hepatic microsomal hydroxylase inducers should be taken into consideration in clinical states in which MP is being used.


Parathyroid hormone status and renal responsiveness in familial hypophosphatemic rickets.

Basal serum and urinary biochemical parameters and their response to PTH or calcium infusion were examined in 14 untreated patients with familial hypophosphatemic rickets (FHR) from 5 kindreds and 9 normal control subjects after a period of dietary equilibration. FHR subjects exhibited significantly elevated basal serum iPTH levels (FHR: 11.4 +/- 0.8, controls: 5.1 +/- 0.5 ng/ml, P less than 0.001) and urinary cAMP excretion (FHR: 7.83 +/- 0.81, controls: 3.78 +/- 0.46 nmol/mg creatinine P less than 0.001). In response to PTH infusion (6 units/kg over 4 hours) FHR subjects exhibited a mean 34% decrease in TRP and a 22-fold increase in cAMP excretion, both comparable to the control response. Calcium infusion (10 mg/kg over 1 h) rapidly suppressed serum iPTH and urinary cAMP values in FHR subjects. However, TRP remained inappropriately low for the level of serum phosphate. Basal and post-calcium infusion serum iPTH levels correlated positively with urinary cAMP in FHR subjects and controls. Pre- and post-calcium infusion iPTH levels correlated with serum calcium in FHR subjects. Mean Salivary phosphate concentration was significantly reduced in FHR subjects (FHR: 12.68 +/- 0.87, controls: 22.47 +/- 2.16 mg/100 ml, P less than 0.001). However, calculated salivary phosphate clearance rates were similar in FHR and control subjects. PTH or calcium infusion did not significantly alter salivary phosphate concentration or clearance rates in either patients or controls. We concluded that untreated FHR patients exhibit a state of mild secondary hyperparathyroidism and an at least normal renal phosphaturic response to PTH. In addition, there is no evidence for increased salivary phosphate excretion in FHR.


Familial incomplete virilization due to partial end organ insensitivity to androgens.

A 16-yr-old 46 XY individual with a familial incomplete male pseudohermaphroditism closely resembling the syndrome described by Gilbert-Dreyfus et al. was studied. The patient's habitus was masculine despite the presence of a small phallus, pseudo-vaginal perineal hypospadias, bifid scrotum, gynecomastia, and diminished virilization. Blood samples obtained at 20-min intervals were submitted to hormone analysis. Episodic fluctuations of plasma gonadotropins with mean values of LH above the normal male range and FSH within normal limits were observed. Moderately elevated plasma testosterone and increased plasma estradiol also showed episodic oscillations. The administration of LH-releasing hormone resulted in a significative increase of plasma LH and FSH. Testicular biopsy revealed the presence of seminiferous tubules with few spermatogonia and no spermatocytes, and normal sertoli and interstitial cells. Gonadal stimulation with hCG for 4 consecutive days induced a significative increase of plasma testosterone and estradiol. The daily administration of 50 mg of testosterone propionate for 3 days neither depressed the circulating levels of gonadotropins nor modified the pulsatile pattern of gonadotropins release. Administration of testosterone and 5alpha-dihydrotestosterone propionate failed to diminish plasma LH and FSH levels. Testosterone administration for 10 weeks also failed to induce virilization. These results are similar to those observed in patients with testicular feminization syndrome, and the underlying abnormality involves a partial defect of the mechanism of action of testosterone rather than decreased androgen biosynthesis. According to a recently proposed classification this individual corresponds to the type 1 incomplete male pseudohermaphroditism.


Heterogeneity of human growth hormone and prolactin secreted in vitro: Immunoassay and radioreceptor assay correlations.

Gel filtration of sera and of in vitro pituitary incubation media from a normal subject and from subjects with pituitary tumors or physiologically elevated growth hormone (hGH) and/or prolactin (hPRL) levels, has been performed. Heterogeneous immunoreactive forms of both hormones with comparable elution profiles in sera and in in vitro incubation media were identified. In each instance the most retarded component ('little hPRL' and 'little hGH') migrated identically with the respective radioiodinated pituitary standard and constituted the major component identified. Several components of intermediate mobility were identified and characterized as 'big hPRL', 'big hGH,' and 'big big hGH'. In addition, a void volume immunoreactive peak was often identified. No interconversion was demonstrated on refiltration of prolactin tumor sera or normal pituitary incubation media. Radioreceptor activity utilizing pregnant rabbit liver membranes for hGH and rabbit mammary membranes for hPRL indicates comparable immunologic and receptor activity in all forms identified following gel filtration of prolactin tumor sera (for hPRL) and normal pituitary incubation media (for hGH). Only for the larger species of HGH, identified by gel filtration of clinical grade hGH, was diminished receptor activity demonstrated. These data suggest that the human pituitary synthesizes and secretes protein hormones of different molecular size according to gel filtration elution volume profiles but with comparable receptor and immunologic assay reactivity. These results support but do not establish the proposal that pituitary hormones are also secreted as prohormones.


