Purification of a soluble rat liver protein that stimulates microsomal 4-methyl sterol oxidase activity.

A soluble rat liver protein that stimulates microsomal methyl sterol oxidase activity has been isolated and purified to homogeneity by salt fractionation, differential heat inactivation, two calcium phosphate gel association, and Sephadex filtration. The protein, as isolated, may be dissociated with detergent into subunits with a molecular weight of approximately 10,300, as determined electrophoretically. In addition to this stimulatory protein, postmicrosomal supernatant fraction of rat liver contains a low molecular weight methyl sterol oxidase inhibitor, possibly cholesterol ester, which is removed during protein purification. Purified soluble protein enhances the observed rate of oxidative attack of the methyl sterol substrates, 4, 4-dimethyl-5alpha-cholest-7-en-3beta-ol and 4alpha-methyl-5alpha-cholest-7-en-3beta-ol. Addition of increasing amounts of either partially purified or homogeneous soluble protein yields hyperbolic stimulation, from which a K'm of about 7 muM has been calculated (based on a monomeric molecular weight of 10,300). Sequential additions of the soluble protein yield equal increments of stimulation. These results are consistent with the suggestion that the soluble protein may be a reactant in the oxidative process. Methyl sterol oxidase is inhitited in vitro by cholesterol, several oxygenated sterols, and cholesterol esters. The extent of inhibition is much greater when the soluble protein is present in the incubation. The inhibition is competitive with respect to methyl sterol substrate; cholesterol succinate, a water-soluble ester, is strongly inhibitory, K'i (ester)/K'm(substrate) approximately 0.2. Since end product inhibition of methyl sterol oxidase may be produced by accumulation of cholesterol or cholesterol metabolites, the soluble protein may participate in regulation of the activity of some microsomal enzymes of cholesterol biosynthesis.


Enzymatic reactions of methionine sulfoximine. Conversion to the corresponding alpha-imino and alpha-keto acids and to alpha-ketobutyrate and methane sulfinimide.

L-Methionine sulfoximine is a substrate of L-amino acid oxidase (Crotalus adamanteus), glutamine transaminase, and gamma-cystathionase. In the reaction catalyzed by L-amino acid oxidase, methionine sulfoximine is converted to aplph-imino-gamma-methylsulfoximinylbutyrate, which undergoes rapid gamma elimination yielding methane sulfinimide and 2-imino-3-butenoic acid. Methane sulfinimide is converted to methane sulfonamide, methane sulfinic acid, and methane sulfonic acid; 2-imino-3-butenoic acid is hydrolyzed to vinylglyoxylate, which polymerizes to an insoluble product. When the reaction is carried out in the presence of semicarbazide, the imine formed initially is quantitatively trapped as alpha-keto-gamma-methylsulfoximinylbutyrate semicarbazone, from which the free alpha-keto acid may be obtained. When the reaction is carried out in the presence of a mercaptan (RSH), a gamma exchange reaction occurs leading to formation of a new alpha-keto acid substituted in the gamma position by an SR-group; thus, alpha-keto-gamma-(beta-hydroxyethiol)butyric acid (S-(hydroxyethyl)-2-keto-4-mercaptobutyric acid) was obtained when L-methionine sulfoximine was oxidized in the presence of 2-mercaptoethanol, and enzymatic transamination of this alpha-keto acid with L-glutamine gave the new amino acid, L-omega-hydroxyethionine. The reaction of L-methionine sulfoximine with gamma-cystathionase yields 1 mol each of alpha-ketobutyrate and methane sulfinimide; the latter is hydrolyzed almost exclusively to methane sulfinic acid. Transamination of L-methionine sulfoximine yields the corresponding alpha-keto acid (alpha-keto-gamma-methylsulfoximinylbutyrate), which is stable. Some of these reactions may occur in vivo, and thus contribute to the toxicity of L-methionine sulfoximine.


Characterization of carnitine acylcarnitine translocase system of heart mitochondria.

Mersalyl inhibited the respiration of heart mitochondria under conditions that required the transport of (-)-carnitine and acyl(-)-carnitines. The exchange of external carnitine and acylcarnitines for intramitochondrial carnitine was also inhibited by mersalyl and 1 mM mersalyl proved suitable for the inhibitor-stop assay of carnitine acylcarnitine translocase. The carnitine-carnitine and (-)-carnitine-acetyl(-)-carnitine exchanges involved a mole to mole exchange. The carnitine-carnitine exchange did not require energy. The carnitine acylcarnitine translocase resembles the Pi transport system in inhibition by mersalyl and N-ethylmaleimide and in lack of a cation requirement for activity; yet the two are not identical inasmuch as operation of only the former transport system was inhibited by long chain acyl(+)-carnitines. Additional results render it improbable that the transport of carnitine and acylcarnitines is catalyzed by any other known mitochondrial transport systems. The carnitine acylcarnitine translocase activity is unlikely to be shared by one of the carnitine acyltransferases because the mersalyl inhibition of carnitine palmitoyltransferase and carnitine acetyltransferase was noncompetitivcase. Rapid acetylation of intramitocondrial free (-)-carnitine occurred when acetyl-CoA was generated intramitochondrially but not with exogenous acetyl-CoA. Theese observations substantiate the view (Pande, S. V. (1975) Proc. Natl. Acad. Sci. U.S.A. 72, 883-887) that a system exists in mitochondria for the transport of carnitine and its esters and that the matrix has a pool of carnitine compounds which has access to that carnitine acyltransferase which is localized on the inner side of the inner mitochondrial membrane.


Use of beta-parinaric acid, a novel fouorimetric probe, to determine characteristic temperatures of membranes and membrane lipids from cultured animal cells.

A naturally occurring fluorescent compound, beta-parinaric acid, was employed as a probe to measure the effects of temperature changes on plasma membrenes, microsomes, and mitochondria and on their respective lipids after isolation form LM cells grown in suspension culture. A computer-centered spectrofluorimenter simultaneously measured the absorbance, absorbance-corrected fluorescence, and relative fluorescence efficiency of beta-parinaric acid incorporated into the membranes or isolated membrane lipids. These parameters were measured as a function of temperature. The probe revealed five characteristic breaks or changes in slope with both the plasma membranes as well as their extracted lipids. These discontinuities occurred at approximately 18, 23, 31, 38, and 43 degrees. The other isolated subcellular organelles, microsomes, and mitochondria, as well as their respective isolated lipids, exhibited approximately the same characteristic temperatures (+/- 1 degree) as plasma membranes. Thus, these data negate one criterion of the theory that an asymmetric distribution of characteristic temperatures exist across the membranes of LM cells.


Physical properties of membranes isolated from tissue culture cells with altered phospholipid composition.

A choline-requiring strain of mouse fibroblast cells (LM cells) was cultured in suspension with choline, N,N'-dimethylethanolamine, N-monomethylethanolamine, or ethanolamine. These choline analogues were incorporated into membrane phospholipids as phosphatidyl-N,N'-dimethylethanolamine, phosphatidyl-N-monomethylethanolamine, and phosphatidylethanolamine. Plasma membranes, microsomes, mitochondria, and their respective lipids were isolated and the characteristic temperatures were determined by using two types of fluorescent probes: (a) beta-parinaric acid, a naturally occurring molecule, and (b) 8-anilino-1-naphthalene sulfonic acid, a synthetic organic fluorophore. A computer-centered spectrofluorimeter capable of simultaneous measurement of absorbance, absorbance-corrected fluorescence, and relative fluorescence efficiency was utilized for on-line measurement of all fluorescence parameters. Plots of absorbance corrected fluorescence or of relative fluorescence efficiency versus temperature revealed the same five characteristic temperatures with both types of probe. These characteristic temperatures were independent of the phospholipid composition of the LM suspension cell membranes or their extracted lipids. Plasma membranes, microsomes, and mitochondria containing analogue phospholipids had similar (+/- 1 degree) characteristic temperatures. The presence of analogue phopholipids altered the binding characteristics of beta-parinaric acid with plasma membranes and plasma membrane lipids of LM suspension cells. The equilibrium dissociation constant of plasma membranes and plasma membrane lipids was decreased 2- and 5-fold, respectively, when the cells had been supplemented with ethanolamine. The minimum number of phospholipid molecules per probe binding site was approximately constant in the intact plasma membrane but increased (2-fold) in the isolated plasma membrane lipids. The presence of analogue phospholipids also altered the interaction of 8-anilino-1-naphthalene sulfonic acid with LM cell membranes. The equilibrium dissociation constant of this probe interacting with mitochondrial lipid was decreased 40% by ethanolamine supplementation. The fluorescent properties of both probes were sensitive to the degree of methylation of the polar head group. The absolute values of absorbance-corrected fluorescence and relative fluorescence efficiency were different for each type of membrane from LM cells even with the same analogue supplement. Thus, it appears that LM cells maintain the characteristic temperatures which are a measure of the physical properties of their membranes, despite large alterations of the phospholipid polar head group composition.


Proteolytic cleavage of horse liver cytochrome b5. Primary structure of the heme-containing moiety.

The amino acid sequence of the NH2-terminal segment of horse cytochrome b5, containing the heme binding site, has been determined. A fragment, representing residues 7 through 90, was obtained by tryptic cleavage of native cytochrome b5. Chymotryptic cleavage of native cytochrome b5 yields a peptide containing residues 1 through 98. Contrary to native cytochrome b5, neither derivative showed binding to horse liver microsomal vesicles. The complete primary structure of the polar moiety has been deducted from automated and manual sequence analysis of peptides obtained from tryptic and chymotryptic digests of native cytochrome and apocytochrome preparations. Glutamyl residues at positions 41, 42, 47, and 48 appear to be replaced by aspartyl residues in some molecules. Such microheterogeneity is not observed at glutamyl residues at other positions. The native cytochrome b5 preparation contains a blocked NH2-terminal residue.


Spectral changes in bovine factor X associated with activation by the venom coagulant protein of Vipera russelli.

