Compressive and diametral tensile strength of titanium-reinforced composites.

This article determines the compressive and diametral tensile strength of two titanium-reinforced composites (Bis-GMA-based), Ti-Core and Flexi-Flow cem with titanium and compares their strengths to dentin and commercially available core materials and cements. In addition scanning electron microscope (SEM) photographs were taken of Ti-Core and Flexi-Flow cem with titanium. Compressive and tensile loading was performed on a modified universal testing apparatus. Ti-Core and Flexi-Flow cem with titanium were measured to have compressive strengths of 41,132 and 41,876 psi and tensile strengths of 5219 and 4930 psi, respectively. Statistically (ANOVA, one-way analysis of variance), these titanium-reinforced composites are stronger in compressive and tensile strength than Ketac-Silver, Fleck's zinc cement, Durelon, Ketac-Cem, and GC Miracle Mix. Both titanium-reinforced composite materials approach the compressive and diametral tensile strengths of dentin (43,100 and 6000 psi). SEMs revealed that the titanium was uniformly and homogeneously interspersed within the resin matrix of the material.


Noncarious cervical lesions: the decision to ignore or restore.

A variety of unusual noncarious cervical lesions (NCL) are depicted that appear to negate W.D. Miller's toothbrush/dentifrice abrasion theory and demonstrate that other factors may be involved in their etiology. Confusion exists in the designation of NCL ever since G.V. Black stated in 1908 that toothbrush/dentifrice abrasion is an erosive effect. Since abrasion and erosion are two distinct activities, it is suggested that dentistry adopt the same terminology as chemical engineering in order to foster improved communication between the sciences. The term "abfraction" has been used to supplant erosion because it seems more appropriate when describing the loss of tooth substance attributable to effects of occlusal loading forces as well as the physiochemical breaking that occurs during stress corrosion. Numerous reasons, based on accepted engineering principles, indicate that NCL should be restored. It is incumbent on dentists to become cognizant of these reasons, since this would help them inform patients of the benefits to be gained by restoring such deficient areas.


Diastema closure: a case report.

This clinical study describes two combined modalities of treatment, orthodontic and porcelain laminate placement, to facilitate diastema closure. Geristore, a dual-cure fluoride-releasing composite was mixed to bond orthodontic brackets in place. H6 elastic bands were used with the orthodontic brackets to close the diastemas sufficiently and to allow the placement of Cerinate porcelain laminates to produce a beneficial cosmetic effect.


Hepatitis B virus-related markers in secondary and in essential mixed cryoglobulinemias: a multicentric study of 596 cases. The Italian Group for the Study of Cryoglobulinemias (GISC).

The aim of this study was to assess the prevalence of HBV-related markers in cryoglobulinemias and the possible association between hepatitis B virus (HBV) and essential mixed cryoglobulinemia (EMC). A retrospective survey of the prevalence of HBV infection in cryoglobulinemic patients was carried out in 596 cases of cryoglobulinemia. On the basis of clinical and laboratory criteria the cases were grouped as secondary to connective tissue disease, to lymphoproliferative disorders, to chronic liver diseases or to chronic infectious diseases. The cases in which an associated disease was not evidenced were considered as essential mixed cryogobulinemias. Subjects in which liver dysfunction was first diagnosed simultaneously with cryoglobulinemia, were considered as a separate group. A greater prevalence of HBsAg and anti-HBc antibodies was found in cryoglobulinemias secondary to chronic liver disease (p < 0.0001) and in those associated with liver involvement at diagnosis (p < 0.05) than that found in EMCs. The prevalence of anti-HBs antibodies did not differ significantly among the groups. Proven contact with the virus, documented by at least one positive marker, was evidenced more frequently in cryoglobulinemias secondary to liver disease than in the other groups (p < 0.01). The prevalence of HBV related markers in EMCs and in hospitalized patients not suffering from diseases associated with cryoglobulin production were similar, and seems to reflect the epidemiological situation of HBV infection in Italy. In conclusion, our findings do not support an association of HBV with EMC.


[The modulator effect of bradykinin with respect to tracheobronchial clearance in healthy subjects].

Bradykinin (Bk), a vasoactive nonapeptide which has been proposed as mediator in the pathogenesis of bronchial asthma, is a potent secretagogue in canine airways and in human nasal mucosa in vivo. A study was carried out in order to evaluate the effect of inhaled Bk on tracheobronchial clearance (TBC), measured by a radioaerosol technique in 10 healthy volunteers. Subjects inhaled Bk (8 mg/mL) or vehicle placebo on two occasions, at least 2 weeks apart, in a double blind and randomized fashion 30 min after radioaerosol inhalation. TBC, expressed as the area under the tracheobronchial radioaerosol retention curve calculated for the first 6 hours (AUC 0-6h), was enhanced in 9 of 10 subjects following inhaled Bk when compared to placebo. The mean values for AUC 0-6h were significantly reduced from 132%.h to 92%.h (p < 0.001) with placebo and Bk respectively. We conclude that Bk has a marked effect on tracheobronchial clearance in normal human airways.


[The humoral and cellular factors of glomerular damage].

The present review summarizes current concepts on glomerular non-inflammatory and inflammatory immune injury. Non-inflammatory glomerular injury can be ascribed to mechanisms involving antibody alone, terminal component of complement (C5b-9) or cytokines having as primary target the glomerular epithelial cells. These mechanisms induce changes in cell cytoskeleton and in surface distribution of adhesion molecules leading to functional alterations which may account for loss of glomerular permselectivity. Inflammatory glomerular injury is mediated by neutrophils, macrophages, platelets and lymphocytes as well as by resident glomerular cells. Cell derived mediators include proteases, reactive oxygen species, cationic proteins, cytokines and growth factors that may variably affect recruitment of inflammatory cells, proliferation of resident glomerular cells and production of extracellular matrix. These alterations may determine proteinuria, impairment of renal function and eventually progression of glomerular injury to sclerosis. The inflammatory type of glomerular injury is mainly initiated by subendothelial-mesangial immune deposits and/or activation of cell mediated immunity. The role of factors such as the nature, intensity and persistence of initiating mechanisms, the types of inflammatory cells involved and the response of intrinsic glomerular cells, in expression and progression of glomerular injury are discussed.


Hematological changes in infectious diseases. Hematological consequences of viral infections (1).

A number of distinct hematological alterations take place in the presence of infectious diseases. This review seeks to provide a diagnostic guide and prognostic criteria for the clinician in the light of the concomitant hematological changes occurring in the course of acute and chronic viral, bacterial, spirochetal and protozoal infections. The authors emphasize the fact that although certain broad principles may be applied to each type of infection, these principles may have to be interpreted with considerable latitude in any individual case, on account of the variable factors of virulence and resistance. The careful examination of blood films and bone marrow smears, supplemented when appropriate by cytochemical reactions, may be useful in the differential diagnosis of various infective states and in assessing both the severity of the pathological condition and the response of the patient.


[Atrial natriuretic factor and pulmonary function].

The aim of this review is to provide a critical and concise discussion of present knowledge on the role of atrial natriuretic factor (ANF) in physiological as well as pathological pulmonary conditions. The lung contributes only to a small extent to the production of circulating ANF; on the other hand, the lung represents the major degrading site of the protein. Plasmatic ANF concentration increment during lung disease may therefore be due to a reduction in ANF plasma removal enzyme rather than to increased ANF production. Lung tissue shows more ANF receptor sites than any other organ. The effect of ANF on bronchial and pulmonary artery muscle lining is particularly evident. In fact ANF administration in asthmatic patients leads to bronchodilation comparable to dilation induced by salbutamol. Furthermore, elevated levels of circulating ANF seem to influence fluid redistribution through alveolar-capillary membrane leading to protein mobilization through the alveolar space. On the contrary, in the cardiomyopathic hamster ANF induces relevant guanylate cyclase activation before the animal has developed hemodynamic changes. Guanylate-cyclase activation may protect the lung through counteracting pulmonary edema formation, as shown by fluid reduction in alveolar spaces following pneumotoxic agents administration. This effect seems independent of natriuretic and hypotensive ANF effects.


Thyroid storm with encephalopathic symptoms due to Graves' disease and inappropriate secretion of thyrotropin.

A 41-year-old woman is described, first hospitalized in the neurosurgical department for a transient ischemic attack with left hemiparesis followed after 6 hours by tonic-clonic seizures starting from the left hemiface and quickly generalized. Brain computed tomography (CT) scan and magnetic resonance imaging were normal. Clinically the patient presented tremor, tachycardia, generalized muscle weakness, and profuse diaphoresis. T4 and T3 were elevated. The patient was transferred from the neurosurgical to the medical department where a thyroid storm due to autoimmune Graves' disease with normal thyrotropin (TSH) values responsive to thyrotropin-releasing hormone (TRH) stimulation was diagnosed. A syndrome of inappropriate secretion of TSH was suspected in an unusual presentation as autoimmune Graves' hyperthyroidism. The TSH alpha-subunit and alpha-subunit/TSH molar ratio were normal, which supported the diagnosis of non-neoplastic inappropriate secretion of TSH. However, severe autoimmune Graves' hyperthyroidism is very rare indeed because autoantibodies to thyroid antigens are generally non-detectable in such patients. Our patient was treated initially with barbiturates, then with dexamethasone, Lugol's solution, methimazole and propranolol. Treatment of this patient proved difficult, and definitive improvement was obtained only after triiodothyroacetic acid administration, but methimazole and propranolol administration could not be discontinued. Fine needle aspiration biopsies of the thyroid in 2 occasions showed follicular or follicular-papillary proliferation with lymphocytic infiltration as in chronic thyroiditis. The patient is now in good clinical conditions and is followed up regularly. Autoimmune Graves' hyperthyroidism may be associated in extremely rare instances with non neoplastic inappropriate secretion of TSH.


The effect of carotid sinus pressure changes on mean circulatory filling pressure in the anaesthetized rabbit.

The effect on mean circulatory filling pressure (MCFP) of changes in pressure within the vascularly isolated carotid sinus has been studied in anaesthetized rabbits. With the sinus exposed to arterial pressure (102 +/- 6 mm Hg; mean +/- SEM) MCFP was 6.18 +/- 0.40 mm Hg. At sinus pressures of 160 and 40 mm Hg, MCFP was 5.71 +/- 0.39 and 6.43 +/- 0.33 mm Hg respectively. The mean difference of 0.72 +/- 0.06 mm Hg between MCFP at these two sinus pressures was significant (P < 0.001). MCFP varied with blood volume when this was increased or reduced by 8%, but at each blood volume level the difference between MCFP at high and low sinus pressure remained significant (P < 0.01). The gradient for venous return, MCFP minus right atrial pressure, was altered by changes in carotid sinus pressure, being 0.60 +/- 0.12 mm Hg less at a sinus pressure of 160 mm Hg than at a sinus pressure of 40 mm Hg. This difference was significant (P < 0.01).