Thyrotropin and prolactin pituitary reserve in the "empty sella syndrome".

Ten patients (8 women, 2 men) with the "empty sella syndrome" were studied to evaluate the pituitary reserve of human thyrotropin (hTSH) and prolactin (hPRL). None of the patients had signs or symptoms of hypopituitarism or primary hypothyroidism. All patients had normal baseline thyroid function tests except for 2 patients with mild elevations in total triiodothyronine as measured by competitive protein displacement assay (T3D). Eight of ten patients had normal hTSH responses to thyrotropin releasing hormone (TRH), whereas the 2 patients with elevated T3D had blunted TRH responses. In the 4 patients studied, the metabolic clearance (MCR) and production rates (PR) of hTSH were normal. In 9 of 10 patients normal baseline serum hPRL levels were detected, and each responded to TRH. In one case serum hPRL was undetectable and failed to respond to TRH. The assessment of other anterior pituitary function revealed few minor abnormalities. In summary, like other anterior pituitary hormones, the pituitary reserve in "empty sella syndrome" of hTSH and hPRL is usually normal. When abnormalities do occur, they are attributable to other co-existent endocrine pathology.


The effect of short-term triiodothyronine administration on thyroxine response to exogenous TSH in man.

In order to examine whether thyroid hormone concentration interfere with the thyroid gland responsivity to TSH, paired studies on the effect of short-term T3 treatment on T4 response to exogenous TSH ( 5 I.U. i.m.) were carried out in 10 euthyroid volunteers. During T3 administration ( 120 mcg/day for 4 days starting 48 hr before TSH injection) a significant decrease in T4 concentrations was observed both prior to and after TSH, with an inhibition of the T4 response ranging from 36-77% as calculated from the area under the response curve. The present data are in agreement with the existence of a "short-loop" thyroid-thyroid regulatory mechanism in man. In fact, the decreased percent rise of T4 after TSH suggests an inhibitory effect of T4 release following TSH, even though a modification of T4 kinetic parameters during T3 administration may account for a portion of the lowering of T4 blood concentrations.


Pattern of plasma testosterone and delta4-androstenedione in normal newborns: Evidence for testicular activity at birth.

Plasma testosterone (T) and delta4-androstenedione (delta) levels were measured by a sensitive and specific radioimmunoassay method in 157 blood specimens from normal neonates. In both sexes at birth plasma T and delta were significantly higher in peripheral than in cord blood and drop within the first week of life. In males a secondary increase in both T and delta had occurred by the 2nd week of life while values continue to decrease in females. The present data demonstrate that testicular activity is still present at birth and suggest that the transient fall in T levels likely due to the removal of chorionic gonadotropin (hCG) secretion is responsible for the secondary activation of the hypothalamic pituitary axis by a negative feed-back mechanism.


Influence of body weight on estradiol metabolism in young women.

The metabolism of estradiol in girls with anorexia nervosa was compared with obese subjects and normal weight, age and sex matched controls. Increasing body weight was accompanied by a sharp decrease in 2-hydroxylation of estradiol and an increase in 16alpha-hydroxylation. These metabolic changes are discussed in terms of the biological activity of 2-hydroxyestrone and estriol.


Effect of temperature on transport and plating media for enteric pathogens.

