Search

Levamisole, a veterinary antihelminthic, was recently recognized as an adulterant in cocaine and is known to cause severe adverse reactions in some cocaine users. Because of the health concerns involving levamisole-adulterated cocaine, we developed a liquid chromatography-tandem mass spectrometry (LC-MS-MS) method for the detection of levamisole in urine. This method was used to determine the prevalence of levamisole in cocaine-positive patient samples. All cocaine-positive urine samples that were sent to the San Francisco General Hospital Clinical Laboratory were tested for levamisole for one month. For LC, an Agilent 1200 series was used with a C(18) column and a gradient of mobile phase A (0.05% formic acid) and B (acetonitrile/methanol). Detection was carried out with an Applied Biosystems QTRAP(®) LC-MS-MS. The levamisole LC-MS-MS method was linear over the range of 5-2500 ng/mL (r > 0.996). Interassay and intraassay CVs were < 6%. The lower limit of detection for levamisole was 0.5 ng/mL. Out of 949 total urine drug screens, 20% were positive for benzoylecgonine, and of those, 88% were positive for levamisole. The high prevalence of levamisole-adulterated cocaine and potential toxicity in cocaine users is a serious public health concern. These findings validate the utility of an LC-MS-MS method for the detection of levamisole.

Background: Vitamin D is an essential nutrient, and there is a growing appreciation of its clinical significance. In addition, there has recently been a discussion as to the best method of measuring this analyte in serum: immunoassay, HPLC or LC-MS/MS. Due to the increased interest in vitamin D, there has been an exponential increase in the number of clinicians testing their patients and therefore also in the volume of this test run in clinical laboratories., Methods: Vitamin D levels were determined by chemiluminescence immunoassay in a reference laboratory and a subset by LC-MS/MS at San Francisco General Hospital., Results: We developed a robust and rapid LC-MS/MS assay to detect 25-hydroxyvitamin D3 and D2 in serum for use in the routine clinical laboratory. Additionally, we determined that 71% of patients served by San Francisco General Hospital have insufficient serum vitamin D levels (≤29 ng/ml) and that these levels are significantly associated with parathyroid hormone levels, total calcium concentration, age and ethnicity., Conclusions: The high degree of vitamin D insufficiency at San Francisco General Hospital may be reflective of the status of the patients served by this county hospital; largely an underserved, multi-ethnic urban population., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Background: Liquid chromatography-mass spectrometry (LC-MS) and tandem LC-MS (LC-MS/MS) are increasingly used in toxicology laboratories as a complementary method to gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-ultraviolet detection (LC-UV) for comprehensive drug screening (CDS). This study was designed to characterize the sensitivity and specificity of three LC-MS(/MS) vendor-supplied methods for targeted CDS and identify the current limitations associated with the use of these technologies., Methods: Five methods for broad spectrum CDS, including LC-UV (REMEDi), full scan GC-MS, LC-MS (ZQ-Mass Detector with MassLynx-software), LC-QTRAP-MS/MS (3200-QTRAP with Cliquid-software) and LC-LIT-MS/MS (LXQ Linear Ion Trap with ToxID-software) were evaluated based on their ability to detect drugs in 48 patient urine samples., Results: The tandem MS methods identified 15% more drugs than the single stage MS or LC-UV methods. Use of two broad spectrum screening methods identified more drugs than any single system alone. False negatives and false positives generated by the LC-MS(/MS) software programs were identified upon manual review of the raw data., Conclusions: The LC-MS/MS methods detected a broader menu of drugs; however, it is essential to establish manual data review criteria for all LC-MS(/MS) drug screening methods. Use of an EI-GC-MS and ESI-LC-MS/MS combination for targeted CDS may be optimal due to the complementary nature of the chromatographic and ionization techniques., (Copyright 2010 Elsevier B.V. All rights reserved.)