Bovine Factor X is a zymogen involved in blood coagulation that is converted to activated Factor X in the presence of Ca(II) by the coagulant protein of Russell's viper venom. To monitor structural transitions in Factor X during conversion to activated Factor X, the ultraviolet absorption, fluorescence emission, and circular dichroism spectra of activated Factor X and Factor X were compared. The ultraviolet absorption difference spectrum in the aromatic region comparing activated Factor X and Factor X indicates minima at 292.5, 285, and 278 nm and a small maximum at 305 nm; these differences are due to tryptophan and tyrosine perturbations. The activation of Factor X at 25 degrees in the presence of 8.3 mM CaCl2 with the use of Factor X:venom coagulant protein in molar rations of 1500:1 yielded a time-dependent increase in this spectrum which was linear for about 60 min and which temporally paralleled the development of activated Factor X activity. The binding of Ca(II) to factor X or activated Factor X is associated with a red-shifted tryptophan difference spectrum; however, this perturbation appears to make only a small contribution to the total perturbation observed during Factor X activation. Solvent perturbation studies in 20% glycerol suggest that an average of 3.1 tryptophan residues and 9.0 tyrosine residues are exposed to solvent in Factor X in 8.3 mM CaCl2 at pH 7.4; an additional 0.5 tryptophan residue and tyrosine reside become exposed to solvent during activation of Factor X in 8.3 mM CaCl2. The activation of Factor X by the venom coagulant protein is associated with a small red shift in the intrinsic tryptophan fluorescence emission spectrum. Far- and near-ultraviolet circular dichroism spectroscopy detected no difference between Factor X and activated Factor X. In summary, the activation of Factor X to activated Factor X appears associated with exposure of tryptophan and tyrosine side chains previously buried within the protein and with minimal changes in the secondary structur. These results suggest that conversion of Factor X to activated Factor X involves functionally important, but structurally subtle, changes in the three-dimentional structure.


Nuclear relaxation and gelation study of the interaction of organophosphates with human normal and sickle hemoglobins. In vitro gelation of sickle oxyhemoglobin in the presence of inositol hexaphosphate.

31P relaxation studies reveal a 3-fold enhancement of the longitudinal relaxation rate of both phosphoryl groups of hemoglobin-bound 2,3-bisphosphoglycerate upon conversion of methemoglobin to fluoromethemoglobin presumably due to an order of magnitude increase in the electron spin relaxation time. The enhancement of the longitudinal components of 31P relaxation (T(-1)1pr) upon binding to hemoglobin is not exchange-limited, since it is more than an order of magnitude smaller than the effect observed on the transverse components (T(-1)2pr). From the observed paramagnetic component, T(-1)1M, of the bound state relaxation rate of 2,3-bisphosphoglycerate, using the correlation time tau s obtained from the frequency dependence of water proton relaxation, we obtained an NMR root mean sixth distance from the four heme iron atoms to each of the 31P nuclei of 24 +/- 1 A. This is in excellent agreement with the x-ray crystallographic determination of this distance of (25 +/- 1) A in the 2,3-bisphosphoglycerate-deoxyhemoglobin complex, indicating that the spatial disposition of the allosteric site in the deoxy and oxy conformations of hemoglobin relative to the various heme irons may be the same, and that the same protein groups may be involved in binding 2,3-bisphosphoglycerate to the two forms of hemoglobin. Water proton relaxation studies reveal the existence of different conformational states of methemoglobins with 2,3-bisphosphoglycerate and inositol hexaphosphate. Inositol hexaphosphate alters the conformation to a strained (T) state with deoxy-like quaternary and tertiary globin structure as indicated by the finding that equimolar amounts of inositol heasphosphate induce gelation in a 4 mM sickle methemoglobin solution at temperatures greater than or equal 24 degrees. More interestingly, oxyhemoglobin S shows an identical thermodynamically reversible gelation behavior, with the same transition temperature (24 degrees), arguing against a mutual coupling of the protein conformation and the heme spin state in functional ferrohemoglobins. High ionic strengths (approximately 1 M) and pH values above neutrality block inositol hexaphosphate induced gelation of sickle met- and oxyhemoglobins. Unlike inositol hexaphosphate, the presence of saturating amounts of 2,3-bisphosphoglycerate does not promote gelation of a 4 mM met- or oxyhemoglobin S solution.


Some properties of purified skeletal muscle alpha-actinin.

Highly purified alpha-actinin can be made by using the low ionic strength extraction procedure previously described (Arakawa N., Robson, R. M., and Goll, D. E. (1970) Biochim. Biophys. Acta 200, 284-295) and then subjecting the crude alpha-actinin fraction obtained with this extraction procedure to successive chromatography on DEAE-cellulose and hydroxyapatite. Hydrozyapatite chromatography specifically removes a protein having a subunit molecular weight of 42,000 on sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. Hydroxyapatite-purified alpha-actinin sediments entirely as a 6.21 S boundary in the analytical ultracentrifuge with no trace of the small 9 to 10 S boundary seen in earlier alpha-actinin preparations purified by DEAE-cellulose chromatography. In 100 mM KCl, 20 mM Tris-acetate, pH 7.5, hydroxyapatite-purified alpha-actinin has a diffusion coefficient (D020,w) of 2.71 X 10(-7) cm2-s-1, an intrinsic viscosity of 20.6 ml-g-1, a molecular weight of 201,000 +/- 4,300 (plus or minus least squares standard error) as determined by sedimentation equilibrium, and a molecular weight of 210,000 as determined by sedimentation diffusion. In 6 M guanidine HCl, hydroxyapatite-purified alpha-actinin has a molecular weight of 106,000 +/- 6,300 as determined by sedimentation equilibrium and a molecular weight of 100,000 as determined by a calibrated 4% agarose gel permeation column. SDS-polyacrylamide gel electrophoresis gives a molecular weight of 96,000 to 100,000 for hydroxyapatite-purified alpha-actinin. Rod-shaped particles 44 X 390 to 400 A are seen in electron micrographs of negatively stained alpha-actinin. By assuming 45% hydration and a molecular weight of 206,000, dimensions of approximately 40 X 500 A can be calculated for the alpha-actinin molecule by using either s 020, w, D 020, w, intrinsic viscosity, or a calibrated 6% agarose gel permeation column. Hydroxyapatite-purified alpha-actinin has an alpha-helical content of 74% as measured by circular dichroism at 208 nm.


Banded figers in high temperature coacervates of elastin peptides.

Electron micrographs of negatively stained coacervates of the synthetic polypentapeptide of propoelastin and of alpha-elastin exhibit banded fibers when the coacervates are formed, stained, and dried at temperatures greater than 50 degrees. This apparent increase in order occurs at the same temperature as an increase in order in aqueous solution and as a change in the volume expansion coefficient of fibrous elastin.


Preparation and characterization of wear debris of orthopedic materials for biocompatibility studies.

In order to test the biocompatibility of wear debris of orthopedic materials, a method has been developed to produce artificial debris of stainless steel, cobalt-chromium alloy, and high-density polyethylene. In this process, called the accelerated rubbing process, two cylindrical blocks of the same alloy were held under normal pressure in triply distilled water. One block remained stationary while the other rotated at approximately 4000 rpm. The water with the metal debris was recirculated between the rubbing surfaces. To prepare debris from polyethylene, a stainless steel block was rubbed over a polyethylene block submersed in liquid nitrogen. The shape of the metal debris was granular, whereas the polyethylene debris had a shred-like shape. The size distributions were determined from scanning electron micrographs, and it was found that the particles of the metal debris (stainless steel or cobalt-chrome alloy) ranged in sizes from 0.1-10 mu with 75% of all particles less than 2 mu in diameter. The size of polyethylene debris, expressed as the volume of an ellipsoid, ranged from 10-20,000 mu3 with 45% of all particles in the range of 400-2500 mu3. The x-ray diffraction pattern revealed that the debris of stainless steel consisted primarily of the face-centered cubic (fcc) alloy with small amounts of body-centered cubic (bcc) alloy and Fe3O4, whereas the cobalt-chrome debris consisted of the fcc alloy with a small amount of Cr2O3. The structure of the polyethylene debris was quite similar to that of the solid sample used in its preparation. The percent crystallinity Pc and the size of the crystalline regions D were slightly smaller in the debris than in the solid sample, i.e., Pc = 52 and 55%, D = 90 and 130 A, in the debris and the solid, respectively. To test the suitability of the artificial debris for biocompatibility studies, stainless steel debris was implanted into knees of adult female New Zealand rabbits. Bacterial challenge studies indicated prolongation, but not increased susceptibility to infection.


Biocompatibility of nitinol alloy as an implant material.

The biocompatibility of nitinol alloy as a potential implant material was investigated through in vivo studies on beagles. A high-purity alloy was fabricated into prototype bone plates and implanted into the femurs of beagles. Commercial cobalt-chromium (Co-Cr) alloy bone plates served as reference controls, an additional control data were obtained from beagles subjected to "sham" operations. The bone plates were removed from the animals and examined after exposures of 3, 6, 12, and 17 months. There was no evidence of either localized or of general corrosion on the surfaces of the bone plates and screws. Gross clinical, radiological, and morphological observations of the tissue at the implantation sites during the autopsies uncovered no signs of adverse tissue reactions resulting from the implants. Histological analyses were performed on samples of muscle and bone adjacent to the implantation sites, and of tissues removed from such organs as the liver, spleen, brain, and kidneys. No significant differences were noted between samples taken from controls and those taken from dogs exposed to the implants. Neutron activation analyses were carried out on suitable samples. The analysis data suggest that there is no metallic contamination in the organs due to the implants; however, there does appear to be some chromium contamination from the Co-Cr alloy implants in the adjacent bone. On the basis of the totality of the data, it is concluded that nitinol alloy is sufficiently compatible with dog tissue to warrant further investigation of its potential as a biomaterial.


In vitro effects of bone cement on S. aureus growth and phagocytosis and glucose metabolism of macrophage: a preliminary study.

A preliminary study of the effects of Surgical Simplex, mixed for various time periods, was performed on S. aureus growth rate, and phagocytosis and glucose metabolism of macrophage in vitro. Quantitatively, no significant effects of the mixture on S. aureus growth rate were observed. The cement mixed for less than 4 min tended to cause adverse effects on phagocytosis rate and hexose monophosphate shunt activity of macrophage. This information may not be directly applicable to in vivo, but in vitro the cement mixture in a certain situation can cause decreased phagocytosis and hexose monophosphate shunt activity of macrophage.


A unified mathematical model for diffusion from drug-polymer composite tablets.

