Long-Term Follow-Up of Right Ventricle to Pulmonary Artery Biologic Valved Conduits Used in Pediatric Congenital Heart Surgery.

Valved conduit reconstruction between the right ventricle (RV) and the pulmonary circulation is often necessary in the surgical treatment of complex congenital heart defects. The aim of this study is to evaluate the long-term performance of the three types of conduits we have used and assess risk factors for conduit failure. Retrospective, single-center review of 455 consecutive pediatric patients with 625 conduits from 1990 to 2019 undergoing RV-to-pulmonary artery (PA) reconstruction with a valved conduit. The three conduit types investigated were pulmonary homograft, aorta homograft, and bovine jugular vein (BJV) graft. Overall patient survival was 91.4%, freedom from conduit replacement (FCR) was 47.4%, and freedom from reintervention (FFR) was 37.8% with a median follow-up of 8.7 years (interquartile range 4.3-13.3 years). For pulmonary homografts, 10-, 20-, and 28-year FCR was 79.6%, 68.6%, and 66.0%, respectively. For aortic homografts, 10-, 20-, and 30-year FCR was 49.8%, 31.5%, and 23.0%, respectively. For BJV grafts, 10- and 19-year FCR was 68.1% and 46.0%, respectively. When controlling for baseline variables, FCR was similar for pulmonary homografts and BJV grafts. Overall patient survival was excellent. Risk factors for conduit failure in patients operated with reconstruction of the RV-PA outflow tract included low age, low weight, small conduit size, and certain cardiac diagnoses. There was no evidence for a shorter life span of the second graft. Pulmonary homografts and BJV grafts performed similarly but the risk of endocarditis was greater in the BJV group.

ID-MAM: A Validated Identity and Multi-Attribute Monitoring Method for Commercial Release and Stability Testing of a Bispecific Antibody.

Post-translational modifications (PTMs) that impact the safety or efficacy of protein therapeutics are critical quality attributes (CQAs) that need to be controlled to ensure product quality. Peptide mapping with online mass spectrometry (MS) is a powerful tool that has been used for many years to monitor PTM CQAs during product development. However, operating peptide mapping methods with high-resolution mass spectrometers in GMP compliant, commercial quality control (QC) labs can be difficult. Peptide mapping is also required as an identity test in several countries. To address these two different needs, we utilized high-resolution peptide mapping for comprehensive characterization during development and then developed and validated a targeted multi-attribute monitoring (MAM) method using the low-resolution Waters QDa MS system with a fully automated data processing workflow that is suitable for identity (ID) testing, sequence variant control, and CQA quantitation in commercial QC labs. The ID-MAM method was validated for the quantitation of three selected PTM CQAs (CDR isomerization, Fc Met oxidation, and CDR Met oxidation) to ensure control of the oxidation and isomerization degradation pathways of a bispecific antibody (BsAb). This ID-MAM method was successfully validated in six labs (three analytical development and three QC labs) across four countries for commercial release and stability testing of a BsAb. CQA results obtained with the ID-MAM method were similar to results obtained using high-resolution peptide mapping, and the method was robust and reproducible. To our knowledge, this ID-MAM method is the first MS-based peptide mapping method implemented in GMP compliant QC labs for commercial release and stability testing of a biotherapeutic.

Association of physical activity metrics with indicators of cardiovascular function and control in children with and without type 1 diabetes.

