Parametric conditional frailty models for recurrent cardiovascular events in the lipid study

Background Analysis of recurrent event data is frequently needed in clinical and epidemiological studies. An important issue in such analysis is how to account for the dependence of the events in an individual and any unobserved heterogeneity of the event propensity across individuals.Methods We applied a number of conditional frailty and nonfrailty models in an analysis involving recurrent myocardial infarction events in the Long-Term Intervention with Pravastatin in Ischaemic Disease study. A multiple variable risk prediction model was developed for both males and females. Results A Weibull model with a gamma frailty term fitted the data better than other frailty models for each gender. Among nonfrailty models the stratified survival model fitted the data best for each gender. The relative risk estimated by the elapsed time model was close to that estimated by the gap time model. We found that a cholesterol-lowering drug, pravastatin (the intervention being tested in the trial) had significant protective effect against the occurrence of myocardial infarction in men (HR¼0.71, 95% CI0.60–0.83). However, the treatment effect was not significant in women due to smaller sample size (HR¼0.75, 95% CI 0.51–1.10). There were no significant interactions between the treatment effect and each recurrent MI event (p¼0.24 for men and p¼0.55 for women). The risk of developing an MI event for a male who had an MI event during follow-up was about 3.4 (95% CI 2.6–4.4) times the risk compared with those who did not have an MI event. The corresponding relative risk for a female was about 7.8 (95% CI 4.4–13.6). Limitations The number of female patients was relatively small compared with their male counterparts, which may result in low statistical power to find real differences in the effect of treatment and other potential risk factors.Conclusions The conditional frailty model suggested that after accounting for all the risk factors in the model, there was still unmeasured heterogeneity of the risk for myocardial infarction, indicating the effect of subject-specific risk factors. These risk prediction models can be used to classify cardiovascular disease patients into different risk categories and may be useful for the most effective targeting of preventive therapies for cardiovascular disease.

A protocol for the combined sub-fractionation and delipidation of lipid-binding proteins by hydrophobic interaction chromatography

Cellular lipids frequently co-purify with lipid binding proteins isolated from tissue extracts or heterologous host systems and as such hinder in vitro ligand binding approaches for which the apo-protein is a prerequisite. Here we present a technique for the complete removal of unesterified fatty acids, phospholipids, steroids and other lipophilic ligands bound to soluble proteins, without protein denaturation. Peroxisome proliferator activated receptor gamma ligand binding domain and intracellular fatty acid binding proteins were expressed in an Escherichia coli host and completely delipidated by hydrophobic interaction chromatography using phenyl sepharose. The delipidation procedure operates at room temperature with complete removal of bound lipids in a single step, as ascertained by mass spectrometry analysis of organic solvent extracts from purified protein samples. The speed and capacity of this method makes it amenable to scale-up and high-throughput applications. The method can also easily be adapted for other lipid binding proteins that require delipidation under native conditions.

Long-term effect of calcium-vitamin D3 fortified milk on blood pressure and serum lipid concenstrations in healthy older men

Background/Objectives:
Some epidemiological and clinical studies have shown that increased dairy consumption or calcium and/or vitamin D supplementation can have a beneficial effect on blood pressure, and lipid and lipoprotein concentrations. The aim of this study was to assess the long-term effects of calcium-vitamin D3 fortified milk on blood pressure and lipid-lipoprotein concentrations in community-dwelling older men.

Subjects/Methods:
This is a substudy of a 2-year randomized controlled trial in which 167 men aged >50 years were assigned to receive either 400 ml per day of reduced fat (approx1%) milk fortified with approximately 1000 mg of calcium and 800 IU of vitamin D3 or to a control group receiving no additional fortified milk. Weight, blood pressure, lipid and lipoprotein concentrations were measured every 6 months. Participants on lipid-lowering (n=32) or antihypertensive medication (n=39) were included, but those who commenced, increased or decreased their medication throughout the intervention were excluded (n=27).

Results:
In the 140 men included in this study (milk, n=73; control, n=67), there were no significant effects of the calcium-vitamin D3 fortified milk on weight, systolic or diastolic blood pressure, total cholesterol, high-density lipoprotein or low-density lipoprotein cholesterol or triglyceride concentrations at any time throughout the intervention. Similar results were observed after excluding men taking antihypertensive or lipid-lowering medication or limiting the analysis to those with baseline calcium intakes <1000 mg per day and/or with hypovitaminosis D (25(OH)D <75 nmol/l).

Conclusions:
Supplementation with reduced-fat calcium-vitamin D3 fortified milk did not have a beneficial (nor detrimental) effect on blood pressure, lipid or lipoprotein concentrations in healthy community-dwelling older men.