The relationship between systolic blood pressure and microvascular resistance in non-diabetic and diabetic subjects.

Skin blood flow and microvascular resistance were measured in the feet of hypertensive and normotensive subjects by the non-invasive transient thermal clearance method. Skin blood flow decreased and microvascular resistance increased as a function of systolic brachial blood pressure for the non-diabetic subjects who were not treated by vasoactive medication. The relationship between blood flow and systolic blood pressure for diabetic patients and for patients who were treated by enalapril was poor. The average resistance for non-diabetic patients treated by enalapril was lower than that of untreated non-diabetic subjects. For diabetic patients no significant difference in resistance between enalapril treated and untreated patients was found. The results demonstrate that for non-diabetic patients the role of microvascular resistance in hypertension is significant while it is less important in diabetic patients.


Cyclic perfusion of the lung by dense gas breathing may reduce the (A-a)DO2.

The decrease in the alveolar-arterial O2 difference (A-a)Do2 in dense atmosphere could be the result of cyclic lung perfusion due to large swing in pleural pressure during breathing cycles /4/. Using a mathematical model of the lung to calculate (A-a)Do2, a slight decrease was demonstrated in (A-a)Do2 as perfusion changed from steady to cyclic. The present study incorporates variable vertical partitioning of perfusion in this model, as a function of instantaneous total blood flow. This demonstrated a reduction of the Po2 difference between the apex and the base of the lung, and a reduction of (A-a)Do2, when perfusion was assumed to be cyclic, of 4.1 to 5.0 torr when blood flow was continuous throughout the breath, and of 5.0 to 5.7 torr when perfusion was assumed to be pulsatile. The results agree with experimental findings: reduction of (A-a)Do2 when breathing dense gas and improved apical perfusion in pulsatile blood flow. Calculation of the spatial VA/Q inequality suggests there is no correlation with (A-a)Do2 reduction. The decrease in (A-a)Do2 is attributed to the mixing of lung capillary blood flows having different O2 saturations.


Evidence against oxidant injury and endotoxin underlying glycerol-induced fatal rhabdomyolysis in rats.

Rhabdomyolysis in experimental animals and in man may be fatal. The mechanism of action causing death in man and in the experimental model of laboratory animals injected with glycerol in a dose causing fatal rhabdomyolysis is unknown. The aim of this study was to examine two possible causes of death following glycerol injection (0.5-2 ml/100 g) to the leg muscle of rats following a period of water deprivation (24-72 h). The first factor examined was free radical formation, via the administration of various antioxidants such as natural antioxidant (NAO - 5 mg/100 g), vitamin E (0.2 mg/100 g each day for 7 days), dimethylthiourea (DMTU - 50 mg/100 g) and superoxide dismutase (SOD - 0.8 mg/100 g). The second factor was a high blood endotoxin level due to the glycerol injection. This possibility was examined by glycerol injection to rats that had developed endotoxin tolerance. The low survival rate (0-30%) of rats receiving a glycerol injection following preventive treatment with antioxidants as well as rats which had developed endotoxin tolerance (0-40%), excludes the possibility that the cause of death in the glycerol model resulting in fatal rhabdomyolysis is due to free radical formation or to a high and unneutralized blood level of endotoxin.


Effects of changes in plasma volume on fatal rhabdomyolysis in the rat induced by glycerol injections.

Rhabdomyolysis can be fatal in both experimental animals and man, but very little is known of the factors causing increased mortality in rhabdomyolysis. The aims of this study were to create an animal model of fatal rhabdomyolysis in rats by a glycerol injection into the leg muscle, and to elucidate some of the factors affecting mortality as a result of rhabdomyolysis formation. In this study, two factors which can result in increased mortality in rats as a result of glycerol injection, were examined. These factors include varying doses of 50% glycerol (0.5-2 ml/100 g) and various stages of dehydration prior to glycerol injection. Dehydration was induced by 1: chronic dehydration, in which the rats underwent water deprivation for a period of 24 to 72 hours prior to injection of glycerol; 2: acute dehydration, by the induction of either diuresis, by injecting sucrose (200-600 mg/100 g) to the femoral vein, or hemorrhage (0.7-2.1 ml/100 g). The results demonstrate that the mortality rate in rats increased in all three models of dehydration as the dose of glycerol injected to the rats increased (above a dose of 0.75 ml/100 g) and as the extent of dehydration increased. Use of a blood substitute before or after glycerol injection in order to compensate for the loss of body fluids did not increase the survival rate of the glycerol-injected rats. In contradistinction, rats treated with non-lethal doses of glycerol exhibited substantial resistance to a second lethal dose of glycerol, injected two weeks following the first injection.


[Evaluation of the antioxidant and antiradical activities of substances and biological objects using iron-initiated chemiluminescence].

Antioxidative and antiradical activity of the following typical inhibitors of free radical reactions were investigated in the model systems: ethanol, ionol, uric acid, superoxide dismutase, albumin. The antioxidative and antiradical activities of these inhibitors were shown to differ in various model systems.


[Possible effects of the influence of dynamic disorder of biological systems on characteristics of intramolecular mobility, determined by Mossbauer spectroscopy].

A model for describing dynamic properties of proteins is proposed. The model involves the distribution over amplitudes and correlation times of intramolecular dynamics. It has been shown that distribution parameters and its temperature dependence have a great influence upon the values of experimental dynamic characteristics. Besides the discrepancy between the real and experimental temperature, dependence of characteristics on intramolecular dynamics can be observed.


[Osmotic regulation of furosemide-sensitive K+ (86Rb+) transport in rat brain synaptosomes].

In the rat brain synaptosomes the furosemide-sensitive component of 86Rb+ uptake constituted 30.8% of the total uptake in the medium containing 132 mM Na+. A decrease in the medium tonicity from 310 to 230 mOsm increased the rate of 86Rb+ uptake from 2.38 +/- 0.58 to 7.12 +/- 0.52 nMoles/mg of protein/min.


[A physical model of gravitational erythrocyte sedimentation].

A model of erythrocyte sedimentation under gravity is proposed. The model is based on two-phase description of the blood and it involves a kinetic equation of erythrocyte aggregation. A method of characteristics is employed for analysing the physical pattern of sedimentation for different hematocrit. An approximate decision for trajectory of the interface between pure plasma and low-located region of the erythrocyte suspension is obtained. By utilizing this decision the simple coefficient of the erythrocyte aggregation ability calculated in terms of hematocrit and erythrocyte sedimentation rate is possible.


[Bifurcation analysis of a mathematical model of the interaction of cytotoxic lymphocytes with tumor cells. The effect of immunologic amplification of tumor growth and its interconnection with other "anomolous" phenomena of oncoimmunology].

Mathematical model of the immune response of the cytotoxic lymphocytes to the nonexponentially growing immunogenic tumour has been studied. Local and global biffurcations for realistic values of the parameters were determined. The connection of the oncoimmunological "anomalous" phenomena (immunostimulation of tumour growth, "sneaking through" of tumour, formation of the tumour "dormant" state) has been shown. The probability of manifestation of these effects in vivo was estimated.


[Dependence of the nerve pulse generation threshold on the ability of forming an input signal based on the Hodgkin-Huxley model].

Different methods of definition of the threshold of the action potential (AP) generation were considered. It was established that the threshold defined as a difference between the critical value of the membrane depolarization and the resting potential was practically independent of the value and duration of an input signal which was given as a stepped function. It was shown that the critical values of the membrane depolarization could be defined by the point of bend, as well as by the point of maximum of the curve curvature on the frontal part of AP.


[Evaluation of the possibilities of modeling biological tissues by the Focker-Planck and Gui-Chapman joint system of equations].

When describing the galvanic effect on biological tissues ion diffusion was taken into account in the Focker-Planck model, in the Gui-Chapman model account was taken of bolzman ion distribution while solving Poisson equation. To specify the models joint solution of these equations ought to be carried out. However, the equation system of Focker-Planck and Gui-Chapman proved to be reestimated, and therefore one can speak only about quasisolution as a solution of an incorrectly stated problem.


[Surface tension of diluted solutions of human tears in the normal state and in various types of eye pathology].

It is shown that the measurement of diluted solutions surface tension of normal tears and tears at different eye pathologies (cataract, glaucoma before and after operation) gave identical results. It is interpreted as a normal function of the lacrimal gland at these pathologies.


[Connection of the carbon isotopic composition of human hair with its functional state].

Temporal variations of carbon isotope composition of hair taken from faces of three men with different health state were studied. Drastic enrichment (up to 20%) of hair carbon in heavy isotope, as compared to the basic line reflecting background isotope variations was discovered in one of the men under test. This enrichment correlated with his worsened state of health. Then carbon isotope composition of hair returned to the background level. Probable explanation of this correlation was suggested in terms of earlier described mechanism of carbon isotope fractionation in a living cell.


[Variability of some isotopic characteristics of carbon in man].

Variability of some carbon isotope characteristics in man (i.e. carbon isotope composition of hair, blood, nails, expiratory carbon dioxide) was studied. Slow (with the period about 20-30 days) oscillations near some average level with a variable amplitude were observed in the isotope composition of the hair carbon. The maximum deviation from the mean level in one person tested was 6%. In the isotope composition of carbon dioxide respired under physical load and under conditions of aerobic metabolism during short periods (up to 15 minutes) there were also found significant variations from the mean level, the maximal ones being 6/1000. An attempt was made to explain the changes observed of isotope characteristics in terms of the principal carbon isotope fractionation cell model proposed earlier.


[A biomechanical invariant of automated movement].

Trained arm movements and two-link arm dynamics and control of arm movements model are investigated. It is found that for these movements the component of the movements energy dissipations in the control force potential is invariable during the outer force actions.


[Mechanical energy expenditure on human movement and the anthropomorphic mechanism].

