Supramolecular Solvent Extraction-Gas Chromatography-Tandem Mass Spectrometry for Detection of Benzodiazepines in Urines.

ABSTRACT: Objective To establish a method using supramolecular solvent and gas chromatography-tandem mass spectrometry （GC-MS/MS） to analyze 9 benzodiazepines in urines. Methods Urine samples containing 9 benzodiazepines reference substance were subjected to liquid-liquid extractions with supramolecular solvent, which consisted of tetrahydrofuran and 1-hexanol. The solvent layer was evaporated to dryness by stream of nitrogen. The residue was reconstituted with methanol, and GC-MS/MS analysis was performed on it. The way of data collection was multiple reaction monitoring （MRM） mode; internal standard method was employed for quantification. Results In urine samples, when the range of mass concentration was 1-100 ng/mL for diazepam, midazolam, flunitrazepam and clozapine, 5-100 ng/mL for lorazepam and alprazolam, 2-100 ng/mL for nitrazepam and clonazepam, and 0.2-100 ng/mL for estazolam, respectively, good linearities were obtained, correlation coefficients were 0.999 1-0.999 9, the lower limits of the quantifications ranged from 0.2 to 5 ng/mL, the extraction recovery rates were 81.12%-99.52%. The intra-day precision ［relative standard deviation （RSD）］ and accuracy （bias） were lower than 9.86% and 9.51%, respectively; the inter-day precision （RSD） and accuracy （bias） were lower than 8.74% and 9.98%, respectively. Nine drugs in urine samples showed good stability at ambient temperature and -20 ℃ within 15 days. The mass concentrations of alprazolam in urine samples obtained from 8 volunteers who took alprazolam tablets orally within 8-72 h after ingestions ranged from 6.54 to 88.28 ng/mL. Conclusion The supramolecular solvent extraction GC-MS/MS method for analysis of 9 benzodiazepines in urines provided by this study is simple, fast, accurate and sensitive, which can provide technical support for monitoring of poisoning by benzodiazepines for clinical treatment and judicial identification.

Metastatic Pleomorphic Adenoma in the infratemporal fossa and neck following total parotidectomy after 30 years.

Metastasising pleomorphic adenoma is rare and may occur years after surgical excision of a pleomorphic adenoma (PA). We present a 61-year-old woman with a right infratemporal PA with metastases to the cervical lymph nodes after 30 years following a total parotidectomy. She was treated successfully with a resection of the tumour with combined neck and mandibulotomy approach along with postoperative radiotherapy given subsequently.

Predictive Accuracy of Mandibular Ramus Flexure as a Morphologic Indicator of Sex Dimorphism in Jordanians / Exactitud Predictiva de la Flexura de la Rama Mandibular como Indicador Morfológico de Dimorfismo Sexual en Jordanos

The present study investigated the validity of mandibular ramus flexure in sex differentiation among young Jordanian adults using Orthopantomographic images in a double blind study. A normative sample comprised of 419 images was selected, of which 126 belonged to male patients and 293 images to females. The exclusion criteria used in the selection involved excluding images that showed marked loss of mandibular molars, over erupted, tilted, anomalous molars and teeth or mandibles associated with pathology. Each image was examined for the presence of a flexure on the posterior margin of the ramus at the occlusal plane. The posterior margin of the ramus was carefully traced graphically and the occlusal plane level was delineated as guided by the height of cusp tips at the occlusal surfaces of the mandibular molars. The ramus flexure was more accurately diagnostic for females (94.6%), than for males (47.6%); this gave rise to an overall diagnostic accuracy of 70.9% which is far below the reported 90.6-99.0% by Loth & Henneberg. Our results, however, agreed with some reported findings of other researchers. In conclusion, ramus flexure provides a moderately acceptable predictive accuracy and could be considered as a supplementary rather than a definitive mean of sex determination. In sexing unknown skeletal remains, it is necessary to employ as many methods or traits as possible instead of relying on one morphological trait.

