GC-MS method for metabolic profiling of mouse femoral head articular cartilage reveals distinct effects of tissue culture and development.

OBJECTIVE: The metabolic profile of cartilage is important to define as it relates to both normal and pathophysiological conditions. Our aim was to develop a precise, high-throughput method for gas/chromatography-mass/spectrometry (GC-MS) semi-targeted metabolic profiling of mouse cartilage. METHOD: Femoral head (hip) cartilage was isolated from 5- and 15-week-old male C57BL/6J mice immediately after death for in vivo analyses. In vitro conditions were evaluated in 5-week-old samples cultured ±10% fetal bovine serum (FBS). We optimized cartilage processing for GC-MS analysis and evaluated group-specific differences by multivariate and parametric statistical analyses. RESULTS: 55 metabolites were identified in pooled cartilage (4 animals per sample), with 29 metabolites shared between in vivo and in vitro conditions. Multivariate analysis of these common metabolites demonstrated that culturing explants was the strongest factor altering cartilage metabolism, followed by age and serum starvation. In vitro culture altered the relative abundance of specific metabolites; whereas, cartilage development between five and 15-weeks of age reduced the levels of 36 out of 43 metabolites >2-fold, especially in TCA cycle and alanine, aspartate, and glutamate pathways. In vitro serum starvation depleted six out of 41 metabolites. CONCLUSION: This study describes the first GC-MS method for mouse cartilage metabolite identification and quantification. We observed fundamental differences in femoral head cartilage metabolic profiles between in vivo and in vitro conditions, suggesting opportunities to optimize in vitro conditions for studying cartilage metabolism. In addition, the reductions in TCA cycle and amino acid metabolites during cartilage maturation illustrate the plasticity of chondrocyte metabolism during development.

C-Reactive Protein Levels and the Risk of Incident Cardiovascular and Cerebrovascular Events in Patients with Obstructive Sleep Apnea.

PURPOSE: Patients with obstructive sleep apnea (OSA) are at increased risk of cardiovascular and cerebrovascular disease (CVD) but it is unclear who are at greatest risk. We determined whether the inflammatory marker, C-reactive protein (CRP), could be a useful prognostic biomarker. METHODS: Adult patients referred for polysomnography (PSG) with OSA were studied. Serum CRP levels were measured using ELISA the morning after PSG. Validated CV events within 4 years of PSG were ascertained by linking to provincial research datasets. RESULTS: 155 patients with OSA (AHI ≥ 5/h) had CRP measured. Median age was 53 and median AHI was 21/h. 10 patients (7.1%) suffered at least one event, but rates varied substantially by CRP (0/35 patients in the lowest quartile, and 7/39 in the highest CRP quartile). In the unadjusted analysis, patients in the highest CRP quartile (≥ 2.38 mg/L) were significantly more likely to suffer an event (odds ratio = 9.72 (95% CI 2.43-38.84), p = 0.001). CRP continued to be a significant predictor after controlling for multiple confounders. OSA severity and desaturation were not significantly associated with prospective events. CONCLUSIONS: In this small preliminary study, OSA patients with an elevated CRP were significantly more likely to suffer a CVD event in the 4 years after PSG. Although these findings need to be confirmed in larger prospective cohorts, CRP may be useful in risk stratifying OSA patients to guide therapy or to identify patients that might be most appropriate for clinical trials of CVD prevention.

The pharyngeal contractile integral is a useful indicator of pharyngeal swallowing impairment.

