High-fat feeding primes the mouse knee joint to develop osteoarthritis and pathologic infrapatellar fat pad changes after surgically induced injury.

OBJECTIVE: Obesity is one of the greatest risk factors for osteoarthritis (OA) and evidence is accumulating that inflammatory mediators and innate immunity play an important role. The infrapatellar fat pad (IPFP) could be a potential local source of inflammatory mediators in the knee. Here, we combine surgical joint damage with high-fat feeding in mice to investigate inflammatory responses in the IPFP during OA development. DESIGN: Mice (n = 30) received either a low-fat diet (LFD), high-fat diet (HFD) for 18 weeks or switched diets (LFD > HFD) after 10 weeks. OA was induced by surgical destabilization of the medial meniscus (DMM), contralateral knees served as sham controls. An additional HFD-only group (n = 15) received no DMM. RESULTS: The most pronounced inflammation, characterized by macrophage crown-like structures (CLS), was found in HFD + DMM mice, CLS increased compared to HFD only (mean difference = 7.26, 95%CI [1.52-13.0]) and LFD + DMM (mean difference = 6.35, 95%CI [0.53-12.18). The M1 macrophage marker iNOS increased by DMM (ratio = 2.48, 95%CI [1.37-4.50]), while no change in M2 macrophage marker CD206 was observed. Fibrosis was minimal by HFD alone, but in combination with DMM it increased with 23.45% (95%CI [13.67-33.24]). CONCLUSIONS: These findings indicate that a high-fat diet alone does not trigger inflammation or fibrosis in the infrapatellar fat pad, but in combination with an extra damage trigger, like DMM, induces inflammation and fibrosis in the infrapatellar fat pad. These data suggest that HFD provides a priming effect on the infrapatellar fat pad and that combined actions bring the joint in a metabolic state of progressive OA.

Human C-reactive protein aggravates osteoarthritis development in mice on a high-fat diet.

OBJECTIVE: C-reactive protein (CRP) levels can be elevated in osteoarthritis (OA) patients. In addition to indicating systemic inflammation, it is suggested that CRP itself can play a role in OA development. Obesity and metabolic syndrome are important risk factors for OA and also induce elevated CRP levels. Here we evaluated in a human CRP (hCRP)-transgenic mouse model whether CRP itself contributes to the development of 'metabolic' OA. DESIGN: Metabolic OA was induced by feeding 12-week-old hCRP-transgenic males (hCRP-tg, n = 30) and wild-type littermates (n = 15) a 45 kcal% high-fat diet (HFD) for 38 weeks. Cartilage degradation, osteophytes and synovitis were graded on Safranin O-stained histological knee joint sections. Inflammatory status was assessed by plasma lipid profiling, flow cytometric analyses of blood immune cell populations and immunohistochemical staining of synovial macrophage subsets. RESULTS: Male hCRP-tg mice showed aggravated OA severity and increased osteophytosis compared with their wild-type littermates. Both classical and non-classical monocytes showed increased expression of CCR2 and CD86 in hCRP-tg males. HFD-induced effects were evident for nearly all lipids measured and indicated a similar low-grade systemic inflammation for both genotypes. Synovitis scores and synovial macrophage subsets were similar in the two groups. CONCLUSIONS: Human CRP expression in a background of HFD-induced metabolic dysfunction resulted in the aggravation of OA through increased cartilage degeneration and osteophytosis. Increased recruitment of classical and non-classical monocytes might be a mechanism of action through which CRP is involved in aggravating this process. These findings suggest interventions selectively directed against CRP activity could ameliorate metabolic OA development.

Variable cartilage degradation in mice with diet-induced metabolic dysfunction: food for thought.

