Quality of development and reporting of dietetic intervention studies in primary care: a systematic review of randomised controlled trials.

BACKGROUND: High-quality research methodologies and clear reporting of studies are essential to facilitate confidence in research findings. The aim of the present study was to conduct an in-depth examination of the methodological quality and reporting of studies included in a recent systematic review of dietitians' effectiveness at providing individualised nutrition care to adult patients. METHODS: The methodological quality and reporting of 27 Randomised Controlled Trials (RCTs) were appraised using the UK Medical Research Council (MRC) Guidelines for complex interventions and the CONSORT checklist for reporting RCTs. A quality appraisal checklist was developed for each guideline/assessment tool aiming to evaluate the extent to which each study met the designated criteria. Excerpts from studies that best addressed criteria were collated to provide exemplary accounts of how criteria may be achieved in future studies. RESULTS: None of the reviewed studies met more than half of the MRC Guidance criteria, indicating that there is clear room for improvement in reporting the methodological underpinnings of these studies. Similarly, no studies met all criteria of the CONSORT checklist, suggesting that there is also room for improvement in the design and reporting of studies in this field. CONCLUSIONS: Dietitians, researchers and journal editors are encouraged to use the results and exemplary accounts from this review to identify key aspects of studies that could be improved in future research. Improving future research will enhance the quality of the evidence-base that investigates the outcomes of dietary interventions involving dietitians.

Migration of formaldehyde from melamine-ware: UK 2008 survey results.

Fifty melamine-ware articles were tested for the migration of formaldehyde - with hexamethylenetetramine (HMTA) expressed as formaldehyde - to see whether the total specific migration limit (SML(T)) was being observed. The SML(T), given in European Commission Directive 2002/72/EC as amended, is 15 mg kg(-1). Fourier transform-infrared (FT-IR) spectroscopy was carried out on the articles to confirm the plastic type. Articles were exposed to the food simulant 3% (w/v) aqueous acetic acid under conditions representing their worst foreseeable use. Formaldehyde and HMTA in food simulants were determined by a spectrophotometric derivatization procedure. Positive samples were confirmed by a second spectrophotometric procedure using an alternative derivatization agent. As all products purchased were intended for repeat use, three sequential exposures to the simulant were carried out. Formaldehyde was detected in the simulant exposed to 43 samples. Most of the levels found were well below the limits set in law such that 84% of the samples tested were compliant. However, eight samples had formaldehyde levels that were clearly above the legal maximum at six to 65 times the SML(T).

Determination of unusual soya and non-soya phytoestrogen sources in beer, fish products and other foods.

Fish and fish products (14 samples), Indian foods and meals (10 samples), spices (30 samples) and beers (10 samples) were analysed for their phytoestrogen content, and a number of significant non-soya sources of dietary phytoestrogens were identified. No isoflavones were detected in unprocessed, farmed or ocean fish, but some samples of processed fish products contained soya isoflavones, which are assumed to come from coatings or protein addition. Additionally, some processed fish products contained, genistein glycocongugates not derived from soya. Genistein was detected in Indian meals such that, for example, a single portion of a vindalooo curry contained 11 mg genistein. The origin was most likely from the spices used, since the analysis of curry powders, chilli powder, crushed red chillies, garam masala and tandoori powder revealed that some contained genistein at more than 100 mg kg(-1). Cumin was the most likely source material, although not all individual samples of cumin tested contained high levels of genistein. Prenylnaringenin phytoestrogens were determined in UK hop-based beers at mean concentrations of 0.21 mg(-1) 6-prenylnaringenin and 0.06mg(-1) 8-prenylnaringenin. The beers also contained traces of daidzein, genistein and biochanin A. The significance of 'hidden soya' in processed foods and these non-soya sources of phytoestrogens is that UK dietary intake of phytoestrogens must be assumed to be higher than estimated previously and that some sources of phytoestrogens remain poorly characterized.

Development and application of an LC-MS method to determine possible migration of mercaptobenzothiazole, benzothiazole and related vulcanization residues from rubber used in contact with food and drink.