BACKGROUND: A limitation to the expanded use of high-resolution pharyngeal manometry (HRPM) in clinical practice is the lack of useful pharyngeal parameters that are easy to interpret, generalizable between patients, and do not require specialized software. In this study, we sought to test the relationship between the pharyngeal contractile integral (PhCI) with videofluoroscopic abnormalities as assessed with the Modified Barium Swallow Impairment Profile© ™. METHODS: Adult dysphagic patients were recruited to undergo simultaneous HRPM and videofluoroscopy during a standardized swallowing protocol. KEY RESULTS: Thirty-six patients were included in the study. The mean PhCI was 247 mm Hg·cm·s (range 2-488 mm Hg·cm·s). The lower pharyngeal total (PT) group (N=20; mean PT=3.9) had a mean PhCI of 299 mm Hg·cm·s, while the higher PT group (N=16; mean PT=12.7) had a mean PhCI score of 188 mm Hg·cm·s (P=.01). There was also a significant negative correlation between normalized PhCI to PT scores (r=-.47; P=.004). Patients with higher PhCIs exhibited less severe penetration-aspiration scores on thin liquids (1.44 vs 3.78; P=.03) and all consistencies combined (1.21 vs 1.99; P=.03). CONCLUSIONS & INFERENCES: The PhCI is a useful indicator of the presence of pharyngeal swallowing impairment and is technically simple to calculate with currently available software programs. Advancement of software is necessary to refine the clinical value of this parameter. High-resolution pharyngeal manometry has the potential to be a valuable adjunct procedure for the evaluation and treatment of dysphagic individuals.

Respiratory-swallowing coordination in normal subjects: Lung volume at swallowing initiation.

This study was designed to investigate the significance of bolus types and volumes, delivery methods and swallowing instructions on lung volume at swallowing initiation in normal subjects in a single experiment using a multifactorial approach. Our broad range goal was to determine optimal lung volume range associated with swallowing initiation to provide training targets for dysphagic patients with disordered respiratory-swallow coordination. Our hypothesis was that swallows would be initiated within a limited range of quiet breathing lung volumes regardless of bolus volume, consistency or task. Results confirmed this hypothesis and revealed that swallows were initiated at mean lung volume=244ml. Cued swallows were initiated at lower quiet breathing volumes than un-cued swallows (cued=201ml; un-cued=367ml). Water boluses were initiated at slightly higher quiet breathing volumes than solids. Data suggest that swallows occur within a restricted range of lung volumes with variation due to instructions, bolus type and other experimental variables.

Risk factors associated with increased prevalence of abdominal aortic aneurysm in women.

BACKGROUND: Four randomized trials of men aged 65-80 years showed that aneurysm-related mortality was reduced by 40 per cent by ultrasound screening. Screening is considered economically viable when the prevalence of abdominal aortic aneurysm (AAA) is 1·0 per cent or higher. This is not the case for women, in whom the prevalence of AAA is less than 1 per cent. The aim of the present investigation was to determine the prevalence of AAA 3·0 cm or larger in women screened with ultrasound imaging, the risk factors associated with AAA in this population, and whether high-risk groups can be identified with an AAA prevalence of 1 per cent or greater. METHODS: Demographic data and risk factors were collected from the first 50 000 women who attended for private cardiovascular screening in the UK. Tests included ultrasound screening for AAA, ankle : brachial pressure index (ABPI), carotid duplex imaging for carotid atherosclerosis, and electrocardiography for atrial fibrillation. RESULTS: AAA was detected in 82 of 50 000 women screened; these aneurysms were rare below the age of 66 years (7 of 24 499). In the 66-85-years age group there were 72 AAAs in 25 170 women (0·29 per cent). Univariable analysis demonstrated that a history of stroke/transient ischaemic attack (TIA), hypertension, smoking, atrial fibrillation, ABPI of less than 0·9 and internal carotid artery stenosis of at least 50 per cent were associated with an increased prevalence of AAA (P < 0·001). In multivariable linear logistic regression of risk factors, age 76 years or more, history of stroke/TIA, hypertension and smoking were independent predictors of AAA. This model had an area under the receiver operating characteristic (ROC) curve (AUC) of 0·711 (95 per cent c.i. 0·649 to 0·772) and could identify 2235 women who had 22 AAAs (prevalence 0·98 per cent). By adding ABPI, atrial fibrillation and carotid stenosis, the prediction improved to an AUC of 0·775 (0·724 to 0·826). This model could identify 3701 women who had 58 AAAs (prevalence 1·57 per cent). CONCLUSION: This report should stimulate consideration of a targeted AAA screening programme for women aged over 65 years.

Percentile distribution of blood pressure readings in 35683 men and women aged 18 to 99 years.

