RESUMO
Paralytic shellfish toxins (PSTs), including gonyautoxins and saxitoxins, are produced by multiple species of microalgae and dinoflagellates, and are bioaccumulated by shellfish and other animals. Human exposure to PSTs typically occurs through ingestion of recreationally harvested contaminated shellfish and results in nonspecific symptomology. Confirmation of exposure to PSTs has often relied on the measurement of saxitoxin, the most toxic congener; however, gonyautoxins (GTXs), the sulfated carbamate derivatives of saxitoxin, may be present in shellfish at higher concentrations. To improve identification of PST exposures, our group has developed an online solid phase extraction hydrophilic interaction liquid chromatography method to identify GTX1-4 in human urine with tandem mass spectrometry. The reportable range varied for each analyte, with all falling within 0.899 and 250 ng/mL in urine with precision <15% and >85% accuracy as determined for all quality control samples. This new online method quantitates GTX1-4 following exposures to PSTs, supporting the work of public health authorities.
Assuntos
Cromatografia Líquida/métodos , Saxitoxina/análogos & derivados , Extração em Fase Sólida/métodos , Espectrometria de Massas em Tandem/métodos , Ensaios de Triagem em Larga Escala/métodos , Humanos , Limite de Detecção , Modelos Lineares , Reprodutibilidade dos Testes , Saxitoxina/química , Saxitoxina/isolamento & purificação , Saxitoxina/urinaRESUMO
This study was conducted among U.S. Army soldiers to evaluate the association between exposure to o-chlorobenzylidene malononitrile (CS riot control agent) and urinary metabolite 2-chlorohippuric acid (CHA) detected in test subjects (n = 87) after completion of Mask Confidence Training. CS exposures ranged 0.086-4.9 mg/m³ ([Formula: see text] = 2.7 mg/m³). CHA levels (corrected for creatinine) at 2-, 8-, 24-, and 30-hr post-exposure resulted in ranges of 94.6-1120 µg/g-cr ([Formula: see text] = 389 µg/g-cr), 15.80-1170 µg/g-cr ([Formula: see text] = 341 µg/g-cr), 4.00-53.1 µg/g-cr ([Formula: see text] = 19.3 µg/g-cr), and 1.99-28.4 µg/g-cr ([Formula: see text] = 10.6 µg/g-cr), respectively. Spearman's correlation revealed CHA levels strongly correlated with time sampled (r = -0.748, p < 0.05) and weakly correlated with CS concentration (r = 0.270, p < 0.05). A linear relationship was observed between CHA, CS concentration, and time of urine sample according to the following regression equation: ln(CHA, µg/g-cr) = 5.423 + 0.316 (CS conc., mg/m³) - 0.002 (time sampled), (R = 0.910, R² = 0.827, p < 0.05). This relationship suggests that CHA has the potential to be an effective retrospective indicator of CS exposure in future biomarker developments.
Assuntos
Hipuratos/análise , Substâncias para Controle de Distúrbios Civis/metabolismo , o-Clorobenzilidenomalonitrila/metabolismo , Adulto , Biomarcadores/urina , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Militares , Exposição Ocupacional/análise , Dispositivos de Proteção Respiratória , Substâncias para Controle de Distúrbios Civis/toxicidade , Fatores de Tempo , o-Clorobenzilidenomalonitrila/toxicidadeRESUMO
Harmful cyanobacterial blooms are becoming more common and persistent around the world. When in bloom, various cyanobacterial strains can produce anatoxins in high concentrations, which, unlike other cyanobacterial toxins, may be present in clear water. Potential human and animal exposures to anatoxins occur mainly through unintentional ingestion of contaminated algal mats and water. To address this public health threat, we developed and validated an LC-MS/MS method to detect anatoxins in human urine to confirm exposures. Pooled urine was fortified with anatoxin-a and dihydroanatoxin at concentrations from 10.0 to 500 ng/mL to create calibrators and quality control samples. Samples were diluted with isotopically labeled anatoxin and solvent prior to LC-MS/MS analysis. This method can accurately quantitate anatoxin-a with inter- and intraday accuracies ranging from 98.5 to 103% and relative standard deviations < 15%, which is within analytical guidelines for mass spectrometry methods. Additionally, this method qualitatively detects a common degradation product of anatoxin, dihydroanatoxin, above 10 ng/mL. We also evaluated a commercial anatoxin-a ELISA kit for potential diagnostic use; however, numerous false positives were detected from unexposed individual human urine samples. In conclusion, we have developed a method to detect anatoxins precisely and accurately in urine samples, addressing a public health area of concern, which can be applied to future exposure events.