In regulated vesicle exocytosis, SNARE protein complexes drive membrane fusion to connect the vesicle lumen with the extracellular space. The triggering of fusion pore formation by Ca(2+) is mediated by specific isoforms of synaptotagmin (Syt), which employ both SNARE complex and membrane binding. Ca(2+) also promotes fusion pore expansion and Syts have been implicated in this process but the mechanisms involved are unclear. We determined the role of Ca(2+)-dependent Syt-effector interactions in fusion pore expansion by expressing Syt-1 mutants selectively altered in Ca(2+)-dependent SNARE binding or in Ca(2+)-dependent membrane insertion in PC12 cells that lack vesicle Syts. The release of different-sized fluorescent peptide-EGFP vesicle cargo or the vesicle capture of different-sized external fluorescent probes was used to assess the extent of fusion pore dilation. We found that PC12 cells expressing partial loss-of-function Syt-1 mutants impaired in Ca(2+)-dependent SNARE binding exhibited reduced fusion pore opening probabilities and reduced fusion pore expansion. Cells with gain-of-function Syt-1 mutants for Ca(2+)-dependent membrane insertion exhibited normal fusion pore opening probabilities but the fusion pores dilated extensively. The results indicate that Syt-1 uses both Ca(2+)-dependent membrane insertion and SNARE binding to drive fusion pore expansion.

Synaptotagmins contain tandem C2 domains and function as Ca(2+) sensors for vesicle exocytosis but the mechanism for coupling Ca(2+) rises to membrane fusion remains undefined. Synaptotagmins bind SNAREs, essential components of the membrane fusion machinery, but the role of these interactions in Ca(2+)-triggered vesicle exocytosis has not been directly assessed. We identified sites on synaptotagmin-1 that mediate Ca(2+)-dependent SNAP25 binding by zero-length cross-linking. Mutation of these sites in C2A and C2B eliminated Ca(2+)-dependent synaptotagmin-1 binding to SNAREs without affecting Ca(2+)-dependent membrane binding. The mutants failed to confer Ca(2+) regulation on SNARE-dependent liposome fusion and failed to restore Ca(2+)-triggered vesicle exocytosis in synaptotagmin-deficient PC12 cells. The results provide direct evidence that Ca(2+)-dependent SNARE binding by synaptotagmin is essential for Ca(2+)-triggered vesicle exocytosis and that Ca(2+)-dependent membrane binding by itself is insufficient to trigger fusion. A structure-based model of the SNARE-binding surface of C2A provided a new view of how Ca(2+)-dependent SNARE and membrane binding occur simultaneously.

The rate of nuclear muon capture by the proton has been measured using a new technique based on a time projection chamber operating in ultraclean, deuterium-depleted hydrogen gas, which is key to avoiding uncertainties from muonic molecule formation. The capture rate from the hyperfine singlet ground state of the microp atom was obtained from the difference between the micro(-) disappearance rate in hydrogen and the world average for the micro(+) decay rate, yielding Lambda(S)=725.0+/-17.4 s(-1), from which the induced pseudoscalar coupling of the nucleon, g(P)(q(2)=-0.88m(2)(micro))=7.3+/-1.1, is extracted.

Synaptotagmin I is considered to be a Ca2+ sensor for fast vesicle exocytosis. Because Ca2+-dependent vesicle exocytosis persists in synaptotagmin I mutants, there must be additional Ca2+ sensors. Multiple synaptotagmin isoforms co-reside on vesicles, which suggests that other isoforms complement synaptotagmin I function. We found that full downregulation of synaptotagmins I and IX, which co-reside on vesicles in PC12 cells, completely abolished Ca2+-dependent vesicle exocytosis. By contrast, Ca2+-dependent exocytosis persisted in cells expressing only synaptotagmin I or only synaptotagmin IX, which indicated a redundancy in function for these isoforms. Although either isoform was sufficient to confer Ca2+ regulation on vesicle exocytosis, synaptotagmins I and IX conferred faster and slower release rates, respectively, indicating that individual isoforms impart distinct kinetic properties to vesicle exocytosis. The downregulation of synaptotagmin I but not synaptotagmin IX impaired compensatory vesicle endocytosis, which revealed a lack of isoform redundancy and functional specialization of synaptotagmin I for endocytic retrieval.

Soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins play key roles in membrane fusion, but their sorting to specific membranes is poorly understood. Moreover, individual SNARE proteins can function in multiple membrane fusion events dependent upon their trafficking itinerary. Synaptosome-associated protein of 25 kDa (SNAP25) is a plasma membrane Q (containing glutamate)-SNARE essential for Ca2+-dependent secretory vesicle-plasma membrane fusion in neuroendocrine cells. However, a substantial intracellular pool of SNAP25 is maintained by endocytosis. To assess the role of endosomal SNAP25, we expressed botulinum neurotoxin E (BoNT E) light chain in PC12 cells, which specifically cleaves SNAP25. BoNT E expression altered the intracellular distribution of SNAP25, shifting it from a perinuclear recycling endosome to sorting endosomes, which indicates that SNAP25 is required for its own endocytic trafficking. The trafficking of syntaxin 13 and endocytosed cargo was similarly disrupted by BoNT E expression as was an endosomal SNARE complex comprised of SNAP25/syntaxin 13/vesicle-associated membrane protein 2. The small-interfering RNA-mediated down-regulation of SNAP25 exerted effects similar to those of BoNT E expression. Our results indicate that SNAP25 has a second function as an endosomal Q-SNARE in trafficking from the sorting endosome to the recycling endosome and that BoNT E has effects linked to disruption of the endosome recycling pathway.

Background & Aims: The risk of colorectal cancer in relatives of patients with adenomatous colonic polyps is not well defined. This study assessed whether finding colonic neoplasia during screening colonoscopy was related to the family history of colorectal cancer among the participants' parents and siblings., Methods: Self-reported family history of colorectal cancer was recorded for all participants in a screening colonoscopy study. The size and location of all polyps were recorded before their removal and histologic examination. Participants were grouped according to the most advanced lesion detected., Results: Three thousand one hundred twenty-one patients underwent complete colonoscopic examination. Subjects with adenomas were more likely to have a family history of colorectal cancer than were subjects without polyps (odds ratio [OR], 1.36; 95% confidence interval [CI], 1.09-1.70). The finding of a small (<1 cm) tubular adenoma as the most advanced lesion was associated with only a modest increase in the OR of colorectal cancer in family members (OR, 1.26; 95% CI, 0.99-1.61), but the presence of an advanced adenoma was associated with a higher OR (OR, 1.62;5% CI, 1.16-2.26). Younger age of adenoma diagnosis was not related to a higher prevalence of a family history of colorectal cancer., Conclusions: Relatives patients with advanced colorectal adenomas have an increased risk of colorectal cancer. Individuals with advanced colorectal adenomas should be counseled about the increased risk of colorectal cancer among their relatives.

The effect of hyperbaric oxygen on epiphyseal ischemia was evaluated using a pediatric rabbit model. Forty-five animals were compared in this study: 23 from a control pilot study and 22 hyperbaric exposed animals. In each animal the right distal femoral and proximal tibial epiphyses were isolated on a popliteal vascular pedicle. The left leg acted as the control. The growth difference between the rabbit's hindlimbs was the means of comparison throughout the groups established. Warm ischemia was induced by applying a vascular clamp to the right popliteal artery for 12 hours (20 animals) and 7 hours (17 animals). The remaining 8 animals underwent a sham operation without interruption of epiphyseal perfusion. On completion of the ischemic period hyperbaric oxygen therapy (HBOT) was performed on 12 12-hour (12h-HBOT) and 10 7-hour (7h-HBOT) animals at 2 atmospheres for 90 minutes twice per day for 4 postoperative days. The animals were killed on either postoperative day 14 or 90. Measurement of longitudinal bone growth was performed on the 90-day animals from serial radiographs at the time of surgery and then at 1 month, 2 months, and 3 months after surgery. There was no significant difference in longitudinal bone growth between the sham-operated and the 7h-HBOT animals at 1, 2, and 3 months. There was a statistically significant difference, however, between the normal growth of the 7h-HBOT group compared with the abnormal growth of the 7-hour, 12-hour, and 12h-HBOT animals. Histology was consistent, with the bone growth data demonstrating relative normalcy of the 7h-HBOT group epiphyseal plates versus severe architectural aberrance and necrosis of the 12h-HBOT group epiphyses. Our experimental data indicate that a clinical trial should be instituted using HBO for pediatric replantation patients when warm ischemia exceeds 7 hours. (J Hand Surg 2000; 25A:159-165., (Copyright 2000 by the American Society for Surgery of the Hand.).)