A method was developed for the analysis of food and drink for residues of specific vulcanization accelerators used to cross-link rubber. The method was applied to the analysis of 236 samples of selected retail foodstuffs that may have been in contact with rubber during their manufacture, transport and storage. The method of analysis involved extraction of the food using acidified solvent and analysis by liquid chromatography/atmospheric pressure chemical ionization-mass spectrometry (LC-APcI-MS). The detection limit depended on the sample type and was in the range 0.005-0.043 mg kg(-1) for 2-mercaptobenzothiazole (MBT) and benzothiazole (BT). The average analytical recovery rate was 82% for MBT and 87% for BT. The analytical method was validated using a blind check sample exercise. For MBT and BT at seven different concentrations in the range 0.1-0.2 mg kg(-1), the laboratory found a mean of 91 and 90% of the expected concentrations, respectively. No trace of MBT or BT was found in any of the retail samples. It is also concluded that no sample contained significant 2-mercaptobenzothiazyl disulphide (MBTS) or N- cyclohexyl-2-benzothiazole sulphenamide (CBS). Both MBTS and CBS are important accelerators used to vulcanize rubber and they break down in foodstuffs to form MBT and BT. The absence of MBT and BT in the foodstuffs therefore also provides proof of the absence of MBTS and CBS.

Sarco/endoplasmic reticulum Ca2+-pump isoform SERCA3a is more resistant to superoxide damage than SERCA2b.

Endo/sarcoplasmic reticulum (ER) Ca2+-pumps are important for cell survival and communication but they are inactivated by reactive oxygen species (ROS). We have previously reported that the Ca2+-pump isoform SERCA3a is more resistant than SERCA2b to damage by peroxide. Since peroxide and superoxide differ in their redox potentials, we now report the effects of superoxide on the two Ca2+-pump isoforms. We isolated microsomes from HEK293 cells transiently transfected with SERCA2b or SERCA3a cDNA. We exposed these microsomes to superoxide which was generated using xanthine plus xanthine oxidase and catalase to prevent accumulation of peroxide due to superoxide dismutation. Superoxide damaged the Ca2+-transport activity of both isoforms but SERCA3a was damaged at higher concentrations of superoxide and upon longer periods of exposures than was SERCA2b. Thus the SERCA3a isoform is more resistant than SERCA2b to inactivation by both superoxide and peroxide.

Composition and role of tapetal lipid bodies in the biogenesis of the pollen coat of Brassica napus.

The composition of the two major lipidic organelles of the tapetum of Brassica napus L. has been determined. Elaioplasts contained numerous small (0.2-0.6 micron) lipid bodies that were largely made up of sterol esters and triacylglycerols, with monogalactosyldiacylglycerol as the major polar lipid. This is the first report in any species of the presence of non-cytosolic, sterol ester-rich, lipid bodies. The elaioplast lipid bodies also contained 34- and 36-kDa proteins which were shown by N-terminal sequencing to be homologous to fibrillin and other plastid lipid-associated proteins. Tapetosomes contained mainly polyunsaturated triacylglycerols and associated phospholipids plus a diverse class of oleosin-like proteins. The pollen coat, which is derived from tapetosomes and elaioplasts, was largely made up of sterol esters and the C-terminal domains of the oleosin-like proteins, but contained virtually no galactolipids, triacylglycerols or plastid lipid-associated proteins. The sterol compositions of the elaioplast and pollen coat were almost identical, consisting of stigmasterol > campestdienol > campesterol > sitosterol >> cholesterol, which is consistent with the majority of the pollen coat lipids being derived from elaioplasts. These data demonstrate that there is substantial remodelling of both the lipid and protein components of elaioplasts and tapetosomes following their release into the anther locule from lysed tapetal cells, and that components of both organelles contribute to the formation of the lipidic coating of mature pollen grains.

A microbore high performance liquid chromatography/electrospray ionization mass spectrometry method for the determination of the phytoestrogens genistein and daidzein in comminuted baby foods and soya flour.

A microbore high performance liquid chromatographic/electrospray/mass spectrometric (HPLC/ESI-MS) method has been developed for the determination of the phytoestrogens daidzein and genistein in soya flours and baby foods. The samples were hydrolysed and extracted with acetonitrile-water prior to analysis. LC was performed on a microbore Primesphere 5C8 column using a water/acetonitrile/acetic acid mobile phase at a flow rate of 60 microliters/min. Atmospheric pressure ionization in the form of pneumatically assisted electrospray mass spectrometry (ESI-MS) was used as the method of detection. The limit of detection was 0.2 mg/ kg for daidzein and 0.7 mg/kg for genistein in the flour and food samples. The method proved both robust and reliable when operated over a long time period (10 days, 463 injections) generating precision data with a coefficient of variation of 4-15%.

Determination of dimetridazole, ronidazole and their common metabolite in poultry muscle and eggs by high performance liquid chromatography with UV detection and confirmatory analysis by atmospheric pressure chemical ionisation mass spectrometry.