Mechanical energy expenditures of the man and anthropomorphic locomotion machine during movement are compared theoretically. Sources of the mechanical energy affecting movement of human's lower extremity are modelled by 8 muscles, 3 of which are the two-joint muscles. The model of the lower extremity of anthropomorphic locomotion machine is moved by joint moments. It was shown that in the same movement the model of the human lower extremity can spend less mechanical energy than that of the model of the anthropomorphic locomotion machine. It is caused by the presence of two-joint muscles in the first model. Such an economy of mechanical energy expenditures realized by the two-joint muscle is possible at simultaneous execution of three conditions: 1) signs of the muscle powers, which are produced by that muscle at both joints, are opposite; 2) moments produced by that muscle at each of both joints have the same direction with the joint moments at these joints; 3) one-joint antagonistic muscles are not active. An expression which makes it possible to estimate the mechanical energy savings by the two-joint muscles during humans' movement was developed.


Sensitive method for the determination of phenytoin in plasma, and phenytoin and 5-(4-hydroxyphenyl)-5-phenylhydantoin in urine by high-performance liquid chromatography.

An isocratic high-performance liquid chromatographic method is described for the quantitative analysis of phenytoin (PHT) in plasma and both phenytoin and its main metabolite, 5-(4-hydroxyphenyl)-5-phenylhydantoin (p-HPPH) in urine. Following ethyl acetate (plasma) and Extrelut-1 (urine) extraction, samples are analysed by means of a reversed-phase column (Nova-Pak C18), using a mobile phase consisting of methanol-water-tetrahydrofuran (40:60:4, v/v/v) with UV detection at 230 nm. The chromatography is complete in 10 min and the results show good precision (RSD 1.23-4.49%) and sensitivity for a linear range of 0.4-4.0 micrograms ml-1 for PHT in plasma, 0.1-1.0 microgram ml-1 for PHT in urine and 0.1-1.2 microgram ml-1 for p-HPPH in urine. The results indicate the method to be suitable for pharmacokinetic studies.


Planar chromatography: current status and future perspectives in pharmaceutical analysis--I. Applicability, quantitation and validation.

The most important features of capillary action planar chromatographic methods are summarized. Certain properties such as ease in operation, sensitivity to experimental conditions, stationary and mobile phase selection, phase system optimization, separation efficiency and detection possibilities are discussed only briefly. Other aspects such as the applicability of planar chromatographic methods in pharmaceutical analysis, quantitation and validation are considered in more detail. The advantages and limitations of different evaluation techniques (visual comparison, spot elution techniques and in situ densitometry) are also discussed. Validation steps specific to planar chromatographic methods are emphasized.


Planar chromatography: current status and future perspectives in pharmaceutical analysis (short review)--II. Special techniques and future perspectives in planar chromatography.

The state of the art of various special analytical planar chromatographic methods is summarized especially for forced-flow planar chromatography (FFPC), overpressured-layer chromatography (OPLC) and rotation planar chromatography (RPC) as well as for the automated multiple development (AMD) technique. The connection between analytical planar and column liquid chromatographic methods and the identification of separated compounds with chromatographic and spectroscopic data are summarized. Some aspects of future perspectives, such as parallel connected multi-layer FFPC and long distance OPLC (LD-OPLC) are given. A combination of OPLC with the AMD method is predicted as the method of the future. Strategies using FFPC techniques are suggested in the form of a flow chart.


Use of TLC and multivariate mathematical statistical methods to study the interaction of monoamine oxidase inhibitory drugs with amino acids.

The interaction of 17 monoamine oxidase inhibitory drugs (propargylamine derivatives) with amino acids was studied by charge-transfer chromatography. The data set was evaluated by principal component analysis (PCA). To assess the effect of the information loss caused by normalization, PCA was separately carried out on the covariance and on the correlation matrix. The strength and selectivity of interaction was separated by the spectral mapping technique. Calculation proved that the amino group of the drugs interacted with the second carboxyl group of dicarboxylic amino acids, and that the interaction was of electrostatic character. This finding made probable the direct interaction between the drugs and the target enzyme or enzymes. The results of both PCA methods were similar. However, coordinates of the spectral map showed only slight correlation with the corresponding coordinates of the two-dimensional nonlinear maps proving the different information content of the methods.


Determination of ascorbic acid and isoascorbic acid by capillary zone electrophoresis: application to fruit juices and to a pharmaceutical formulation.

Capillary zone electrophoresis was applied to the determination of ascorbic and isoascorbic acid, analysing the various parameters of influence such as the separation voltage, the buffer pH and concentration, the type of separation capillary or the loading conditions. Both analytes could be adequately determined within 5 min. The proposed method uses a 20 cm x 25 microns i.d. coated column, 0.1 M phosphate buffer pH 5.0, 8 kV separation voltage and light absorption detection at 265 nm. Linear calibration curves were obtained in the 0-1 mg mi-1 range, with detection limits of 0.5 micrograms ml-1. This method proved to be very rapid, simple and practical for the qualitative and quantitative determination of ascorbic acid in lemon and orange juices, as well as in a commercially available pharmaceutical formulation.


The determination of the geometric isomers and related impurities of dothiepin in a pharmaceutical preparation by capillary electrophoresis.

The application of capillary zone electrophoresis in the assay of the tricyclic antidepressant drug, dothiepin, in tablets is discussed. The method developed for dothiepin which exists as the cis- and trans-isomers and contains two major related impurities, an 11-oxo compound and a propanamine, utilizes inclusion complexation with beta-cyclodextrin. For optimization of the method the structured procedure of factorial design was used; the electrolyte solution was 50 mM disodium hydrogen phosphate with 10 mM beta-cyclodextrin-propan-1-ol(90:10, v/v). Good precision (RSD = 1.06%, n = 6), linearity (y = 26.84x + 2.25), and correlation (r = 0.999, n = 7) was obtained for trans-dothiepin. The reproducibility of tablet extraction was also acceptable (RSD = 0.77%, n = 6); the recovery of the trans-isomer was 98% (w/w) and the level of cis-isomer in tablets of dothiepin (75 mg) was 5.58% (w/w).


The automation of dissolution testing of solid oral dosage forms.

Dissolution testing of solid oral dosage forms plays a very important part both in the development of new products and in quality control. A fully automated system for dissolution testing known as AUTO DISS is presented and its components are described. On-line determination of active ingredient concentration is possible with the aid of an integrated automatic sampler in combination with various measuring instruments (UV-vis spectrometry, liquid chromatography and flow injection analysis). The suitability of the system is demonstrated by determination of the dissolution of brotizolam from tablets by FIA and of bepafant from capsules by diode-array spectroscopy.


Selectivity manipulation in micellar electrokinetic chromatography.

Micellar electrokinetic chromatography (MEKC) permits the separation of electrically neutral analytes by chromatographic principles in a capillary electrophoresis system. The most effective way to obtain high resolution in MEKC is to increase the separation factor, as in conventional chromatography. The separation factor in MEKC depends on the molecular structure of the micelle and hence on the surfactant used, the pH of solution, and the nature of any additives to the micellar solution. The hydrophilic moieties of surfactant molecules generally affect selectivity more than do the hydrophobic moieties. Chiral surfactants enable the enantiomeric separation of mixtures of chiral solutes to be achieved. Mixed micelles consisting of ionic and nonionic surfactants display different selectivity from that of single ionic micelles. Additives such as cyclodextrins, ion-pair reagents, urea, organic solvents and metals can also serve as useful modifiers of the micellar solution for improving separation. In particular, cyclodextrins are useful for the separation of aromatic isomers and enantiomers. A general introductory guide to the design of successful separations by MEKC is proposed, based primarily on the author's work.


The use of microdialysis for the determination of plasma protein binding of drugs.

Microdialysis sampling was used for the determination of the protein binding and the free therapeutic drug concentration of drugs in plasma in vitro. Several drugs with varying extent of protein binding and for which the plasma monitoring is important were studied. To mimic the in vivo situation, an artificial blood vessel was constructed and filled with spiked plasma circulating at the flow rate of human blood at 37 degrees C. The microdialysis probe (16 mm membrane length, 20000 MW cut off) was placed in the vessel and perfused with 0.9% NaCl at 5 microliters min-1. Dialysates were collected every 10 min and were analysed by reversed-phase LC with UV detection. The free concentration of the drug was calculated by correcting the concentration in the dialysate for the recovery of the probe, which was also determined in the artificial blood vessel after the experiment. The data confirm that microdialysis is a valid alternative technique for the determination of protein binding or free therapeutic plasma concentration of drugs on a comparative basis. Reference to literature values indicates that the results of the proposed method correspond reasonably well with accepted values.


Determination of compounds with anticholinesterase activity in commercial drugs by a new enzyme sensor.

A suitable enzyme sensor for the analysis of anticholinesterase compounds of pharmaceutical interest is described. It is based on the competitive inhibiting properties of these compounds on the enzyme butyrylcholinesterase and it is constituted by a hydrogen peroxide amperometric electrode modified by a superimposed Nylon membrane containing two chemically immobilized biological mediators (butyrylcholinesterase and choline oxidase). Some applications to the analysis of several pharmaceutical forms containing different compounds showing anticholinesterase activity are also reported and evaluated.


Analytical comparison of an enzyme-amperometric method for chlorocresol determination in ointments with colorimetry and liquid chromatography.

The direct determination of chlorocresol in n-hexane extracts of commercial ointments was successfully performed using an enzyme-amperometric probe for analysis of phenols and working in previously characterized and optimized non-aqueous solvents. The analytical data obtained were compared with those found by using classical HPLC or chemical spectrophotometric method for determination of phenols.


PVC membrane electrode without inner reference solution for the direct determination of ephedrine in pharmaceutical preparations.

A PVC membrane electrode, without inner reference solution, based on an ion association extraction system responding to ephedrine is described. It incorporates an ephedrine-tetrakis (4-chlorophenyl)borate ion-pair complex in 2-nitrophenyloctyl ether. The prepared electrode exhibits a near Nernstian response (57.5 mV per decade) over the concentration range of 2 x 10(-5)-10(-1) M ephedrine in solutions of pH 2.5-9. The reproducibility of the electrode potentials were +/- 1 mV by day during at least 6 months. Response time was about 6 s for ephedrine concentrations between 10(-5) and 10(-1) M. Determinations of ephedrine in pharmaceutical preparations (tablets, nasal drops and syrups) by direct potentiometry gave an average recovery of 99.1% (w/w) and a mean standard deviation of 1.9% (w/w).


Electrochemical behaviour of tizanidine at solid electrodes.