OBJECTIVE: Little is known about the role of physical activity accumulation in cardiovascular disease risk for children with type 1 diabetes. Improved insight to identify factors of influence in key health outcomes could be provided by considering the entire physical activity profile. METHODS: Pulse wave velocity (PWV), augmentation index and heart rate variability (HRV) were assessed cross-sectionally in children with (n = 29, 12.1 ± 2.1 years) and without (n = 19, 12.1 ± 2.1 years) type 1 diabetes. Time spent sedentary and in each physical activity intensity, intensity gradient and average acceleration were derived from seven consecutive days of monitoring with wrist-worn accelerometry. Comparison between groups and influence of physical activity accumulation on cardiovascular metrics were explored with linear mixed models. RESULTS: Diabetic children demonstrated a higher PWV and a greater volume of light physical activity (p < 0.01), a more negative intensity gradient (p < 0.01), a lower average acceleration and less time in bouted moderate-to-vigorous physical activity (MVPA; p < 0.05). Overall, intensity gradient was strongly correlated with average acceleration, MVPA and bouted MVPA (r2 = 0.89, r2 = 0.80, r2 = 0.79, respectively; all p < 0.05), while average acceleration was correlated with MVPA and bouted MVPA (r2 = 0.85, r2 = 0.83, respectively; p < 0.05). Accounting for disease status, intensity gradient and average acceleration were significant predictors of HRV indices (p < 0.05) and PWV (p < 0.01, p < 0.05, respectively). CONCLUSION: Overall, MVPA was most associated with central stiffness, highlighting the importance of meeting activity guidelines. Diabetic children demonstrated poorer cardiovascular health than their counterparts, likely attributable to a lower intensity and physical activity volume, identifying physical activity intensity as a key target for future interventions.

Implementation of a High-Resolution Liquid Chromatography-Mass Spectrometry Method in Quality Control Laboratories for Release and Stability Testing of a Commercial Antibody Product.

Liquid chromatography-mass spectrometry (LC-MS) has been widely used throughout biotherapeutic development. However, its implementation in GMP-compliant commercial quality control (QC) laboratories remains a challenge. In this publication, we describe the covalidation and implementation of an automated, high-throughput, and GMP compliant subunit LC-MS method for monitoring antibody oxidation for commercial product release and stability testing. To our knowledge, this is the first report describing the implementation of a high-resolution LC-MS method in commercial QC laboratories for product release and stability testing in the biopharmaceutical industry. This work paves the road for implementing additional LC-MS methods to modernize testing in commercial QC with more targeted control of product quality.

Alcoholism and nutrition: a review of vitamin supplementation and treatment.

PURPOSE OF REVIEW: This is a review of the research on the effectiveness of vitamin supplementation for alcoholism and alcohol-related illnesses. The focus is on research, both clinical and basic on alcohol treatment and nutritional effectiveness of these vital nutrients. RECENT FINDINGS: Most of the research involves basic experiments exploring the impact of vitamin depletion or deficits on physiological systems, especially liver and brain, in rodents. These often include behavioral measures that use cognitive, learning/memory and motivation experiments that model clinical studies. These provide support for hypotheses concerning the impact of such deficiencies in clinical populations. Clinical studies are rare and involve evaluation of the outcome of supplementation usually in the context of a treatment program. Specific vitamins, dosages and treatment programs vary. Deficiencies in retinoids (vitamin A), thiamine (B1) and niacin (B3) are the most frequently investigated. However, there is a greater need for further research on other vitamins, and for more uniform supplementation and treatment procedures. SUMMARY: The literature is primarily basic research on specific vitamins. There are very significant findings with individual vitamin supplementation and combinations that show promise of our understanding of the role of vitamins in the disease of alcoholism and its treatment.

Long-term results of aortic arch reconstruction with branch pulmonary artery homograft patches.

BACKGROUND: Homograft tissue is an important reconstructive material used in the surgical correction of a variety of congenital heart defects. The aim of this study is to evaluate the long-term outcome of pulmonary artery (PA) branch patches used in the reconstruction of the thoracic aorta in children. METHODS: Retrospective review of 124 consecutive pediatric patients undergoing corrective surgery for their congenital heart defects between 2001 and 2016. Survival, reoperation, and reintervention data were collected, as well as imaging data to assess for presence of recoarctation, dilation, or aneurysm formation in the area of patch reconstruction. RESULTS: Overall 15-year survival was 83.9% and 15-year freedom from reintervention in the area of patch reconstruction was 89.2%. Rates of mortality (0%), cardiac transplantation (0%), and reoperation (0.8%) attributable to the area of patch reconstruction were low. The frequency of catheter-based intervention in the area of patch reconstruction was 9.7%; such interventions were successful in all but one patient, who ultimately underwent successful surgical aortoplasty. CONCLUSIONS: Homograft patches harvested from PA branches are an effective reconstructive material used for reconstruction of the aorta in small children. Long-term results show no risk of aneurysm formation and low rates of stenosis formation.