A method was developed for the determination of the nitroimidazole compounds dimetridazole (DMZ) and ronidazole (RNZ) and their common metabolite, 2-hydroxymethyl-1-methyl-5-nitroimidazole (2-OH-M). Extracts obtained from a clean-up process using strong cation exchange (SCX) solid phase extraction (SPE) can be analysed either by high performance liquid chromatography with UV detection (HPLC-UV) or by high performance liquid chromatography with atmospheric pressure chemical ionisation mass spectrometry (HPLC-APCI-MS) as a confirmatory method. Up to 20 samples can be extracted in approximately 4 h. The HPLC-UV analysis had a limit of detection of 0.5 microgram kg-1. Validation in chicken muscle fortified at a concentration of 5 micrograms kg-1 gave recoveries of 75% DMZ, 77% RNZ and 81% 2-OH-M with RSDs of 16.4, 11.3 and 14.0%, respectively (n = 17). Validation in egg fortified at the same concentration gave recoveries of 77% DMZ, 80% RNZ and 80% 2-OH-M, with RSDs of 14.9, 22.0 and 18.2%, respectively (n = 18). The limit of detection of the HPLC-APCI-MS method was 0.1 microgram kg-1 for DMZ and RNZ and 0.5 microgram kg-1 for 2-OH-M. This method gave mean recoveries in fortified egg samples of 65% DMZ, 87% RNZ and 75% 2-OH-M with RSDs of 22, 11 and 14%, respectively (n = 10). The ratios of the peak areas of the molecular ion and a fragment ion were monitored as added confirmation of the presence of the analyte. Both the HPLC-UV screening procedure and the HPLC-APCI-MS confirmatory method have subsequently been used for the analysis of several hundred samples as part of UK surveillance programmes.

Screening and confirmation of triphenylmethane dyes and their leuco metabolites in trout muscle using HPLC-vis and ESP-LC-MS.

An extraction and clean-up protocol for the determination of Malachite Green and Crystal Violet and the corresponding leuco compounds in trout muscle has been developed. Final determination is by HPLC with visible (screening) or ESP-MS (confirmation) detection. In both cases lead(IV) oxide was used on-line to oxidise the leuco compounds back to the parent after chromatographic separation and prior to detection. The procedure was validated down to 2 micrograms kg-1. Intra- and inter-batch precision was measured at 3 levels for all compounds. Recoveries were in the range 66-116% with RSD of 1-17% for determination by HPLC with visible detection. For LC-MS determination, recoveries were in the range 61-94% with RSD of 4-15%. Limited surveillance data indicated that Malachite Green usage was more effectively monitored by including the leuco compound as well as the parent (9 positives for the leuco compound as opposed to 1 for Malachite Green out of 31 samples analysed).

Determination of the pesticide fenbutatin oxide in tomatoes, cucumbers and bananas by high performance liquid chromatographic/atmospheric pressure chemical ionization-mass spectrometry.

A rapid method, using high-performance liquid chromatography (HPLC)/ atmospheric pressure chemical ionization-mass spectrometry, has been developed for the determination of fenbutatin oxide in tomatoes, cucumbers and bananas. Samples were homogenized with sodium carbonate and ethyl acetate, filtered through sodium sulphate, concentrated and solvent exchanged into acetonitrile prior to analysis. HPLC was performed on a Hypercarb column with 10:90 acetic acid (5% v/v glacial acetic acid in water)/acetonitrile at a flow rate of 1 mL/min. Positive ionization selected-ion monitoring was performed on the 7 isotopic cluster ions from the tris(2-methyl-2-phenylpropyl) tin fragment. A comparison of solvent-based and extract-based standards showed that tomato and cucumber matrices had a slight enhancement effect on the signal intensity, whereas the banana matrix exerted a signal suppression effect. Calibration was linear over the range 0.25-5.0 ng/microL. The mean spike recoveries (extracts spiked at 0.5 mg/kg) were 88% for tomatoes and 80% for both cucumbers and bananas with relative standard deviations of 6%, 7% and 8% respectively. Limits of detection were commodity dependent and ranged from 0.06-0.12 ng/microL (equivalent to 0.01-0.02 mg/kg in the crop). Ionization was stable for long analytical time periods.

Extraction of maleic hydrazide residues from potato crisps and their determination using high-performance liquid chromatography with UV and atmospheric pressure chemical ionisation mass spectrometric detection.