Assuntos
Toxinas de Cianobactérias , Espectrometria de Massas em Tandem , Tropanos , Água , Animais , Humanos , Cromatografia Líquida , Ensaio de Imunoadsorção EnzimáticaRESUMO
RATIONALE: Although use is prohibited, concerns remain for human exposure to nerve agents during decommissioning, research, and warfare. High-resolution mass spectrometry (HRMS) was compared to tandem mass spectrometry (MS/MS) analysis for the quantitation of five urinary metabolites specific to VX, Russian VX, soman, sarin and cyclosarin nerve agents. The HRMS method was further evaluated for qualitative screening of metabolites not included in the test panel. METHODS: Nerve agent metabolites were extracted from urine using solid-phase extraction, separated using hydrophilic interaction chromatography and analyzed using both tandem and high-resolution mass spectrometry. MS/MS results were obtained using selected reaction monitoring with unit resolution; HRMS results were obtained using a mass extraction window of 10 ppm at a mass resolution of 50 000. The benchtop Orbitrap HRMS instrument was operated in full scan mode, to measure the presence of unexpected nerve agent metabolites. RESULTS: The assessment of two quality control samples demonstrated high accuracy (99.5-104%) and high precision (2-9%) for both HRMS and MS/MS. Sensitivity, as described by the limit of detection, was overlapping for both detectors (0.2-0.7 ng/mL). Additionally, the HRMS method positively confirmed the presence of a nerve agent metabolite, not included in the test panel, using the accurate mass and relative retention time. CONCLUSIONS: The precision, accuracy, and sensitivity were comparable between the current MS/MS method and this newly developed HRMS analysis for five nerve agent metabolites. HRMS showed additional capabilities beyond the current method by confirming the presence of a metabolite not included in the test panel.
Assuntos
Substâncias para a Guerra Química/análise , Substâncias para a Guerra Química/metabolismo , Espectrometria de Massas em Tandem/métodos , Humanos , Compostos Organofosforados/metabolismo , Compostos Organofosforados/urina , Compostos Organotiofosforados/metabolismo , Compostos Organotiofosforados/urina , Sarina/metabolismo , Sarina/urina , Soman/metabolismo , Soman/urinaRESUMO
BACKGROUND: Fixation of the baseplate to the glenoid for the Reverse Shoulder Prosthesis (DJO Surgical, Austin, TX, USA) requires secure screw purchase to avoid excessive micromotion and baseplate failure. The best screw length for fixation is unknown. In addition, excessively long screws or a plunge of the drill bit during baseplate insertion could injure surrounding structures. METHODS: Reverse Shoulder Prosthesis baseplates were inserted in 10 fresh-frozen shoulders by use of a 6.5-mm central screw and four 5.0-mm peripheral locking screws placed 90° to the baseplate. The top superior screw was placed into the base of the coracoid, corresponding to the 1-o'clock position in a right shoulder. The distances to surrounding vital structures were recorded, screws were removed, and screw hole lengths were measured to determine the most effective lengths in different parts of the glenoid scapula. RESULTS: The screw length was 30 mm for the superior screw holes, 28 mm for the inferior screw holes, 13 mm for the anterior screw holes, and 15 mm for the posterior screw holes. The central screw trajectory was through the anterior cortex. The anterior screw trajectory violated the subscapularis belly in all specimens. The posterior screw touched the suprascapular nerve or artery in 3 of 10 specimens. DISCUSSION: The superior and inferior screws have the longest bony fixation. Drill bit plunge during placement of the anterior screw poses a risk to the subscapularis muscle. Drilling for the posterior screw risks injury to the suprascapular nerve and artery at the spinoglenoid notch. CONCLUSIONS: The posterior screw should be placed with care to avoid neurovascular complications.