High-throughput LC-MS quantitation of cell culture metabolites.

Monitoring cell culture metabolites, including media components and cellular byproducts, during bio manufacturing is critical for gaining insights into cell growth, productivity and product quality. Historically, cell culture metabolite analysis was a complicated process requiring several orthogonal methods to cover the large number of metabolites with diverse properties over wide concentration ranges. These off-line analyses are time consuming and not suitable for real time bioreactor monitoring. In this study, we present a high-throughput LC-MS method with a 17-min cycle time that is capable of simultaneously monitoring 93 cell culture metabolites, including amino acids, nucleic acids, vitamins, sugars and others. This method has high precision and accuracy and has been successfully applied to the daily profiling of bioreactors and raw material qualification. Information obtained in these studies has been used to identify limiting amino acids during production, which guided adjustments to the feed strategy that prevented the potential misincorporation of amino acids. This type of metabolite profiling can be further utilized to build predictive process models for adaptive feedback control and pave the road for continuous manufacturing and real-time release testing.

Use of In Vitro Systems To Model In Vivo Degradation of Therapeutic Monoclonal Antibodies.

Major degradation pathways such as deamidation, isomerization, oxidation, and glycation may be accelerated after administration of antibody therapeutics to the patient. Tracking in vivo product degradation is important because certain post-translational modifications can inactivate the protein and reduce product efficacy. However, in vivo characterization of protein therapeutics is not routinely performed because of technical challenges and limited sample availability. In vitro models offer several potential advantages, including larger sample supplies, simpler and faster methods for sample preparation and analysis, and the potential to distinguish differences in product degradation from differences in product clearance. In this study, we compared the rates of in vivo product degradation using mAb1 recovered from clinical serum samples with the rates of in vitro product degradation using mAb1 recovered from spiked phosphate buffered saline (PBS) and spiked human serum samples to determine if results from the in vitro model systems could be used to predict the in vivo results. The antibody samples were characterized by peptide mapping or intact mass analysis to quantify multiple quality attributes simultaneously, including deamidation, isomerization, oxidation, N-terminal pyroglutamate formation, and glycation. It was clearly demonstrated that both the spiked PBS and spiked serum models were effective in predicting in vivo results for deamidation, isomerization, N-terminal pyroglutamate formation and glycation, whereas only the spiked serum model was effective in predicting in vivo results for oxidation.

Pulmonary oxygen uptake and muscle deoxygenation kinetics during heavy intensity cycling exercise in patients with emphysema and idiopathic pulmonary fibrosis.

BACKGROUND: Little is known about the mechanistic basis for the exercise intolerance characteristic of patients with respiratory disease; a lack of clearly defined, distinct patient groups limits interpretation of many studies. The purpose of this pilot study was to investigate the pulmonary oxygen uptake ([Formula: see text] O2) response, and its potential determinants, in patients with emphysema and idiopathic pulmonary fibrosis (IPF). METHODS: Following a ramp incremental test for the determination of peak [Formula: see text] O2 and the gas exchange threshold, six emphysema (66 ± 7 years; FEV1, 36 ± 16%), five IPF (65 ± 12 years; FEV1, 82 ± 11%) and ten healthy control participants (63 ± 6 years) completed three repeat, heavy-intensity exercise transitions on a cycle ergometer. Throughout each transition, pulmonary gas exchange, heart rate and muscle deoxygenation ([HHb], patients only) were assessed continuously and subsequently modelled using a mono-exponential with ([Formula: see text] O2, [HHb]) or without (HR) a time delay. RESULTS: The [Formula: see text] O2 phase II time-constant (τ) did not differ between IPF and emphysema, with both groups significantly slower than healthy controls (Emphysema, 65 ± 11; IPF, 69 ± 7; Control, 31 ± 7 s; P < 0.05). The HR τ was slower in emphysema relative to IPF, with both groups significantly slower than controls (Emphysema, 87 ± 19; IPF, 119 ± 20; Control, 58 ± 11 s; P < 0.05). In contrast, neither the [HHb] τ nor [HHb]:O2 ratio differed between patient groups. CONCLUSIONS: The slower [Formula: see text] O2 kinetics in emphysema and IPF may reflect poorer matching of O2 delivery-to-utilisation. Our findings extend our understanding of the exercise dysfunction in patients with respiratory disease and may help to inform the development of appropriately targeted rehabilitation strategies.