El presente estudio investigó la validez de flexura de la rama mandibular en la diferenciación sexual entre adultos jóvenes jordanos utilizando imágenes de ortopantomografías, en un estudio doble ciego. Se seleccionó una muestra normativa compuesta de 419 imágenes, de las cuales 126 pertenecían a hombres y 293 a mujeres. Se excluyeron imágenes que mostraron una pérdida marcada de los molares mandibulares, dientes sobre erupcionados o inclinados, molares anómalos y los dientes o mandíbulas con enfermedad. Cada imagen fue examinada para detectar la presencia de un ángulo en el margen posterior de la rama en el plano oclusal. El margen posterior de la rama mandibular fue trazado gráficamente y el plano oclusal se delineó estableciendo como guía la altura de las puntas cuspídeas en las superficies oclusales de los molares mandibulares. La flexura de la rama mandibular fue más precisa en el diagnóstico de las mujeres (94,6%) que para los hombres (47,6%); con una precisión diagnóstica global del 70,9%, la cual es inferior a la reportada por Loth & Henneberg entre 90,6­99,0%. Nuestros resultados, concuerdan con algunos hallazgos reportados por otros investigadores. En conclusión, la flexura de la rama mandibular proporciona una exactitud de predicción moderadamente aceptable y podría ser considerada como un complemento en lugar de un medio definitivo de la determinación del sexo. En restos óseos desconocidos, es necesario emplear la mayor cantidad de métodos o rasgos posibles que depender de un solo rasgo morfológico.

Efficiency of irrigation water application in sugarcane cultivation in Pakistan.

BACKGROUND: Diminishing irrigation water supplies are threatening the sustainability of irrigated agriculture in Pakistan. Within the context of dwindling water resources and low agricultural water productivity, it is imperative to improve efficiency in agricultural production and to make efficient use of available water resources. This study employs a non-parametric approach to estimate the extent of technical and irrigation water efficiency in sugarcane cultivation in Pakistan. RESULTS: The mean technical efficiency score is 0.96 for tube-well owners whereas it is 0.94 for water buyers. The mean irrigation water efficiency score is 0.86 for tube-well owners whereas it is 0.72 for water buyers. We find that across all farms, 59% of the tube-well owners and 45% of the water buyers are fully technically efficient, whereas only 36% of the tube-well owners and 30% of the water buyer are fully efficient in irrigation water use. CONCLUSIONS: This study finds that sugarcane growers are operating at fairly high technical efficiency levels. But, there is considerable potential to improve irrigation water efficiency. This study proposes expanding the role of agricultural extension services from merely agronomic grounds to guide farmers to undertake cost benefit analysis of the available production technology, would help achieve higher efficiency levels.

Burnout among clinical dental students at Jordanian universities.

Dentistry is a profession demanding physical and mental efforts as well as people contact, which can result in burnout. The level of burnout among 307 clinical dental students in 2 Jordanian universities was evaluated using the Maslach Burnout Inventory survey. Scores for the inventory's 3 subscales were calculated and the mean values for the students' groups were computed separately. Dental students in both universities suffered high levels of emotional exhaustion and depersonalization. The dental students at the University of Jordan demonstrated a significantly higher level of emotional exhaustion than their counterparts at the Jordan University of Science and Technology.

Burnout among clinical dental students at Jordanian universities

Dentistry is a profession demanding physical and mental efforts as well as people contact, which can result in burnout. The level of burnout among 307 clinical dental students in 2 Jordanian universities was evaluated using the Maslach Burnout Inventory survey. Scores for the inventory's 3 subscales were calculated and the mean values for the students' groups were computed separately. Dental students in both universities suffered high levels of emotional exhaustion and depersonalization. The dental students at the University of Jordan demonstrated a significantly higher level of emotional exhaustion than their counterparts at the Jordan University of Science and Technology

Coinheritance of two rare genodermatoses (Papillon-Lefèvre syndrome and oculocutaneous albinism type 1) in two families: a genetic study.

The co-occurrence of two rare recessive genetic conditions in apparently unrelated individuals or families is extremely rare. Two geographically distant and apparently unrelated families were identified in which individuals were simultaneously affected by two rare recessive mendelian syndromes, Papillon-Lefevre syndrome and type 1 oculocutaneous albinism. The families were tested for mutations in the causative genes, cathepsin C (CTSC) and tyrosinase (TYR), respectively, by direct sequencing. To assess the relationship of the two families, both families were tested for polymorphisms at eight microsatellite markers spanning both CTSC and TYR loci. Independent mutations (c.318-1G-->A and c.817G-->C/p.W272C) were identified in CTSC and TYR, respectively, that were shared by the affected individuals in both families. The two affected genes lie close together on chromosome bands 11q14.2-14.3, and studies with linked genetic markers suggested that the families shared a small chromosomal segment carrying both mutations that had been transmitted intact from a remote common ancestor. The co-occurrence of the two rare diseases in multiple families depends on their shared chromosomal location, but not on any shared pathogenic mechanism.