Study Design: This study compared the efficacy of characterized 50/50 hydroxyapatite/beta-tricalcium phosphate ceramics of 30%, 50%, and 70% porosity and autograft to promote interbody spinal fusion at C2-C3 and C5-C6 in 24 goats: 12 at 3 months and 12 at 6 months., Objectives: Radiographs, histology, dual energy x-ray absorptiometry analysis, and biomechanical testing were used to evaluate the ability of the 30%, 50%, and 70% porous 50/50 hydroxyapatite/beta-tricalcium phosphate ceramics and autograft to promote cervical interbody fusion., Summary of Background Data: The conundrum in the use of calcium phosphates for interbody fusion is what porosity is most effective to promote ingrowth yet strong enough to resist compressive stresses found in the spine? It is known that the ability for bone ingrowth increases and the compressive strength decreases as porosity of the ceramic is increased. Dense ceramics remain intact but may be surrounded by fibrous tissue. Porous ceramics have good ingrowth but may fracture., Methods: Radiographs were evaluated for fusion and fracture or collapse of the ceramics or autograft. Dual energy x-ray absorptiometry was used to evaluate the fusion mass. Treated motion segments underwent biomechanical testing to quantify the flexibility of the segment. Undecalcified and decalcified histologic analysis were performed to evaluate the presence or absence of a bony union., Results: Thirty percent, 50%, and 70% porous ceramics had better radiographic fusion scores than the autograft at 3 and 6 months. Incidence of ceramic fracture did not increase with porosity and was equivalent to the collapse of autograft, although ceramics maintained disc height when fracture occurred. No statistically significant differences were found between autograft and the porous ceramics with biomechanical testing and peri-implant bone mineral density values as measured by dual energy x-ray absorptiometry. At 3 months, histologic analysis showed a union rate of 0% for autograft and 30% porous ceramic, 67% for 50% porous ceramic, and 83% for 70% porous ceramic. At 6 months, the union rate was 67% for the 30%, 50%, and 70% porous ceramics and 50% for autograft., Conclusions: Thirty percent, 50%, and 70% porous ceramics performed equal to or better than autogenous bone after 3 and 6 months. There may be promise for the use of 50/50 hydroxyapatite/beta-tricalcium phosphate in spine surgery as the need to harvest autograft from the iliac crest is obviated, and complications and cost associated with the harvest are avoided.

A novel canine tibia model was used to evaluate four bone graft materials: autologous cortical bone, allograft cortical bone, hydroxyapatite/tricalcium phosphate (HA/TCP) ceramic granules, and a HA/TCP and collagen composite. Mechanical material properties were assessed using custom-designed stainless steel plugs for control of graft volume and interface surface area. These plugs held the bone graft materials in the cortex of the tibia shaft and allowed in vivo mechanical testing. After 6 months of ad lib weight bearing, the grafts were harvested and tested in torsion. The samples in each animal were compared with the test plugs into which new bone had grown without the addition of graft. Control bone peak shear strength averaged 47 (+/- 8.3) MPa (6.78 +/- 1.2 kpsi). Compared on the basis of peak torque, stiffness, and energy to peak torque, no significant differences were found among any of the graft materials or control bone. Histologic examination revealed the materials to be osteoconductive with the extensive formation of dense, compact cancellous bone. The new bone in the autograft and allograft samples completely filled the available space, whereas gaps persisted in the synthetic ceramics.