Peptide and small molecule inhibitors of HECT-type ubiquitin ligases.

The human genome encodes several hundred E3 ubiquitin ligases containing RING domains, and around 28 containing HECT domains. These enzymes catalyze the transfer of ubiquitin from E2 enzyme thioesters to a huge range of substrates and play crucial roles in many cellular functions. This makes them attractive potential therapeutic targets. However, they have proven difficult to inhibit: very few good inhibitors exist for RING domain ligases, and none have been described for HECT ligases. Here we show that bicyclic peptides isolated by phage display [Heinis C, Rutherford T, Freund S, Winter G (2009) Nat Chem Biol. 5(7):502-507] can target the E2 binding sites on the HECT domains of Smurf2, Nedd4, Mule/Huwe1, and WWP1, and thus act as specific inhibitors of these enzymes in vitro. By screening for displacement of one of these peptides from Smurf2, we were able to identify a small molecule, heclin (HECT ligase inhibitor), which inhibits several HECT ligases in tissue culture cells. In vitro, heclin does not block E2 binding but causes a conformational change that results in oxidation of the active site Cys. This demonstrates that HECT domains are potentially druggable and provides molecules that may be of experimental use. Heclin kills HEK293 cells growing in culture, consistent with an essential role for HECT ligase activity in mammalian cells.

Understanding the Impact of Methionine Oxidation on the Biological Functions of IgG1 Antibodies Using Hydrogen/Deuterium Exchange Mass Spectrometry.

Hydrogen/deuterium exchange mass spectrometry (HDX MS) was used in two case studies to evaluate the impact of methionine (Met) oxidation on the biological functions of IgG1 antibodies. In the first case study, linear correlations were observed between the oxidation of the conserved Fc methionine residues and the loss of neonatal Fc receptor (FcRn) binding and complement-dependent cytotoxicity (CDC) activity. Both heavy chain (HC) residues Met257 and Met433 were located near the FcRn binding interface as indicated by HDX MS and structural modeling; however, HC Met257 oxidation was further demonstrated to have a more significant impact on FcRn binding than HC Met433 oxidation. In addition, oxidation of HC Met257 and HC Met433 could disrupt protein conformation at the CH2-CH3 interface and prevent IgG oligomerization, which is needed for C1q binding and subsequent CDC activity. In the second case study, HDX MS demonstrated that oxidation of the two complementary determining region (CDR) methionine residues had little or no impact on antigen binding of the antibody. Together, these results suggested that HDX MS is a powerful tool for evaluating the impact of individual post translational modifications (PTMs) on the biological activities of antibodies, even when the PTM levels are relatively low. The high selectivity and sensitivity of this method makes it a valuable tool for assisting the critical quality attributes (CQAs) assessment of antibodies.

High-Throughput, Automated Protein A Purification Platform with Multiattribute LC-MS Analysis for Advanced Cell Culture Process Monitoring.

The levels of many product related variants observed during the production of monoclonal antibodies are dependent on control of the manufacturing process, especially the cell culture process. However, it is difficult to characterize samples pulled from the bioreactor due to the low levels of product during the early stages of the process and the high levels of interfering reagents. Furthermore, analytical results are often not available for several days, which slows the process development cycle and prevents "real time" adjustments to the manufacturing process. To reduce the delay and enhance our ability to achieve quality targets, we have developed a low-volume, high-throughput, and high-content analytical platform for at-line product quality analysis. This workflow includes an automated, 96-well plate protein A purification step to isolate antibody product from the cell culture fermentation broth, followed by rapid, multiattribute LC-MS analysis. We have demonstrated quantitative correlations between particular process parameters with the levels of glycosylated and glycated species in a series of small scale experiments, but the platform could be used to monitor other attributes and applied across the biopharmaceutical industry.