The percentile distribution of blood pressure (BP) with regard to age, sex and cardiovascular risk factors is unknown. We aimed to provide epidemiological data for a comprehensive description of the BP distribution across a wide age-range. We used data from the German Metabolic and Cardiovascular Risk Project (GEMCAS), a cross-sectional study with 35 683 participants aged 18-99 years, conducted during October 2005 in 1511 randomly selected general practices in Germany. BP and waist circumference were measured, data on lifestyle, cardiovascular disease (CVD) risk factors and medication assessed. In men, we found even in the lowest percentile (5th) a gradual increase of the systolic BP from the lowest to the highest age group of 10 mm Hg, all other percentile groups an increase of 20 mm Hg. In women, this increase ranged from 15 mm Hg (5th percentile) to 40 mm Hg (95th percentile). In a subgroup of participants with no antihypertensive usage (n=22 550) and no CVD/CVD risk factors (n=13 297), we still observed a distinct age-related increase of BP readings. Our study provides detailed information on the population distribution of BP readings in both sexes and also among very old individuals. The results are useful in a public health context to plan gender- and age-specific prevention strategies.

Joint recommendations for reporting carotid ultrasound investigations in the United Kingdom.

At present in the United Kingdom a number of different criteria are used to grade disease in carotid ultrasound investigations. One main cause of this has been the difference in the method of grading angiograms used in the NASCET and ECST large carotid surgery trials. It is desirable that all centres reporting carotid ultrasound investigations report to the same standard. This paper presents recommendations for the reporting of ultrasound investigations of the extra cranial arteries produced by a Joint Working Group formed between the Vascular Society of Great Britain and Ireland, and the Society for Vascular Technology of Great Britain and Ireland. The recommended criteria are based on the NASCET method of grading carotid bulb disease. Key recommendations include recording peak systolic velocity (PSV) and end-diastolic velocity (EDV) in both internal and distal common carotid arteries; measuring all velocities at a Doppler angle of 45-60 degrees; the use of internal carotid PSV of >1.25 ms(-1) and >2.3 ms(-1) and a Peak Systolic Velocity Ratio of >2 and >4 to indicate >50% and >70% stenosis respectively; and the use of the St Mary's Ratio to grade >50% stenoses in deciles. General recommendations are also given for the acquisition, interpretation and reporting of the data.

Decreased skeletal muscle mitochondrial DNA in patients treated with high-dose simvastatin.

Statins are generally well tolerated, but can cause myopathy and have been associated with mitochondrial abnormalities. The aim of this study was to determine whether muscle mitochondrial DNA (mtDNA) levels are altered during statin therapy. We retrospectively quantified mtDNA in 86 skeletal muscle biopsy specimens collected as part of a previously published clinical trial of high-dose simvastatin or atorvastatin versus placebo.

Longitudinal blood flow in shared (arteriovenous anastomoses) and non-shared cotyledons in monochorionic placentae.

OBJECTIVES: In this study we aimed to quantitate monochorionic twin placental blood flow in vivo through arterio-venous anastomoses (AVA) and corresponding vessels within normal cotyledons. METHODS: The topography of chorionic plate vasculature was mapped using colour Doppler in ten monochorionic diamniotic twin (MCDA) pregnancies. Cotyledonary flow was derived by insonation of chorionic veins draining normal (n=10) and paired control shared cotyledons (n=10). Venous volume flow was calculated from five determinations of vessel diameter and three of time average mean velocity (TAMV). Measurements were repeated every 2-4 weeks from 18 until 32 weeks' gestation. RESULTS: Blood flow through non-shared and shared cotyledons increased with gestation (p<0.0001). Median flow at 28 weeks through shared cotyledons was 16 ml/min (15-21) (median, interquartile range), lower than in shared cotyledons (31, 25-35) (p<0.001), as was median volume flow across gestation calculated as area under the curve (shared cotyledons 126 (122-167), control cotyledons 269 (214-274), p=0.01). However, velocity was similar, with the difference due to smaller vein diameters draining shared compared to normal cotyledons (mean 3.6mm (SD 0.8) vs. 4.5mm (0.8), p=0.004). Ex vivo quantitation of insonated cotyledons and of all cotyledons confirmed the difference in vein diameter in the placentae studied. CONCLUSIONS: Blood flow through shared cotyledons was lower across gestation than through paired normal cotyledons in the placenta studied due to the smaller diameter of the AVA vessels. The size of AVAs rather than simply their presence and direction may contribute to determining transfusional imbalance in monochorionic twins.