The purpose of this study was to determine the optimal ratio of calcium hydroxyapatite (HA) to beta tricalcium phosphate (beta TCP) in a biphasic porous calcium phosphate (BCP) ceramic for effective repair of periodontal osseous defects. Defects were surgically produced in beagle dogs and made chronic for 4 months to simulate periodontal disease. Mucoperiosteal periodontal flaps were reflected, followed by osseous defect debridement and root planing. Specially prepared ceramic with different HA/beta TCP ratios were implanted into the prepared defects. The sites were allowed to heal for 6 months, animals were euthanized, and site-blocks were removed for histological study. During the follow-up phase, scaling and polishing were done once a month, and standardized probing attachment levels were recorded pre- and 6-months postoperatively. The Duncan's multiple range test showed that all the treatments produced statistically significant higher gain in probing attachment levels than the control group (0HA/0 beta TCP) (P < 0.05). Among the 7 "active" treatment groups, 2 (65/35 and 85/15) had significantly higher gain in probing attachment levels than those in 3 groups (50/50, 100/0, and 0/100) (P < 0.05). Histologically, higher HA ratio (but not 100% HA) showed accelerated new bone formation and new attachment levels. Based on histological results, the 85HA/15 beta TCP ratio appears to demonstrate greater gain in attachment level and bone regeneration in the treatment of periodontal osseous defects.

Tricalcium phosphate ceramic, with 50% porosity and 400 to 500 micrometer pore diameter, was used to augment the edentulous alveolar ridge of 10 adult mongrel dogs. The implants were evaluated histologically at different time intervals (7, 30, 90, 180, and 360 days). Preoperative and postoperative blood chemistry studies were also evaluated. The results showed that, other than for the expected acute nonspecific inflammatory response due to the surgery, the material was well tolerated by the tissues and was nontoxic. Bone and soft tissues grew into the pores, thereby creating an excellent biomechanical bond between the ceramic implant and surrounding structures. Preoperative and postoperative blood chemistry studies demonstrated no significant change.

In six patients, porous tricalcium phosphate ceramic (400 to 500 micrometer pore diameter and 50% pore volume) was used to repair naturally occurring periodontal osseous defects. These patients were evaluated clinically, radiographically and hematologically. The clinical evaluation indicated that there was no adverse inflammatory response at the implant site except the anticipated immediate postsurgical trauma. Radiographically, there was a significant increase in bone height with a mean gain of 5.2 mm. Whether or not this gain consisted of bone and/or ceramic is yet to be conclusively determined. The SMA-12 blood chemistry study revealed that no significant change occurred pre- or post-operatively (P less than 0.01). Although the number of patients in our study was limited, the results are very promising. The ceramic is nontoxic to human tissue and repair of the periodontium is most likely obtainable, the desired objective being the restoration of the natural state of the periodontium.

A normal 1-year-old infant palate and a newborn cleft palate were studied grossly, radiographically, and microscopically for histological structure in hard palate osteotomy sites. A correlated study of biopsies from an osteotomy site seven days postoperatively and another at twelve months following osteotomy was also made. The findings showed that successful osteotomy in infant cleft palate surgery translocates autogenous fibrous bone and osteogenic cells into a cleft-bridging position. Woven fibrous bone rapidly forms across the cleft, and then matures by lamellar bone replacement and haversian system remodeling. Both normal and cleft palates have a rich anastomosis of microscopic blood supply that is vitally important in the remodeling process. These factors may help to explain successful long-term results in early cleft palate osteotomy surgery.

Two mongrel adult dogs were used in this study. The mandible was edentulated unilaterally and then augmented with porous tribasic calcium phosphate ceramic. To determine the effects of a denture over the augmented site, a unilateral distal-extension removable partial denture was constructed and inserted. At the end of the 6-month period, clinical evaluation revealed a healthy residual alveolar ridge tissue with no inflammatory reaction due to the implant. Histologic examination confirmed the clinical findings. There was no inflammation, bone and soft tissue grew into the pores, and no bone resorption occurred. It is concluded therefore that porous tribasic calcium phosphate ceramic is a potential material for residual alveolar ridge augmentation.