Influence of antenatal physical exercise on haemodynamics in pregnant women: a flexible randomisation approach.

BACKGROUND: Normal pregnancy is associated with marked changes in haemodynamic function, however the influence and potential benefits of antenatal physical exercise at different stages of pregnancy and postpartum remain unclear. The aim of this study was therefore to characterise the influence of regular physical exercise on haemodynamic variables at different stages of pregnancy and also in the postpartum period. METHODS: Fifty healthy pregnant women were recruited and randomly assigned (2 × 2 × 2 design) to a land or water-based exercise group or a control group. Exercising groups attended weekly classes from the 20th week of pregnancy onwards. Haemodynamic assessments (heart rate, cardiac output, stroke volume, total peripheral resistance, systolic and diastolic blood pressure and end diastolic index) were performed using the Task Force haemodynamic monitor at 12-16, 26-28, 34-36 and 12 weeks following birth, during a protocol including postural manoeurvres (supine and standing) and light exercise. RESULTS: In response to an acute bout of exercise in the postpartum period, stroke volume and end diastolic index were greater in the exercise group than the non-exercising control group (p = 0.041 and p = 0.028 respectively). Total peripheral resistance and diastolic blood pressure were also lower (p = 0.015 and p = 0.007, respectively) in the exercise group. Diastolic blood pressure was lower in the exercise group during the second trimester (p = 0.030). CONCLUSIONS: Antenatal exercise does not appear to substantially alter maternal physiology with advancing gestation, speculating that the already vast changes in maternal physiology mask the influences of antenatal exercise, however it does appear to result in an improvement in a woman's haemodynamic function (enhanced ventricular ejection performance and reduced blood pressure) following the end of pregnancy. TRIAL REGISTRATION: ClinicalTrials.gov NCT02503995. Registered 20 July 2015.

Diagnostic ultrasound features and outcome of restrictive foramen ovale in fetuses with structurally normal hearts.

Intrauterine foramen ovale (FO) restriction in association with congenital heart disease (CHD) carries a poor prognosis. However, in the absence of CHD, the clinical importance of restrictive FO in the fetus is not well understood. We evaluated the antenatal prevalence, clinical presentation, diagnostic ultrasound features, and outcome of restrictive FO in fetuses without CHD. We reviewed the echocardiographic and clinical records of 23 fetuses diagnosed with a restrictive FO and structurally normal heart between 2001 and 2012. The atrial septum, dimensions of cardiac structures, left and right cardiac output and Doppler interrogation of cardiac flows were examined. The clinical outcomes of all fetuses with restrictive FO were analysed. Restrictive FO was identified in 23 of 1,682 (1.4%) fetuses with no CHD. Enlarged right heart structures (100%), hypermobile or redundant primum atrial septum (91%), increased right-to-left ventricular cardiac output ratio (91%), and posteriorly angulated ductus arteriosus (68%) were the most common echocardiographic findings associated with this rare phenomenon. Additional noncardiac systemic abnormalities were identified in 13 (56%) babies. Seven (30%) neonates developed persistent pulmonary hypertension, and 7 infants died. Antenatal restrictive FO is an underrecognised entity despite being a common cause of right heart dilatation in the fetus. In the absence of CHD, restrictive FO is well tolerated antenatally, but its frequent association with noncardiac abnormalities and pulmonary hypertension in the neonate are noteworthy.

Targeted CQA analytical control strategy for commercial antibody products: Replacing ion-exchange chromatography methods for charge heterogeneity with multi-attribute monitoring.