Laboratory and non-laboratory-based risk prevention models for secondary prevention of cardiovascular disease : the LIPID study

Aims : The aims of this study were to examine whether risk prediction models for recurrent cardiovascular disease (CVD) events have prognostic value, and to particularly examine the performance of those models based on non-laboratory data. We also aimed to construct a risk chart based on the risk factors that showed the strongest relationship with CVD.

Methods and results : Cox proportional hazards models were used to calculate a risk score for each recurrent event in a CVD patient who was enrolled in a very large randomized controlled clinical trial. Patients were then classified into groups according to quintiles of their risk score. These risk models were validated by calibration and discrimination analyses based on data from patients recruited in New Zealand for the same study. Non-laboratory-based risk factors, such as age, sex, body mass index, smoking status, angina grade, history of myocardial infarction, revascularization, stroke, diabetes or hypertension and treatment with pravastatin, were found to be significantly associated with the risk of developing a recurrent CVD event. Patients who were classified into the medium and high-risk groups had two-fold and four-fold the risk of developing a CVD event compared with those in the low-risk group, respectively. The risk prediction models also fitted New Zealand data well after recalibration.

Conclusion : A simpler non-laboratory-based risk prediction model performed equally as well as the more comprehensive laboratory-based risk prediction models. The risk chart based on the further simplified Score Model may provide a useful tool for clinical cardiologists to assess an individual patient's risk for recurrent CVD events.

Lipid composition of cashmere goat fibres

This study examined the differences in the chemical composition, particularly fatty acids, of the lipid extracted from the fibre of bucks, does and castrated goats. The study provides a more detailed understanding of the chemical composition of buck fibre lipid and how it varies throughout the year, and also details the effect of body region and nutrition on the production and chemical composition of lipid from buck fibre. Lipid was extracted with either petroleum ether (non-polar) or chloroform/methanol azeotrope (polar) and analysed by gas chromatography and gas chromatography-mass spectrometry. The more polar solvent system extracted larger amounts of lipid and more of each individual fatty acid. The following buck specific ethyl branched fatty acids were identified: 2-ethylhexanoic, 4-ethylhexanoic, 2-ethyloctanoic, 4-ethyloctanoic, 6-ethyloctanoic, 2-ethyldecanoic, 4-ethyldecanoic, 2-ethyldodecanoic, 6-ethyldodecanoic, 4-ethyldodecanoic, 2-ethyltetradecanoic, 6-ethyltetradecanoic, 4-ethyltetradecanoic, 2-ethylhexadecanoic and 4-ethyloctadecanoic acids. Of these buck specific fatty acids only 4-ethylhexanoic (T), 4-ethyloctanoic, 4-ethyldecanoic, 4-ethyldodecanoic, 6-ethyldodecanoic (T), 4-ethyltetradecanoic, 2-ethylhexadecanoic (T) and 4-ethylhexadecanoic acids have been previously identified or tentatively identified (T) in buck fibre extracts. This shows that the chemical composition of buck fibre lipid is more complex than previously reported, and that it may be more difficult than previously thought to artificially duplicate the odour of the buck. Buck fibre samples had lower average concentrations of 2-methylpropanoic, 2-methylbutanoic, iso-pentadecanoic, anteiso-pentadecanoic, iso-hexadecanoic, anteiso-heptadecanoic, iso-octadecanoic and anteiso-nonadecanoic acids as compared with fibre samples from does, spayed does, or wethers that were castrated at one month of age. The reduced concentrations of these fatty acids in buck fibre extracts were likely to be due to the synthesis of ethyl branched derivatives of iso and anteiso fatty acids. Buck fibre samples had higher concentrations of benzoic acid as compared with fibre samples from does, spayed does, or wethers that were castrated at one month of age. The significance of these results is that non buck specific fatty acids may also make a contribution to the odour of bucks. When fibre samples were collected at various times throughout the year, it was found that the bucks had increased amounts of lipid and ethyl branched fatty acids in fibre samples shorn from March to September, as compared with fibre samples shorn in November and January. The increase in the amount of lipid and ethyl branched fatty acids corresponded with both the rutting period of the buck and the period when the buck odour was increased. This suggests that ethyl branched fatty acids could be pheromones. The variation in lipid content and fatty acid composition was also examined between fibre samples collected from different body regions of the buck during April, as alterations in sebaceous gland activity around the neck during rutting have been reported. It was found that the average amount of lipid in the neck region of the bucks was not statistically higher than the average amounts in the midside and hind regions. However, the ethyl branched fatty acid concentrations were statistically higher in the fibre from around the neck as compared with the fibre from the other body regions, which is consistent with the odour of the buck being most pronounced around the head and neck region. The lipid content and composition of fibre samples from bucks fed high and low quality diets (lucerne and pangola grass, respectively) was examined to determine the effect of nutrition on buck specific components. The high quality diet increased the amount of lipid and ethyl branched fatty acids in fibre samples collected in April from the neck, midside and hind regions, as compared with fibre samples from the corresponding body regions from bucks fed the low quality diet. Thus it may be possible for the pheromone levels of bucks to be increased by simply providing them with good nutrition. The lipid content and ethyl branched fatty acid concentrations of fibre samples increased earlier in the year for the lucerne fed bucks as compared with the pangola grass fed bucks. The lucerne fed bucks had increased concentrations of ethyl branched fatty acids in fibre samples shorn during December to June (6 months) whereas the pangola grass fed bucks had increased concentrations of ethyl branched fatty acids in fibre samples shorn during April to August (4 months). These observations show that good nutrition can result in both the earlier production of ethyl branched fatty acids and an extended period when ethyl branched fatty acids are produced. This suggests that nutrition can be used to manipulate pheromone levels in the buck. The period when the ethyl branched fatty acids were increased corresponded with the period when the plasma luteinizing hormone (LH) and testosterone concentrations, odour and sebaceous gland volume of the bucks were increased, which supports the assumption that ethyl branched fatty acids are involved in odour production and act as pheromones.