The derivation and experimental verification of a unified mathematical model for the estimation of drug release rate from drug-polymer composite tablets are presented. Cylindrical coordinates are utilized in the solution of the diffusion equation for a three-dimensional system. The model is applicable to tablets that range from the shape of a flat disk (radius greater than thickness) to that of a cylindrical rod (radius less than thickness). The general solution for the fraction of drug released at a time t is (see article). This approach to a three-dimensional system, utilizing cylindrical coordinates, presents a comprehensive method for the estimation of drug release rates from sustained release tablets with drug distributed homogeneously throughout a polymer matrix. The calculated and experimental drug diffusion rate of pyrimethamine from pyrimethamine-silicone rubber composite tablets that range in shape from that of a disk to a cylinder, and of hydrocortisone from EVA, polycaprolactone, and PVA terpolymer, are compared.


Polymer-ceramic composite for tooth-root implant.

A new polymer-ceramic composite suitable for tooth-root implants has been developed in this study. This material exhibited the desirable combination of good mechanical properties, controlled porosity, and ease of processing. A thermal processing technique was utilized to polymerize acrylic acid (AA) in the presence of either 0.3 or 0.05 mu alumina particles. Porosity and pore size distribution were influenced by the alumina particle size and the processing technique. For a 50 vol % AA solution, the composite had an average compressive strength of 18,000 psi and 38% porosity when 0.3 mu filler particles were used. In comparison, the 0.05 mu alumina-filled composite had an average compressive strength of 28,000 psi with a 15% porosity. Data on the physical and structural characteristics of the composite are presented in this study. Based on these results, the composite material shows good potential for use in tooth-root implants as well as other orthopedic implant applications.


In vitro corrosion of dental amalgam phases.

The effects of corrosion on the major phases of dental amalgam microstructure have been studied in vitro on samples prepared by electroplating mercury on Ag3Sn. Tests were made in synthetic saliva, and samples were examined in a scanning electron microscope before and after the exposure. Simple immersion had little effect on the phases. Contact of amalgam with gold increased the probability of deterioation of the gamma2 phase. By anodic polarization beyond the breakdown potential, the gamma2 phase was selectively dissolved. Under conditions simulating a crevice, the gamma2 phase was dissolved and the gamma1 phase suffered morphological changes.


Fracture characteristics of acrylic bone cements. I. Fracture toughness.

The vital first phase of the overall materials study to protract the life of the total joint replacements is the identification of the fracture toughness and fatigue properties of bone cements. Information gained from fatigue testing, performed in a manner which simmulates in vivo conditions, and fracture toughness, which is a measure of the propensity of a crack to propagate, is the first step towards the prediction of the life of the total joint replacement. This study is concerned with the fracture toughness of Zimmer and Simplex-P cold-curing bone cements. Following cement fabrication conditions which closely approximate clinical procedures, fracture toughness testing was conducted on cement specimens which were immersed in bovine serum at 37 degrees C in order to simulate in vivo conditions. In addition, a similar study was completed on specimens, tested in air at ambient temperature for purposes of comparison. Results of this procedure, when analyzed by a Student's t-test at the 95% confidence level with eight degrees of freedom, indicate that both Zimmer and Simplex-P exhibit a higher fracture toughness in the simulated physiological environment. In order to determine whether the addition of barium sulfate to these cements compromises the fracture toughness, the above described testing rationale was repeated, indicating the existence of a complicated relationship between the different testing environments and barium sulfate. The importance of these results lies in the fact that an increased fracture toughness indicates that a cement will inherently exhibit a greater degree of resistance to the propagation of cracks, which could contribute to the ultimate failure of the total joint replacement.


Congenital dislocation of the hip. Use of the Pavlik harness in the child during the first six months of life.

From 1968 to 1972, twenty-three infants under six months old with twenty-seven dislocated hips were treated with a Pavlik harness. All the dislocations except three were successfully reduced. Only one child required hospitalization. All the patients were followed for more than two years. All but three of the hips were clinically and roentgenographically normal at follow-up, and none had avascular necrosis. In infants, the Pavlik harness successfully utilizes the principle of reduction in flexion, avoiding forced abduction.


Results of phalangectomy of the fifth toe for hammertoe. The Ruiz-Mora procedure.

The Ruiz-Mora procedure has been advocated for the correction of hammertoe deformities and congenital overlapping of the fifth toe, primarily on empirical grounds. This is a review of forty-one such procedures in which the operation was used for correction of a hammertoe deformity of the fifth toe with a painful corn overlying the proximal interphalangeal joint. Twenty-two patients with thirty-one procedures were followed for an average of 3.5 years. All of the patients had complete relief of symptoms and correction of deformity. However, in 23 per cent of the patients a painful prominent head of the fifth metartarsal or a bunionette developed, and in 32 per cent a hammertoe deformity of the adjacent fourth toe developed with a painful corn either over the proximal interphalangeal joint or on the tip of the fourth toe. These problems seem to be related to excessive shortening of the the fifth toe. Less bone resection (resection of only the head and neck of the proximal phalanx) might be preferable and might not lead to these complications.


The wick catheter technique for measurement of intramuscular pressure. A new research and clinical tool.

The wick catheter technique was developed in 1968 for measurement of subcutaneous pressure and has been modified for easy intramuscular insertion and continuous recording of interstitial fluid pressure in animals and humans. Studies in dogs of the anterolateral compartment of the leg in simulation of the compartment syndrome showed the technique to be accurate and reproducible. The wick catheter technique is capable of important clinical applications in the diagnosis and treatment of acute and chronic compartment syndromes.


Prevention of fatal pulmonary thromboembolism by heparin prophylaxis after surgery for hip fractures.

From 1960 through 1975, 337 patients with surgically treated acute fracture of the hip received subcutaneously administered heparin to prevent thromboembolic disease according to various regimens. Four hundred and three patients received no heparin. The incidence of fatal pulmonary embolism was 3.5 per cent in the 403 patients who reveived no heparin and 0.0 per cent in the 147 patients who were treated by the currently used regimen of prophylaxis, as follows: With the dose modified according to the coagulometer-test time, patients received 2,500 units on admission and every six hours until the day before operation. Then they were given 5,000 to 10,000 units eight to ten hours before surgery and 2,500 units every six hours after surgery until they were fully mobilized.


Thromboembolism following total hip-replacement arthroplasty. The efficicy of dextran-aspirin and dextran-warfarin in prophylaxis.

The efficacy of two antithrombotic regimens, combined dextran and aspirin and combined dextran and warfarin, was analyzed by comparing the incidence of thromboembolism following total hip replacement in two groups of similar patients. Of the 427 who received dextran and aspirin, 7 per cent had thromboembolic complications, including one case of fatal pulmonary embolus and one case of recurrent emboli that required vena caval ligation, and 15 per cent had wound-healing complications. Of the 197 patients who received dextran and warfarin, 5 per cent had thromboembolism and 24 per cent had wound healing complications. Although both prophylactic regimens seemed effective, dextran and aspirin appeared less effective in reducing thromboembolic complications than dextran and warfarin, but there were fewer wound complications in that group. One-fourth of the patients on dextran-warfarin were not adequately anticoagulated despite close supervision. In forty-five patients with a history of thromboembolism who were excluded from the study and analyzed separately, warfarin alone and the two described regimens were equally ineffective in preventing thromboembolism, and the incidence of thromboembolic complications was high. Dextran-aspirin and dextran-warfarin appear to be satisfactory and relatively simple methods of thromboembolic prophylaxis.


A clinical evaluation of aspirin prophylaxis of thromboembolic disease after total hip arthroplasty.

Prophylaxis of thromboembolic disease using aspirin was studied prospectively in 340 patients undergoing total hip replacement, excluding those with prior thromboembolic disease. Clinical criteria were used to diagnose thrombophlebitis, while clinical criteria, roentgenography, blood-gas determinations, electrocardiography, and pulmonary vascular scanning were used to diagnose pulmonary emboli. Five patients had signs of symptoms of pulmonary emboli, all confirmed by pulmonary vascular scan. No fatal pulmonary emboli occurred. Twenty-six patients had thrombophlebitis. Without simultaneous control group, a reduction in postoperative thromboembolic disease in these patients receiving aspirin was not proved. However, the observed low incidence of clinically evident thromboembolic manifestations suggests that aspirin may be a simple and useful prophylactic agent in these patients.


Thromboembolic disease in patients undergoing total knee replacement.

In a prospective study fo the incidence of deep-vein thrombosis in thirty patients undergoing total knee replacement, all patients had clinical examinations and 125I fibrinogen scanning, while those suspected of having deep venous thrombosis also had confirmatory venography. Sixteen (53 per cent) of the thirty patients had thromboembolic disease; nine had thrombi only in the limb operated on; four had bilateral deep venous thrombi; and three had pulmonary embolism. In nine patients who took aspirin regularly the incidence of thromboembolism was 11 per cent, while in the eight who did not take aspirin or any other antiplatelet drug the incidence was 88 per cent, a difference which was highly significant (p = 0.003).


Venous thrombosis after total hip replacement. Combined monitoring as a guide for prophylaxis and treatment.

Monitoring of 140 patients after hip-replacement arthroplasty using both impedance plethysmography and 125I fibrinogen scanning of the lower extremities showed that half had no evidence of thrombosis, one-quarter had moderate or extensive thrombosis, and one-quarter had abnormal scans only. In the patients with only abnormal scans the process appeared to resolve spontaneously. The findings by impedance plethysmography therefore appeared to differentiate non-invasively between thromboses that would and would not resolve, and it is suggested that for some patients a monitoring regimen may be preferable to routine prophylaxis.


Cuff-impedance phlebography and 125I fibrinogen scanning versus roentgenographic phlebography for diagnosis of thrombophlebitis following hip surgery. A preliminary report.

Seventy-eight limbs of forty-eight patients undergoing total hip replacement were studied by roentgenographic phlebography, cuff-impedance phlebography, and 125I fibrinogen scanning. Compared with roentgenographic phlebography, cuff-impedance phlebography detected seven of ten thrombi in the thigh but none of nine thrombi in the calf and popliteal veins, giving an over-all accuracy of 80 per cent. The 125I fibrinogen scanning technique detected none of the ten thrombi in the thigh and seven of the nine in the calf and popliteal veins, giving an over-all accuracy of 78 per cent. Combining the results of the two techniques, fourteen of the nineteen thrombi were detected. Cuff-impedance phlebography appears to be a useful method for the diagnosis of thrombi in the thigh after hip surgery.