Antimony-resistant Leishmania donovani in eastern Sudan: incidence and in vitro correlation

A longitudinal study was done in a leishmaniasis -endemic region in eastern Sudan during the period November 2001-February 2003 to determine the incidence of failure of sodium stibogluconate treatment. We studied 820 confirmed visceral leishmaniasis patients. All were treated with sodium stibogluconate, 20 mg/kg body weight for at least 28 days. Parasites were isolated from lymph node aspirates from 22 participants identified as relapsed patients. All isolates were typed as Leishmania donovani based on polymerase chain reaction [PCR] amplification of parasite kDNA. Six parasites showed in vitro resistance to sodium stibogluconate using murine J774 macrophage amastigote testing method. The resistant isolates showed different restriction profiles when the amplified kDNA PCR products were digested with ALU1 restriction enzyme, indicating that resistance was mediated by different parasite clones

Oral health status of workers exposed to acid fumes in phosphate and battery industries in Jordan.

OBJECTIVES: To investigate the prevalence and nature of oral health problems among workers exposed to acid fumes in two industries in Jordan. SETTING: Jordan's Phosphate Mining Company and a main private battery factory. DESIGN: Comparison of general and oral health conditions between workers exposed to acid fumes and control group from the same workplace. SUBJECTS AND METHODS: The sample consisted of 68 subjects from the phosphate industry (37 acid workers and 31 controls) drawn as a sample of convenience and 39 subjects from a battery factory (24 acid workers and 15 controls). Structured questionnaires on medical and dental histories were completed by interview. Clinical examinations were carried out to assess dental erosion, oral hygiene, and gingival health using the appropriate indices. Data were statistically analysed using Wilcoxon rank-sum test to assess the significance of differences between results attained by acid workers and control groups for the investigated parameters. RESULTS: Differences in the erosion scores between acid workers in both industries and their controls were highly significant (P<0.05). In both industries, acid workers showed significantly higher oral hygiene scores, obtained by adding the debris and calculus scores, and gingival index scores than their controls (P<0.05). The single most common complaint was tooth hypersensitivity (80%) followed by dry mouth (77%) on average. CONCLUSION: Exposure to acid fumes in the work place was significantly associated with dental erosion and deteriorated oral health status. Such exposure was also detrimental to general health. Findings pointed to the need of establishing appropriate educational, preventive and treatment measures coupled with efficient surveillance and environmental monitoring for detection of acid fumes in the workplace atmosphere.

Fluoride release from glass ionomer restorative materials and the effects of surface coating.

OBJECTIVES: This in vitro study on fluoride (F) release from conventional and metal-reinforced glass ionomer investigated the following: (1) the release of F in deionized water compared to artificial saliva, (2) the effect of various surface coatings on F release, (3) the uptake of released F by hydroxyapatite, (4) the expression of the release data in a mathematical model, (5) F content in the powders and set materials, and (6) surface morphology of varnished and resin-coated specimens. METHODS: Glass ionomer Ketac-Fil (KF), Fuji II (FJ), and Ketac-Silver (KS) were mixed according to the manufacturers' instructions, and prepared into specimens of 137.8 mm2 surface area. All three specimens were suspended in 50 ml of deionized water, artificial saliva, or aqueous solution of hydroxyapatite and submitted to constant agitation at 37 degrees C. In a separate experiment, the specimens were coated with varnish or light-cured bonding resin and tested for F release in solutions similar to those for uncoated specimens. The release of F occurred for 28 days. The concentration of F was measured with F-ion-specific electrode. RESULTS: All tested products showed a strong initial rate of F release which decreased with time until it reached a relatively steady rate after two weeks. The F released from KF and FJ was comparable in both pattern and magnitude. They released approximately four times more F than KS. In all cases, the release of F in artificial saliva was significantly (p < 0.001) less than in deionized water. Surface coating the specimens significantly reduced the F release ( p < 0.05 top < 0.001, depending on the product and type of coating). The inhibitory effect of coating markedly decreased with time. Resin coating reduced F release more than varnish in KF and KS, but not for FJ. Essentially, all F released in aqueous solution was taken up by the hydroxyapatite, with FJ ranking the highest in increasing hydroxyapatite F concentration. Over the 28 days, the quantities of F released from FJ, KF, and KS were, respectively, 3.8, 2.3, and 1.0% of the total F content in the specimens. The F concentration in the set KS was 53.9 and 72.5% of that found in KF and FJ, respectively. The release data as a function of time were best described by the power curve. Micromorphological examinations revealed remnants of surface coatings on specimens after 14 days storage in artificial saliva. CONCLUSIONS: Glass ionomer cements released significantly less F in artificial saliva than in deionized water. Surface coating the specimens substantially reduced F release. These clinically relevant factors were not considered by many in vitro release studies which overestimate the F availability from glass ionomers. A recall appointment 24 h after the placement of glass ionomer restoration should be given for surface finishing.