The effect of wide variations in incubation temperatures and long periods of incubation on transport and enrichment broths and plating media was determined by exhaustive analysis of 132 diarrheal stools for salmonellae and shigellae. Homogenized stools were streaked onto eosin methylene blue (EMB), Salmonella-Shigella (SS), and xylose lysine deoxycholate (XLD) agar plates, and into saline, Cary-Blair (CB) transport medium, and Selenite F and gram-negative (GN) enrichment broths. Incubation temperatures were compared at 20 C, 35 C, 40 C and ambient, and over a range of 4 to 52 C for media incubated in an insulated picnic cooler in an auto trunk. At 1, 2, 4, and 7 days the plates were observed, and the broths were subcultured. Each stool was streaked to 12 plates for 48 observations and pickings, and to 48 tubes, subcultured to 192 plates, for a total of 240 observations for pathogens. Analysis of data from 6,246 Salmonella-positive plates showed direct streaking to be most effective after 2 days of incubation, but broths were equally effective at 1 or 2 days. By day 4 many plates were overgrown, and both plates and broths showed diminution of positives by about 10% and at day 7, 19%. The 2,434 Shigella-positive plates were more demanding in all times and temperatures of incubation than salmonellae. Although at day 2 best results were obtained on direct streaking, shigellae die-offs in broths were excessive, with positive declining 23.7% by day 2, 49% by day 4, and 60% by day 7. Direct plating of both pathogens was poor at 20 C with about 48% success, but salmonellae preferred higher temperatures (35 and 40 C), whereas shigellae chose 35 C and ambient, which averaged 28 C for the 10-month study. Temperature was immaterial to salmonellae in broths with ambient slightly better than 35 C, but shigellae preferred 20 C and showed a 50% failure rate at 40 C, ambient being equal to 35 C. The preferential rank of broths in efficacy was GN greater than selenite greater than saline greater than CB greater than direct for salmonellae; for shigellae, GN greater than saline greater than direct greater than CB greater than selenite, with selenite proving to be unsuitable for shigellae. Plating media preferences were XLD greater than EMB greater than SS. Ten of 39 shigellae strains could not be recovered from the selenite and SS media combination, the many replications notwithstanding. The effectiveness of salmonellae and shigellae detection at ambient temperatures in Louisiana during the 10-month study period, as compared to controlled incubation temperatures, indicates that satisfactory enteric bacteriology can be done in warm climates without constant temperature incubators.


Production of influenza virus complement-fixing antibody in mouse ascitic fluid.

Suspension cultures of BHK-21/13s cells infected with either A/Hong Kong/8/68 or B/Massachusetts/3/66 strains of influenza were used to prepare type-specific influenza ribonucleoprotein-immunizing antigens. These antigens were used in comparing four immunization schedules in the production of immune mouse ascitic fluids. Large volumes of ascitic fluids were obtained by these schedules. These fluids contained type-specific complement-fixing antibody and were devoid of nonspecific host tissue or heterologous cross-reactions.


Effects of atmosphere of incubation and of routine subcultures on detection of bacteremia in vacuum blood culture bottles.

Studies comparing isolation rates of bacteria and yeasts from vented and unvented vacuum blood culture bottles containing soybean-casein digest broth showed significantly more frequent and more rapid recovery of Candida and Pseudomonas from the vented bottle and no other statistically significant differences between the two. Subculture of bottles on the day of their collection was shown to accelerate recovery of 48% of positive cultures by day 1. A second subculture of known positive cultures yielded additional organisms in 1.5% of cultures.


Differential medium for detecting dental plaque bacteria resembling Actinomyces viscosus and Actinomyces naeslundii.

A medium for detecting colonies of Actinomyces viscosus and Actinomyces naeslundii in dental plaque samples was developed. The medium (CNAC-20) contains 20.0 mug of 3CdSO4-8H2O per ml of Columbia CNA agar base. Laboratory strains of A. viscosus grew on CNAC-20 in characteristic round, white, smooth, opaque colonies. Increasing the cadmium concentration impaired the growth of some A. viscosus strains. Stock strains of A. naeslundii and A. israelii grew in colonies of similar white, opaque morphology. The few strains of other gram-positive plaque bacteria that grew on CNAC-20 had colonies easily distinguished from those of A. viscosus. Most of the bacterial strains freshly isolated from Actinomyces-like colonies on CNAC-20 that had been inoculated with human dental plaque samples were found to have cultural characteristics consistent with previous descriptions of A. viscosus or A. naeslundii. CNAC-20 may facilitate investigations into the relationship of microaerophilic Actinomyces with the etiology of dental diseases.


A radioimmunoassay for serum rat thyroglobulin. Physiologic and pharmacological studies.