OBJECTIVE: Human cohort studies have demonstrated a role for systemic metabolic dysfunction in osteoarthritis (OA) pathogenesis in obese patients. To explore the mechanisms underlying this metabolic phenotype of OA, we examined cartilage degradation in the knees of mice from different genetic backgrounds in which a metabolic phenotype was established by various dietary approaches. DESIGN: Wild-type C57BL/6J mice and genetically modified mice (hCRP, LDLr-/-. Leiden and ApoE*3Leiden.CETP mice) based on C57BL/6J background were used to investigate the contribution of inflammation and altered lipoprotein handling on diet-induced cartilage degradation. High-caloric diets of different macronutrient composition (i.e., high-carbohydrate or high-fat) were given in regimens of varying duration to induce a metabolic phenotype with aggravated cartilage degradation relative to controls. RESULTS: Metabolic phenotypes were confirmed in all studies as mice developed obesity, hypercholesteremia, glucose intolerance and/or insulin resistance. Aggravated cartilage degradation was only observed in two out of the twelve experimental setups, specifically in long-term studies in male hCRP and female ApoE*3Leiden.CETP mice. C57BL/6J and LDLr-/-. Leiden mice did not develop HFD-induced OA under the conditions studied. Osteophyte formation and synovitis scores showed variable results between studies, but also between strains and gender. CONCLUSIONS: Long-term feeding of high-caloric diets consistently induced a metabolic phenotype in various C57BL/6J (-based) mouse strains. In contrast, the induction of articular cartilage degradation proved variable, which suggests that an additional trigger might be necessary to accelerate diet-induced OA progression. Gender and genetic modifications that result in a humanized pro-inflammatory state (human CRP) or lipoprotein metabolism (human-E3L.CETP) were identified as important contributing factors.

Dermal, inhalation, and internal exposure to 1,6-HDI and its oligomers in car body repair shop workers and industrial spray painters.

OBJECTIVES: To study inhalation and dermal exposure to hexamethylene diisocyanate (HDI) and its oligomers as well as personal protection equipment (PPE) use during task performance in conjunction with urinary hexamethylene diamine (HDA) in car body repair shop workers and industrial spray painters. METHODS: Personal task based inhalation samples (n = 95) were collected from six car body repair shops and five industrial painting companies using impingers with di-n-butylamine (DBA) in toluene. In parallel, dermal exposure was assessed using nitril rubber gloves. Gloves were submerged into DBA in toluene after sampling. Analysis for HDI and its oligomers was performed by LC-MS/MS. Urine samples were collected from 55 workers (n = 291) and analysed for HDA by GC-MS. RESULTS: Inhalation exposure was strongly associated with tasks during which aerosolisation occurs. Dermal exposure occurred during tasks that involve direct handling of paint. In car body repair shops associations were found between detectable dermal exposure and glove use (odds ratio (OR) 0.22, 95% confidence interval (CI) 0.09 to 0.57) and inhalation exposure level (OR 1.34, 95% CI 0.97 to 1.84 for a 10-fold increase). HDA in urine could be demonstrated in 36% and 10% of car body repair shop workers and industrial painting company workers respectively. In car body repair shops, the frequency of detectable HDA was significantly elevated at the end of the working day (OR 2.13, 95% CI 1.07 to 4.22 for 3-6 pm v 0-8 am). In both branches HDA was detected in urine of approximately 25% of the spray painters. In addition HDA was detected in urine of a large proportion of non-spray painters in car body repair shops. CONCLUSION: Although (spray) painting with lacquers containing isocyanate hardeners results in the highest external exposures to HDI and oligomers, workers that do not perform paint related tasks may also receive a considerable internal dose.

Determination of kava lactones in food supplements by liquid chromatography-atmospheric pressure chemical ionisation tandem mass spectrometry.

Reversed-phase liquid chromatography and detection with atmospheric pressure chemical ionisation tandem mass spectrometry was used for the determination of kava extracts in herbal mixtures. One percent of kava extract can be detected, corresponding to approximately 0.05-0.2 mg/g of the individual kava lactones kavain, dihydrokavain, yangonin, desmethoxyyangonin, methysticin and dihydromethysticin. Reliable quantification is obtained from concentrations of 0.25-1 mg/g, depending on the compound. At these concentration levels, the relative standard deviations were 10-14%. Validation showed good linearity and recoveries for all the kava lactones with the exception of yangonin. During method development, degradation of yangonin was observed. The degradation product was identified by nuclear magnetic resonance (NMR) as cis-yangonin. The method was applied to the analysis of commercial herbal products available in the Dutch market before and after market restrictions of kava-containing preparations. The results showed that even though 'old' products contained kava extract, the new formulations were negative on kava lactones. cis-Yangonin was also present in the herbal products.