Long-term results in the treatment of femoral-shaft fractures in young children by immediate spica immobilization.

Manual traction reduction without anesthesia and immediate immobilization in a spica cast were used to treat eighty-five children ranging in age from birth to ten years old who had closed femoral-shaft fractures and no associated injuries. Up to two centimeters of overriding, 30 degrees of anterior angulation, and 15 degrees of medial angulation were accepted. Any angulation in excess of these amounts, or lateral or posterior angulation, was corrected by wedging the cast at the fracture site. Further telescoping of the fracture fragments in the case was attributed to the child pressing the foot against the bottom of the cast and was prevented by removing the sole of the cast. Of the seventy-five children examined two to eighteen years after fracture, none had any residual skeletal deformity or joint stiffness. The length discrepancies of the fractured limbs ranged from 1.7 centimeters of shortening to 0.9 centimeter of overgrowth.


Results of proximal tibial osteotomy. The effects of tibiofemoral angle, stance-phase flexion-extension, and medial-plateau force.

Forty-eight knees were evaluated after proximal tibial osteotomy, performed for varus deformity to determine the desired amount of correction of the deformity, the effect of osteotomy on knee motion during gait and one medial-plateau force during standing, and the relationships between these factors and the result. Correction of the tibiofemoral angle to 5 degrees of genu valgum or more produced the best and most lasting results. Stance-phase flexion-extension increased the rotation decreased in knees with good results while the other gait parameters were not significantly changed. Medial-plateau force was decreased by successful tibial osteotomy. The knees with the best and most lasting results had 7 degrees of stance-phase flexion-extension or more during walking and either a valgus tibiofemoral angle of 5 degrees or more or a medial-plateau force of 50 per cent of body weight or less.


The arterial supply of the developing proximal end of the human femur.

By means of perfusion studies, an analysis was made of the arterial supply to the proximal end of the femur in 150 specimens from autopsied fetuses and children, aged from twenty-six weeks of gestation to fourteen years and eight months old. All died of diseases which did not involve the hip joint. Two anastomotic rings were found: an extracapsular one formed by the medial and lateral femoral circumflex arteries, and a subsynovial intra-articular ring at the articular cartilage-neck junction. The intra-articular rings in males were discontinuous more often than in females. A three-plane analysis of totally-cleared specimens demonstrated that the epiphyseal plate constituted an absolute barrier to blood flow between the epiphysis and metaphysis in all but two of the 124 barium sulphate-perfused specimens examined. A smaller number of ascending cervical arteries crossed the anterior and medial surfaces of the mid-neck in the specimens from three to ten-year-old white children than in those from newborn to two-year-old white and black children. This finding may be important for the etiology of Legg-Perthes disease. No differences with respect to age, sex, or race were found in the arteries of the ligamentum teres.


Triple tenodesis of the knee. A soft-tissue operation for the correction of paralytic genu recurvatum.

There are two types of genu recurvatum: one with bone deformity, which responds well to osteotomy, and one with posterior soft-tissue laxity with secondary bone changes. To date, no reliable treatment short of arthrodesis has been effective for the second type. An operation for this type of genu recurvatum (post-poliomyelitic) is described: a soft-tissue reconstruction of the lax tissues posterior to the knee joint done in three layers. The operation was done on sixteen knees in fourteen patients, with an average follow-up of four years and three months. The average recurvatum before surgery was 42 degrees and at the time of follow-up it was 6 degrees. Preliminary surgery is often required and precise surgical technique and prolonged bracing after surgery also are needed. All but one patient was made brace-free provided the limb operated on was not flail.


Gliding function following flexor-tendon injury. A biomechanical study of rat tendon function.

Gliding function of flexor tendons one hour to eight weeks after a standard injury was studied in rat digits by determining terminal force of flexion, tendon excursion, and work of flexion using a tensile testing machine. A rapid decrease in gliding occurred within hours of injury, indicating that the postoperative hematoma and edema restrict gliding long before collagenous adhesions are synthesized. The improvement in gliding function with time implies that fibrous adhesions can be remodeled. Surgical methods and therapeutic agents, therefore, should be directed toward limiting early tissue injury and seeking ways to enhance the late remodeling of fibrous peritendinous adhesions along functional lines.


Ruptures of the tendo achillis. An objective assessment of surgical and non-surgical treatment.

A comparative study is presented of seventy-nine patients with rupture of the tendo achillis, forty-eight of whom were treated surgically and thirty-one, non-surgically. With one exception, all were healthy, athletic individuals. Twenty-two per cent of the patients had an initial misdiagnosis. The patients treated surgically were more satisfied with the results of their treatment. Strength, power, and endurance as measured on the Cybex II dynamometer revealed that the patients treated non-surgically attained only 72 per cent of normal strength and 70 per cent of normal power and endurance as compared with the surgically treated patients. Wound complications occurred in only two patients and no reruptures occurred in the surgically treated group, whereas there were nine rereptures in the group not treated surgically.


Osteolytic form of Paget's disease. Differential diagnosis and pathogenesis.

Of fourteen patients with Paget's disease for whom adequate roentgenograms and pathological material were available, six had lesions showing significant or predominant osteolysis. Based on a study of these six patients and a review of the literature, the following mechanisms were identified as causes of the so-called osteolytic type of the disease: (1) an "early" destructive Paget's lesion; (2) "advanced" Paget's disease with secondary degenerative changes; (3) "seeding" of an independent osteolytic lesion, particularly tumor in pre-existing Paget's disease; (4) sarcomatous transformation; and (5) immobilization after fracture. An osteolytic lesion in a patient without other bone disease may be due to Paget's disease, while an associated lytic lesion may be the result of a variety of conditions. Accurate pathological diagnosis is essential.


A comparison of computer asisted tomography (EMI scanner) with conventional neuroradiologic methods in the investigation of patients clinically suspected of intracranial tumor.

Computerized tomography has been used as the initial radiologic investigation among 212 intracranial tumor suspects. Ninety-five of these patients were ultimately shown to have tumors as a result of all their investigations. Eighty tumors were clearly defined by EMI scanning, four further scans were suspicious but not unequivocally abnormal and six scans showed complications of the tumor rather than the lesion itself. Thus, only five scans were falsely negative in the presence of a tumor. The false positive and false negative rates for EMI scanning have been compared with conventional techniques and computerized tomography shown to compare favourably with angiography or encephalography.


Angiographic assessment of pancreatic pseudocyst: a reappraisal.

The correct diagnosis was established by angiography in 16 of 21 patients with pancreatic pseudocyst. The characteristic angiographic abnormalities are arcuate stretching of intrapancreatic arteries combined with a filling defect in the parenchymal phase of the angiogram. In the five false negative patients, the diagnosis was not made either because of the small size of the pseudocyst or a primary extrapancreatic location of the lesion. Because of this high false negative rate and the accuracy of B-mode ultrasonic scanning in abdominal cystic lesions, ultrasound should be the primary diagnostic modality in patients with suspected pancreatic pseudocysts.


Mönckeberg's arteriosclerosis revisited: or silver vessels among the old.

This study showed that Mönckeberg's arteriosclerosis was found in 23 patients over the age of 60. Diabetes or probable diabetes mellitus was found in 22 out of 23 of these patients. It is concluded that roentgenographic demonstration of MAS in this age group is highly specific for diabetes mellitus. Further, a pilot study suggests that if MAS is present, without demonstrable plain film evidence of coexistent IAS, the chances of the patient having elevated cholesterol seems to be approximately one quarter that of the general population over 60. His chances of an elevated triglyceride appear to be about half that of the same population group.


Gastric and small bowel polyps in Gardner's syndrome and familial polyposis coli.

The adenomas of Familial Polyposis Coli and of Gardner's syndrome are widely thought to affect the large bowel only. However, this report and a critical review of the literature shows that of the few hundred reported cases of these diseases, at least thirty-six have also shown gastric and/or small bowel polyps. The majority have been concentrated in stomach, duodenum and ampulla of Vater and the latter two areas have shown the highest incidence of carcinoma. It seems that gastric polyps are especially associated with Familial Polyposis Coli, and duodeno-ampullary ones with Gardner's syndrome.


Structure of the cell envelope of Halobacterium halobium.

The structure of the isolated cell envelope of Halobacterium halobium is studied by X-ray diffraction, electron microscopy, and biochemical analysis. The envelope consists of the cell membrane and two layers of protein outside. The outer layer of protein shows a regular arrangement of the protein or glycoprotein particles and is therefore identified as the cell wall. Just outside the cell membrane is a 20 A-thick layer of protein. It is a third structure in the envelope, the function of which may be distinct from that of the cell membrane and the cell wall. This inner layer of protein is separated from the outer protein layer by a 65 A-wide space which has an electron density very close to that of the suspending medium, and which can be etched after freeze-fracture. The space is tentatively identified as the periplasmic space. At NaCl concentrations below 2.0 M, both protein layers of the envelope disintegrate. Gel filtration and analytical ultracentrifugation of the soluble components from the two protein layers reveal two major bands of protein with apparent mol wt of approximately 16,000 and 21,000. At the same time, the cell membrane stays essentially intact as long as the Mg++ concentration is kept at treater than or equal to 20 mM. The cell membrane breaks into small fragments when treated with 0.1 M NaCl and EDTA, or with distilled water, and some soluble proteins, including flavins and cytochromes, are released. The cell membrane apparently has an asymmetric core of the lipid bilayer.


Environmentally induced changes in mitochondria and endoplasmic reticulum of Saccharomyces carlsbergensis yeast.

The effects of culture environment on the volume density and surface density of mitochondria and endoplasmic reticulum in a facultative yeast were studied. When compared with cells grown aerobically on a nonrepressive substrate, cells grown in the absence of oxygen showed a sharp reduction in both volume density of mitochondria and surface density of the inner mitochondrial membrane (imm) in the remaining mitochondrial profiles. Use of fermentable (repressive) substrates under aerobic conditions restricted the volume density of mitochondria to a much greater extent than the surface density of imm. The range of mitochondrial volume densities in these experiments was 4-11%. Surface density of endoplasmic reticulum (ER) was sensitive to growth rate and in particular to changes in oxygen tension, showing large fluctuations during both anaerobic and aerobic adaptation. These fluctuations in ER are discussed in relation to the known role of this organelle in lipid metabolism.