The electrochemical behaviour of tizanidine [5-chloro(delta-2-imidazolinyl-2-amino)-4-benzothiadiazole-2,1,3], a centrally-active skeletal muscle relaxant has been investigated in aqueous media at the carbon paste electrode (CPE). Cyclic voltammetry at different pH values, controlled potential coulometry and comparative studies on three structurally related molecules have permitted identification of the oxidation site of tizanidine and suggest possible oxidation products in acidic media. The electrochemical reduction at the CPE occurred in one irreversible step and the reduction product (diamine derivative) was detected and characterized on the positive going scan in cyclic voltammetry. Quantitative measurements of tizanidine within the range 2 x 10(-5) M and 1 x 10(-4) M have been realized at the CPE using the differential pulse technique.


Novel flow injection analysis systems for drug analysis.

Flow injection analysis (FIA) has become a versatile tool for rapid and automated analyses. As its capabilities have increased, so have the complexity and operation of the apparatus. We have been investigating ways to simplify both the apparatus and the application of injection techniques. A novel cam-driven syringe pump and the development of sequential injection analysis (SIA) are reviewed, and some applications presented. Flow injection coulometric titrations are presented as a means to further alleviate reagent stability and calibration problems. These systems have potential for automatically carrying out many drug assays.


The determination of the preservative, chlorocresol, in a pharmaceutical formulation by flow injection analysis.

A flow injection analysis (FIA) procedure is described for the determination of chlorocresol in a parenteral pharmaceutical formulation. The product is directly injected into a carrier stream of water and subsequently reacted with a reagent stream of nitrous acid. The resulting brown nitro-derivative is determined spectrophotometrically at 400 nm. The method has been validated and should be applicable to chlorocresol in other pharmaceutical products and to compounds containing a phenolic ring, assuming absence of matrix interference.


Spectrophotometric determination of promazine with an oxidative column in FIA manifolds.

A simple flow-injection spectrophotometric method for the determination of promazine is described. The two proposed procedures are based on the oxidation of analyte with a manganese dioxide column. Concentrations of promazine in the ranges 2-20 and 1-6 are determined with a relative standard deviation of 1.0%. The injection rates are 62 and 80 samples h-1, respectively. The influence of foreign species and the determination of promazine in a pharmaceutical formulation are also reported.


Liquid chromatographic separation of phenolic drugs using catalytic detection: comparison of an enzyme reactor and enzyme electrode.

A catalytic detection system based on the use of immobilized tyrosinase and two different transducers (photometric and electrochemical) is described. Comparison between tyrosinase immobilized in a packed-bed reactor and at the surface of a graphite electrode is discussed in terms of sensitivity in a flow injection system. The enzyme electrode configuration gives the highest sensitivity for the quantitation of dopamine. For the immobilized tyrosinase reactor with photometric detection the range for dopamine is linear up to 0.75 mM (136 micrograms ml-1) and the immobilized tyrosinase reactor with electrochemical detection and the tyrosinase electrode extends this dynamic range to 1 mM (181 micrograms ml-1). Liquid chromatographic separation and post-column detection using the tyrosinase electrode is shown for spiked samples of serum.


The use of near infrared spectroscopy in the quality control laboratory of the pharmaceutical industry.

The suitability of NIR spectroscopy as an alternative to several compendial test methods is discussed. Using ampicillin trihydrate as an example it is demonstrated that eight quality criteria are controlled by recording the NIR spectrum of a batch sample and calculating its Conformity Index.


Spectrofluorimetric determination of sulphonamides in pharmaceutical compounds and foods.

Spectrofluorimetry and room temperature photochemically-induced fluorescence (RTPF) have been applied to the determination of sulphacetamide (SAC), sulphaguanidine (SG) and sulphamethazine (SMT) in milk and pharmaceutical formulations. The methods are suitable for determining 0.02-0.10 micrograms ml of SAC, 0.10-0.50 micrograms ml of SG, and 0.40-1.00 micrograms of SMT.


Fluorescence enhancement of two terpenes commonly present in essential oils.

The fluorescence spectra of anethole and eugenol dissolved in methanol-aqueous binary systems with the addition of alpha- and beta-cyclodextrin were studied. Observed enhancement of the fluorescence intensity is possibly due to the higher quantum yield of the cyclodextrin-hydrocarbon inclusion complexes. The measured fluorescence intensities for eugenol and anethole in the presence of alpha- and beta-cyclodextrin were processed using principal component analysis. The results obtained suggest 1:1 and 2:1 complexation, as confirmed by a double reciprocal plot for terpenes complexed to cyclodextrins. In both cases (anethole and eugenol) detection limits were improved after addition of cyclodextrins. This phenomenon can be applied for improvement of direct fluorescence and HPLC-fluorescence assays.


A biotin-avidin based screening test for methamphetamine in urine.

A biotin-avidin based screening test for methamphetamine in urine samples has been developed. The assay method utilizes the immunoprecipitin reaction between an antibody to methamphetamine and a conjugate prepared by complexing avidin with a biotinyl amphetamine derivative. The rates of the immunoprecipitin reaction is monitored on the Beckman ARRAY 360 nephelometer. Methamphetamine inhibits the precipitin reaction, and the extent of inhibition allows the quantitation of methamphetamine in the urine samples. Using a cut-off value of 0.7 microgram ml-1, the assay correctly predicted 83 of 84 samples (98.8%) confirmed to be positive by GC-MS (> 500 ng ml-1). Of 59 GC-MS confirmed negative samples, 46 samples were found to be negative by this method as compared to 34 samples determined with the EMIT assay. Within-run and between-run relative standard deviations near the cut-off value were less than 4%. Cross-reactivity with amphetamine was < 7%.


Analysis of mono-, di- and triglycerides in pharmaceutical excipients by capillary supercritical fluid chromatography.

Mono-, di- and triglycerides are important components of oils, fats and other natural products. Since in general fatty acids are mixtures and glycerol can be differently substituted, finger-prints of the composition are suitable for better characterization. Since capillary supercritical fluid chromatography (SFC) employing carbon dioxide as mobile phase is compatible with flame ionization detection, it is possible to analyse many solutes at trace levels. Supercritical carbon dioxide offers higher solute diffusivity compared with the inert carrier gas conventionally used in gas chromatography and has a lower viscosity than the liquid solvents used in HPLC. Thus, glycerides of fatty acids can be separated and eluted at a lower temperature and with shorter analysis time in SFC. In this study the analysis of mono-, di- and triglyceride mixtures in several pharmaceutical excipients is reported using capillary SFC. Quantitative analysis is possible on the basis of a response factor established for each analyte. The accuracy of the method and its advantages are demonstrated.


Estimation of impurity profiles of drugs and related materials. Part 9: HPLC investigation of flumecinol.

Two HPLC systems have been developed for the investigation of flumecinol (3-trifluoromethyl-alpha-ethyl-benzhydrol). The reversed-phase system (LiChrosorb RP-18; methanol-water, 7:3, v/v) enables the isolation and identification of the impurities. The chiral system (Chiralcel OD; hexane-2-propanol, 98:2, v/v) separates the enantiomers of flumecinol and its impurities. The potential of spectral convolution in peak identification in HPLC impurity profiling is demonstrated by an example involving the identification of the 4'-methyl-analogue of flumecinol.


Assessment of chromatographic peak purity of drugs by multivariate analysis of diode-array and mass spectrometric data.

Numerous multivariate chemometric approaches have been developed for LC-UV data acquired using a diode-array detector (DAD), but these methods have not been widely exploited for LC-MS data. Principal component analysis (PCA) and subsequent axis rotation within the reduced factor space are assessed for LC-DAD and LC-MS data as approaches for estimating the number of components (i.e. the rank of the data) under a single chromatographic peak for compounds whose UV-spectra are very similar. Multivariate techniques for LC-DAD data are shown to suffer from inherent limitations of sensitivity for the minor components. The novel technique in LC-MS of plotting the rotated PCA data in two-dimensional factor space generates characteristic ion clusters, giving a visual criterion of peak purity. Single ion chromatograms produced subsequently confirm the profile of each coeluting component and give evidence of the degree of peak overlap. The application of this new chemometric technique to the detection of low levels of coeluting impurities by LC-MS is discussed as a novel approach for the validation of LC separations in pharmaceutical research and development.


Liquid chromatography for the quantitative analysis of antibiotics--some applications using poly(styrene-divinylbenzene).

Problems arising from the microbiological assay of antibiotics are discussed. The existence of several systems to express the potency leads to confusion. The use of potency to express the content of bulk products can lead to difficulties in the interpretation of the content of pharmaceutical preparations. Such problems can be avoided if the content of antibiotics is expressed as percentage weight in weight. This involves the application of selective assay methods such as liquid chromatography. The reproducibility of liquid chromatography depends largely on the quality of the stationary phase. Use of poly(styrene-divinylbenzene) as the stationary phase can lead to good reproducibility as is reported for erythromycin and for the tetracyclines, of which minocycline is discussed as an example.


Analysis of erythromycin estolate by liquid chromatography.

A method is described for the determination of erythromycin estolate by liquid chromatography. A C18 reversed-phase column (25 x 0.46 cm i.d.) was used with acetonitrile-tetrabutylammonium sulphate (pH 6.5, 0.2 M)-phosphate buffer (pH 6.5, 0.2 M)-water [x:5:5:(90-x), v/v/v/v] as mobile phase. The proportion of acetonitrile (x) has to be adapted to the type of stationary phase used. For RSil C18 LL 42.5% (v/v) was used. The column was heated at 35 degrees C, the flow rate was 1.5 ml min-1 and UV detection was performed at 215 nm. The main component, erythromycin A propionate, was separated from all other components which were present in commercial samples. The impurities most frequently observed were the propionate ester of erythromycin C and the amide N-propionyl-N-demethyl-erythromycin A. Erythromycin A was shown to be present in specialties.


Dependence of the retention of some barbituric acid derivatives on a porous graphitized carbon column on their physicochemical parameters.

The retentions of 45 barbituric acid derivatives were determined on a porous graphitized carbon column (PGC) in unbuffered methanol-water eluent mixtures at various organic phase concentrations and the retention data were correlated with the various hydrophobic and electronic parameters of barbituric acid derivatives. Each derivative showed symmetric peaks in each eluent proving the good separation characteristics of the PGC column without buffering the eluent. Significant linear correlations were found between the logk' value and the concentration of the organic mobile phase in the eluent. Stepwise regression analysis indicated that the retention of barbituric acid derivatives is mainly governed by the electronic parameters, and the lipophilicity of various substituents did not affect significantly the retention, although the eluents were typical reversed-phase eluents.


Multidrug assay method for antimalarials.