Surface behaviour and lipid interaction of Alzheimer beta-amyloid peptide 1-42: a membrane-disrupting peptide

Amyloid aggregates, found in patients that suffer from Alzheimer's disease, are composed of fibril-forming peptides in a β-sheet conformation. One of the most abundant components in amyloid aggregates is the β-amyloid peptide 1–42 (Aβ 1–42). Membrane alterations may proceed to cell death by either an oxidative stress mechanism, caused by the peptide and synergized by transition metal ions, or through formation of ion channels by peptide interfacial self-aggregation. Here we demonstrate that Langmuir films of Aβ 1–42, either in pure form or mixed with lipids, develop stable monomolecular arrays with a high surface stability. By using micropipette aspiration technique and confocal microscopy we show that Aβ 1–42 induces a strong membrane destabilization in giant unilamellar vesicles composed of palmitoyloleoyl-phosphatidylcholine, sphingomyelin, and cholesterol, lowering the critical tension of vesicle rupture. Additionally, Aβ 1–42 triggers the induction of a sequential leakage of low- and high-molecular-weight markers trapped inside the giant unilamellar vesicles, but preserving the vesicle shape. Consequently, the Aβ 1–42 sequence confers particular molecular properties to the peptide that, in turn, influence supramolecular properties associated to membranes that may result in toxicity, including: 1), an ability of the peptide to strongly associate with the membrane; 2), a reduction of lateral membrane cohesive forces; and 3), a capacity to break the transbilayer gradient and puncture sealed vesicles.

Neurotoxic, redox-competent Alzheimer's --amyloid is released from lipid membrane by methionine oxidation

The amyloid β peptide is toxic to neurons, and it is believed that this toxicity plays a central role in the progression of Alzheimer's disease. The mechanism of this toxicity is contentious. Here we report that an Aβ peptide with the sulfur atom of Met-35 oxidized to a sulfoxide (Met(O)Aβ) is toxic to neuronal cells, and this toxicity is attenuated by the metal chelator clioquinol and completely rescued by catalase implicating the same toxicity mechanism as reduced Aβ. However, unlike the unoxidized peptide, Met(O)Aβ is unable to penetrate lipid membranes to form ion channel-like structures, and β-sheet formation is inhibited, phenomena that are central to some theories for Aβ toxicity. Our results show that, like the unoxidized peptide, Met(O)Aβ will coordinate Cu₂₊ and reduce the oxidation state of the metal and still produce H2O2. We hypothesize that Met(O)Aβ production contributes to the elevation of soluble Aβ seen in the brain in Alzheimer's disease.

Qualitative and quantitative analysis of lipid classes in fish oils by thin-layer chromatography with an Iatroscan flame Ionization detector (TLC-FID) and liquid chromatography with an evaporative light scattering detector (LC-ELSD)

Combined effects of hydrogen and air flow rates on the peak response of selected neutral lipid classes (triacylglycerol, diacylglycerol, monoacylglycerol, free fatty acids, and ethyl esters) were studied to optimize and calibrate the Iatroscan Mk-6s Chromarod system for the qualitative and quantitative analysis of lipid classes by thin-layer chromatography (TLC) with flame ionization detection in fish oil during the transesterification process. Air flow rate of 2 L/min, hydrogen flow rate of 150-160 mL/min, and scan rate of 30 s/rod were found to be the optimum conditions. All samples were also analyzed by high performance liquid chromatography (HPLC) with evaporative light scattering detection. Quantitative results obtained by TLC with the flame ionization detection method were comparable to those obtained from HPLC with evaporative light scattering detection.