A double antibody radioimmunoassay has been developed to measure thyroglobulin in rat (RTg) serum. The lowest detectable quantity measurable was 5.0 ng/ml. Specificity was documented by: (a) fall in serum RTg to undetectable levels after thyroid ablation; (b) the fact that L-thyroxine, D-thyroxine, L-triiodothyronine, D-triiodothyronine, triiodothyroacetic acid, tetraiodothyroacetic acid, triiodothyropropionic acid, moniodotyrosine, diiodotyrosine, and human thyroglobulin (HTg) in concentrations up to 40,000 ng per tube did not cross-react in the assay; (c) the demonstration that constant levels of serum RTg were observed while varying amounts of serum (criterion of parallelism) were introduced in the assay. The mean RTg concentration in tail vein blood of adult Sprague-Dawley rats were 101.5 +/- 13.0 ng/ml (SEM) (n=21); values ranged from 12.0 to 258.0 ng/ml. Chronic administration of a high-iodine diet (HID) did not affect serum thyroglobulin levels. Chronic administration of a low-iodine diet (LID) and propylthiouracil (PTU) led to a statistically significant increase in serum RTg that was accompanied by a significant rise in serum thyrotropin (rTSH). Serum thyroxine (T4) administered to normal rats for 14 days (20 mug/day subcutaneously) depressed serum RTg concentration from a mean level of 119.4 +/- 17.5 ng/ml (n=19) to a mean of 35.0 +/- 0.27 ng/ml (n=19) (P less than 0.001). While rats were on continuous T4 suppression, bovine thyroid-stimulating hormone (bTSH) given intravenously (2 IU) resulted in a mean maximal increment of RTg of 332.0 +/- 81.5 ng/ml (n=6) at 24 h. IgC-(LATS) long-acting thyroid stimulatory injected intravenously resulted in a mean maximal increment of RTg concentration at 96 h of 87.2 +/- 14.3 ng/ml (n=5). Normal IgG had no statistical significant effect of RTg levels at any time after the injection.


Partial purification and characterization of contact activation cofactor.

The contact phase of intrinsic clotting involves Factor XI, Factor XII, Fletcher factor, and a fourth activity that we call contact activation cofactor (CAC). All four of these activities are reduced or absent in Dicalite-adsorbed plasma. A modified activated partial thromboplastin time assay for CAC has been defined by using a substrate of Dicalite-adsorbed plasma combined with partially purified sources of Factors XI and XII, and Fletcher factor. The following properties of CAC in plasma have been determined by using the assay: it is stable up to 60 min at 56 degrees C; gradually loses activity at 80 degrees C; is stable between pH 6 and 9; is precipitated by ammonium sulfate between 40% and 50% saturation; is slightly adsorbed by A1(OH)3; and is eluted from DEAE-cellulose after the major protein peaks. A purification procedure has been devised that separates CAC from other known clotting factors. Isolated CAC was less stable than CAC in plasma, but in the presence of dilute human serum albumin it retained full activity for 80 min at 56 degrees C. On gel filtration CAC had an apparent mol wt of 220,000 daltons. These properties are consistent with those described for Fitzgerald factor, which further supports the conclusion that CAC and Fitzgerald factor represent the same activity. Isolated CAC promoted the generation of activated Factor XI (XIa) in a mixture containing purified Factor XI, Factor XII, and kaolin. The amount of Factor XIa generated was proportional to the amount of added CAC. No time-consuming reaction between Factor XI or Factor XII and CAC could be demonstrated.


25-hydroxycholecalciferol stimulation of muscle metabolism.

Intact diaphragms from vitamin D-deficient rats were incubated in vitro with [3H]leucine. Oral administration of 10 mug (400 U) of cholecalciferol 7 h before incubation increased leucine incorporation into diaphragm muscle protein by 136% (P less than 0.001) of the preparation from untreated animals. Nephrectomy did not obliterate this response. ATP content of the diaphragm muscle was also enhanced 7 h after administration of the vitamin. At 4 h after administration of cholecalciferol, serum phosphorus concentration was reduced by 0.7 mg/100 ml (P less than 0.025) and the rate of inorganic 32PO4 accumulation by diaphragm muscle was increased by 18% (P less than 0.025) over the untreated animals. Increasing serum phosphate concentration of the vitamin D-deficient animals by dietary supplementation with phosphate for 3 days failed to significantly enhance leucine incorporation into protein. However, supplementation of the rachitogenic, vitamin D-deficient diet with phosphorus for 3 wk stimulated the growth of the animal and muscle ATP levels. This increase in growth and muscle ATP content attributed to the addition of phosphorus to the diet was less than the increase in growth and muscle ATP levels achieved by the addition of both phosphorus and vitamin D to the diet. To eliminate systemic effects of the vitamin, the epitrochlear muscle of the rat foreleg of vitamin D-depleted rats was maintained in tissue culture. Addition of 20 ng/ml of 25-hydroxycholecalciferol (25-OHD3) to the medium enhanced ATP content of the muscle and increased leucine incorporation into protein. Vitamin D3 at a concentration of 20 mug/ml and 1,25-dihydroxycholecalciferol (1,25-(OH)2D3) at a concentration of 500 pg/ml were without effect. Analysis of muscle cytosol in sucrose density gradients revealed a protein fraction which specifically bound 25-OHD3 and which demonstrated a lesser affinity for 1,25-(OH)2D3. These studies suggest that 25-OHD3 may influence directly the intracellular accumulation of phosphate by muscle and thereby play an important role in the maintenance of muscle metabolism and function.