A general separation strategy, involving solid-phase extraction followed by reversed-phase ion-pairing HPLC with an organic counter ion for a set of 11 widely used antimalarial drugs and metabolites has been developed. The basis underlying the separation has been explored and work, including quantitative data, has been carried out on illustrative separations which form the basis of novel quantitative assays of groups of antimalarials which are relevant to current prophylaxis and treatment of malaria.


HPLC analysis of imidazole antimycotic drugs in pharmaceutical formulations.

Reversed-phase HPLC on different column packing materials (Hypersil C-18, Spherisorb-CN, Chromspher-B) is used to obtain selective separations of imidazole antimycotic drugs, such as ketoconazole, clotrimazole, tioconazole, bifonazole, isoconazole, econazole, miconazole and fenticonazole. The use of a post-column on-line photochemical reactor is shown to be useful for the enhancement of the sensitivity of the HPLC analysis with UV detection. The proposed HPLC methods are applied to the analysis of commercial dosage forms (creams) with solid-phase extraction (SPE) procedure, using a diol sorbent, being found to be a convenient technique for the sample preparation giving quantitative drug recovery.


Design of chiral LC separations for calcium antagonists on alpha 1-acid glycoprotein and ovomucoid columns.

Three chiral calcium antagonist drugs, gallopamil and two dihydropyridine derivatives, have been successfully separated within short retention times using both the alpha 1-acid glycoprotein chiral stationary phase (Chiral-AGP) and the ovomucoid column (Ultron ES-OVM). Aqueous buffer at defined pH is modified by the addition of an organic component, in order to modulate the retention properties of each system. Optimization of pH and organic modifier is carried out using the modified simplex method, with Kaiser's peak separation function as a criterion. The influence of pH and percentage of organic modifier on retention, selectivity, resolution and column performance are discussed for the two dihydropyridines analysed on Chiral-AGP and Ultron ES-OVM stationary phases. A new method is proposed as a new chiral system suitability test for these protein-based phases, utilizing a racemic mixture of closely eluting verapamil enantiomers as a probe.


Nadolol: high-pressure liquid chromatographic methods for assay, racemate composition and related compounds.

High-pressure liquid chromatographic (HPLC) methods have been developed for the determination of drug content, racemate A and related compounds in nadolol raw materials. The method for drug content and related substances resolved seven related compounds and several unknown impurities from the drug. The minimum quantifiable levels were 0.05% or less for four of the seven related compounds and 0.3% or less for the remainder. Total impurities in eight raw material samples ranged from 0.06 to 0.96% and assay levels ranged from 98.7 to 101.0%. The method was adapted for the determination of nadolol racemate A by a change in mobile phase composition. One raw material sample contained less than 40% of racemate A. Two samples which had a granular appearance were variable in racemate A content. The methods were adapted for the determination of drug and racemate A in nadolol tablets. Drug content in three tablet samples was between 96.2 and 98.4% and racemate A content was about 52%.


Indirect detection of anti-acetylcholinesterase compounds in microcolumn liquid chromatography using packed bed reactor with immobilized human red blood cell acetylcholinesterase and choline oxidase.

The inhibiting compounds were separated by micro-column liquid chromatography in the mobile phase containing the natural substrate acetylcholine. A home-made packed bed microbioreactor system containing immobilized enzyme acetylcholinesterase (ACHE) in human red blood cell membrane and choline oxidase (CHO) from alcaligenes was used for the post-column conversion of acetylcholine to hydrogen peroxide which was detected by an electrochemical detector. The inhibition effect of the solutes caused a decrease in the acetylcholinesterase activity, a decrease in the formation of hydrogen peroxide and also a decrease in the response corresponding to the concentration of the solutes. The rate of the enzyme regeneration was also recorded. The micro-system was compared with a conventional LC system comprising commercially prepared enzyme reactor. The stability of the enzymes is at least 3 weeks at ambient temperature. The limit of detection depends on biological activity of inhibition and for galanthamine was 1 pmol.


Determination of verapamil and norverapamil in human plasma by liquid chromatography: comparison between a liquid-liquid extraction procedure and an automated liquid-solid extraction method for sample preparation.

A conventional liquid-liquid extraction (LLE) procedure with high-performance liquid chromatography (HPLC) has been developed for the determination of verapamil and its main metabolite, norverapamil, in plasma. After addition of the internal standard, plasma samples were basified with phosphate buffer (pH 9.0) and extracted with a mixture of cyclohexane-dichloromethane. After centrifugation, the organic layer was separated and the analytes were extracted back into a 0.1 N sulphuric acid solution containing 2-aminoheptane. An aliquot of this aqueous phase was then injected directly onto the HPLC column. This LLE procedure has been compared with an automated liquid-solid extraction (LSE) method that has been developed in parallel. Good linearity was obtained using both extraction methods. The absolute recoveries for the two analytes were ca 95% with the automated LSE procedure and slightly lower (ca 84%) for the LLE method. The automated method gives better results with respect to detectability and precision, but the LLE procedure is simpler to develop, requires much less expensive equipment, and remains a useful alternative when the number of samples to be analysed is limited.


Analysis of cocaine, benzoylecgonine, ecgonine methyl ester, ethylcocaine and norcocaine in human urine using HPLC with post-column ion-pair extraction and fluorescence detection.

The measurement of cocaine and its major metabolites has been achieved by an HPLC method that compensates for their different solubilities and detection properties. Although ecgonine methyl ester is a major metabolite it is generally not measured by HPLC because it is poorly detectable by UV, and its water solubility makes recovery from urine difficult. Using modified solid-phase extraction procedures recoveries of 85% for ecgonine methyl ester, 97% for cocaine, 106% for benzoylecgonine and 80% for ethylcocaine have been obtained from urine. Increased chromatographic retention and detection sensitivity has been obtained by formation of the t-butyldimethylsilyl derivative of ecgonine methyl ester which was found to be stable in the HPLC mobile phase for at least 1 week. Alkylation of norcocaine and benzoylecgonine has improved their detection sensitivity and also chromatographic resolution. All calibrations were linear over the range 200-1000 ng ml-1 in urine with correlation coefficients > 0.99.


Solid-phase extraction and optimized separation of doxorubicin, epirubicin and their metabolites using reversed-phase high-performance liquid chromatography.

A reversed-phase isocratic high-performance liquid chromatographic method is described in which a formal structured procedure was applied to predict the mobile phase composition giving optimal baseline resolution of the clinically important anticancer agents doxorubicin and 4'-epidoxorubicin (epirubicin), their principal metabolites, and daunorubicin (internal standard). These formal statistical procedures included the simultaneous techniques of solvent selectivity triangle and factorial design for range-finding preliminary studies, followed by use of the modified simplex, a sequential procedure. These were used to select the parameters of organic modifier, buffer strength and pH necessary for use with a Spherisorb ODS 1 column, to achieve optimal separation of eight anthracycline solutes. Ultraviolet and fluorescence detection was used (lambda ex = 254 nm, lambda em = 560 nm), and the latter gave a low detection limit for doxorubicin in serum of 1 ng ml-1. The optimal mobile phase composition was determined to be acetonitrile-0.06 M Na2 HPO4 containing 0.05% (v/v) triethylamine adjusted to pH 4.6 with 0.03 M citric acid (35:65, v/v). A solid-phase extraction method was developed to enable the selective isolation of anthracyclines by adsorption onto C8 Bond-Elut cartridges, and is based on extraction of serum spiked with a mixture of the anthracycline solutes. The anthracyclines were eluted using acetonitrile-0.2 M Na2 HPO4 containing 0.05% (v/v) triethylamine adjusted to pH 3.6 with 0.1 M citric acid (67.5:32.5, v/v). Reproducible recoveries for doxorubicin (94 +/- 8%) and for epirubicin (96 +/- 8%) were obtained (n = 5). In particular, recoveries for the 7-deoxyaglycone metabolite (99%) were higher than other extraction methods cited.(ABSTRACT TRUNCATED AT 250 WORDS)


Determination of a renin inhibitor in plasma by solid-phase extraction using acetone as protein binding displacer followed by on-line high-performance liquid chromatography.

H 218/54 is a potent inhibitor of human renin activity (pIC50 = 8.3 at pH 6) and is therefore a potential agent for blood pressure reduction. This lipophilic compound is highly bound to plasma proteins, e.g. 99.7% in rats and 99.6% in humans. For pharmacokinetic studies, a quantitative assay for 3H-H 218/54 in plasma has been developed. On top of an AASP phenyl solid-phase cartridge 70 microliters of rat plasma or 1 ml of cynomolgus plasma was mixed with 200 microliters of water containing 20% acetone. The acetone displaced the substance from plasma proteins without precipitation of the sample and clogging of the extraction column. The mixture was passed through the cartridge, which adsorbed 3H-H 218/54. The cartridge was placed in an AASP autosampler connected to a reversed-phase LC system, with a Vydac C-18 column and CH3CN-H2O-TFA (60:40:0.1, v/v/v) as mobile phase. The effluent from the separation column was collected in fractions for radioactivity counting. Recovery, as measured after adding various amounts of tritium-labelled H 218/54 to blank plasma followed by repeated analysis of the samples, was close to 100% with relative standard deviations between 1.4 and 3.0%. At the lowest level tested, 200 dpm per sample, the recovery was 120% with a relative standard deviation of only 10%. The sensitivity of the method will depend on the specific radioactivity of the dose given.


Reversed-phase liquid chromatography of the opioid peptides. 3. Development of a microanalytical system for opioid peptides involving microbore liquid chromatography, post-column derivatization and laser-induced fluorescence detection.

A microanalytical system has been developed for the determination of peptides in small samples. Naphthalene-2,3-dicarboxaldehyde-beta-mercaptoethanol (NDA-BME) was used as the labelling reagent system as an alternative to NDA-cyanide (NDA-CN) because of the faster labelling when CN was replaced by a thiol. The fluorescence characteristics of the NDA-thiol adducts, N-substituted 1-alkylthiobenz[f]isoindoles (TBIs), were found to be different from the previously described cyanobenz[f]isoindole (CBIs) adducts formed by the reaction of primary amines with NDA-CN. The excitation maximum of the TBI adducts was at 460 nm, which was closer to the 457.9 nm argon-ion laser line, than the 440-nm maximum of the CBI adduct. The limit of detection for leucine enkephalin was 36 fmol (S/N = 3) and linearity was proven for greater than 2 orders of magnitude, from 45 fmol to 9 pmol for an injection volume of 60 nl. The detectability was limited by the high background noise produced by the post-column derivatization system. The utility of the system was demonstrated for the analysis of methionine enkephalin and its potential oxidation products, using leucine enkephalin as a suitable internal standard.