Comparative effects of tuna oil and salmon oil on liver lipid metabolism and fatty acid concentrations in rats

The comparative effect of tuna oil (TO) and salmon oil (SO) on the plasma and liver lipid and fatty acid compositions in Sprague Dawley rats was investigated. The total triacylglycerol (TG) and total cholesterol (TC) concentrations in liver was significantly decreased in the TO group; TG level in liver was also significantly decreased in the SO group. The mRNA expression of HMG-CoA reductase in liver was significantly down-regulated in the TO and SO groups relative to the control group. The plasma TG and TC were decreased in TO, but not in SO; plasma low-density lipoprotein and very low-density lipoprotein levels in TO and SO were decreased compared with the control group. The total n-3 polyunsaturated fatty acid (PUFA) in plasma and liver phospholipids was significantly elevated in the TO and SO. Docosahexaenoic acid (22:6n-3) and eicosapentaenoic acid (20:5n-3) in tissues were significantly increased in the TO and SO, respectively. In this study, TO had a more beneficial effect on liver TC and plasma TG, TC, high-density lipoprotein in rats than SO. The likely mechanism for lowering liver and plasma cholesterol by n-3 PUFA is to suppress the mRNA expression of gene encoding HMG-CoA reductase responsible for cholesterol biosynthesis.

PRACTICAL APPLICATIONS

The beneficial effects of n-3 polyunsaturated fatty acids (PUFAs) from fish and fish oil on human health is derived from their role in modulating membrane lipid composition and affecting metabolic and signal-transduction pathways. In the present study, we demonstrated that n-3 PUFA, docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) from tuna and salmon oils can be effectively incorporated into tissue membranes. Tuna oil rich in DHA has more beneficial effect on liver total cholesterol (TC) and plasma triglyceride, TC and HDL in rats than salmon oil, which is rich in EPA. The present data could provide information for the potential application of fish oils as components of functional food, and selected for fortification with different fish oils.

Effects of rosiglitazone on intramyocellular lipid accumulation in Psammomys obesus

Objective: To examine the effects of rosiglitazone in intramyocellular lipid (IMCL) content in diabetic Psammomys obesus using novel electron microscopy technologies.

Background: P. obesus is an unique polygenic model of obesity and type 2 diabetes. Male diabetic P. obesus were treated daily with 5 mg/Kg Rosiglitazone by oral gavage for 14 days. Data were compared with a group of age-matched diabetic P. obesus treated with saline vehicle.

Methods: Assessment of insulin resistance and adiposity were determine before and after the treatment period by oral glucose tolerance test (oGTT) and dual energy X-ray absorptiometry (DEXA) analysis. We used a new scanning electron microscopy technology, (WETSEM) to investigate the effects of rosiglitazone administration on IMCL content, size and distribution in red gastrocnemius muscle.

Results: Rosiglitazone treatment improved glucose tolerance in P. obesus with no difference in the overall body fat content although a significant reduction in subscapular fat mass was observed. Rosiglitazone changed the distribution of lipid droplet size in skeletal muscle. Treated animals tended to have smaller lipid droplets compared with saline-treated controls.

Conclusions: Since smaller IMCL droplets are associated with improvements in insulin sensitivity, we propose that this may be an important mechanism by which rosiglitazone affects glucose tolerance.

Can dietary lipid source circadian alternation improve omega-3 deposition in rainbow trout?

With the salmonid industry currently exploiting the vast majority of globally available fish oil, there is the need to optimise fish oil utilisation by increasing its efficiency in terms of transferring the health-promoting long chain omega-3 fatty acids (n−3 LC-PUFA) into farmed fish flesh. The aim of this study was to evaluate if dietary fatty acid deposition is affected by the time of feeding, and hence identify possible innovative feeding strategies towardsmore efficient use of dietary fish oil. Over a period of 12 weeks, three diets with different lipid sources, canola oil (CO), fish oil (FO) or a 50/50 blend of the two oils (Mix), were alternated daily and fed to rainbow trout (Oncorhynchus mykiss). Six treatments were administered to fish, reference treatment (REF, continuously fed FO), control treatment (CT, continuously fed Mix), am canola oil ration (amCOR), pm canola oil ration (pmCOR), am canola oil satiation (amCOS) and pm canola oil satiation (pmCOS). Fish received either the CO diet in the am or pm feeds and received the FO diet at the opposite time. A significant increase in growth and feed consumption was noted in the pmCOS treatment. Fillet fatty acid profile was modified by associated feeding schedules and was generally reflective of dietary fatty acid profile. No significant increases in n−3 LCPUFA deposition were observed. However, both linoleic acid (18:2n−6) and α-linolenic acid (18:3n−3) contents were significantly higher in pmCOR compared to amCOR and CT. The results of the present study suggest the existence of cyclical circadian patterns in fatty acid deposition in rainbow trout.