Safety and efficacy of rosuvastatin therapy for the prevention of hyperlipidemia in adult cardiac transplant recipients.

BACKGROUND: Hyperlipidemia after orthotopic heart transplantation (OHT) is associated with immunosuppression. Many OHT patients have increased lipid levels above published guidelines despite treatment with high doses of statins. Treatment with rosuvastatin (ROS) in OHT patients has not yet been evaluated. Therefore, we assessed its efficacy and safety in an OHT population. METHODS: Twenty-one OHT recipients, median age 66 years, whose lipid levels were sub-optimal on the highest tolerated doses of statins, received ROS in addition to standard immunosuppression. Total cholesterol (TC), low-density lipoprotein (LDL-C) and high-density lipoprotein cholesterol (HDL-C), triglycerides (TG), liver transaminases (AST) and creatinine kinase (CK) were measured before and during treatment with ROS. RESULTS: After 6 weeks on an average ROS dose of 10 mg/day, a TC:HDL-C ratio of <4 was reached in 76% of patients, and 70% of patients reached an LDL-C level of <2.5 mmol/liter (100 mg/dl). TC decreased to <5.2 mmol/liter (200 mg/dl) in 80% of patients and TG decreased to <2 mmol/liter (175 mg/dl) in 61% of patients. Except for the HDL-C increase, all changes were statistically significant. The decrease in the median TC:HDL-C ratio between baseline and 6 weeks was also statistically significant (p = 0.001). There were no significant changes in CK or AST levels, and no clinical evidence of myositis. One patient developed myalgia and 2 were withdrawn from the study because of mild elevation of CK (<3-fold upper limit of normal [ULN]). CONCLUSIONS: In the setting of tertiary referral centers, ROS appears to be safe and effective in lowering LDL-C in OHT recipients in whom treatment with other statins failed to achieve target LDL-C. No evidence of liver or muscle dysfunction was noted. Long-term studies are needed to ascertain the effect of ROS therapy on incidence of coronary artery disease (CAD) in this population.

A technique for the measurement of patellar tracking during weight-bearing activities using ultrasound.

A new fixation device, the femoral clamp, was developed in this study for the ultrasound measurement of patellar medio-lateral motion during sitting and squatting knee flexion/extension. Seventeen subjects, 6 males, 11 females, aged between 18 and 40 years were recruited for the test. Results showed that the patella moved medially then laterally from extension to flexion when sitting. Weight-bearing knee motion produced a more laterally tracked patella without the presence of the initial medial patellar translation. The tracking patterns of the patellae were similar regardless of knee movement direction. The patellar lateral position was greatly affected by the movement task (p < 0.0005), and was also influenced by gender for maximum medial position (p < 0.05). The reproducibility of the measurement was between 0.29 and 0.90 for the intra-rater and 0.34-0.75 for the inter-rater reliability. The accuracy of the ultrasound measurement was validated by interventional magnetic resonance (iMR) imaging of the patella and the mean error of the measurement was 1.4 +/- 3.2 mm. Although further research is needed to improve the accuracy and reliability of this method, it has demonstrated the feasibility of obtaining patellar tracking data during load-bearing activities.

Accuracy of an electromagnetic three-dimensional ultrasound system for carotid artery imaging.