Determination of medroxyprogesterone acetate in serum by HPLC with peroxyoxalate chemiluminescence detection using a fluorogenic reagent, 4-(N,N-dimethylaminosulphonyl)-7-hydrazino-2,1,3-benzoxadiazole.

The high-performance liquid chromatographic determination of medroxyprogesterone acetate (MPA) with peroxyoxalate chemiluminescence (PO-CL) detection is described. The spiked serum containing MPA was extracted on Bond-Elut C18 columns and derivatized with 4-(N,N-dimethylaminosulphonyl)-7-hydrazino-2,1,3-benzoxadiazole (DBD-H). The hydrazone of MPA with DBD-H was confirmed to be a mono-DBD-derivative. The reaction mixture was separated by direct injection onto a C18 analytical column, and quantified by PO-CL detection. The linear range of the standard curve, in serum, was 15.6-96.6 ng ml-1 with a detection limit of 9 ng ml-1 using only 100 microliters of serum, while the detection limit of standard MPA derivatized with DBD-H was 8.7 fmol per injection. The relative standard deviation of the method was 7.4% at 19.3 ng and 1.7% at 77.3 ng ml-1.


Endocrinology of calcium metabolism in amphibians, with emphasis on the evolution of hypercalcemic regulation in tetrapods.

Amphibians are the most diverse group as regards the endocrinology of Ca homeostasis because in them only "true" parathyroid glands made first appearance but its involvement varies greatly according to their phyletic affinities and habitats. Presence of ultimobranchial gland, parathyroid and renal 25-hydroxyvitamin-D-1-hydroxylase enzyme in anurans represents their tetrapod pattern of Ca regulation. Among "higher" urodeles, both parathyroid and pituitary are involved in hypercalcemic regulation whereas in "lower" urodeles (some of them are "aparathyroid" too), pituitary alone appears to discharge this function. The latter situation is analogous to the fish. Apodans do possess ultimobranchial and parathyroid glands but the problem of their Ca metabolism has not yet been explored. This review deals exhaustively with the endocrinology of Ca metabolism in amphibians. Also, the evolution of hypercalcemic regulation in tetrapods will be discussed in the light of recent developments.


Hermaphroditism in young mouse chimeras.

Among 23 5-day old mouse chimeras, 11 were sex chimeras. Four of them were hermaphrodites, six were males and one was female. In the gonads of three hermaphrodites fragments of ovarian tissue were very small. Growing oocytes were observed not only in ovotestes but also in testes. The course of transformation of ovotestes into testes is discussed.


Reconstruction of midface bone defects with vitallium micromesh.

Complex midface defects following trauma and tumor resection present a challenging problem for maxillofacial surgeons. Regardless of the cause of these defects, restoration of accurate contour is essential to achieve both proper function and cosmetically acceptable appearance. Traditionally, bone grafts have been the preferred method of reconstruction. We present the first report of the use of Vitallium micromesh in reconstruction of midface bone and cartilaginous defects in 3 patients. In 1 patient, bone loss resulted from trauma; in the other 2, loss was due to tumor resection. In all 3 patients, Vitallium micromesh was used successfully as a replacement for the bony skeleton without morbidity or complications. Use of this material is contingent on its rigid fixation. We do not recommend use of this technique in the absence of rigid fixation.


Soft-tissue alterations associated with acute, extended open reduction and internal fixation or orbital fractures.

Soft-tissue alterations associated with radical degloving of the overlying soft tissues during extended open reduction and rigid internal fixation of facial fractures involving 51 orbits in 36 patients were studied. In all patients, the blepharoplasty skin muscle flap incision was employed in conjunction with, as necessary, a gingivobuccal sulcus and coronary incisions. Patients were evaluated between 6 and 20 months after surgery. Physical examination and standardized photographs were used for assessment. Ectropion developed in 2 lids (4%), and 10 lids (20%) had increased scleral show. Thirteen of the 51 lids (25%) revealed lateral canthal displacement; all 13 lids underwent canthal stripping. Cheek pad displacement developed in 8 patients (22%), all of whom had complete maxillary degloving without soft-tissue resuspension. No patient had frontal nerve palsy. Depression in the temporal area was noted in all patients in whom the temporalis was mobilized. Remote incisions and soft-tissue degloving used for extended open reduction and internal fixation are associated with morbidity that can be minimized by meticulous technique and soft-tissue repositioning at closure.


Packed capillary liquid chromatography coupled to fluorescence detection: application to human blood samples for the determination of glutathione.

The present investigation analyses the potentials of capillary chromatography using packed fused silica capillaries filled with 5 microns RP-18 for the fluorescence determination of glutathione in human blood samples. Adaptation of conventional HPLC equipment for miniaturized chromatographic assays proved successful. Sample preparation was relatively simple, though care should be taken in sample handling. The thiolic compound mercaptoethanol was used as internal standard. Qualitative determinations were based on standard addition providing increased peak heights at identical retention times. Quantitative determinations gave linear calibration curves, with a standard glutathione recovery of 98.9% and an intra-assay reproducibility of 3.3%. The glutathione values measured appeared within the normal range of 0.9-1.7 mmol glutathione per litre of blood.


Influence of the maxillary canine on mandibular fracture.

The length of the root of the mandibular canine tooth has been considered by many authors as being a source of weakness in the mandible. It has also been suggested that a direct blow or a bending force around this tooth can result in traumatic injury. We advance a theory that implicates the maxillary canine tooth as directly contributing to the mandibular canine region fracture pattern.


Liquid chromatographic method for separation and determination of elaiophylin in biotechnology processes of salinomycin production.

An LC method for the quick and precise quantitative determination of elaiophylin in biotechnology processes of salinomycin production was prepared. A mobile phase of methanol 0.04 M diammonium hydrogen phosphate pH 6.0-ethyl acetate (55:35:10, v/v/v) and an elevated column temperature optimized the chromatography. Separation of elaiophylin was obtained in 4-6 min. Positive identification and peak purity of elaiophylin have been carried out by photodiode array (PDA) detection. An agreement between the PDA spectra of samples and the elaiophylin standard were obtained.


Neurological deterioration after lumbar cerebrospinal fluid drainage.

Large-bore lumbar spinal fluid drainage is used frequently as part of the preoperative and intraoperative management of patients undergoing cranial base tumor resection. Such drainage allows displacement of the brain with minimal force, thereby potentially decreasing retraction damage to it. We document 2 patients in whom serious complications resulted from lumbar drainage systems. These patients deteriorated into a coma state following cerebrospinal fluid (CSF) drainage. Reinfusion of synthetic CSF solutions caused a brisk return to normal neurological status. These plus other potential complications associated with lumbar drainage, such as persistent CSF leaks into the back and soft-tissue nerve root injury, warranted abandoning the lumbar cistern drainage route of CSF drainage in favor of drainage directly from the intracranial compartment. Depending on the particular operation performed, drainage of CSF near the cribriform plate, the suprachiasmatic cistern, or from the sylvian fissure may be effective sites for CSF drainage. Unlike lumbar drainage, intracranial CSF drainage does not have the added risk of promoting cerebral herniation.


Total temporomandibular joint reconstruction with a Delrin titanium implant.

Numerous alloplastic materials have been used for partial or total reconstruction of the temporomandibular joint in patients with fibrous or bony ankylosis, severe degenerative joint disease, or traumatic deformity, and for reconstruction following tumor resection and corrective surgery following multiple arthroplasties. We have had initial success with the use of a polyoxymethylene (Delrin) condylar head affixed to a pure titanium mesh for these reconstructive procedures. This versatile prosthesis does not require use of a separate glenoid fossa prosthesis. The surgical technique, as well as a historical perspective on alloplastic total joint prostheses used to date, is described in several patients.


Prevention of cranial reossification after surgical craniectomy.

An experimental study in rats was designed to see whether the pericranium, used as a graft over the dura, can prevent or delay osseous reunion after craniectomy. A 10 x 3-mm section of the frontal and parietal bones, including the coronal suture line, was resected in 15 rats. Pericranium was then used as a free graft on one-half of the defect to cover the dura and separate it from the cut edges of the craniectomy. The other side acted as a control. All animals were killed 2 months after operation, the skull was removed, and any remaining bony defect on either side of the midline was revealed. The area of residual defect in 13 specimens was determined by statistical image analysis using the Oxford Modular Cataract Image Analysis System (Oxford, England) and Wilcoxon's rank sum test and showed a highly significant difference between the treated and the untreated sides (p = 0.002).


The frontal tiara.

A technique for the construction of a frontal bar and a forehead plate as a single unit is described. This technique could be used in any procedure in which a frontal bandeau is advanced and the forehead needs to be remodeled. By using microscrew fixation, the bulkiness of miniplates and wires is eliminated.


Recipient vessels for microvascular transplants in patients with hemifacial microsomia.

Hemifacial microsomia is a developmental abnormality involving structures derived from the first and second branchial arches. Microvascular transplants are increasingly being used to improve facial contour in patients with this condition. We have reviewed 9 patients with this abnormality to determine which recipient vessels have been used. In 6 patients, the facial vessels were located and used for flap revascularization. In 3 patients, the facial vessels could not be identified intraoperatively, and the occipital artery and a branch of the external jugular vein were used as recipient vessels. In this series of hemifacial microsomia patients, therefore, the facial vessels could not be located in 3 patients. We recommend that surgeons performing microsurgical transplants in cases of hemifacial microsomia be prepared to explore the external carotid and external jugular systems for recipient vessels.


Effects of resorbable fixation on craniofacial skeletal growth: a pilot experimental study.

The effects of fixation composed of resorbable polymers on craniofacial development was investigated in an animal model. Fourteen rabbits had amalgam markers placed bilaterally at the lambdoid, coronal, and frontonasal sutures at 28 days of age. Seven animals (group I) were not plated and served as controls. The other 7 animals (group II) were fixed across the left coronal suture with a 4-hole, compressive resorbable plate. After 6 months, intermarker distances (growth) were measured radiographically, and the amount of fixation degradation was determined by extracted plate weights. Resorbable plating across the coronal suture resulted in local marker constriction adjacent to fixation. Compensatory lengthening of the distal aspect of the frontal bone occurred, however resulting in unaltered total cranionasal lengths compared with that of control animals. Plate degradation occurred with a mean decrease of 7% from their original weights. Molecular weight analysis of the plate differences, however, was not done. This initial study indicates that a large plate size and slow resorption properties of polymer fixation can have a similar restrictive effect on bone growth as that of metal fixation. Whether thinner plates or more rapidly resorbing polymers will permit normal growth in this animal model awaits further testing.