Novel monitoring concepts to acquire new water quality knowledge.

Two novel water quality monitoring concepts were developed: the HPLC-fingerprint for the monitoring of yet unidentified pollutants and the HPLC-Toxprint for the recognition of (unknown) toxic or genotoxic compounds. The paper describes applications of both concepts. The HPLC-fingerprint is used for the evaluation of the overall water quality in addition to the monitoring of individual pollutants. Based on their occurrence (frequency, concentration, location) a listing of unknown priority pollutants is set up. Participating waterworks monitor for these compounds using a dedicated HPLC-DAD library that contains the required compound data (UV-absorbance spectrum, retention time index). In five years of experience with this concept, the HPLC-fingerprint was also found very suitable for the retrieval of new priority pollutants in existing HPLC-fingerprint databases, providing historic data on these new compounds. The HPLC-fingerprint concept was also used as an Early Warning System for accidental spills or sabotage. The HPLC-Toxprint was successfully applied in identifying genotoxicity (in the umu-test) in various waste water samples. By the application of LC-MS/MS genotoxicity could be assigned to acridine-derivatives in one of these wastewaters. To enable the evaluation of drinking water resources, the sensitivity of the HPLC-Toxprint was improved, now allowing the detection of pollutants with a 10% genotoxic potential as compared to 2-aminoanthracene (the positive control compound) at concentrations as low as 0.1 microg/l.

Surface and wastewater quality monitoring: combination of liquid chromatography with (geno)toxicity detection, diode array detection and tandem mass spectrometry for identification of pollutants.

Identification of unknown water pollutants with liquid chromatography and tandem mass spectrometry (LC-MS-MS) is often more complex and time consuming than identification with gas chromatography and mass spectrometry (GC-MS). In order to focus the identification effort on relevant compounds, unknown peaks need to be selected carefully. Based on its frequency of occurrence in the LC-Diode Array Detection (LC-DAD) chromatograms of surface and infiltrated waters, an unknown peak was selected for identification with LC-MS-MS. This compound was identified as hexamethoxymethylmelamine (HMMM), a chemical often used in the coating industry. This is the first time the presence of this chemical in surface waters has been reported. In addition to HMMM, two other structurally related compounds were found to be present in the investigated surface water. A standard mixture of HMMM and its by-products did not exhibit (geno)toxicity under the test conditions applied in this study. In another example, a genotoxic fraction of an industrial wastewater was isolated and examined by LC-MS-MS using a modern quadrupole-orthogonal acceleration-time-of-flight mass spectrometer (Q-TOF). Four compounds were detected. The structures of two compounds present are proposed to be 9-amino-2-hydroxy-acridine and 9-hydroxy-acridine-N-oxide or its structural isomer dihydroxy-acridine. Confirmation with standards could not be carried out, as pure compounds are not available. The other two compounds (structural isomers) could not be identified based on the data available within this study.

Screening and identification of unknown contaminants in water with liquid chromatography and quadrupole-orthogonal acceleration-time-of-flight tandem mass spectrometry.

In order to assess and maintain the quality of surface waters, target compound monitoring is often not sufficient. Many unknown micro-contaminants are present in water, originating in municipal, industrial or agricultural effluents. Some of these might pose a risk to drinking water production and consequently to human health. The possibilities of screening surface water and identification of these non-target water pollutants with modern data acquisition possibilities of hybrid quadrupole-orthogonal acceleration time of flight mass spectrometers (Q-TOF), such as data-dependent MS to MS/MS switching were investigated. Using model compounds, a procedure for the liquid chromatography-tandem mass spectrometry (LC-MS/MS) screening of water extracts was developed, enabling the detection and identification of compounds at levels < or = 0.25 microg/l in surface water. Based on the accurate mass the elemental compositions for the precursor and product ions are calculated. The calculated chemical formulae are searched against the Merck index, the NIST library, an own database containing about 2,500 water pollutants (pesticides and other contaminants) as well as a CI-CID library containing tandem MS spectra of about 100 water contaminants. The developed approach was applied for the identification of unknown compounds, present in native surface water extract. For three of these compounds, structures were proposed. Confirmation of the proposed structures with standards was beyond the scope of this study.