Freehand, three-dimensional (3-D) ultrasound (US) systems, which incorporate an electromagnetic tracking device to register the 3-D spatial location of images acquired using a standard linear array scan-probe, are a flexible and cost-effective solution for many clinical applications. The reconstruction accuracy of one such system was investigated by using a precision-made phantom. The error in 3-D distance measurements, under conditions appropriate to US investigations of the carotid arteries, was found to be -0.45 +/- 1.30 mm, equivalent to -0.53 +/- 3.39% (mean +/- SD). The results are relevant to data acquired using a single sweep scan and for distances in the range 25.00 to 79.06 mm. Both the overall accuracy and precision in point-target location were found to be relatively unaffected by scan depth, and the precision of point-target location was found to be poorest in the elevational direction. In conclusion, the system tested in our laboratory performed with high accuracy, adopting a setup and scan-sweep identical to that used for imaging of the carotid arteries in 3-D.

PKA, PKC, and AKAP localization in and around the neuromuscular junction.

BACKGROUND: One mechanism that directs the action of the second messengers, cAMP and diacylglycerol, is the compartmentalization of protein kinase A (PKA) and protein kinase C (PKC). A-kinase anchoring proteins (AKAPs) can recruit both enzymes to specific subcellular locations via interactions with the various isoforms of each family of kinases. We found previously that a new class of AKAPs, dual-specific AKAPs, denoted D-AKAP1 and D-AKAP2, bind to RIalpha in addition to the RII subunits. RESULTS: Immunohistochemistry and confocal microscopy were used here to determine that D-AKAP1 colocalizes with RIalpha at the postsynaptic membrane of the vertebrate neuromuscular junction (NMJ) and the adjacent muscle, but not in the presynaptic region. The labeling pattern for RIalpha and D-AKAP1 overlapped with mitochondrial staining in the muscle fibers, consistent with our previous work showing D-AKAP1 association with mitochondria in cultured cells. The immunoreactivity of D-AKAP2 was distinct from that of D-AKAP1. We also report here that even though the PKA type II subunits (RIIalpha and RIIbeta) are localized at the NMJ, their patterns are distinctive and differ from the other R and D-AKAP patterns examined. PKCbeta appeared to colocalize with the AKAP, gravin, at the postsynaptic membrane. CONCLUSIONS: The kinases and AKAPs investigated have distinct patterns of colocalization, which suggest a complex arrangement of signaling micro-environments. Because the labeling patterns for RIalpha and D-AKAP 1 are similar in the muscle fibers and at the postsynaptic membrane, it may be that this AKAP anchors RIalpha in these regions. Likewise, gravin may be an anchor of PKCbeta at the NMJ.

Development of three-dimensional power Doppler ultrasound imaging of fetoplacental vasculature.

To develop an off-line system for three-dimensional (3-D) ultrasound (US) reconstruction of fetoplacental vasculature using colour segmentation and reconstruction software and to determine sources of error in fully freehand ultrasound image acquisition. US images were acquired freehand with the Acuson Sequoia (5C 2-MHz transducer) using power Doppler. After digital transfer to a personal computer, CQ Analysis software (Kinetic Imaging Ltd, Liverpool, UK) was used to segment the colour information from these images, and the resulting 8-bit grey-scale images were used for 3-D rendering using commercial software (VoxBlast, Vaytek Inc., Fairfield, IA, USA). 2-D scanning, software and freehand acquisition accuracy were assessed using a linear test rig and distance and volume phantoms (Dansk Phantom Service Ltd); 2-D scanning accuracy was within 1.3%, and software reconstruction accuracy within 1% for x and y planes and up to 3% for the z plane. Fully freehand acquisition was associated with a 12% to 18% mean percentage error in distance measurement in the plane of acquisition. Volumetric reconstruction inaccuracy was between 1.5% and 19.7% for precisely separated images and between 16.2% and 39.2% for fully freehand image acquisition. Rendered 3-D US vascular images clearly delineated vascular anatomy within the placenta and cord. Fully freehand 3-D US does have a role in off-line reconstruction of vascular anatomy, although variability in the z plane precludes its use for volumetric measurement. (E-mail: a.welsh@ic.ac.uk)

Parity and carotid artery atherosclerosis in elderly women: The Rotterdam Study.