Assuntos
Artroplastia de Substituição/métodos , Parafusos Ósseos , Articulação do Ombro/cirurgia , Dissecação , Humanos , Desenho de PróteseRESUMO
To combat the ongoing opioid epidemic, our laboratory has developed and evaluated an approach to detect fentanyl analogs in urine and plasma by screening LC-QTOF MS/MS spectra for ions that are diagnostic of the core fentanyl structure. MS/MS data from a training set of 142 fentanyl analogs were used to select the four product ions and six neutral losses that together provided the most complete coverage (97.2%) of the training set compounds. Furthermore, using the diagnostic ion screen against a set of 49 fentanyl analogs not in the training set resulted in 95.9% coverage of those compounds. With this approach, lower reportable limits for fentanyl and a subset of fentanyl-related compounds range from 0.25 to 2.5 ng/mL in urine and 0.5 to 5.0 ng/mL in plasma. This innovative processing method was applied to evaluate simulated exposure samples of remifentanil and carfentanil in water and their metabolites remifentanil acid and norcarfentanil in urine. This flexible approach enables a way to detect emerging fentanyl analogs in clinical samples.
Assuntos
Cromatografia Líquida/métodos , Fentanila , Espectrometria de Massas em Tandem/métodos , Fentanila/análogos & derivados , Fentanila/análise , Humanos , Íons/química , Medicamentos Sintéticos/análiseRESUMO
Human exposures to fentanyl analogs, which significantly contribute to the ongoing U.S. opioid overdose epidemic, can be confirmed through the analysis of clinical samples. Our laboratory has developed and evaluated a qualitative approach coupling liquid chromatography and quadrupole time-of-flight mass spectrometry (LC-QTOF) to address novel fentanyl analogs and related compounds using untargeted, data-dependent acquisition. Compound identification was accomplished by searching against a locally-established mass spectral library of 174 fentanyl analogs and metabolites. Currently, our library can identify 150 fentanyl-related compounds from the Fentanyl Analog Screening (FAS) Kit), plus an additional 25 fentanyl-related compounds from individual purchases. Plasma and urine samples fortified with fentanyl-related compounds were assessed to confirm the capabilities and intended use of this LC-QTOF method. For fentanyl, 8 fentanyl-related compounds and naloxone, lower reportable limits (LRL100), defined as the lowest concentration with 100 % true positive rate (n = 12) within clinical samples, were evaluated and range from 0.5 ng/mL to 5.0 ng/mL for urine and 0.25 ng/mL to 2.5 ng/mL in plasma. The application of this high resolution mass spectrometry (HRMS) method enables the real-time detection of known and emerging synthetic opioids present in clinical samples.