High-performance liquid chromatography--ToxPrint: chromatographic analysis with a novel (geno)toxicity detection.

In order to aid the monitoring of the overall quality of (surface) waters a new analytical approach has been developed, combining on-line solid-phase extraction, HPLC separation and effect-related detection. Compounds present in surface water or wastewater samples are extracted on-line with Oasis [poly(divinylbenzene-co-N-vinylpyrrolidone)] material and directly fractionated by reversed-phase HPLC. The eluent of the total chromatogram is collected on a microtitre plate in fractions of 1 min each. After evaporation and re-dissolvation in a suitable solvent, the (geno)toxicity of the individual fractions before and after enzymatic activation with S9, is determined with the umu test. In this way, harmful compounds can be detected and localized in the HPLC-diode array detection trace even without their identity and exact concentration being known at that moment. The method was developed using two test compounds, 4-nitroquinoline-N-oxide and 2-aminoanthracene. Compounds with mutagenic properties comparable to those of the test compounds can be detected from 0.1 microg/l, which is a concentration relevant for surface waters. The new analytical approach was successfully applied to various types of model samples, as well as real wastewater.

Screening for transformation products of pesticides using tandem mass spectrometric scan modes.

The applicability of tandem mass spectrometric (MS-MS) scan modes such as constant neutral-loss and precursor-ion scanning to screen for unknown transformation products (TPs) of pesticides at environmentally relevant concentrations (low-microng/l level) is studied. The selection of the MS-MS scan modes is based on the product-ion scan of the parent pesticide, and TPs are detected which are unaltered in the part of the structure concerned. The screening approach is applied to a surface water sample spiked with atrazine and three known TPs at a level of 3 microg/l to study the possibility to extract the TPs from the total ion chromatogram. Next, the approach is used to identify unknown TPs formed after (bio)degradation of two test compounds, fenchlorazole-ethyl (FCE) and furathiocarb (FTC). By using the precursor-ion scan mode, two TPs were detected after biodegradation of FCE, fenchlorazole-methyl and fenchlorazole; in surface water only fenchlorazole was found. The constant neutral-loss scan mode was used to identify carbofuran as TP of FTC. The added value of the proposed procedure is the increased selectivity at the cost of sensitivity. Best results are, therefore, obtained for samples which contain large amounts of matrix constituents.

Determination of the herbicide amitrole in water with pre-column derivatization, liquid chromatography and tandem mass spectrometry.

Amitrole is a widely used polar herbicide, difficult to isolate from water. Due to its persistence, it can easily pollute ground and surface waters used in drinking water production. A fully automated on-line SPE-HPLC (solid-phase extraction-high-performance liquid chromatography) method with atmospheric pressure chemical ionisation-tandem mass spectrometry detection is described for the determination of amitrole. A pre-column derivatization with 9-fluorenylmethoxycarbonyl chloride directly in the native aqueous sample allows an enrichment step by SPE and HPLC separation. Due to the sensitivity of tandem mass spectrometric detection, a limit of detection and quantification as low as 0.025 microg/l was achieved in drinking water and ground and surface water. Based on the constant ratio of two selected product ions together with the retention time, the identification is very selective and quantitation is very reliable. The performance characteristics of the described method fully meet the requirements set by the EU Drinking Water Directive: recoveries of >95% in drinking water and >75% in surface water were achieved, as well as RSD values for repeatability of <9% in drinking water and <12% in surface water (determined at a spiking level of 0.1 microg/l). The method was successfully applied to real samples of ground and surface water with actual concentration up to 1.1 microg/l.