BACKGROUND AND PURPOSE: It has been postulated that physiological changes in the cardiovascular system, lipids, and glucose metabolism during pregnancy may increase subsequent risk of cardiovascular disease. Examination of the association between parity and risk factors for atherosclerosis may contribute information regarding possible mechanisms. METHODS: The relationship of parity with cardiovascular risk factors and the presence of carotid atherosclerosis was examined in the Rotterdam Study, a population-based study comprising 4878 women aged 55 years and older. Carotid atherosclerosis was assessed by ultrasonographic detection of plaques in the common carotid artery and bifurcation. Logistic regression models were used to compute odds ratios and 95% confidence intervals, adjusted for confounding factors. RESULTS: Parity was inversely associated with high-density lipoprotein cholesterol, and alcohol intake. Parity was positively associated with body mass index, total/HDL cholesterol ratio, insulin resistance, age at menopause, and socioeconomic status. Relative to nulliparous women, parous women had 36% (9% to 71%) greater risk of carotid atherosclerosis, rising to 64% in women with >/=4 children (19% to 127%). Adjustment for known cardiovascular risk factors, including insulin resistance and current lipid levels, did not diminish the magnitude of this association. CONCLUSIONS: Data demonstrated that there is a positive association between parity and risk of carotid artery plaques in elderly women and, further, that high parity is associated with lower HDL cholesterol levels and higher glucose/insulin ratios long after childbearing has ceased.

Optimisation and evaluation of an electromagnetic tracking device for high-accuracy three-dimensional ultrasound imaging of the carotid arteries.

Electromagnetic tracking devices provide a flexible, low cost solution for three-dimensional ultrasound (3-D US) imaging. They are, however, susceptible to interference. A commercial device (Ascension pcBIRD) was evaluated to assess the accuracy in locating the scan probe as part of a digital, freehand 3-D US imaging system aimed at vascular applications. The device was optimised by selecting a measurement rate and filter setting that minimised the mean deviation in repeated position and orientation measurements. Experimental evaluation of accuracy indicated that, overall, absolute errors were small: the RMS absolute error was 0.2 mm (range: -0.7 to 0.5 mm) for positional measurements over translations up to 90 mm, and 0.2 degrees (range: -0.8 to 0.9 degrees ) for rotational measurements up to 30 degrees. In the case of position measurements, the absolute errors were influenced by the location of the scanner relative to the scan volume. We conclude that the device tested provides an accuracy sufficient for use within a freehand 3-D US system for carotid artery imaging.

Oxidative modification and inactivation of the proteasome during coronary occlusion/reperfusion.

Restoration of blood flow to ischemic myocardial tissue results in an increase in the production of oxygen radicals. Highly reactive, free radical species have the potential to damage cellular components. Clearly, maintenance of cellular viability is dependent, in part, on the removal of altered protein. The proteasome is a major intracellular proteolytic system which degrades oxidized and ubiquitinated forms of protein. Utilizing an in vivo rat model, we demonstrate that coronary occlusion/reperfusion resulted in declines in chymotrypsin-like, peptidylglutamyl-peptide hydrolase, and trypsin-like activities of the proteasome as assayed in cytosolic extracts. Analysis of purified 20 S proteasome revealed that declines in peptidase activities were accompanied by oxidative modification of the protein. We provide conclusive evidence that, upon coronary occlusion/reperfusion, the lipid peroxidation product 4-hydroxy-2-nonenal selectively modifies 20 S proteasome alpha-like subunits iota, C3, and an isoform of XAPC7. Occlusion/reperfusion-induced declines in trypsin-like activity were largely preserved upon proteasome purification. In contrast, loss in chymotrypsin-like and peptidylglutamyl-peptide hydrolase activities observed in cytosolic extracts were not evident upon purification. Thus, decreases in proteasome activity are likely due to both direct oxidative modification of the enzyme and inhibition of fluorogenic peptide hydrolysis by endogenous cytosolic inhibitory protein(s) and/or substrate(s). Along with inhibition of the proteasome, increases in cytosolic levels of oxidized and ubiquitinated protein(s) were observed. Taken together, our findings provide insight into potential mechanisms of coronary occlusion/reperfusion-induced proteasome inactivation and cellular consequences of these events.