125I-labeled human epidermal growth factor. Binding, internalization, and degradation in human fibroblasts.

125I-labeled human epidermal growth factor (hEGF) binds in a specific and saturable manner to human fibroblasts. At 37 degrees C, the cell-bound 125I-hEGF initially may be recovered in a native form by acid extraction; upon subsequent incubation, the cell-bound 125I-hEGF is degraded very rapidly, with the appearance in the medium of 125I-monoiodotyrosine. At 0 degrees C, cell-bound 125I-hEGF is not degraded but slowly dissociates from the cell. The data are consistent with a mechanism in which 125I-hEGF initially is bound to the cell surface and subsequently is internlized before degradation. The degradation is blocked by inhibitors of metabolic energy production (azide, cyanide, dinitrophenol), some protease inhibitors (Tos-Lys-CH2Cl, benzyl guanidobenzoate), a lysosomotropic agent (chloroquine) various local anesthetics (cocaine, lidocaine, procaine), and ammonium chloride. After the binding and degradation of 125I-hEGF the fibroblasts are no longer able to rebind fresh hormone. The binding capacity of these cells is restored by incubation in a serum-containing medium; this restoration is inhibited by cycloheximide or actinomycin D.


Involvement of bristle coat structures in surface membrane formations and membrane interactions during coenocytotomic cleavage in caps of Acetabularia mediterranea.

In the multinucleate cap rays of the green alga Acetabularia mediterranea the cell surface increases dramatically within a short time period during the final stages of coenocytotomic cleavage. In early stages of cyst formation the cytoplast is traversed by numerous large and prolate cleavage vesicles which are characterized by typical columellar or spinous coat structures. The cleavage vesicles are closely associated with the surface of plastids and, to a lesser degree, of mitochondria. This intimate association seems to be mediated by regularly spaced, densely stained intermembranous cross-bridge structures and is maintained throughout cleavage. These cleavage vesicles contain a finely fibrillar material structurally similar to the hyaline layer of mucilage that fills the space between the plasma membrane and cell wall. They line up with invaginations of the plasmalemma and vacuole membranes and, together with smaller vesicles interspersed, constitute preformed "perforation lines" for the final separation of the coenoblast portions. Equidistantly spaced plaques of attachment of such vesicles with surface membrane are described. We hypothesize (a) that the cleavage vesicle membrane is the immediate precursor to the new postcoenocytotomic surface membrane, (b) that the cleavage vesicle coat structures are integrated into the subsurface coat of the plasma membrane, (c) that growth of the laterally attached cleavage vesicles by intussusception of small fuzzy-coated vesicles is confined to their "free ends," (d) that the intermembranous cross-bridge elements are related to bristle coat structures and play a role in the establishment of the cleavage lines, and (e) that the coenocytotomic cleavage process is organized so that adjacent plastids are separated in a way that guarantees the inclusion of several plastids in each cyst.


Variations in distribution of con A receptor sites and anionic groups during red blood cell differentiation in the rat.

The distribution of receptors for concanavalin A (Con A) and anionic groups on plasma membranes of developing blood cells was investigated in the rat. Glutaraldehyde-fixed bone marrow and circulating blood cells were exposed to ferritin-conjugated Con A or positively chaged ferric oxide (CI) and processed for electro n microscopy. The frequency of Con A and CI binding sites varied during different erythroid developmental stages and amont different leukoid cell types. There was a constant inverse relationship between Con A and CI binding sites. Among leukoid cells, Con A binding was high on plasma cells and macrophages, lower on neutrophils and lymphocytes, and still lower on eosinophils and basophils; CI binding was highest in the latter and lowest in plasma cells and macrophages. Among erythroid cells, there was a progressive increase in Con A and a decrease in CI binding after successive divisions of erythroblasts, and a progressive decrease in Con A and an increase in CI binding upon maturation of the orthochromatic erythroblast to the reticulocyte. The most pronounced modification in distribution of these sites occurred during nuclear expulsion: that protion of the plasma membrane surrounding the extruded nucleus was heavily labeled by Con A (up to four times that of the orthochromatic erythroblast) whereas the reticulocyte had considerably fewer sites. The situation was reversed with CI. The results suggest that the concentration of nonsialated glycoproteins (to which Con A binds) varies inversely to that of sialoproteins (to which CI binds) in the membrane of the differentiating erythroid cell. The remarkable changes observed at the time of nuclear extrusion suggest that there is either local modification of glycosylated groups with removal of sialyl residues from the membrane surrounding the extruded nucleus of selective segregation of membrane glycoproteins leading to a high concentration of sialoproteins (glycophroin) in the membrane of the mature erythrocyte.


Redistribution of surface anionic sites on the luminal front of blood vessel endothelium after interaction with polycationic ligand.

The ability of anionic groups on the luminal surface of blood vessels to redistribute by lateral migration under the influence of multivalent ligands was analyzed by electron microscopy, using cationized ferritin (CF). In vitro interaction of blood vessel segments with CF results in rapid aggregation of most anionic sites on the luminal fromt of the endothelium, followed by internalization or detachment of the CF patches, leaving most of the luminal surface devoid of anionic sites. Further incubation of such endothelial cells without CF results in regeneration of binding capacity for the polycationic label. Transport of CF, but not of native ferritin, across the endothelium by vesicle transport, followed by exocytosis of the interiorized CF clusters on the tissue front of the endothelium, was also observed. The possibility that such activities in the blood vessels in vivo may be associated with local changes in the normal distribution of the surface anionic sites as well as in accumulation of debris in the subendothelial layers of the vessels is suggested.


Paramyosin in invertebrate muscles. II. Content in relation to structure and function.

By quantitative sodium dodecyl sulfate-polyacrylamide gel electrophoresis, paramyosin:myosin heavy chain molecular ratios were calculated for three molluscan muscles:Aequipecten striated adductor, Mercenaria opaque adductor, and Mytilus anterior byssus retractor; and four arthropodan muscles:Limulus telson, Homarus slow claw. Balanus scutal depressor, and Lethocerus air tube retractor. These ratios correlate positively with both thick filament dimensions and maximum active tension development in these tissues. The role of paramyosin in these muscles is discussed with respect to the following characteristics: force development, "catch," and extreme reversible changes in length.


Relationship of transformation, cell density, and growth control to the cellular distribution of newly synthesized glycosaminoglycan.

Mouse 3T3 cells and their Simian Virus 40-transformed derivatives (3T3SV) were used to assess the relationship of transfromation, cell density, and growth control to the cellular distribution of newly synthesized glycosaminoglycan (GAG). Glucosamine- and galactosamine-containing GAG were labeled equivalently by [3H=A1-glucose regardless of culture type, allowing incorporation into the various GAG to be compared under all conditions studied. Three components of each culture type were examined: the cells, which contain the bulk of newly synthesized GAG and are enriched in chondroitin sulfate and heparan sulfate; cell surface materials released by trypsin, which contain predominantly hyaluronic acid; and the media , which contain predominantly hyaluronic acid and undersulfated chondroitin sulfate. Increased cell density and viral transformation reduce incorporation into GAG relative to the incorporation into other polysaccharides. Transformation, however, does not substantially alter the type or distribution of newly synthesized GAG; the relative amounts and cellular distributions were very similar in 3T3 and 3T3SV cultures growing at similar rates at low densities. On the other hand, increased cell density as well as density-dependent growth inhibition modified the type and distribution of newly synthesized GAG. At high cell densities both cell types showed reduced incorporation into hyaluronate and an increase in cellular GAG due to enhanced labeling of chondroitin sulfate and heparan sulfate. These changes were more marked in confluent 3T3 cultures which also differed in showing substantially more GAG label in the medium and in chondroitin-6-sulfate and heparan sulfate at the cell surface. Since cell density and possibly density-dependent inhibition of growth but not viral transformation are major factors controlling the cellular distribution and type of newly synthesized GAG, differences due to GAG's in the culture behavior of normal and transformed cells may occur only at high cell density. The density-induced GAG alterations most likely involved are increased condroitin-6-sulfate and heparan sulfate and decreased hyaluronic acid at the cell surface.


Cytochalasin B inhibits actin-related gelation of HeLa cell extracts.

When the 100,000 g supernatant fraction (extract) of HeLa cells lysed in a buffer containing sucrose, ATP, DTE, EGTA, imidazole, and Triton X-100 is incubated at 25 degrees C, it gels, and actin and a HMWP are progressively enriched in the extract and in gel isolated from extract. CB (greater than or equal to 0.25 muM) inhibits gelation and specifically lowers the concentrations of actin and the HMWP in the fraction which sediments at 100,000 g after incubation. These results indicate that actin and HMWP are partly disaggregated by cytochalasin treatment, and thus that their aggregation is related gelation. Inasmuch as previous results showed that actin is present and HMWP is enriched in the plasma membrane fraction of HeLa cells, the results also point to a possible relation between plasma membrane-associated gel and in vivo effects of CB.


Absence of rDNA amplification in the uninucleolate oocyte of the cockroach Blattella germanica (Oorthoptera: Blattidae).

Amplification of the genes coding for ribosomal RNA oocurs in the oocytes of a wide variety of organisms. In oocytes of various species of crickets (Orthoptera: Gryllidae) the amplified DNA is contained in a large extrachromosomal DNA body. Multiple nucleoli form about the periphery of the DNA body during the diplotene stage of meiosis I. In contrast to the general pattern of orthopteran oocytes, oocytes of the cockroach Blattella germanica demonstrate a single large nucleolus instead of many nucleoli. In order to determine whether the genes coding for rRNA are amplified in the oocytes of B. germanica, the relative amount of rDNA in oocytes was compared with the rDNA content of spermatocytes and somatic cells. An extrachromosomal DNA body similar to that present in crickets is not present in B. germanica. A satellite DNA band which contains nucleotide sequences complementary to rRNA accounts for approximately 3-5% of the total DNA in somatic and in male and female gametogenic tissues. Female cells contain approximately twice as much rDNA as do male cells. An XX-XO sex-determining mechanism is operative in B. germanica. In situ hybridization with rRNA indicates that the nucleolar organizer is located on one end of the X chromosome and that oocytes do not contain more than twice the amount of rDNA found in spermato cytes. The data indicate that rDNA is not amplified in the uninucleolate oocyte of B germanica.