Peptide mapping with mass spectrometry (MS) is an important tool for protein characterization in the biopharmaceutical industry. Historically, peptide mapping monitors post-translational modifications (PTMs) of protein products and process intermediates during development. Multi-attribute monitoring (MAM) methods have been used previously in commercial release and stability testing panels to ensure control of selected critical quality attributes (CQAs). Our goal is to use MAM methods as part of an overall analytical testing strategy specifically focused on CQAs, while removing or replacing historical separation methods that do not effectively distinguish CQAs from non-CQAs due to co-elution. For example, in this study, we developed a strategy to replace a profile-based ion-exchange chromatography (IEC) method using a MAM method in combination with traditional purity methods to ensure control of charge variant CQAs for a commercial antibody (mAb) drug product (DP). To support this change in commercial testing strategy, the charge variant CQAs were identified and characterized during development by high-resolution LC-MS and LC-MS/MS. The charge variant CQAs included PTMs, high molecular weight species, and low molecular weight species. Thus, removal of the IEC method from the DP specification was achieved using a validated LC-MS MAM method on a QDa system to directly measure the charge variant PTM CQAs in combination with size exclusion chromatography (SE-HPLC) and capillary electrophoresis (CE-SDS) to measure the non-PTM charge variant CQAs. Bridging data between the MAM, IEC, and SE-HPLC methods were included in the commercial marketing application to justify removing IEC from the DP specification. We have also used this MAM method as a test for identity to reduce the number of QC assays. This strategy has received approvals from several health authorities.

Cardiac Metastasis Presenting as ST-Elevation Myocardial Infarction.

A 52-year-old woman with a history of lung cancer presented with progressive shortness of breath. Her ECG showed evidence of ST-elevation myocardial infarction (STEMI) though no evidence of obstructive coronary artery disease (CAD) was seen on coronary angiography. Further imaging with CT and cardiac MRI (CMRI) demonstrated tumor, likely metastatic cancer, within myocardial tissue. This case is demonstrative of the possible relationship between ST-segment elevation on ECG and corresponding tumor invasion and highlights the differential diagnoses of STEMI, including cardiac metastasis.

Evaluation and comparison of patch materials used for pulmonary arterioplasty in pediatric congenital heart surgery.

Objective: To evaluate the long-term performance of the patch materials we have used to augment the pulmonary arterial tree across a wide spectrum of diagnoses and anatomical locations. Methods: Retrospective, single-center review of 217 consecutive pediatric patients at a tertiary referral center from 1993 to 2020 who underwent patch arterioplasty of the pulmonary arterial tree from the pulmonary bifurcation to the distal pulmonary arterial branches. Reintervention data were collected and analyzed. Lesion-specific anatomy and other variables were analyzed as risk factors for reintervention. Results: There were 280 total operations performed (217 initial operations and 63 reoperations) and 313 patches used. The patches used were autologous pericardium (166, 53.0%), pulmonary homograft (126, 40.3%), and a heterogeneous group of other materials (21, 6.7%). Overall patient survival was 86.2%, freedom from reoperation was 81.0% and freedom from reintervention (FFR) was 70.6%, with a median follow-up of 13.8 years (interquartile range, 6.3-17.9 years). For all patches, 10-, 20-, and 27-year FFR was 76.6%, 70.6%, and 70.6%, respectively. FFR was similar among all 3 patch type groups (P = .29). Multivariable Cox regression analysis showed that diagnoses of pulmonary atresia with ventricular septal defect and major aortopulmonary collateral arteries and hypoplastic left heart syndrome, patches placed at initial cardiac operation, and increasing number of cardiac operations were risk factors for reintervention. Conclusions: Autologous pericardium and pulmonary homograft patches performed similarly. Although patch type conferred no difference in need for reintervention, other risk factors did exist. Namely, diagnoses of pulmonary atresia with ventricular septal defect and major aortopulmonary collateral arteries and hypoplastic left heart syndrome, patch placement at a patient's first cardiac operation, and increasing number of cardiac operations were risk factors for reintervention.

Influence of exercise intensity on pulmonary oxygen uptake kinetics in young and late middle-aged adults.