Determinants of human adipose tissue lipoprotein lipase. Effect of diabetes and obesity on basal- and diet-induced activity.

The role of insulin in the regulation of human adipose tissue lipoprotein lipase was evaluated. Adipose tissue heparin-releasable lipoprotein lipase (thought to be related to peripheral clearance of plasma triglycerides) was low in insulin-deficient, untreated hyperglycemic diabetic subjects (P less than 0.001) and treatment of hyperglycemia returned the activity to normal. In chronic hyperinsulinism, represented by obesity, heparin-releasable activity among control subjects was correlated to percent of ideal body weight (r=0.53, P less than 0.05) and to fat cell size (r=0.61, P less than 0.02). Acetone-ether powder lipoprotein lipase activity (presumed to reflect total tissue enzyme) was also related to percent of ideal body weight (r=0.76, P less than 0.001 for controls; r=0.67, P less than 0.05 for diabetics) and to fat cell size (r=0.71, P less than 0.01 for controls; r=0.85, P less than 0.01 for diabetics. Postprandial-stimulated insulin secretion was related to diet-induced changes in lipoprotein lipase in control subjects; both were dependent upon the amount of dietary carbohydrate. In contrast, the diabetic patients with low insulin responses, failed to increase lipoprotein lipase activity with feeding. The changes in heparin-releasable (r=0.66, P less than 0.01) and acetone-ether powder (r=0.69, P less than 0.01) activity during feeding were related to the percent increase in plasma insulin. Thus, insulin appears to be important in the regulation of human adipose tissue lipoprotein lipase activity. Elevated insulin levels in obesity and increased insulin secretion after eating were associated with increased lipoprotein lipase activity. Defects in insulin secretion, both in postabsorptive and postprandial states, are associated with low adipose tissue lipoprotein lipase and may lead to hypertriglyceridemia in diabetic man.


Transcobalamins I and II as natural transport proteins of vitamin B12.

There are two conflicting theories of how plasma vitamin B12 (B12) is transported in man: (a) by two distinct transport proteins, transcobalamins I and II (TC I and II), each having a specific role and time of function; and (b) by three active transport proteins, TC I, II, and III, that take up B12 randomly in proportion to the unsaturated amounts of each. To test these theories a man was given 1.12 mug, 229 muCi, of [57Co]B12 mixed with food. Blood samples were taken several times on the 1st day and at lengthening intervals up to day 51. The amount of TC II-B12 was measured in each sample by: gel filtration and by precipitation with (NH4)2SO4. Total serum R-B12 was then separated into TC I and TC III by: (a) a single step anion exchange system and (b) isoelectric focusing (IEF). As the B12 was being absorbed, 92-95% of that in venous blood was carried by TC II. Absolute and percentage transport by TC II declined sharply during the first 24 h; between days 7 and 51 20-33% of the label was on TC II, and the rest was carried by R-type binders. Absolute transport by TC I did not reach a maximum until after day 1 and before day 3. Transport by an alpha2 R-type binder, TC III, could not be demonstrated. TC I was isoelectrically heterogenous, with the components focusing between pH 2.9 and 3.35. It was concluded that (a) TC II is the dominant carrier of B12 immediately after absorption; (b) maximum transport by TC I requires the passage of time after absorption; (c) after the absorbed B12 reaches equilibrium with the total body B12, about one fourth of the plasma B12 is carried by TC II and three fourth by TC I; and (d) TC I and TC II are the only functional transport proteins of plasma B12.


Effect of insulin-glucose infusions on plasma glucagon levels in fasting diabetics and nondiabetics.