Regulation of NCAM by growth factors in serum-free myotube cultures.

Regulation of the neural cell adhesion molecule (NCAM) was examined in primary cultures of chick skeletal muscle grown in serum-free defined medium. Relative levels of NCAM (per microgram protein) increased 20-30% in myotubes grown on Matrigel, a reconstituted basement membrane preparation, compared to those grown on collagen; total NCAM levels on Matrigel were increased 40-55% due to the additional increase in total protein. A dose dependent increase in relative NCAM levels in myotubes grown on Matrigel in defined medium was observed with the addition of adsorbed horse serum, while relative NCAM levels in myotubes grown on collagen were unaffected by altering the serum concentration. Thus, extracellular matrix molecules and soluble factors exert trophic effects on myotube NCAM expression. Similar developmental changes in the expression of the different molecular size forms of NCAM occurred in myotubes grown on collagen and Matrigel: levels of 150K and 135K Mr forms decreased during development, while 125K remained prominent in older myotubes. Relative NCAM levels were specifically enhanced 11-26% by several factors: nerve growth factor, thyroxine, insulin-like growth factor II, dibutyryl cyclic AMP, veratridine (a sodium ion channel agonist), and nisoldipine (a calcium ion channel agonist). Total protein and overall myotube development in serum-free cultures were enhanced by fetuin, insulin-like growth factor II, acidic fibroblast growth factor, calcitonin gene-related peptide, dibutyryl cyclic AMP, and veratridine. Thus, changes in extracellular matrix, intracellular calcium, and sodium ions, as well as extracellular trophic factors, such as nerve growth factor, thyroxine, and insulin-like growth factor II, may regulate muscle NCAM expression during embryonic development.

Matrigel enhances myotube development in a serum-free defined medium.

Previously reported serum-free defined media for muscle cell culture require supplementation with hormones, purified growth factors or attachment factors. This report describes a culture system that enhances embryonic chick, skeletal muscle cell growth and differentiation in a serum-free defined medium, without added specialized trophic factors. Myoblasts adhered more to and proliferated more rapidly on a reconstituted basement membrane substrate, Matrigel, than on rat-tail collagen. Matrigel contains several basement membrane attachment molecules which apparently obviate the need for added purified attachment factors. Matrigel also appeared to play a trophic role in subsequent development by enabling the serum-free growth of myotubes which suggests that Matrigel mediates the cellular interaction of growth or attachment factors. Collagen, on the other hand, did not support serum-free myotube growth. Supplementation of defined medium with increasing levels of horse serum enhanced total protein in myotubes grown on both substrates; protein was higher in Matrigel cultures for each medium tested. The serum-free defined medium supported complete morphological differentiation of myotubes grown on Matrigel and maintained myotube cultures up to 22 days. Fibroblast proliferation was higher in cultures on collagen in defined medium with high serum levels, but was virtually eliminated in cultures on Matrigel in serum-free defined medium. The culture system described supports the differentiation of embryonic muscle cells in a simple, serum-free defined medium, thus providing an in vitro model of developing myotubes which should be particularly useful for studies of regulation mediated by extracellular factors.

A new method for evaluation of urinary autocrine motility factor and tumor cell collagenase stimulating factor as markers for urinary tract cancers.