It is unclear whether pulmonary oxygen uptake (Vo2) kinetics demonstrate linear, first-order behavior during supra gas exchange threshold exercise. Resolution of this issue is pertinent to the elucidation of the factors regulating oxygen uptake (Vo2) kinetics, with oxygen availability and utilization proposed as putative mediators. To reexamine this issue with the advantage of a relatively large sample size, 50 young (24 ± 4 yr) and 15 late middle-aged (54 ± 3 yr) participants completed repeated bouts of moderate and heavy exercise. Pulmonary gas exchange, heart rate (HR), and cardiac output (Q) variables were measured throughout. The phase II τ was slower during heavy exercise in both young (moderate: 22 ± 9; heavy: 29 ± 9 s; P ≤ 0.001) and middle-aged (moderate: 22 ± 9; heavy: 30 ± 8 s; P ≤ 0.001) individuals. The HR τ was slower during heavy exercise in young (moderate: 33 ± 10; heavy: 44 ± 15 s; P ≤ 0.05) and middle-aged (moderate: 30 ± 12; heavy: 50 ± 20 s; P ≤ 0.05) participants, and the Q τ showed a similar trend (young moderate: 21 ± 13; heavy: 28 ± 16 s; middle-aged moderate: 32 ± 13; heavy: 40 ± 15 s; P ≥ 0.05). There were no differences in primary component Vo2 kinetics between age groups, but the middle-aged group had a significantly reduced Vo2 slow component amplitude in both absolute (young: 0.25 ± 0.09; middle-aged: 0.11 ± 0.06 l/min; P ≤ 0.05) and relative terms (young: 15 ± 10; middle-aged: 9 ± 4%; P ≤ 0.05). Thus Vo2 kinetics do not demonstrate dynamic linearity during heavy intensity exercise. Speculatively, the slower phase II τ during heavy exercise might be attributable to reduced oxygen availability. Finally, the primary and slow components of Vo2 kinetics appear to be differentially influenced by middle age.

Mineral partitioning in milk and milk permeates at high temperature.

The soluble phase of milk was separated at 20 and 80 °C using ultrafiltration. The resulting permeates were then subjected to further ultrafiltration and dialysis at close to these two temperatures. It was found that pH, Ca2+ and soluble Ca decreased as the separation temperature increased both in original UF permeates and in dialysates obtained from these permeates, but P decreased only slightly. The major reason for these changes was due to the precipitation of calcium phosphate/citrate complexes onto the casein micelle with concomitant release of H+. The pH of both permeates and dialysates from milk at 20 °C were slightly higher than for milk. When UF permeates collected at 20 and 80 °C, were each dialysed at both these temperatures, the dialysate collected at 80 °C showed much less temperature dependence for pH and ionic calcium compared with that collected at 20 °C. This is in contrast to milk, which shows considerable temperature dependence for pH and ionic calcium. Further experiments revealed that the pH and Ca2+ concentration of permeates showed high temperature dependence above the temperature at which they were separated, but a much lower temperature dependence below that temperature. These findings suggest that dialysis and UF of milk at high temperature provide the best means yet for estimating the pH and ionic calcium of milk at that temperature.

Altered hepatic lipid metabolism in C57BL/6 mice fed alcohol: a targeted lipidomic and gene expression study.

Chronic alcohol consumption is associated with fatty liver disease in mammals. The object of this study was to gain an understanding of dysregulated lipid metabolism in alcohol-fed C57BL/6 mice using a targeted lipidomic approach. Liquid chromatography tandem mass spectrometry was used to analyze several lipid classes, including free fatty acids, fatty acyl-CoAs, fatty acid ethyl esters, sphingolipids, ceramides, and endocannabinoids, in plasma and liver samples from control and alcohol-fed mice. The interpretation of lipidomic data was augmented by gene expression analyses for important metabolic enzymes in the lipid pathways studied. Alcohol feeding was associated with i) increased hepatic free fatty acid levels and decreased fatty acyl-CoA levels associated with decreased mitochondrial fatty acid oxidation and decreased fatty acyl-CoA synthesis, respectively; ii) increased hepatic ceramide levels associated with higher levels of the precursor molecules sphingosine and sphinganine; and iii) increased hepatic levels of the endocannabinoid anandamide associated with decreased expression of its catabolic enzyme fatty acid amide hydrolase. The unique combination of lipidomic and gene expression analyses allows for a better mechanistic understanding of dysregulated lipid metabolism in the development of alcoholic fatty liver disease.