Assuntos
Analgésicos Opioides/sangue , Analgésicos Opioides/urina , Cromatografia Líquida de Alta Pressão , Fentanila/sangue , Fentanila/urina , Espectrometria de Massas por Ionização por Electrospray , Detecção do Abuso de Substâncias/métodos , Espectrometria de Massas em Tandem , Analgésicos Opioides/síntese química , Cromatografia Líquida de Alta Pressão/normas , Fentanila/análogos & derivados , Fentanila/síntese química , Humanos , Limite de Detecção , Padrões de Referência , Reprodutibilidade dos Testes , Espectrometria de Massas por Ionização por Electrospray/normas , Detecção do Abuso de Substâncias/normas , Espectrometria de Massas em Tandem/normasRESUMO
The applications of polysaccharide phenyl carbamate derivatives as chiral stationary phases (CSPs) for capillary electrochromatography (CEC) are often hindered by longer retention times, especially using a normal-phase (NP) eluent due to very low electroosmotic flow (EOF). Therefore, in this study, we propose an approach for the aforementioned problems by introducing two new types of negatively charged sulfate and sulfonated groups for polysaccharide CSPs. These CSPs were utilized to pack CEC columns for enantioseparation with a NP eluent. Compared to conventional cellulose tris(3,5-dimethylphenyl carbamate) or CDMPC CSPs, the sulfated CDMPC CSP (sulfur content 4.25%, w/w) shortened the analysis time up to 50% but with a significant loss of enantiomeric resolution (approximately 60%). On the other hand, the sulfonated CDMPC CSP (sulfur content 1.76%, w/w) not only provided fast throughput but also maintained excellent resolving power. In addition, its synthesis is much more straightforward than the sulfated one. Furthermore, we studied several stationary phase parameters (CSP loading and silica gel pore size) and mobile phase parameters (including type of mobile phase and its composition) to evaluate the throughput and enantioselectivity. Using the optimized conditions, a chiral pool containing 66 analytes was screened to evaluate the enantioselectivity under three different mobile phase modes (i.e., NP, polar organic phase (POP) and reversed-phase (RP) eluents). Among these mobile phase modes, the RP mode showed the highest success rate, whereas some degree of complementary enantioselectivity was observed with NP and POP. Finally, the feasibility of applying this CSP for CEC-MS enantioseparation using internal tapered column was evaluated with NP, POP and RP eluents. In particular, the NP-CEC-MS provided significantly enhanced sensitivity when methanol was replaced with isopropanol in the sheath liquid. Using aminoglutethimide as model chiral analyte, all three modes of CEC-MS demonstrated excellent durability as well as excellent reproducibility of retention time and enantioselectivity.
Assuntos
Eletrocromatografia Capilar/métodos , Polissacarídeos/química , Espectrometria de Massas por Ionização por Electrospray/métodos , Sulfatos/química , Ácidos Sulfônicos/química , Celulose/química , Concentração de Íons de Hidrogênio , Íons , Fenilcarbamatos/química , Porosidade , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , EstereoisomerismoRESUMO
This work focuses on the simultaneous analysis of beta-blockers with multiple stereogenic centers using capillary electrochromatography-mass spectrometry (CEC-MS) with a vancomycin stationary phase. The critical mobile phase variables (composition of organic solvents, acid/base ratios) as well as column temperature and electric field strength, effecting enantioresolution and analysis time were first optimized sequentially. Next, to achieve global optimum a multivariate D-optimal design was used. Although multivariate approach did not improve enantioresolution any further, analysis time was significantly reduced. Under optimum CEC-MS conditions, all stereoisomers were resolved with resolution in the range 1.0-3.1 in less than 60 min with an average signal-to-noise (S/N) greater than 1000. The developed CEC-MS method has the potential to emerge as a screening method for analysis of multiple chiral compounds using a single protocol using the same column and mobile phase conditions, thus reducing the operation time and cost.
Assuntos
Antagonistas Adrenérgicos beta/isolamento & purificação , Eletrocromatografia Capilar/métodos , Labetalol/isolamento & purificação , Nadolol/isolamento & purificação , Espectrometria de Massas por Ionização por Electrospray/métodos , Análise Multivariada , Solventes , Estereoisomerismo , TemperaturaRESUMO
AIM: An immunomagnetic capture protocol for use with LC-MS was developed for the quantitation of saxitoxin (STX) in human urine. MATERIALS & METHODS: This method uses monoclonal antibodies coupled to magnetic beads. STX was certified reference material grade from National Research Council, Canada. Analysis was carried out using LC-MS. RESULTS: With an extraction efficiency of 80%, accuracy and precision of 93.0-100.2% and 5.3-12.6%, respectively, and a dynamic range of 1.00-100 ng/ml, the method is well suited to quantify STX exposures based on previously reported cases. CONCLUSION: Compared with our previously published protocols, this method has improved selectivity, a fivefold increase in sensitivity and uses only a third of the sample volume. This method can diagnose future toxin exposures and may complement the shellfish monitoring programs worldwide.