Over the past several years, many tumor markers, including cell surface antigens, T-antigen, ras p55, and ras p52 proteins, have been studied as potential tumor markers of bladder cancer. The lack of specificity and inconsistency of these markers led us to develop a new method for studying the urinary excretion of autocrine motility factor (uAMF) and tumor cell collagenase stimulating factor (TCSF) in 24-hour and first morning voided specimens. AMF is a glycoprotein secreted by the malignant cells and is responsible for cell locomotion, a key event in invasion and metastases of the malignant cells. TCSF is a membrane bound glycoprotein of tumor cells that stimulates fibroblast collagenase production. We have utilized an enzyme-linked immunoabsorption assay to detect the levels of uAMF and TCSF in urine samples collected from normal volunteers, patients with benign diseases, and patients with bladder cancer. Our data indicate that urinary concentrations of uAMF and TCSF are elevated in patients with bladder cancer. Furthermore, the levels of uAMF and TCSF are more elevated in invasive tumors as compared with benign counterparts. We have localized uAMF and TCSF in bladder cancer cells, utilizing immunohistologic techniques.

Plasminogen activator secretion by established lines of human ovarian carcinoma cells in vitro.

Ten human ovarian carcinoma cell lines (A121, A121[as], Caov-3, Caov-4, NIH:OVCAR-3, OVCA 420, OVCA 429, OVCA 432, OVCA 433, and SK-OV-3) were examined for secretion of plasminogen activators (PAs) using a chromogenic PA assay and SDS-PAGE zymography. PA activity was detected in conditioned media from all 10 cell lines. PA levels secreted by the 10 individual lines in a 24-hr period spanned a large range, with the extremes being 8 and 5244 milliPloug units (mPU)/10(6) cells for SK-OV-3 and OVCA 420 cells, respectively. Secreted PAs were identified as urokinase (UK)-like or tissue plasminogen activator (tPA)-like using dual criteria of comigration with UK or tPA standards on SDS-PAGE zymography and fibrin-dependence characteristics. Using both criteria, all 10 cell types produced UK-like activity, while tPA-like activity was produced by only 5 of the lines: A121[as], Caov-3, NIH:OVCAR-3, OVCA 429, and OVCA 433. Two additional cell lines produced PA activities that were tPA-like if judged by only one of the two criteria. Thus, Caov-4 cells produced a PA which comigrated with tPA, yet displayed no fibrin-dependent characteristics. Conversely, SK-OV-3 cells produced a fibrin-dependent PA, yet a band comigrating with tPA was not seen on SDS-PAGE zymography. Two lines derived from primary and ascitic sites from the same patient (A121 and A121[as], respectively) produced PAs with markedly different characteristics. Thus, PA produced by A121 cells was 100% UK-like, while that produced by A121[as] cells was greater than 90% tPA-like. Also, the total PA activity secreted by A121 cells was four times that secreted by A121[as] cells. In addition to bands comigrating with UK or tPA, all of the cell lines except Caov-3 and NIH:OVCAR-3 displayed higher molecular weight PA activities suggestive of the SDS-stable complexes between PAs and PA inhibitors reported in other cell types. While our results indicate that PA production may be a general characteristic of ovarian carcinoma cells in culture, individual patterns of UK and tPA production appear to be complex and vary from cell line to cell line. The precise characteristics of PA production in a given cell line may therefore depend on currently unidentified characteristics of the original tumor.

Hormonal regulation of CA125 tumor marker expression in human ovarian carcinoma cells: inhibition by glucocorticoids.