The effect of the intravenous infusion of insulin plus glucose on plasma glucagon levels was studied in hyperglycemic fasting adult-type and juvenile-type diabetics and compared with fasting nondiabetics. Adult-type diabetics were given insulin for 2 h at a rate of 0.03 U/kg-min, raising their mean insulin to between 25 and 36 muU/ml; glucagon declined from a base-line value of 71+/-2 (SEM) to 56+/-1 pg/ml at 120 min (P less than 0.001). In juvenile-type diabetics given the same insulin-glucose infusion, glucagon declined from a base-line level of 74+/-8 to 55+/-5 pg/ml at 120 min (P less than 0.05). The absolute glucagon values in the diabetic groups did not differ significantly at any point from the mean glucagon levels in nondiabetics given insulin at the same rate plus enough glucose to maintain normoglycemia. When glucagon was expressed as percent of baseline, however, the normoglycemic nondiabetics exhibited significantly lower values than adult-type diabetics at 90 and 120 min and juvenile-type diabetics at 60 min. In nondiabetics given insulin plus glucose at a rate that caused hyperglycemia averaging between 134 and 160 mg/dl, glucagon fell to 41+/-7 pg/ml at 120 min, significantly below the adult diabetics at 90 and 120 min (P less than 0.01 and less than 0.05) and the juvenile group at 60 min (P less than 0.01). The mean minimal level of 39+/-2 pg/ml was significantly below the adult (P less than 0.001) and juvenile groups (P less than 0.05). When insulin was infused in the diabetic groups at a rate of 0.4 U/kg-min together with glucose, raising mean plasma insulin to between 300 and 600 muU/ml, differences from the hyperglycemic nondiabetics were no longer statistically significant. It is concluded that, contrary to the previously reported lack of insulin effect in diabetics during carbohydrate meals, intravenous administration for 2 h of physiologic amounts of insulin plus glucose is accompanied in unfed diabetics by a substantial decline in plasma glucagon. These levels are significantly above hyperglycemic nondiabetics at certain points but differ from normoglycemic nondiabetics only when expressed as percent of the baseline. At a supraphysiologic rate of insulin infusion in diabetics, these differences disappear.


Heme synthetase deficiency in human protoporphyria. Demonstration of the defect in liver and cultured skin fibroblasts.

The final step in heme biosynthesis is chelation of porphyrin with Fe++ catalyzed by the mitochondrial enzyme heme synthetase. We have employed a sensitive radiochemical assay for this enzyme, using 59Fe and deuteroporphyrin or protoporphyrin as substrates. In this method iron is maintained in the ferrous state, oxygen is excluded from the incubation system, and labeled heme product is extracted into ethyl acetate. This assay has been used to measure the activity of heme synthetase in homogenates of liver, obtained by needle biopsy, and in sonicates of human skin fibroblasts, cultured in vitro. In addition, activity of the first enzyme of the heme synthetic pathway, delta-aminolevulinic acid synthetase, has been measured in fibroblast lysates. Lysates of fibroblasts from eight patients with protoporphyria had activities of delta-aminolevulinic acid synthetase which did not differ significantly from those of eight normal fibroblast lines, whereas activity of heme synthetase, with either deuteroporphyrin or protoporphyrin as substrate, was markedly decreased in sonicates of skin fibroblasts from these patients, the mean being 8% of control with deuteroporphyrin and 14% with protoporphyrin as substrate. In homogenates of liver from five patients with protoporphyria, activity of heme synthetase was also significantly less than that found in six patients without prophyria, the mean being 13% of control with protoporphyrin as substrate. These results provide evidence that decreased activity of heme synthetase is the basic defect in the heme synthetic pathway in protoporphyria. This deficiency is probably responsible for protoporphyrin accumulation and hence the biochemical and clinical features observed in protoporphyria.


Effects of low-level lead exposure on pyrimidine 5'-nucleotidase and other erythrocyte enzymes. Possible role of pyrimidine 5'-nucleotidase in the pathogenesis of lead-induced anemia.

Similarities between lead-induced anemia and a new hereditary erythorenzymopathy involving pyrimidine-specific 5'-nucleotidase prompted studies of the effects of lead on this and other erythrocyte enzymes. In vitro incubations of normal mature erythrocytes demonstrated that significant inhibition of pyrimidine 5'-nucleotidase occurred in the presence of lead at concentrations that had minimal effects on many other erythrocyte enzymes assayed simultaneously. Similarly, subjects with chronic lead intoxication secondary to industrial exposure exhibited substantial and consistent impairment of erythrocyte pyrimidine-5'-nucleotidase activity. Results suggest that lead-induced deficiency of this enzyme in maturing erythroid elements could, if sufficiently severe, result in induction of basophilic stippling and premature erythrocyte hemolysis analogous to that encountered in the genetically induced enzyme-deficiency syndrome.