Assuntos
Cromatografia Líquida/métodos , Testes Imunológicos , Saxitoxina/urina , Intoxicação por Frutos do Mar/urina , Espectrometria de Massas em Tandem/métodos , Anticorpos Monoclonais/imunologia , Calibragem , Humanos , Imãs , Microesferas , Padrões de Referência , Saxitoxina/química , Saxitoxina/normas , Intoxicação por Frutos do Mar/diagnóstico , Fatores de TempoRESUMO
Tetrodotoxin (TTX) is an extremely potent paralytic toxin responsible for yearly illness and death around the world. A clinical measurement is necessary to confirm exposure because symptoms of TTX intoxication cannot be distinguished from other paralytic toxins. Our group has developed an online solid phase extraction hydrophilic interaction liquid chromatography (HILIC) method for the analysis of TTX in human urine with tandem mass spectrometry. The reportable range for the method was 2.80 - 249 ng/mL in urine with precision and accuracy within 15% as determined for all quality control samples. No isotopically-labeled internal standard is available for TTX; thus a surrogate internal standard, voglibose, was investigated to compensate for matrix effects and ionization suppression. However, upon evaluation, voglibose was ineffective for this purpose. This new online method rapidly identifies TTX, facilitating the work of public health authorities and providing support to monitoring programs worldwide.
Assuntos
Cromatografia Líquida/métodos , Ensaios de Triagem em Larga Escala , Extração em Fase Sólida/métodos , Espectrometria de Massas em Tandem/métodos , Tetrodotoxina/urina , Calibragem , Humanos , Limite de Detecção , Padrões de ReferênciaRESUMO
Saxitoxin (STX) and neosaxitoxin (NEO) are potent neurotoxins that cause paralytic shellfish poisoning (PSP). PSP typically occurs through the ingestion of bivalve shellfish that have consumed toxin producing dinoflagellates. Due to initial presentation of symptoms being nonspecific, a clinical measurement is needed to confirm exposure to these toxins. Our group has developed an online solid phase extraction hydrophilic interaction liquid chromatography (HILIC) method for the analysis of STX and NEO in human urine with tandem mass spectrometry. A unique feature of this online method is the incorporation of a new synthetic (15)N4-STX labeled internal standard used for quantitation. Manual sample preparation time was reduced by approximately 70% for 98 urine samples as compared to a previously reported method. The lowest reportable limit for STX was improved from 5.0 ng/mL to 1.01 ng/mL and from 10.0 ng/mL to 2.62 ng/mL for NEO. Three analysts validated the method with 20 calibration curves total over 30 days with precision and accuracy within ±15% for all QCs. This new online method rapidly identifies STX and NEO exposure with improved sensitivity, which can facilitate the work of public health authorities to confirm the cases of PSP, complementing the many shellfish monitoring programs worldwide.
Assuntos
Neurotoxinas/urina , Saxitoxina/análogos & derivados , Saxitoxina/urina , Intoxicação por Frutos do Mar/urina , Métodos Analíticos de Preparação de Amostras , Automação Laboratorial , Calibragem , Cromatografia Líquida de Alta Pressão , Humanos , Interações Hidrofóbicas e Hidrofílicas , Limite de Detecção , Neurotoxinas/química , Neurotoxinas/toxicidade , Isótopos de Nitrogênio , Reprodutibilidade dos Testes , Saxitoxina/química , Saxitoxina/toxicidade , Sensibilidade e Especificidade , Intoxicação por Frutos do Mar/diagnóstico , Intoxicação por Frutos do Mar/etiologia , Extração em Fase Sólida , Espectrometria de Massas em Tandem , ToxicocinéticaRESUMO
Two types of automated solid phase extraction (SPE) were assessed for the determination of human exposure to fentanyls in urine. High sensitivity is required to detect these compounds following exposure because of the low dose required for therapeutic effect and the rapid clearance from the body for these compounds. To achieve this sensitivity, two acceptable methods for the detection of human exposure to seven fentanyl analogs and three metabolites were developed using either off-line 96-well plate SPE or on-line SPE. Each system offers different advantages: off-line 96-well plate SPE allows for high throughput analysis of many samples, which is needed for large sample numbers, while on-line SPE removes almost all analyst manipulation of the samples, minimizing the analyst time needed for sample preparation. Both sample preparations were coupled with reversed phase liquid chromatography and isotope dilution tandem mass spectrometry (LC-MS/MS) for analyte detection. For both methods, the resulting precision was within 15%, the accuracy within 25%, and the sensitivity was comparable with the limits of detection ranging from 0.002ng/mL to 0.041ng/mL. Additionally, matrix effects were substantially decreased from previous reports for both extraction protocols. The results of this comparison showed that both methods were acceptable for the detection of exposures to fentanyl analogs and metabolites in urine.