A method was required for the determination of maleic hydrazide residues in potato crisps. A published method for the extraction of the analyte from onions and potatoes was evaluated and found to be inappropriate due to the inability of the extracting solvent to penetrate the oily matrix. A method was developed to overcome this problem; the resulting recovery data (mean = 92.9%, R.S.D. = 8.3%, n = 16) confirmed its efficiency, and was used to analyse 48 retail potato crisp samples. To confirm possible residues identified by screening with HPLC-UV, an HPLC-atmospheric pressure chemical ionization MS method was developed. There was good agreement between the data obtained from the two detection techniques (R2 = 0.978, slope = 1.11).

Analytical methods for the determination of sterigmatocystin in cheese, bread and corn products using HPLC with atmospheric pressure ionization mass spectrometric detection.

Methods have been developed for the determination of sterigmatocystin in bread, maize and cheese using HPLC linked to mass spectrometry (MS) atmospheric pressure chemical ionization for detection and concurrent confirmation of sterigmatocystin at levels down to less than 5 micrograms/kg. Candidate extraction methods were initially checked for recovery and reproducibility by spiking commodities at a level of 200 micrograms/kg of sterigmatocystin and using HPLC with post-column derivatization. Recovery was found to be greater than 90% for bread and maize, and 75% for cheese. Mass spectrometer conditions for detecting sterigmatocystin were established by injecting solutions directly into the mass spectrometer. Extracts of bread, maize grits and cheese prepared by the candidate extraction methods were examined using HPLC/MS using samples spiked at a level of 20 micrograms/kg of sterigmatocystin. Results for bread and maize samples showed that the extraction procedure recovered more than 90% of added sterigmatocystin and produced extracts free of interference from co-extractives, with limits of detection of less than 2 micrograms/kg for both commodities. The HPLC/MS results for cheese extracts gave lower average recoveries of 55%. These results were also more variable. However, the apparent limit of detection for sterigmatocystin in cheese was still about 4 micrograms/kg.

Determination of the pesticide diflubenzuron in mushrooms by high-performance liquid chromatography-atmospheric pressure chemical ionisation mass spectrometry.

A method using high-performance liquid chromatography-atmospheric pressure chemical ionisation mass spectrometry (HPLC-APCI-MS) has been developed and validated for the determination of the insecticide diflubenzuron [1-(4-chlorophenyl)-3-(2,6-difluorobenzoyl)urea] in mushrooms. Samples were homogenised with acetone, extracted into dichloromethane-cyclohexane and further cleaned-up by size-exclusion chromatography (SEC). HPLC was performed on an ODS column with methanol-water at 1 ml/min. The limit of detection was 0.02 ng/microl (equivalent to 0.017 mg/kg in the crop). The calibration was linear over the range 0.025-1.0 ng/microl. Recovery of diflubenzuron from spiked mushrooms (0.06-0.58 mg/kg) was 85.5% with a relative standard deviation of 14.5% (n = 56).

Intracellular targeting of antigens internalized by membrane immunoglobulin in B lymphocytes.

An important function of membrane immunoglobulin (mIg), the B cell antigen receptor, is to endocytose limiting quantities of antigen for efficient presentation to class II-restricted T cells. We have used a panel of mIg mutants to analyze the mechanism of mIg-mediated antigen presentation, and specifically to explore the ability of mIg to target internalized antigen to intracellular processing compartments. Transfected mIgs carrying substitutions for the transmembrane Tyr587 residue fail to efficiently present specifically bound antigen. However, these mutants internalize antigen normally, and their defect cannot be attributed to a lack of mIg-associated Ig alpha/Ig beta molecules. A novel functional assay for detecting antigenic peptides in subcellular fractions shows that wild-type mIg transfectants generate class II-peptide complexes intracellularly, whereas only free antigenic peptides are detectable in the mutant mIg transfectants. Furthermore, an antigen competition assay reveals that antigen internalized by the mutant mIgs fails to enter the intracellular processing compartment accessed by wild-type mIg. Therefore, mIg specifically targets bound and endocytosed antigen to the intracellular compartment where processed peptides associate with class II molecules, and the transmembrane Tyr587 residue plays an obligatory role in this process. Targeting of internalized antigen may be mediated by receptor-associated chaperones, and may be a general mechanism for optimizing the presentation of specifically bound and endocytosed antigens in b lymphocytes and other antigen-presenting cells.

Detection of functional class II-associated antigen: role of a low density endosomal compartment in antigen processing.