Plate fixation of premaxillomaxillary suture and compensatory midfacial growth changes in the rabbit.

It has been suggested that rigid fixation of the developing craniofacial skeleton may lead to altered craniofacial growth. However, recent experimental studies have shown that microplate fixation of slow-growing calvarial sutures resulted in regional growth disturbances but had little effect on overall craniofacial growth. The present study was designed to assess the effects of microplate fixation of the more rapidly growing facial sutures on compensatory midfacial growth in the rabbit. Twenty-two 1.5-week-old rabbits were randomly divided into 2 groups: sham controls (n = 11) and animals with bilateral premaxillomaxillary suture fixation (n = 11). Fixation was accomplished using an intraoral approach, a 6-mm straight Luhr microplate, and two 2-mm long self-tapping screws across each suture. Serial lateral head radiographs were collected at 1.5, 3.5, 6, 12, and 18 weeks of age. Results revealed that by 3.5 weeks, animals with rigid fixation showed significantly shortened premaxillary lengths (p < 0.05), class III occlusal relationships, decreased midfacial heights, and abnormal palatocranial base angles compared with sham control animals. By 12 weeks of age, "catch-up" growth was evident in most dimensions in animals with fixation compared with shams. Gross examination of the cleaned and dried skulls revealed bony bridging across the premaxillomaxillary suture and osseous fracture lines extending superiorly from the site of screw fixation. Results demonstrated that rigid fixation of a rapidly growing facial suture did not impair long-term midfacial growth in the rabbit model and suggest that compensatory changes may have occurred at fracture lines from the self-tapping screws in these animals.


Histological and radioisotopical analysis of the skull graft: an experimental study in rabbits.

The purpose of this study was to develop an experimental model in rabbits that would permit us to analyze skull reconstruction and enable the study of bone autografts, osteofacial flaps, and other materials. We use the rabbit model to study the early integration of the skull autograft and to demonstrate the relationship between radioisotopical and histological analyses.


The serratus anterior free tissue transfer for craniofacial reconstruction.

The serratus anterior muscle was used as a free tissue transfer to reconstruct complex craniofacial defects in 5 patients. Serratus anterior muscle alone and serratus anterior muscle with rib were the transfers made. All flaps survived and scapular winging did not occur. The serratus anterior muscle has several advantages for the reconstruction of medium-sized craniofacial defects. Because of its position, a two-team approach is possible. It has a consistent pedicle anatomy and low donor site morbidity. It has a large caliber vessel and a long pedicle. When compared with the commonly employed rectus abdominis flap for moderate-sized defects, the serratus muscle offers greater versatility in design and has the option of incorporating bone and innervated muscle without increasing significant donor site morbidity.


The radial forearm flap in intraoral reconstruction after a pull-through operation.

Free fasciocutaneous forearm flaps, based on the radial artery, were used to replace the tissue defect caused by pull-through operations for cancer of the floor of the mouth in 11 patients. There was no total or partial flap failure. The greatest advantage of the method is that restriction of the tongue movement is less when compared with the results of other methods. The thin forearm flaps are adaptable and pliable. The newly developed lower alveolar crest and the sublingual sulcus make prosthetic rehabilitation possible.


Chondromyxoid fibroma of the zygoma.

A case of chondromyxoid fibroma (CMF) involving the zygoma is presented. To our knowledge, this case report represents the first description of CMF involving an extragnathic site in the facial skeleton exclusive of the cranium. Total excision and immediate reconstruction were done with alloplastic material fixated with microplates and screws.


Mandibular joint dysfunction otalgia caused by a bomb explosion wave.

Four hundred ninety-five patients were exposed to bomb explosion waves in civilian and war areas during the Iraq/Iran conflict. These patients were referred to the Oral and Maxillofacial Surgery Clinic after complaining of temporomandibular joint dysfunction with otalgia. Treatment ranged from conservative measurement to active surgery. Generally, response was good.


Rigid internal fixation of a displaced mandibular coronoid fracture.

A report of the open treatment of the extremely rare displaced coronoid fracture of the mandible is presented. The previously predicted but undocumented oblique submuscular fracture from the depth of the mandibular notch to a point immediately behind the distal end of the alveolar process allowed displacement to occur. Use of plate and screw fixation for this exceedingly unusual injury avoided maxillomandibular fixation and was of benefit to this seizure-prone patient.


Plaque-induced marginal tissue reactions of osseointegrated oral implants: a review of the literature.

An intimate contact between bone and titanium implants was first demonstrated in 1969, and since then the bone-implant interface of osseointegrated implants has been investigated extensively. However, investigations of the marginal tissues and the microflora associated with osseointegrated implants have almost exclusively been carried out over the last decade. This review covers the clinical, radiographic, histologic, and microbiologic studies of marginal tissues of osseointegrated oral implants. In general, successfully osseointegrated implants exhibit low amounts of plaque concomitant with the absence of marginal inflammation. However, plaque accumulation may cause inflammatory reactions around the implants, sometimes giving rise to mucosal hyperplasia. Apparently, keratinized mucosa is not a requisite for the maintenance of peri-implant health if oral hygiene is adequate, but the presence of peri-implant keratinized mucosa is generally advocated. Alveolar bone loss around successful implants is minimal, but significant focal loss may occur due to plaque-induced inflammation or perhaps repeatedly extensive implant load. The progression of plaque-induced alveolar bone loss of osseointegrated implants may be different from that of teeth. It is unknown whether simultaneous marginal inflammation and excessive implant load further increase the loss of alveolar bone height. Both the light microscopic and ultrastructural characteristics of marginal tissues of implants and teeth are similar except for a lack of root cementum with inserting gingival collagen fibers of implants. Clinical inflammatory reactions are histologically characterized by an increased number of inflammatory cells infiltrating the connective tissue. The scattered subgingival microbiota associated with osseointegrated implants surrounded by healthy or slightly inflamed marginal tissues is similar to that of teeth with healthy gingiva. The microbiota associated with implants affected by marginal inflammation and bone loss is complex and consists predominantly of gram-negative anaerobic rods; this, again, is a similarity to periodontal disease.


Antimicrobial treatment of peri-implant infections.

The purpose of this study was to investigate the possibility of antimicrobial treatment of peri-implant infections associated with a periodontitis-like subgingival microbiota. Nine partially or fully edentulous patients with titanium hollow cylinder implants were selected which showed loss of bone and probing depths > or = 5 mm on one or several implants after at least 6 months following installation. They also yielded subgingival microbial samples with > or = 10(6) CFU/ml, including > or = 20% gram-negative anaerobic bacteria. The treatment included mechanical cleaning, irrigation of all peri-implant pockets > 3 mm with 0.5% chlorhexidine and systemic antimicrobial therapy (1000 mg ornidazole for 10 consecutive days). After therapy, bleeding scores decreased immediately and, over a one-year observation period, remained significantly lower than before treatment. A significant gradual reduction in mean probing depths was detected over this one-year period; only one case showed no improvement of local probing depth. Microbiological parameters indicated an instantaneous quantitative and qualitative change following treatment. Subsequently, several of these parameters tended to shift back towards pretreatment values. In the second half of the observation period, however, this tendency was reversed, and levels significantly different from baseline were eventually established. This study demonstrated that treatment aiming at reducing the subgingival bacterial mass and suppressing the anaerobic segment had a beneficial effect in patients suffering from peri-implantitis.


Supracrestal circular collagen fiber network around osseointegrated nonsubmerged titanium implants.

Eight non-submerged titanium implant screws were placed in the first upper molar edentulous sites of monkeys and subsequently kept loaded with single crown prosthesis 1 month following implantation. The animals were killed after a further 14 months and specimens including implant and adjacent teeth were processed for light and electron microscopy. Histological pictures of all samples showed the neck and most of the screw body to be surrounded by new bone. The soft tissue surrounding the implant post included pocket epithelium and supra-crestal connective tissue displaying collagen fiber bundles comparable to gingival ligaments. These peri-implant collagen fiber bundles arose from the neighboring alveolar crest, root cementum of adjacent teeth or, superficially, from the epithelium and followed a circular array around the implant neck.


Speech in connection with maxillary fixed prostheses on osseointegrated implants: a three-year follow-up study.

In 21 individuals, edentulous in the upper jaw, the speech function was evaluated. In an earlier study, registrations were made when the patients wore complete dentures in the upper jaw and 3-6 months after they had been treated with fixed prostheses on osseointegrated implants in the maxilla. These patients were re-examined after 3 years, as reported in this follow-up study. An expert group as well as a non-expert group participated in a perceptual analysis. Acoustic, audiological and cast analyses were performed. In addition, a questionnaire was filled in. The results indicated that, after initial phonetic problems, 94% of the individuals considered themselves free from speech problems at the 3-year follow-up. The patients' experience of the change of the s-sound corresponded well with the judgement of both the expert and the non-expert groups. Difficulties with pronouncing the s-sound were associated with decreased bite force, the number of occluding contacts, the frontal width of the fixed prosthesis and tenderness at palpation of the masticatory muscles. These oral functional factors will contribute to the s-sound production.


Radiographic evaluation of crestal bone levels adjacent to nonsubmerged titanium implants.

Several parameters have been described to determine success or failure in long-term evaluations of dental implants. One of these parameters is the observation of changes in peri-implant bone levels. Studies on submerged implants have analyzed the bone level changes in the pre- and post-loading phases. No such data exist for intentionally nonsubmerged implants. The purposes of this study were: (1) to test the applicability and reproducibility of a simple computer-assisted method in the evaluation of changes in peri-implant bone levels; (2) to establish a baseline for the longterm radiographic follow-up; and (3) to evaluate changes in crestal bone levels adjacent to nonsubmerged ITI implants between the 1-year and 2-year examination. Standardized periapical radiographs were obtained from 80 implants at the 1-year and 2-year follow-up examinations after their placement. The implants were located in different jaw areas of 55 patients and supported single crowns or short-span fixed partial dentures. For each implant, the distance from implant shoulder to first crestal bone contact (DIB) was measured at the proximal surfaces with a digitizer/computer assembly. Statistically significant greater mean DIB were found at the 1-year (baseline) evaluation for: (1) maxillary sites overall (4.10 x 1.02 mm (SD)) compared with mandibular sites overall (3.61 +/- 1.03 mm); (2) maxillary anterior sites (4.08 +/- 0.97 mm) compared with mandibular posterior sites (3.60 +/- 1.05 mm); and (3) maxillary posterior sites (4.13 +/- 1.12 mm) compared with mandibular posterior sites. No statistically significant changes in DIB occurred in any of the jaw locations between the 1-year and 2-year evaluations.(ABSTRACT TRUNCATED AT 250 WORDS)


The macroscopic, microscopic and spectrometric effects of various chemotherapeutic agents on the plasma-sprayed hydroxyapatite-coated implant surface.