Assuntos
Analgésicos Opioides/química , Analgésicos Opioides/urina , Fentanila/análogos & derivados , Fentanila/urina , Extração em Fase Sólida/métodos , Analgésicos Opioides/metabolismo , Cromatografia Líquida de Alta Pressão/métodos , Fentanila/metabolismo , Humanos , Limite de Detecção , Espectrometria de Massas em Tandem/métodosRESUMO
To fulfill the ever growing demand for rapid chiral analysis, this research presents an approach for highthroughput enantiomeric separations and sensitive detection of model chiral analytes using capillary electrochromatography (CEC) with UV and MS detection. This was achieved utilizing a short 7 cm CEC columns packed with cellulose tris (3,5-dimethyl-phenylcarbamate) (CDMPC) or sulfonated cellulose tris (3,5-dimethylphenylcarbamate) (CDMPC-SO3) chiral stationary phases (CSPs) applying outlet side injections in CEC-UV. The separation performance was compared between CDMPC and CDMPC-SO3 CSPs for rapid enantio-separation in CEC-UV mode. In addition, using a high sensitivity UV-flow cell in combination with outlet side injections, the S/N and hence the limit of detection of chiral drug could be improved. The 7-cm packed column was also used with traditional inlet injections for CEC coupled to a low-cost single-quadrupole MS. While outlet side injection was not possible in CEC-MS due to instrumentation constraints, the combined use of a short 7 cm column packed with CDMPC-SO3 CSP provided several fold higher throughput. Both CEC-UV and CEC-MS with short packed bed has the potential for a simple, sensitive and cost-effective method for enantiomeric drug profiling in biological samples.
RESUMO
The work presented here demonstrates the incorporation of vinylbenzyl trimethylammonium (VBTA) as a novel positively charged achiral co-monomer to a glycidyl methacrylate-beta cyclodextrin (GMA/ß-CD) based monolith, providing anion exchange sites with reversed electroosmotic flow (EOF) for capillary electrochromatography (CEC). The monolithic phases, GMA/ß-CD-VBTA and GMA/ß-CD (without co-monomer) were characterized by scanning electron microscopy, optical microscopy, pressure drop/flow-rate curves and nitrogen adsorption analysis. After optimizing the stationary phase and mobile phase parameters, chiral separations of 41 pairs of structurally diverse anionic chiral analytes were compared individually using the GMA/ß-CD-VBTA and GMA/ß-CD monolithic columns. The GMA/ß-CD-VBTA monolith chiral stationary phase separated significantly more acidic compounds compared to the GMA/ß-CD column. To-date there has been limited work in the development of chiral monolithic column for CEC-mass spectrometry (MS). Because of good electrodriven flow characteristics, which allow the column to maintain a stable current in the absence of outlet vial, GMA/ß-CD-VBTA column was successfully coupled to single quadrupole mass spectrometer for CEC-MS of several chiral test compounds. In addition, the same monolithic CEC column when coupled to a triple quadrupole MS instrument, two orders of magnitude higher sensitivity was observed compared to a single quadrupole MS instrument.