We have developed a functional assay to identify processed antigen in subcellular fractions from antigen-presenting cells; stimulatory activity in this assay may be caused by either free peptide fragments or by complexes of peptide fragments and class II molecules present on organellar membrane sheets and vesicles. In addition, we have developed a functional assay to identify proteolytic activity in subcellular fractions capable of generating antigenic peptides from intact proteins. These techniques permit the direct identification of intracellular sites of antigen processing and class II association. Using a murine B cell line stably transfected with a phosphorylcholine (PC)-specific membrane-bound immunoglobulin (Ig), we show that PC-conjugated antigens are rapidly internalized and efficiently degraded to generate processed antigen within an early low density compartment. Proteolytic activity capable of generating antigenic peptide fragments from intact proteins is found within low density endosomes and a dense compartment consistent with lysosomes. However, neither processed peptide nor peptide-class II complexes are detected in lysosomes from antigen-pulsed cells. Furthermore, blocking the intracellular transport of internalized antigen from the low density endosome to lysosomes does not inhibit the generation of processed antigen. Therefore, antigens internalized in association with membrane Ig on B cells can be efficiently processed in low density endosomal compartments without the contribution of proteases present within denser organelles.

Determination of the pesticides diflubenzuron and clofentezine in plums, strawberries and blackcurrant-based fruit drinks by high performance liquid chromatographic/atmospheric pressure chemical ionization-mass spectrometry.

A high performance liquid chromatographic/atmospheric pressure chemical ionization-mass spectrometric (HPLC/APCI-MS) method has been developed for the determination of the pesticides diflubenzuron (1-(4-chlorophenyl)-3-(2,6-difluorobenzoyl)urea) and clofentezine (3,6-bis(2-chlorophenyl)-1,2,4,5-tetrazine) in plums, strawberries and blackcurrant-based fruit drinks. Samples were homogenized with acetone, extracted into dichloromethane + cyclohexane and cleaned-up by high performance gel permeation chromatography. HPLC was performed on an ODS column with methanol + water at 1 mL/min. Detection was by negative-ion selected-ion monitoring APCI-MS. Comparison of response with solvent and matrix-matched standards showed some enhancement of response for the latter, and these standards were consequently used for quantification. The calibration was linear over the range 0.05-0.50 ng/microL in all three matrices. The mean overall recovery of diflubenzuron and clofentezine from spiked extracts (0.086 mg/kg) in all three matrices was 76% and 70% respectively with relative standard deviations of 15% and 12% respectively (n = 12). The limit of detection was both compound and commodity dependent and ranged from 0.01-0.05 ng/microL, equivalent to 0.003-0.014 mg/kg in the crop.

AMPA antagonists: do they hold more promise for clinical stroke trials than NMDA antagonists?

The cytoprotective effects of MK-801 and NBQX, selective N-methyl-D-aspartate (NMDA) and alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor antagonists, respectively, were compared both singularly and in combination in models of transient severe forebrain and transient focal cerebral ischemia. After 10 minutes of four-vessel occlusion ischemia, the sodium salt of NBQX (30 mg/kg IP) given at the time of reperfusion and, subsequently, 15 and 30 minutes later produced a dramatic reduction in CA1 hippocampal necrosis at 7 days. This effect was not obtained with the intraperitoneal administration of either MK-801 (1 mg/kg x 3) or the combination of both NBQX and MK-801 given at the same time intervals. This effect of intraperitoneal NBQX alone was reproduced in a two-vessel occlusion/hypotension model using this same drug administration. Delayed treatment with both NBQX and GYKI 52466, but neither MK-801 nor the combination of NBQX and MK-801 given after a delay, produced a significant reduction in the mean volume of neocortical infarction after transient focal ischemia. We conclude that the AMPA receptor may play a more important role than the NMDA receptor in both selective ischemic necrosis of hippocampal neurons and in neocortical infarction. AMPA antagonists should be subjected to clinical stroke trials.

Multiplane fracture of the distal humerus.

A unique but complex intraarticular fracture of the distal humerus is described. The term multiplane fracture has been applied to highlight a fracture pattern that features not only the well recognized T fracture lines in the sagittal and horizontal planes but also a coronal fracture of the trochlea. The Herbert screw was used in five cases to achieve stable fixation of the purely articular coronal fracture. At a follow-up of 20 months, on average, all fractures healed with an acceptable functional result. No radiographic evidence of avascular necrosis was seen in the follow-up radiographs.