The purpose of this research was to determine the nature of the residual hydroxyapatite (HA)-coated implant surface after treatment with various chemotherapeutic modalities, including: citric acid, chlorhexidine gluconate, hydrogen peroxide, tetracycline HCl, stannous fluoride, polymyxin B and a prototype plastic Cavitron tip. Implant surfaces were evaluated macroscopically, microscopically (scanning electron microscopy (SEM)) and spectrometrically (energy-dispersive spectrometry and X-ray diffraction). HA-substrate bond strength and dissolution testing was also performed for surfaces treated with a supersaturated citric acid solution. All treatments left either microscopic residues or a loss of surface roughness when viewed on SEM. A 30- to 60-s application of citric acid left a significantly greater coating thickness than all other treatments, whereas a 3-min application of citric acid removed significantly more HA than untreated controls. Significant changes in Ca/P ratios were seen with most treatments. The clinical significance of this phenomenon is not known. No treatments altered the crystallinity of the residual HA coating. A 1-min application of citric acid did not significantly alter the tensile bond strength of the coating to the substrate. The clinical significance of these findings is not known at present. However, when taken with results from previous studies, it appears that in treating the infected HA-coated implant surface, a 30- to 60-s application of citric acid (pH 1) may be beneficial in detoxifying the HA coating prior to regenerative procedures. Further in vitro and in vivo studies are necessary to evaluate the biological response to citric acid when used to detoxify the infected implant surface.


Processing endopeptidase deficiency in neurohypophysial secretory granules of the diabetes insipidus (Brattleboro) rat.

The homozygote Brattleboro rat exhibits a hereditary diabetes insipidus due to a deficiency of vasopressin, the antidiuretic hormone. It has previously been shown that in this animal a single nucleotide deletion in the provasopressin gene leads to a mutant precursor with a C-terminal amino acid sequence different from that of the wild-type. However the N-terminal region including the hormone moiety, the processing signal as well as the first two-thirds of the neurophysin is entirely preserved and absence of maturation has to be explained by an additional cause. We show here that the neurohypophysis of the homozygote Brattleboro rat, in contrast to the adenohypophysis, displays a significant decrease in the Lys-Arg processing endopeptidase activity when compared to the heterozygote or the wild-type Wistar. It is suggested that hypothalamic vasopressinergic neurons of the homozygote Brattleboro rat display a deficiency in the processing enzyme in contrast to the oxytocinergic neurons in which processing of prooxytocin is normal.


G-proteins mediate inhibition and activation of Ca(2+)-induced exocytosis from SLO-permeabilized peptidergic nerve endings.

In SLO-permeabilized isolated nerve endings from the rat neurohypophysis, GTP, guanosine 5'[y-thio]triphosphate (GTPyS) and guanosine 5'(beta y-imido]triphosphate (GMPPNP) inhibit the Ca(2+)-evoked vasopressin release. Pretreatment with pertussis toxin enhances the inhibitory effects of both GTP-analogues. Omission of Mg2+ overcomes the effect of GMPPNP and reverses the inhibitory effect of GTP and GTPyS. In the absence of Mg2+, GTP and GTPyS now potentiate Ca(2+)-evoked secretion.


The human placental transferrin receptor: reconstitution into liposomes and electron microscopy.

Human transferrin receptor was isolated from Triton X-100 solubilized placental plasma membranes by a rapid one-step chromatographic procedure based on immunoadsorption of the receptor-transferrin complex on anti-transferrin Sepharose and lectin-affinity on wheat germ agglutinin. Following exchange of Triton X-100 with CHAPS or n-octylglucoside, the purified receptor was incorporated into egg phosphatidylcholine liposomes upon detergent removal by dialysis (lipid/protein ratio 15:1 to 45:1 (w/w)). Reconstitution of the receptor was confirmed by trypsin cleavage to dissociate the large extracellular receptor domain from the liposomal membranes. Electron micrographs of the receptor-lipid recombinants negatively stained with sodium sillicotungstate, showed that the receptor molecules distributed very inhomogeneously on the liposomes, most receptors being clustered. Single copies of the receptor were seen as elongate structures (5 x 10 nm) oriented with their long axis parallel to the liposome surface and separated from this by a 2-3 nm gap. This result provides evidence for a narrow connecting link between the globular extracellular receptor domain and the membrane spanning segment.


Processing and characterization of the low density lipoprotein receptor in the human colonic carcinoma cell subclone HT29-18: a potential pathway for delivering therapeutic drugs and genes.

Low density lipoprotein (LDL) processing has been investigated in the subcloned human colonic carcinoma cell line HT29-18. LDL binding at 4 degrees C was a saturable process in relation to time and LDL concentration. The Kd for LDL binding was 11 micrograms/ml. ApoE-free HDL3 or acetylated LDL did not significantly compete with 125I-LDL binding, up to 500 micrograms/ml. 125I-LDL binding was decreased by 70% in HT29-18 cells preincubated for 24 hours in culture medium containing 100 micrograms/ml unlabelled LDL. Ligand blotting studies performed on HT29-18 homogenates using colloidal gold labelled LDL indicated the presence of one autoradiographic band corresponding to an apparent molecular weight of 130 kDa, which is consistent with the previously reported molecular weight of the LDL receptor in human fibroblasts. At 37 degrees C, 125I-LDL was actively internalized by HT29-18 cells and lysosomal degradation occurred as demonstrated by the inhibitory effect of chloroquine. LDL uptake and degradation by HT29-18 cells also resulted in a marked decrease in endogenous sterol synthesis. These data demonstrate that the HT29-18 human cancerous intestinal cells are able to specifically bind and internalize LDL, and that LDL processing results in down-regulation of sterol biosynthesis. Thus, intestinal epithelial cells possess specific LDL receptors that can be exploited to accomplish drug delivery and gene transfer via the receptor-mediated endocytosis pathway.


Exo-endocytosis in isolated peptidergic nerve terminals occurs in the sub-second range.

Exo- and endocytotic processes induced by depolarization of isolated neurosecretory nerve terminals show a close temporal correlation, which suggests a short time of integration of the neurosecretory granule membrane with the plasma membrane. In order to determine minimal time requirements for exocytosis-coupled endocytosis to occur, we have analyzed by electron microscopy uptake of horseradish peroxidase (HRP) as a fluid phase marker at the onset of depolarization. We have applied rapid mixing and sampling (quenched flow) to assess events in sub-second time periods after stimulation. A significant number of labelled endocytotic vacuoles was observed during the first second of depolarization. This number then further increased by a factor of about 2 (within 5 s) and 4 (within 50 s). Thus, as for exocytosis, the rate of endocytosis decreased considerably during prolonged stimulation. These data indicate i) that a substantial proportion of secretory granules undergoes exocytosis very shortly after stimulation, and ii) that, following exocytosis, the minimal time required for consecutive membrane retrieval is in the sub-second range.


Pulse shape analysis of RBC micropore flow via new software for the cell transit analyser (CTA).

The Cell Transit Analyser (CTA) provides a means to rapidly measure the deformability of large numbers of individual cells. It combines many of the advantages of micropipette studies with the simplicity and speed of filtrometry methods by measuring the duration of each resistive pulse generated as a cell passes through one of 30 identical micropores in a membrane. However, in our opinion, the potential of the system is limited by the microcomputer and software supplied for data analysis. We have therefore written new software for a more-powerful microcomputer to examine the shape of each resistive pulse rather than just the duration. Seven new parameters are derived, which provide additional information regarding the passage of cells through the pores. In particular, the contribution of the entry and exit phases of the cell transit are evident in the rise time and fall time of the pulses. The software is user-friendly and allows the analysis of each pulse to be reviewed, which aids understanding of the system and helps to avoid errors in interpreting the data.


Development of viscoelasticity in heated hemoglobin solutions.

Several previous studies have shown that exposure of RBC to temperatures in the range 47 to 48.8 degrees C causes an irreversible alteration of RBC rheological properties; membrane elasticity and viscosity are both increased and greater pressure is required for RBC entry into pipettes. While it has been tacitly assumed that these rheologic alterations are membrane specific, no data on heat-treated hemoglobin (Hb) solutions appear to exist. The present study was thus designed to evaluate the effects of heat-treatment (48.8 +/- 0.1 degrees C, 3 to 20 min) on the viscoelasticity of Hb solutions (30 to 45 g/100 ml) prepared from normal human RBC. Measurements of the viscous component (VC) and elastic component (EC) were made at 25 degrees C using Couette (GDM) and capillary (OCR-D) rheometers; shear rates ranged from 1 to 200 s-1. All unheated Hb solutions were Newtonian and did not exhibit elasticity. However, after 3 min of heating, an elastic component was measurable. Both VC and EC increased with heating time in a power law fashion. VC continued to exhibit Newtonian behavior, whereas the magnitude of EC was an inverse function of shear rate and directly related to Hb concentration and treatment time. A relaxation function applied to our data suggests a first order reaction. These results indicate that both cytoplasmic and membrane viscoelasticity should be considered in order to fully comprehend the rheologic behavior of heat-treated RBC.


Cell-free plasma layer in cerebral microvessels.

Two diameters of vessel and red cell column in cerebral microvessels (> 29.8 microns in diameter) of cat were measured together with red cell velocity, using a two fluorescent tracer method. A fluorescein isothiocyanate (FITC)-labeled red cell was adopted as a flow tracer to measure the cell velocity with a dual window technique. Based on the fluorescence image, the red cell column diameter was measured. Plasma was stained with rhodamine-B isothiocyanate (RITC)-labeled dextran to measure the vessel diameter. The thickness of the cell-free plasma layer could be determined from the difference of the two diameters. The obtained thickness of the cell-free layer was not described by a simple function of vessel diameter or red cell velocity; it was dependent on the pseudo shear rate defined by the ratio of cell velocity to vessel radius. The layer thickness increased with a decrease in the pseudo shear rate.