Assuntos
Ácidos/química , Eletrocromatografia Capilar/instrumentação , Eletrocromatografia Capilar/métodos , Espectrometria de Massas/métodos , beta-Ciclodextrinas/química , EstereoisomerismoRESUMO
A novel procedure was developed for the fabrication of a fritless packed column for the coupling of capillary electrochromatography (CEC) to mass spectrometry (MS). The process involved the formation of internal tapers on two separate columns. Once the internal tapers are formed and the columns are packed, the untapered ends of each column were joined together by a commercially available connector. Several advantages of the fritless columns are described. First, the design used here eventually eliminates the need for any frits thus reducing the possibility of bubble formation seen with fritted packed columns. In addition, this is the first report in which the internal tapers are formed at both the inlet and outlet column ends making the fritless CEC-MS column more robust compared to only one report with externally tapered counterparts. Second, a comparison of internally tapered single frit packed CEC-MS (previously developed in our laboratory) column versus fritless CEC-MS column reported here shows that the latter provides better efficiency, suggesting no dead volume with equally good sensitivity and chiral resolution of (±)-aminoglutethimide. The fritless column procedure is universal and was used to prepare a series of columns with a variety of commercially available packing material (mixed mode strong cation exchange, SCX; mixed mode strong anion exchange, SAX; C-18) for the separation and MS detection of short chain non-chromophoric polar amines, long chain nonchromophic anionic surfactant as well as oligomers of non-chromophoric non-ionic surfactants, respectively. The fritless columns showed good intra-day repeatability and inter-day reproducibility of retention times, chiral and achiral resolutions and peak areas. Very satisfactory column-to-column and operator-to-operator reproducibility was demonstrated.
Assuntos
Eletrocromatografia Capilar/instrumentação , Espectrometria de Massas/instrumentação , Ácidos Alcanossulfônicos/análise , Aminas/análise , Aminoglutetimida/análise , Eletrocromatografia Capilar/métodos , Cetomacrogol/química , Desenho de Equipamento , Modelos Lineares , Espectrometria de Massas/métodos , Modelos Químicos , Análise Multivariada , Reprodutibilidade dos Testes , Estereoisomerismo , Tensoativos/químicaRESUMO
BACKGROUND: We examined the effects of ultra-high molecular weight polyethylene (UHMWPE) particles on mononuclear cell proinflammatory gene expression in a novel murine model. We hypothesized that mononuclear cell gene transcription of tumor necrosis factor-alpha (TNF-alpha), interleukin-1 beta (IL-1beta), interleukin-6 (IL-6) and macrophage chemoattractant protein-1 (MCP-1) would be upregulated by the addition of polyethylene particles in this murine intramedullary rod model. MATERIAL AND METHODS: The model involved a stainless steel Kirschner wire inserted retrograde with a line-to-line fit in bilateral femora of C57bl/6 mice. Additionally, the right femora were injected with 3 x 10(9) UHMWPE particles. Mononuclear marrow cells were isolated by bone marrow aspiration and Ficoll-Paque centrifugation at 2, 4 and 10 weeks post-surgery. Total RNA was isolated and real-time RT-PCR was performed to quantify gene expression. Histological specimens of explanted femora were also analyzed to track the changes in periprosthetic tissue. RESULTS: UHMWPE particles stimulated gene transcription in mononuclear cells when examined at 2, 4 and 10 weeks post-surgery, compared to the rod-only group. Relative levels of IL-1beta and MCP-1 mRNA increased in a linear fashion over the 10-week time-course. IL-6 mRNA showed increased expression which peaked at 4 weeks. TNF-alpha mRNA expression was variable and reached a minimum at 4 weeks. The addition of UHMWPE particles stimulated ingress of macrophages and multinuclear cells of macrophage origin to the bone-implant interface. INTERPRETATION: In this model, a single bolus of UHMWPE particles had a long-term effect on gene transcription in mononuclear cells which perpetuated a chronic inflammatory state. This murine model of intramedullary particle-induced inflammation simulates periprosthetic events associated with implant wear in humans, and may contribute to a more mechanistic understanding of wear-debris associated prosthesis failure.