Urea synthesis after oral protein ingestion in man.

In an attempt to assess hepatic functional capacity, hourly urea production, and corresponding serum amino acid concentrations after the ingestion of single protein meals (60, 120, and 240 g of protein) were evaluated in 18 normal subjects and in 8 patients with liver disease. In normal subjects, the relationship between urea production and serum amino acid concentration was linear (urea production in milligram urea nitrogen/kilogram lean body mass/hour = 6.3 times mg amino acid nitrogen/100 ml minus 20.5 SE of the estimate 6.9, r = 0.74, P less than 0.001), and variation of protein intake from 50 to 150 g/day for 3 days before testing did not change this relationship. The patients demonstrated impairment of urea synthesis proportional to the clinical severity of their liver disease. The potential clinical applications of these findings need to be determined.


Prostaglandin-stimulated bone resorption by rheumatoid synovia. A possible mechanism for bone destruction in rheumatoid arthritis.

Synovial tissue from patients with rheumatoid arthritis was maintained in organ culture for 3-14 days. Conditioned media from these synovial cultures contained bone resorption-stimulating activity, measured in vitro by using calcium release from mouse calvaria as the assay system. The synovial cultures also produce prostaglandin E2 (PGE2) as measured by serologic methods. The production of both the bone resorption-stimulating activity and PGE2 was inhibited by more than 90% by treatment of the synovial cultures with indomethacin (5 mug/ml). In contrast, treatment of the synovial cultures with colchicine (0.1 mug/ml) caused a marked and parallel increase in the concentration of both bone resorption-stimulating activity and PGE2 in the conditioned media. The bone resorption-stimulating activity was quantitatively extracted into diethyl ether. Within the limits of experimental error, all of the bone resorption-stimulating activity in medium was accounted for by its content of PGE2, itself a potent osteolytic factor. We conclude that the bone resorption-stimulating activity produced by rheumatoid synovia in culture is PGE2.


Effect of corticosteroids on the human monocyte IgG and complement receptors.

A quantitative in vitro assay was employed to directly assess the effect of corticosteroids on the IgG and complement receptor function of human  mononuclear phagocytic cells. In this system corticosteroids were solubilized with cholesterol-phospholipid sonicated dispersions before exposure to mononuclear cells. Solubilized corticosteroids at concentrations between 10(-4) and 10(-3) M inhibited both IgG and complement receptor activity in a dose-response fashion. Inhibition was dependent upon the time of interaction of the mononuclear cells with corticosteroids and was half-maximal by 15 min. The inhibitory effect at all concentrations of hydrocortisone was partially overcome by increasing the number of IgG molecules per erythrocyte. Hydrocortisone also inhibited the binding of erythrocytes coated with both IgG and C3, despite the fact that when both were on the erythrocyte surface a synergistic effect on binding to mononuclear cells was observed. At the steroid concentrations employed, the capacity of mononuclear cells to exclude trypan blue and to take up latex particles and neutral red was unaffected. Mineralocorticoids also inhibited receptor activity, but the sex hormones were less effective. These studies demonstrate an effect of steroid hormones on cell membrane receptor function, and they suggest that an inhibition of the recognition system for IgG and C3 in vivo may explain, in part, the effect of corticosteroids in man.


Pulmonary mechanics by spectral analysis of forced random noise.

The magnitude (Zrs) and phase angle (thetars) of the total respiratory impedance (Zrs), from 3 to 45 Hz, were rapidly obtained by a modification of the forced oscillation method, in which a random noise pressure wave is imposed on the respiratory system at the mouth and compared to the induced random flow using Fourier and spectral analysis. No significant amplitude or phase errors were introduced by the instrumentation. 10 normals, 5 smokers, and 5 patients with chronic obstructive lung disease (COPD) were studied. Measurements of Zrs were corrected for the parallel shunt impedance of the mouth, which was independently measured during a Valsalva maneuver, and from which the mechanical properties of the mouth were derived. There were small differences in Zrs between normals and smokers but both behaved approximately like a second-order system with thetars = 0 degree in the range of 5--9 Hz, and thetars in the range of +40 degrees at 20 Hz and +60 degrees at 40 Hz. In COPD, thetars remained more negative (compared to normals and smokers) at all frequencies and crossed 0 between 15 and 29 Hz. Changes in Zrs, similar in those in COPD, were also observed at low lung volumes in normals. These changes, the effects of a bronchodilator in COPD, and deviations of Zrs from second-order behavior in normals, can best be explained by a two-compartment parallel model, in which time-constant discrepancies between the lung parenchyma and compliant airway keep compliant greater than inertial reactance, resulting in a more negative phase angle as frequency is increased.