Two years after pelvic irradiation for prostatic cancer, bilateral protrusio acetabuli and collapse of the right femoral head requiring prosthetic arthroplasty developed in a 73-year-old man with chronic rheumatoid arthritis. There was no evidence of metastases. Histologic evaluation showed empty lacunae in the bone but no evidence of obliterative endarteritis. Osteonecrosis and pathologic fractures constitute a rare complication of high voltage irradiation.

To correlate the MR appearance of the disk with its fibrous structure, we studied the lumbar intervertebral disks in 10 cadavers with MR, CT, cryomicrotome anatomic sections, and, in selected disks, with histologic and dried sections. In MR images the predominantly fibrous tissues such as Sharpey's fibers had a low signal intensity while the fibrocartilagenous tissues with a mucoid matrix in the intervertebral disk had a high signal intensity. In the equator of the adult disk was a well-defined fibrous plate that contained collagenous, elastic, and reticular fibers with little ground substance. This plate appeared to develop progressively from the periphery of the nucleus toward the center, starting in the second decade of life. The fibrous plate was also distinguished as a lower signal intensity in the MR images.

Recent studies have shown that porous calcium phosphates provide a scaffold for bony ingrowth in various anatomic sites. In this preliminary investigation a type of porous calcium phosphate ceramic was implanted in the vertebral columns of 21 rabbits; the ceramic was studied as a possible bone graft substitute for use in spinal fusions. Animals were killed at three, six, eight, 12, and 24 weeks. Roentgenograms and histologic sections of the implant site were then obtained. Bony tissue ingrowth was progressing during the study period. Woven bone was found by six weeks after implantation, and lamellar bone was evident at 12 weeks. By 12 weeks the bony ingrowth had reached the central portion of the ceramic block. There was no fibrous tissue barrier to normal ossification. This type of calcium phosphate ceramic is compatible with bony ingrowth when implanted in the vertebral column of rabbits.

Tricalcium phosphate ceramic of hydroxyapatite structure with 50% porosity and 800- to 1000-mum pore diameter was implanted in surgically produced infrabony defects in dogs. The defects were evaluated histologically at different time intervals, 1, 2, 4, 8, 16, and 24 weeks. The results show that the ceramic is well tolerated by the tissue and yields no toxic reactions. Bone ingrowth into the pores and repair of the periodontium are clearly demonstrated. No significant hematological changes were observed.

The healing of surgically induced defects in mature rabbits' hyaline cartilage was examined histologically and biochemically. The subchondral bone underneath the lesion was the source of repair. At the end of the first month, mushroom-shaped chondroid buds were seen sprouting from the subchondral bone. By the tenth week, these buds fused to a cartilagenous plug filling the lesion. The repaired cartilage synthesized Type II collagen.

The relative ability of the calcium chelates of calcium disodium ethylenediaminetetraacetate (EDTA) and calcium trisodium ethylenetriaminepentaacetate (DTPA) to protect mice against lethal doses of Clostridium perfringens alpha-toxin was investigated. Their protective ability was assayed by the increase in survival time of mice which had been given large doses of toxin, and by determining the median protective dose of chelate that would protect mice against toxin at a minimum lethal dose of two. In both assay procedures, intraperitoneal, intravenous, and intracutaneous injections of toxin were utilized, and with each toxin injection route the protective ability of the chelate was determined with the three routes of injection. DTPA was 10 to 20 times more effective than EDTA with both types of assay procedure and with all injection routes. DTPA may be superior to EDTA as a protective agent not only because it binds zinc to a greater extent, but also because of its greater retention in the body and its ability to gain entrance into cells. It appears that DTPA may be of value as a therapeutic agent in gas-gangrene.

The toxicity of Clostridium perfringens was determined in mice utilizing three different routes of inoculation. There was marked variation in the minimum lethal dose with the different routes; the largest amount was required for the intracutaneous route, less for the intravenous route, and least with the intraperitoneal route. The relationship of toxicity to the lecithinase content of different toxin preparations was assayed. It was found that the toxicity of toxin preparations in different states of purification was not correlated with their lecithinase content.