Vitreous body collagen. Evidence for a dual origin from the neural retina and hyalocytes.

Two different cells types have been shown to synthesize embryonic chick vitreous collagen (vitrosin) at different stages of development. Identification of vitrosin was established by labeling the embryos in ovo [3H]proline at stages 23 and 28 and separating the extracted vitreous collagen alpha-chains by carboxymethylcellulose chromatography. The labeled collagen consisted predominately of alpha 1 chains, indicating a molecule in the form of a trimer of identical chains designated (alpha 1)3. The molecular weight of the labeled chains measured approximately 95,000 daltons by molecular sieve chromatography, and contained 41% of their imino acid as 4-hydroxyproline. To establish which eye tissues synthesize vitrosine, the collagens produced in organ culture by the isolated neural retina, lens and vitreous body from stages 26-27, 29-30, and 40 were examined. At the two earlier stages, only the neural retina synthesized large quantities of (alpha 1)3 collagen whereas the lens and the cells within the vitreous body itself synthesized relatively small amounts of collagen characterized by an alpha 1:alpha 2 ratio of about 2:1. At stage 40, however, the cells of the vitreous body itself synthesized the greatest quantities of collagen, which now was predominantly an (alpha 1)3 type molecule. Stage 40 neural retina and lens synthesized lesser amounts of collagen with an alpha 1:alpha 2 ratio of 2 to 3:1. Chick vitrosin thus appears to be synthesized by the neural retina in early embryonic stages, whereas the major contribution derives from cells within the vitreous body in later development.


Intercellular adhesive selectivity. III. Species selectivity of embryonic liver intercellular adhesion.

A species difference in the intercellular adhesive selectivity of mixtures of embryonic liver cells is reported. This is first quantitative assessment of species differences in the intercellular adhesive properties of embryonic cells. A collecting aggregate assay, a new double-label assay procedure, and histological and autoradiographic procedures were used to elucidate the intercellular adhesive selectivity of developing mammalian and avian liver cells. Evidence is presented that the reported adhesive differences are not due to the different cell types composing the respective embryonic mammalian and avian livers. Finally, such heterolgous-homotypic selectivity of adhesion is not a property of all tissues, since it is shown that developing brain cells (mesencephalon) do not exhibit the avove intercellular adhesive selectivity (mammalian vs. avian). These findings provide further support for the hypothesis that generic identity as well as cell type may play an important part in determining the intercellular adhesive behavior of heterologous-homotypic mixtures of embryonic cells. A possible evolutionary divergence of morphogenetic mechanisms is discussed.


Interrelationships between water and cellular metabolism in Artemia cysts. V. 14CO2 incorporation.

The ability of cysts of the brine shrimp, Artemia salina, to incorporate 14CO2 into organic compounds soluble in cold-trichloroacetic acid was examined over a broad range of cellular water concentrations. Carbon dioxide was not incorporated by cysts containing less than about 0.3 g H2O/g dried cysts, the "critical hydration" for CO2-fixation. This relationship held whether the cysts were hydrated from the liquid or the vapor phase. The incorporation of radioactivity was shown to be due exclusively to metabolic activity in the cellular component of the cyst. Above the critical hydration, the amount of 14CO2 incorporated was a function of cyst water content, but the kinds of metabolites labelled with this precursor, and their relative proportions, were found to be similar in cysts of greatly different hydration. Almost all of the radioactivity was associated with amino acids, Krebs cycle intermediates and related acids, and pyrimidine nucleotides. The fact that the pathway involved with CO2-fixation, and subsequent metabolism of the fixation products are all initiated in cysts containing as little as 0.3 g H2O/g is particularly noteworthy since this hydration level is well within the range of the amounts of "bound water" described in the literature for a wide array of cells and tissues.


Endotoxin-induced size change in bone marrow progenitors of granulocytes and macrophages.

Injection of 5 mug endotoxin to adult C57BL mice caused a marked increase in the sedimentation velocity of granulocytic and macrophage progenitor (colony-forming) cells in the bone marrow. This change was maximal two days after injection and was not accompanied by corresponding changes in total marrow nucleated cell populations. The endotoxin-induced shift was not dependent on the presence of the thymus but did not occur in mice challenged after preinjection with endotoxin. No changes in buoyant density, cell cycle status, pattern of differentiation and responsiveness of granulocytic and macrophage progenitor cells were observed after the injection of endotoxin. The increased sedimentation velocity of progenitor cells appears to indicate an increase in cell volume but the mechanisms involved have not been identified.


Stimulation of DNA synthesis, cell multiplication, and ornithine decarboxylase in 3T3 cells by multiplication stimulating activity (MSA).

Multiplication stimulating activity (MSA) has been purified from the conditioned media of rat liver cells in culture by a modification of the procedure of Dulak and Temin. Purified MSA stimulates [3H] thymidine incorporation into DNA in subconfluent, serum starved 3T3 cells. Cell cycle analysis by the flow microfluorometer shows that the [3H] thymidine incorporation data reflects DNA synthesis. MSA also stimulates the multiplication of serum starved subconfluent 3T3 cells. MSA is approximately 10-fold less active in 3T3 cells than in chick embryo fibroblasts in stimulating [3H] thymiding incorporation into DNA. MSA causes a 2--10-fold increase in ornithine decarboxylase (ODC) activity in 3T3 cells and the dose response curve parallels the dose response curve for [3H] thymidine incorporation into DNA. The Km of ODC for ornithine is 0.12 mM. There is a 30% decrease in the activity of ornithine transaminase (OTA) during the time period in which MSA causes an increase in ODC activity. Insulin also stimulates [3H] thymidine incorporation into DNA, cell multiplication and ODC activity over the same concentration range as shown for MSA, however, the extent of stimulation by insulin is less than that observed following MSA addition.


Potassium transport in normal and transformed mouse 3T3 cells.

The components of unidirectional K influx and efflux have been investigated in the 3T3 cell and the SV40 transformed 3T3 cell in expontntial and stationary growth phase. Over the cell densities used for transport experiments the 3T3 cell goes from exponential growth to density dependent inhibition of growth (4 X 10(4) to 4 X 10(5) cell cm-2) whereas the SV40 3T3 maintains exponential or near exponential growth (4 X 10(4) to 1 X 10(6) cell cm-2). In agreement with previous observations, volume per cell and mg protein per cell decrease with increasing cell density. Thus, transport measurements have been expressed on a per volume basis. Total unidirectional K influx and efflux in the 3T3 cell is approximately double that of the SV40 3T3 cell at all cell densities investigated. Both cell types have similar volumes initially and show similar decreases with increasing cell density. Thus, in this clone of the 3T3 cell SV40 transformation specifically decreases unidirectional K flux. The magnitude of the total K flux does not change substantially for either cell line during transition from sparse to dense cultures. However, the components of the K transport undergo distinct changes. Both cell lines possess a ouabain sensitive component of K influx, presumably representing the active inward K pump. Both also possess components of K influx and efflux sensitive to furosemide. The data suggest this component represents a one-for-one K exchange mechanism. The fraction of K influx mediated by the ouabain sensitive component is reduced to one half its value when exponential versus density inhibited 3T3 cells are compared (63% versus 31% of total influx). No comparable drop occurs in the SV40 3T3 cell at equivalent cell densities (64% versus 56% of total influx). Thus, the pump mediated component of K influx would appear to be correlated with growth. In contrast, the furosemide sensitive component represents approximately 20% of the total unidirectional K influx and efflux in both cell lines in sparse culture. At high cell densities, where growth inhibition occurs in the 3T3 cell but not the SV40 3T3, the furosemide sensitive component doubles in both cell lines. Thus, the apparent K-K exchange mechanism is density dependent rather than growth dependent.


Growth of cultured cells from patients with ataxia-telangiectasia.

Fibroblast cultures derived from skin biopsies of patients with ataxia-telangiectasia had doubling times (mean of five lines: 29.9 +/- 0.6 hours) significantly longer than normal controls (mean of ten lines: 22.4 +/- 0.5 hours).


Alpha-aminoisobutyric acid transport into human glia and glioma cells in culture.

The AIB transport into human glia and glioma cells in culture has been studied. Because of the high affinity of AIB to the plastic culture dishes, a special washing technique had to be developed. With this technique, it was possible to perform transport experiments in a single plate containing about one million cells. The cells were viable, intact and adhered to the supporting medium throughout the experiment. The AIB transport into both types of cells was Na+-dependent and showed saturation kinetics when the small component of the transport due to diffusion had been subtracted. The AIB transport capacity of neoplastic glioma cells was 3.6 times higher than that of glia cells. This difference was related to the Vmax-values for the two types of cells. The apparent Km-values were the same. Inhibition experiments with other amino acids support the view that AIB is transported via System A in both glia and glioma cells. Sulfhydryl reagents (ethacrynic acid and NEM) and cytochalasin B clearly inhibited the AIB transport into glia cells whereas the effect on glioma cells was minimal.


Different recovery patterns of mouse haemopoietic stem cells in response to cytotoxic agents.

The response and subsequent recovery of mouse haemopoietic progenitor cells (spleen colony forming cells and agar colony forming cells) has been studied following two cytotoxic agents. Busulphan was administered to normal mice and vinblastine to mice where the progenitor cell proliferation rate had been increased by a period of continuous gamma-irradiation. With both these agents there is a difference between the response of the spleen colony forming cells and the agar colony forming cells during the first five days. They then recover together, but much more slowly after fusulphan than after vinblastine even though their proliferation rate is increased. The rate of progenitor cell recovery after busulphan is increased if the progenitor cells are depleted further by vinblastine. However, methotrexate, which severely depletes the peripheral blood count and bone marrow cellularity but not the progenitor cells, has no effect on the recovery following busulphan. These results suggest that following cytotoxic agents the agar colony forming cells ("committed" stem cells) are not self-maintaining but are dependent on a supply of cells from the pluripotential spleen colony forming cells. In addition it appears that the depletion of the progenitor cells of the bone marrow and not the depletion of the maturing cells, provides a stimulus for stem cell recovery.


A possible involvement polyamines in the initiation of DNA synthesis by human WI-38 and mouse BALB/3T3 cells.