Biochemical determinants of 5-fluorouracil response in vivo. The role of deoxyuridylate pool expansion.

5-Fluorodeoxyuridine monophosphate (FdUMP), the active metabolite of 5-fluorouracil (5-FU), is a tight-binding inhibitor of thymidylate synthetase, the enzyme which converts dUMP to TMP. Newly developed assays for FdUMP and dUMP were utilized to assess the competitive roles played by these nucleotides in determining the inhibition of TMP synthesis in mice bearing the P1534 ascites tumor. After 5-FU administration, levels of FdUMP reached a dose-dependent peak within 6 h in the ascites tumor and in bone marrow, and declined thereafter in a biphasic manner with an initial t 1/2 of 6 h and a final t 1/2 of 7-9 days. In duodenal mucosa, FdUMP levels were 1.8-2-fold higher than in the other tissues, but elimination was much more rapid. Simultaneous with the fall in FdUMP a progressive accumulation of the competitive substrate dUMP was observed in each tissue after 5-FU; and peak dUMP levels coincided with recovery of thymidylate synthesis, as determined by the incorporation of [3H]deoxyuridine into DNA. In vitro experiments with partially purifed thymidylate synthetase revealed and initial competitive interaction of dUMP and FdUMP, which, at high concentrations of dUMP was capable of markedly slowing the rate of irreversible inactivation of enzyme by FdUMP. These studies were found to be quantitatively consistent with a two-phase model of enzyme inactivation involving an initial competition between dUMP and FdUMP, with subsequent irreversible inactivation of enzyme by covalent linkage to the inhibitor. Recovery of thymidylate synthesis after 5-FU appears to result from both a fall in intracellular levels of inhibitor and a progressive accumulation of the competitive substrate dUMP.


Hypoxanthine-guanine phosphoribosyltransferase. Characterization of a mutant in a patient with gout.

The mutation in a young gouty male with a partial deficiency of hypoxanthine-guanine phosphoribosyltransferase has been evaluated. The serum uric acid was 11.8 mg/100 ml, and the urinary uric acid excretion was 1,279 mg/24 h. Erythrocyte hypoxanthine-guanine phosphoribosyltransferase was 34.2 nmol/h/mg, adenine phosphoribosyltransferase was 36.5 nmol/h/mg and phosphoribosylpyrophosphate was 2.6 muM. Hypoxanthine-guanine phosphoribosyltransferase from peripheral leukocytes and cultured diploid skin fibroblasts was within the normal range, but enzyme activity in rectal mucosa was below the normal range. Initial velocity studies of the normal enzyme and the mutant enzyme from erythrocytes with the substrates hypoxanthine, guanine, or phosphoribosylpyrophosphate showed that the Michaelis constants were similar. Product inhibition studies distinguished the mutant enzyme from the normal enzyme. Hyperbolic kinetics with increasing phosphoribosylpyrophosphate were converted to sigmoid kinetics by 0.2 mM GMP with the mutant enzyme but not with the normal enzyme. The mutant erythrocyte hypoxanthine-guanine phosphoribosyltransferase was inactivated normally at 80 degrees C and had a normal half-life in the peripheral circulation. The mol wt of 48,000 was similar to the normal enzyme mol wt of 47,000. With isoelectric focusing, the mutant erythrocyte enzyme had two major peaks with isoelectric pH's of 5.50 and 5.70, in contrast to the isoelectric pH's of 5.76, 5.82, and 6.02 of the normal isozymes. Isoelectric focusing of leukocyte extracts from the patient revealed the presence of the mutant enzyme. Cultured diploid fibroblasts from the propositus appeared to function normally, as shown by the inability to grow in 50-100 muM azaguanine and by the normal incorporation of [14C]hypoxanthine into nucleic acid. In contrast, erythrocytes from the patient displayed abnormal properties, including the increased synthesis of phosphoribosylphyrophosphate and elevated functional activity of orotate phosphoribosyltransferase and orotidylic decarboxylase. These unique kinetic, physical, and functional properties provide support for heterogeneous structural gene mutations in partial deficiencies of hypoxanthine-guanine phosphoribosyltransferase.