The CA125 tumor marker is an antigenic determinant present on a high-molecular-weight glycoprotein expressed by more than 80% of newly diagnosed nonmucinous epithelial ovarian cancers. OVCA 433 human ovarian carcinoma cells express the CA125 marker at the cell surface and release large quantities of this antigen into culture medium. Here we show that release of CA125 by OVCA 433 cells is 90 to 95% inhibited by treatment with 1 x 10(-7) M dexamethasone, as determined using a biotin-based enzyme-linked immunosorbent assay utilizing OC125 monoclonal antibodies to CA125. The relative cell surface density of CA125 is also decreased following dexamethasone treatment as determined by immunofluorescence techniques using OC125 monoclonal antibodies. Inhibition of CA125 expression is specific for glucocorticoids, such as cortisol and dexamethasone, and does not occur with estrogens, progestins, androgens, or mineralocorticoids. CA125 inhibition is also dependent on the concentration of steroid used, with half-maximal and maximal inhibition by dexamethasone occurring at about 3 x 10(-9) M and 1 x 10(-7) M, respectively. Previous work has shown that OVCA 433 cells are growth inhibited by glucocorticoids and contain 14,000 glucocorticoid receptors per cell with an affinity for dexamethasone (Kd = 6.6 x 10(-9) M) which corresponds well with the concentration required for half-maximal CA125 inhibition. This correspondence, together with the specificity of CA125 inhibition for glucocorticoids, suggests that this effect is mediated by glucocorticoid receptors and is a specific biological effect of glucocorticoids on this cell type. Our results demonstrate glucocorticoid inhibition of CA125 expression by ovarian carcinoma cells and suggest that endogenous or therapeutically administered glucocorticoids can influence CA125 production by tumors in vivo.

Glucocorticoid sensitivity of OVCA 433 human ovarian carcinoma cells: inhibition of plasminogen activators, cell growth, and morphological alterations.

OVCA 433 human ovarian carcinoma cells secrete large amounts of plasminogen activator (PA), which consists of immunologically identifiable urokinase (UK) and tissue-type PA (tPA). Total extracellular PA activity is 95% inhibited by treatment of cells with 1 X 10(-7) M dexamethasone (Dex) for 3 days. This inhibition is both time and concentration dependent, with half-maximal inhibition occurring after 1.5 days with 1 X 10(-7) M Dex, or with 1 X 10(-9) M Dex for 3 days, respectively. Interestingly, the loss of UK activity precedes the loss of tPA activity, such that half-maximal inhibition of the two PA types occurs at 1 and 2 days, respectively. Dex treatment leads to approximately 50% inhibition of cell growth and pronounced morphological alterations, including marked enlargement, flattening, and multinucleation. Treatment of the cells with other classes of steroid hormones, i.e., estrogens, progestins, androgens, and mineralocorticoids, is without effect on UK and tPA activities, cell growth, or morphology. OVCA 433 cells contain about 14,000 nuclear glucocorticoid receptors (GR) per cell (measured at 37 degrees C), with an average affinity (Kd) for [3H]Dex of 6.6 X 10(-9) M. Only active glucocorticoids compete with [3H]Dex for nuclear GR binding sites. Our results demonstrate steroid-specific glucocorticoid responsiveness of ovarian carcinoma cells, a tumor cell type not usually considered hormonally responsive. Since almost 90% of ovarian carcinoma tumor biopsies contain GR (M. C. Galli, et al., Cancer (Phila.), 47: 1297-1302, 1981), it is possible that glucocorticoid sensitivity could be exploited clinically, particularly following the almost universal development of resistance to the chemotherapeutic drugs commonly used in this disease.

Occupational chronic exposure to metals. I. Chromium exposure of stainless steel welders--biological monitoring.

External and internal chromate exposure of 103 stainless steel welders who were using manual metal are welding (MMA), metal inert gas welding (MIG) and both methods, were measured by ambient and biological monitoring. At the working places the maximum chromium trioxide concentrations were 80 micrograms/m3. The median values were 4 micrograms/m3 (MMA) and 10 micrograms/m3 (MIG). The median chromium concentrations in erythrocytes, plasma and urine of all welders were less than 0.60, 9.00 and 32.50 micrograms/l. For biological monitoring purposes, chromium levels in erythrocytes and simultaneously in plasma seem to be suitable parameters. According to our results, chromium levels in plasma and urine in the order of 10 and 40 micrograms/l seem to correspond to an external exposure of 100 micrograms chromium trioxide per cubic metre, the technical guiding concentration (TRK-value). Chromium concentrations in erythrocytes greater than 0.60 micrograms/l indicate an external chromate exposure greater than the TRK-value.

The level of residual monomer in self-curing denture-base materials.

Because residual monomer may cause tissue irritation, we investigated two self-curing acrylic denture-base materials--one a compression type, and the other a pourable material--by means of gas-liquid chromatography. Both exhibited higher residual monomer levels than did heat-cured acrylics, with thick sections having lower values than did thin sections. The pourable material showed lower values than did the compression variety.