Changing the medium, or adding fresh serum, induces a large proportion of the proliferatively quiescent cells in confluent monolayers of human WI-38 and mouse BALB/3T3 cells to initiate a growth-division cycle. Exposure at the time of the medium change or serum addition to MGBG (methyl glyoxal bis [guanylhydrazone]), an inhibitor of spermidine and spermine synthesis and function, reduces or stops the subsequent flow of cells into the DNA-synthetic phase, without grossly affecting RNA synthesis. Mediation of MGBG action by an actual or functional shortage of spermidine or spermine (but not putrescine), and consequently an involvement of these polyamines in DNA synthesis, is strongly suggested by the reduction of the inhibitor's effectiveness by a brief (1-hour), early prereplicative exposure of the treated cells to exogenous spermidine and spermine (but not putrescine).


Human erythroid colony formation in vitro: evidence for clonal origin.

Human marrow cells, suspended in methylcellulose medium containing erythropoietin, give rise to discrete colonies of hemoglobin synthesizing cells. The presumption that such colonies originate from single progenitor cells has been tested directly in females with X-chromosome inactivation mosaicism using glucose-6-phosphate dehydrogenase (G-6-PD) as a marker. When individual colonies were grown from marrow cells obtained from two black females heterozygous for G-6-PD, only one or the other isoenzyme type was observed, but not both. These results are most consistent with the interpretation that human erythroid colonies arise from single cells.


The ordered columnar structure of mouse filiform papillae.

Examination of carefully oriented 1-mum and 5-mum sections of mouse dorsal tongue together with scanning electron microscope observations indicates a high degree of cellular organization in the papillae. It has been suggested that each filiform papilla consists of 2 dominant and 2 minor columns of cells. Labelling patterns of the basal cells have been investigated in relation to these columns.


Sulphure in epidermal keratohyalin granules: a quantitative assay by x-ray microanalysis.

The elemental composition of different types of keratohyalin granules from the epidermis of newborn and adult rats was studied by means of an EMMA-4 analytical electron microscope, equipped with an energy-dispersive X-ray spectrometer. An absolute quantitation of the sulphur concentration in keratohyalin granules was performed. The results demonstrate that epidermal keratohyalin granules are chemically heterogeneous. A type of keratohyalin granule present in the nuclei and cytoplasm of epidermal cells from both newborn and adult rats - termed single granules - is rich in sulphur, having a content of 2-5-3-6%. Other types of keratohyalin granules, which differ in newborn and adult rats, contain a sulphur-poor component; they often have a sulphur-rich component as well. The sulphur-poor keratohyalin contains 0-6-0-9% sulphur. It is suggested that the sulphur-rich keratohyalin granules are the source of the peripheral envelope protein of cornified cells.


Rapid modification of the morphology of cell contact sites during the aggregation of limpet haemocytes.

Haemocytes suspended in the blood of the limpet Patella vulgata rapidly form microspikes and aggregate in response to withdrawal from the haemocoel. It has previously been suggested that the contacts between colliding cells in shaken suspension are stabilized by the rapid spreading of participant cells over each other's surfaces by a microspike-dependent process. In this communication we report that an electron-microscopic study of the contacts formed between cells in the early stages of aggregation suggests that intercellular contact elicits a rapid response from the organelles concerned with cell motility. This response leads to the rapid progression of each initial collision through 3 major categories of contact morphology, namely cell body/lamella, lamella/lamella and cell body/cell body. There is a concomitant increase in the area of mutually contacting surface between participating cells. On the basis of these results, a model is constructed for the participation of sub-surface components in the process of cellular aggregation. The sequence of development of contact found between aggregating cells is envisaged to be a consequence of cellular locomotory activities resembling those by which these cells spread on to a solid substrate. Included in this process is an element of intercellular recognition. It is suggested that the involvement of motile elements in cell aggregation is a general feature of aggregating cells. This would permit a re-interpretation of aggregation experiments which show specificity of cell adhesion, for total adhesive interactions between cells would depend not only on the adhesiveness per unit area of contacting cell surface but also on the extent or rate of formation of flat contact areas. The latter element could be regulated by a recognition system between contacting cells independently of the force per unit area which holds the 2 surfaces together.


Some ultrastructural aspects of mitosis and early differentiation in human foetal lung.

In rapidly dividing human foetal lung cells there is evidence of synthesis of extra membrane prior to mitosis. During prophase the nuclear envelope breaks down and is conserved as long double or complex stacks during the following stages of mitosis. It is suggested that the membrane is retained partly for incorporation into the daughter cell nuclear envelopes and partly for the formation of RER. The relevance of the observations to the biochemical data in the literature concerning the formation of intracellular membranes is discussed briefly.


The nucleolus organizer and the synaptonemal complex in Endymion non-scriptus (L.).

Stages of meiosis from the bluebell Endymion non-scriptus (L.) were studied by electron microscopy. The nucleolus went through the process of segregation at the beginning of meiosis with the movement to its surface of a pale-staining region. This region was shown to be the same as that called the 'L zone' or lacunae of nucleoli. Its chromosomal nature was strongly suggested by the presence of the synaptonemal complex within it. This demonstrated that the pale-staining region of nucleoli is the nucleolus organizer and almost certainly the chromosome region containing the ribosomal cistrons, and justifies the use of these terms to describe the structure when seen inside the nucleolus. The relationship between this zone and the heterochromatic knob called the nucleolar organizing body in maize by other workers is discussed.


[Compression of retropancreatic common bile duct by tuberculous lymph nodes causing obstructive icterus].

Tuberculosis of the hepatic lymph nodes is among the rare causes of obstructive jaundice and portal hypertension. The authors report a case of compression of the bile duct by retroduodeno-pancreatic tuberculous lymphadenopathy, and recall the difficulty of diagnosis before operation. They emphasize the necessity of simple surgical measures. Removal of the lymph nodes is dangerous and should not be attempted. A biliary by-pass operation should be carried out and its type depends on the precise site of the compression. Antituberculous drugs are then given for 18 months after operation and lead to cure.


[Traumatic rupture of the cervical trachea. Emergency diagnosis and treatment].

The authors report 3 cases of traumatic rupture of the cervical trachea including one associated with total division of the oesophagus. They were impressed by the lack of precision and rapidity of the first surgical measures. They then give an account of the therapeutic gestures which are possible in a non-specialised unit.


[Protrusion of the femoral vessels into the obturator foramen associated with fracture of the pelvis. Surgical treatment].

The authors were led to operate for a severe ischemic syndrome complicating fracture of the pelvis, a patient who presented with curious mobilisation of the femoral vessels in the obturator foramen with arterial and venous compression and who benefitted from treatment by simple reduction.


[Supra-condylar fractures in the child].

The author recalls the technical rules for reduction of supra-condylar fractures in children by the method of percutaneous nailing. Complications and failures of this method are rare and usually due to mistakes. The treatement of sequelae is then considered briefly.


[Aneurysms, late complications at the level of anastomosis of an arterial bypass].

The authors report 12 cases of aneurysm occuring in anastomoses in 9 operated patients. The delay of onset of these lesions was long, 9 years on average. The fraquency appears low, slightly greater than 1 p. 100 in all arterial plastic operations. Among the 10 treated lesions half were false aneurysms due to breakdown of the suture and half were true aneurysms due to progression of the arterial dystrophy. All cases were treated by dissection and insertion of a new prosthesis between the old prosthesis and a healthy area, beyond the anastomosis. Out of 9 patients, there were 8 good immediate results and one failure due to infection at the level of the prosthesis. Among the various methods of prevention, avoiding infection may be the most important.


[Critical study of electric activity of the small intestine in the dog].

Thanks to a cross-circulation, the authors caused to survive in the abdomen of a sacrificed dog, the whole of the small intestine, the motor activity of which was analysed by electro-enterography. This experimental preparation thus eliminates polygraphic recordings of all potential variations outside the small intestine, e.g. stomach, colon, diaphragm, muscle, myocardium. One thus obtains very pure tracings of the variations of potential originating in the small intestine, recorded from bipolar serous intestinal electrodes, they show the activity of the small intestine, consisting of a permanent basal electric rhythm on which are grafted spikes contemporary of motor phenomena. In the abdominal skin there were recorded only a cutaneous slow wave which was contemporary with the deep motor phenomena.


[Efficiency of revascularization of the liver by intra-hepatic implantation of the splenic vascular pedicle].

Experiments carried out in 12 dogs with ligature of the hepatic lobar artery, show that intra-hepatic implantation of the splenic artery, or vascular pedicle, offers limited revascularisation. This produces a fall in blood flow in the implanted artery, reduction in glycogen and hepatic A.T.P., associated with a fall in the total protein content and hepatosine. At the same time, although there is a rich network of neoformed vessels, the hepatic transaminase and lactate activities undergo a pronounced rise.


High-performance liquid chromatography of proteins.

Proteins were separated on microparticulate bonded phase steric exclusion and anion-exchange chromatography supports. A post-column enzyme detector was developed which gives a specific and sensitive response for enzymes. The three iso-enzymes of creatine phosphokinase were separated and assayed in 4 min and the five isoenzymes of lactic dehydrogenase in 6 min.


A low-cost gradient system for high-performance liquid chromatography. Quantitation of complex pharmaceutical raw materials.

A device is described that makes use of an eight-port motor valve to generate step gradients on the low-pressure side of a piston pump with a low dead volume. Such a gradient device with an automatic control unit, which also permits repetition of previous steps, can be built for about half the cost of a gradient system with two pumps. Applications of this gradient unit to the separation of complex mixtures of glycosides and alkaloids are discussed and compared with separations systems using two high-pressure pumps. The gradients that are used on reversed-phase material with solvent mixtures of water and completely miscible organic solvents are suitable for quantitative routine control of pharmaceutical products. The reproducibility of retention data is excellent over several months and, with the use of loop injectors, major components can be determined quantitatively with a reproducibility of better than 2% (relative standard deviation). The step gradient selector valve can also be used as an introduction system for very large sample volumes. Up to 11 can be injected and samples with concentrations of less than 1 ppb can be determined with good reproducibilities.


Anodic coulometric detection with a glassy carbon electrode in combination with reversed-phase high-performance liquid chromatography. Determination of blood levels of perphenazine and fluphenazine.

A detector for liquid chromatography, based on anodic electrochemical oxidation at a glassy carbon electrode, is used in combination with reversed-phase liquid chromatography for the determination of the long-acting tricyclic psychotropic drugs fluphenazine and perphenazine in blood. Completely porous microparticulate silica material, modified so as to give a methyl silica, was used as the stationary phase and buffered mixtures of methanol and water as the mobile phase. The low detection limit and the selectivity of the coulometric detector made possible the determination of concentrations of 1-20 ppb of the compounds of interest. An extraction procedure was in order to obtain sufficient selectivity. Data for the precision, detection limit and yield of the extraction procedure are given. A number of serum levels of perphenazine after oral administration and of fluphenazine after i.m. doses of fluphenazine decanoate are stated.


High-performance liquid chromatographic analysis of porphyrins in clinical materials.

Methods for the isolation of porphyrins as their methyl esters from porphyric urine and faeces as well as other biological materials are described. Quantitative analyses can be carried out by high-performance liquid chromatography (HPLC), using appropriate internal standards; hence excretion patterns in the various types of porphyria can be obtained which may facilitate clinical diagnosis more effectively than the earlier qualitative thin-layer chromatographic methods. Use of the newer microparticulate column packing materials has improved the efficiency of the HPLC analyses, and enables the more convenient isochratic elution techniques to be used (rather than gradient elution). Separations of some porphyrin isomers on these columns are also described.


High-pressure liquid chromatography of steroids secreted by human adrenal and testis cells in monolayer culture.

Reversed-phase high-pressure liquid chromatography with gradient elution on Zorbax-ODS columns has been used to separate, identify, and measure, spectrophotometrically, the steroids secreted by both human adrenal and testis cells in primary monolayer culture. Three related systems using exponential concave gradients have been developed with the specific objective of resolving the steroids produced by these two tissues. A methanol-water gradient has been used to separate most adrenal steroids, an acetonitrile-water gradient to separate testis steroids, and a dioxane-water gradient to separate polar steroids, including aldosterone. These three systems together permit the resolution of at least 43 naturally occurring steroids, plus four synthetic steroids with adrenocortical activity, with overall total elution times of 1 h or less for each system. Retention data for these steroids are given and the separation of steroids in the biological samples illustrated.


Quantitative determination of low levels of daunomycin and daunomycinol in plasma by high-performance liquid chromatography.

A high-performance liquid chromatographic method for the measurement of daunomycin and its main metabolite, daunomycinol, at low concentrations in plasma is described. Quantitative determinations were obtained by the use of adriamycin, another cystostatic anthracycline antibiotic, as an internal standard. The separations were carried out on a 5-mum silica microsphere column with a quaternary solvent mixture as eluent. The components in the eluted peaks were detected by visible absorption at 490 nm and there were no interfering peaks. The advantages of the method are specificity, sensitivity, minimal pre-analysis sample work-up and small sample size. The method is sensitive for plasma levels of daunomycin and daunomycinol above 10 ng/ml. Experimental animal data are presented to illustrate the application of the method.


Separation of catecholamines and their metabolites by adsorption, ion-pair and soap chromatography.

The separation of catecholamines and their metabolites has been carried out by high-performance liquid chromatography using three systems: liquid-solid adsorption, ion-pair partition, and soap chromatography. In the last newly developed technique a reversed-phase packing is used in conjunction with an aqueous organic eluent containing a detergent. The detergent is chosen so that its ion can form ion-pairs with ions of the solutes. Soap chromatography proved the best technique in terms of column efficiency (giving 3000-5000 plates in 125 mm), resolution and sensitivity of detection. Noradrenaline, adrenaline and dopamine, their 3-O-methyl derivatives, L-3,4-dihydroxyphenylalanine, homovanillic acid and other related compounds could be separated in less than 10 min at the 10-50-ng level on columns whose plate heights were in the range of 20-40 mum. The dependence of retention on the concentration of organic modifier and on detergent concentration for three anionic detergents is reported. The method is applied to the direct analysis of urine and the potential of the method for such analyses, especially of pathological urines is demonstrated. Soap chromatography is likely to enlarge the scope of application of high-performance liquid chromatography to biochemical analysis. It is a powerful method for the separation of ionizable compounds which could replace conventional ion-exchange chromatography.


Determination of naloxone and naltrexone as perfluoroalkyl ester derivatives by electron-capture gas-liquid chromatography.

An electron-capture gas chromatographic method is described for the determination of naloxone and naltrexone as the perfluoroalkyl esters. Each compound serves as internal standard for determination of the other. The method permits quantitation of 2-100 ng of either compound. Conditions for derivatization with heptafluorobutyric anhydride (HFBA), pentafluoropropionic anhydride (PFPA), and trifluoroacetic anhydride (TFAA) have been investigated. When catalyzed with pyridine, derivatization with HFBA and PFPA at 70 degrees gives naloxone and naltrexone triesters. Evidence for triester formation was obtained from gas chromatography-methane chemical ionization mass spectrometry and infrared spectral analysis. It was found that both the HFB and PFP triesters are suitable for quantitation of the narcotic antagonists, the HFB derivatives having greater stability than the PFP derivatives. The TFA derivatives are substantially less stable.


Quantitative determination of hexosamines in glycoprotein by ion-exchange chromatography.

An ion-exchange chromatographic method for the quantitative determination of glucosamine and galactosamine in glycoproteins is described. The hexosamines are completely separated from interfering peptides of the acid hydrolysate using a small column of cation exchanger in a manner described earlier by Boas. Chromatographic separation of the hexosamine fraction and the determination of glucosamine and galactosamine are accomplished on an amino acid analyzer with ninhydrin.


New method for the preparation of highly stable polysiloxane-coated glass open-tubular capillary columns and application to the analysis of hormonal steroids.

A new approach to the preparation of methylsiloxane-coated glass open-tubular capillary columns is described. A siloxane polymeric mixture is first prepared by hydrolysis of dimethyldichlorosilane. The polymer is then coated on to the previously etched glass wall by a base-catalyzed reaction to yield a non-polar system of high stability and showing satisfactory chromatographic properties. This type of column has been used for the high-resolution separations of complex hormonal steroid mixtures. The flexibility of this method should permit the preparation of capillary systems with a wide range of polarity.


Rapid quantitative microanalysis of ketones in urine by gas chromatography-mass fragmentography.

A quantitative method is described for determination of 4-heptanone and its precursor in urine. The method involves a single-step extraction of urine with cyclohexane and the analysis of 1 mul of the extract by gas chromatography-mass fragmentography. The detection limit is 8-10(-12)g, corresponding to 2 mug per 1000 ml of urine. The method is linear in the concentration range of 7.6-10(-9)-1.5-10(-5)g/ml and the relative standard deviations for the individual values are between 1.2 and 10%. Approximately 40 samples can be analyzed per day or 1 sample in 7-8 min. By varying the experimental conditions, the method is applicable to other ketones.


Column elution and concentration of volatile compounds in biological fluids.

A new solvent elution method is described for the isolation and concentration of volatile metabolites in biological fluids. The procedure involves elution of the sample with a solvent through a micro-column of an adsorbent into glass-wool and evaporation of the excess solvent in a stream of helium at room temperature. The volatiles are revocered by subsequent heat desorption into a chromatogrpaphic system. Both elution and evaporation are made in a single step using a novel sampling device. Since the technique is based on liquid-solid adsorption chromatography, the adsorbent-solvent system which gives the maximum yield of volatile constituents was studied. The method is simple, highly efficient and reproducible, requiring only small volumes of biological fluids (less than 100 mul).


Profiles of volatile metabolities in body fluids.

A method for the analysis of volatile metabolites present in plasma, urine, breast milk and amniotic fluid collected from mother-infant pairs has been developed which requires only 100 mul of plasma, 3 ml of urine, 20 mul of breast milk and 500 mul of amniotic fluid. After extraction with diethyl ether, the volatile compounds were absorbed on glass wool in a special concentration tube and subsequently desorbed and transferred to a 100-m nickel capillary column for analysis by gas chromatography and gas chromatography-mass spectrometry. The separations, carried out by temperature programming, were complete in 90 min.


Picogram analysis of free triiodothyronine and free thyroxine hormones in serum by equilibrium dialysis and electron capture gas chromatography.

A methodology involving equilibrium dialysis and gas chromatography has been developed to measure concurrently the concentration of the dialyzable (i.e., free) fractions of thyroxine and 3,5,3'-triiodothyronine in serum. The electron capture response of the N,0-diheptafluorobutyryl methyl ester derivatives of these hormones allows their detection and quantitation in the picogram range (detection limit approx. 0.2pg). Purification of the dialysate is achieved using a cation-exchange column which is pretreated with diiodothyronine. Adsorption problems on the glassware are eliminated by silanization. The methodology also resolves 3,3',5'-triiodothyronine (reverse T3).


Determination of hydralazine and its metabolites by gas chromatography-mass spectrometry.

Metabolism of the vasodilator hydralazine was investigated by in vivo and in vitro studies. Standards to identify metabolic products were synthesized. Determination and quantification of hydralazine and its metabolites were accomplished by gas chromatography-mass spectrometry. A deuterium-labeled internal standard was used for quantification. 14C-labeled internal standards were synthesized and used to demonstrate recoveries from the biological samples.


Gas chromatographic study of free polyols and aldoses in cataractous human lens tissue.

An analytical procedure for the qualitative and quantitative analysis of human lens tissue for polyol and aldose content is described. Profile samples are obtained by direct derivatization of lyophilized lenses. The components are analyzed as per-O-acetylpolyols (from the polyols) and per-O-acetylaldononitriles (from the aldoses). This procedure converts each component into a single derivative and terminal dissymmetry for each aldose is retained. The derivatives form in quantitative yield, give good chromatographic peaks, are thermally stable and readily volatilized. They are not subject to adsorption on gas chromatographic columns and are suitable for both qualitative and quantitative analytical studies. Six non-cataractous lenses and fourteen lenses from patients with senile cataract (in seven instances complicated by diabetic pathology) were analyzed. Thermostable borosilicate glass open-tubular capillary columns, coated with the nonpolar phase SE-30, and containing dispersed particles of silanized silicic acid, were used for the gas chromatographic separations. The results are discussed in relation to what is known from earlier studies of human and animal cataracts. A gas chromatographic method for determining the polyol and aldose excretion levels of controlled diabetics is also reported along with a typical metabolic profile.