Genetic analysis of familial hypercholesterolaemia in Western Australia.

OBJECTIVE: To determine the spectrum of mutations associated with familial hypercholesterolaemia (FH) and their detection rate in the FH Western Australia (FHWA) Program. METHODS: Mutation testing of the LDLR gene, plus select regions in APOB and PCSK9, was performed in the first 343 patients considered to be phenotypic index cases of FH and classified on the basis of the Dutch Lipid Clinic Network Criteria (DLCNC) score as "possible", "probable", or "definite" FH. RESULTS: Overall, 86 different pathogenic (or likely pathogenic) mutations were identified in 129 patients, including four compound heterozygotes manifesting a more severe clinical phenotype. Fourteen of these mutations were novel and twelve (9.6%) were large deletions/duplications of the LDLR. The most common mutations were the familial defective apoB-100 mutation APOB p.Arg3527Gln (7.2%) and an LDLR intron 3 splice site mutation c.313 + 1G > A (4.8%). While 70% of 'definite' FH patients were found to carry a mutation, only 29% of 'probable' and 11% of 'possible' FH patients were mutation-positive. CONCLUSION: This information provides a useful DNA database on which to base ongoing cascade screening for FH and future research into the genetic aetiology of FH in Western Australia. These findings suggest genetic testing should be prioritised to those with high DLCNC scores and offers a cost-effective family screening method from FH index cases, leading to detection of other previously undiagnosed and younger family members, enabling early instigation of intervention and preventative measures for premature coronary heart disease.

Familial hypercholesterolaemia: a model of care for Australasia.

Familial hypercholesterolaemia (FH) is a dominantly inherited disorder present from birth that causes marked elevation in plasma cholesterol and premature coronary heart disease. There are at least 45,000 people with FH in Australia and New Zealand, but the vast majority remains undetected and those diagnosed with the condition are inadequately treated. To bridge this major gap in coronary prevention the FH Australasia Network (Australian Atherosclerosis Society) has developed a consensus model of care (MoC) for FH. The MoC is based on clinical experience, expert opinion, published evidence and consultations with a wide spectrum of stakeholders, and has been developed for use primarily by specialist centres intending starting a clinical service for FH. This MoC aims to provide a standardised, high-quality and cost-effective system of care that is likely to have the highest impact on patient outcomes. The MoC for FH is presented as a series of recommendations and algorithms focusing on the standards required for the detection, diagnosis, assessment and management of FH in adults and children. The process involved in cascade screening and risk notification, the backbone for detecting new cases of FH, is detailed. Guidance on treatment is based on risk stratifying patients, management of non-cholesterol risk factors, safe and effective use of statins, and a rational approach to follow-up of patients. Clinical and laboratory recommendations are given for genetic testing. An integrative system for providing best clinical care is described. This MoC for FH is not prescriptive and needs to be complemented by good clinical judgment and adjusted for local needs and resources. After initial implementation, the MoC will require critical evaluation, development and appropriate modification.

Effect of dietary fat to produce non-alcoholic fatty liver in the rat.

BACKGROUND AND AIM: Non-alcoholic steatohepatitis (NASH) belongs to a spectrum of non-alcoholic fatty liver disease (NAFLD). Oxidative stress is hypothesized to play an important role in the progression of the disease. We used the Lieber/DeCarli model for NASH to investigate the mechanisms involved in its progression. METHODS: Male Sprague-Dawley rats were fed standard (35% of energy from fat) or high fat (71% of energy from fat) liquid diets, ad libitum or two-thirds of the amount consumed ad libitum initially for 3 weeks and then extended to 5 weeks. RESULTS: Steatosis was absent in rats at 3 weeks feeding, but by 5 weeks, the high fat/ad lib group showed microvesicular steatosis and foci of macrovesicular steatosis without inflammation. Serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels were not different. By 5 weeks feeding, hepatic triglycerides were highest in the high fat ad lib group and the ad lib groups were higher compared with their restricted groups. The oxidative stress marker, hydroxyalkenal (HAE) was decreased in the standard ad lib compared with the high fat ad lib group. Liver mRNA of interleukin-6, haem oxygenase-1, and markers of endoplasmic stress: C/EBP homologous protein (CHOP), glucose responsive protein-78 (GRP78) and spliced X-box DNA binding protein (spliced XBP1) were similar in the ad lib groups. CONCLUSIONS: Extending the feeding period of the high fat/ad lib diet for 5 weeks placed our rats with Type I to II NAFLD compared to the more progressed Type III state previously obtained after 3 weeks feeding. The milder condition obtained raised the prospect of genetic modifiers present in our rats that resist disease progression.

Chylomicrons in disease-future challenges Invited keynote address.

It has taken many years to unravel the pathological implications of postprandial dyslipidaemia. Life-style and pharmacological interventions to reduce risk of atherosclerosis have developed empirically, without clear understanding of the underlying mechanisms. Acceptance of the premise that chylomicrons play a key role in atherogenesis brings new understanding to the mechanisms underlying empirical risk-reduction strategies. Raised LDL cholesterol while definitely associated with increased risk, is not present in many patients with established atherosclerosis. A large body of research points to a clear association of atherosclerosis with postprandial dyslipidaemia, not only for coronary disease but also for carotid lesions. Bayesian considerations imply that the majority of the community atherosclerosis burden is attributable to disordered chylomicron metabolism. Risk reduction by life-style interventions such as consumption of low fat meals, Mediterranean diets, low cholesterol intake, weight reduction and exercise can all be explained rationally in terms of the impact of these interventions on chylomicron metabolism. Diabetes is strongly associated with postprandial dyslipidaemia. Needed are long-term controlled trials of life-style interventions and management of dyslipidaemia, with endpoints of progression or regression of atherosclerosis. The paradigm that postprandial dyslipidaemia underlies much of the atherosclerosis burden should become a focus of drug development. Agents that reduce absorption of triglycerides and cholesterol will generally be beneficial. Probucol markedly affects chylomicron metabolism. The mechanism for optimization by Probucol is unclear but appears to be unrelated to effects on oxidation. Adrenergic agonists and thyromimetics are also likely to be beneficial. Statins and fish oils appear beneficial but data are limited. Long-term intervention studies are needed with appropriate attention to improvement in postprandial dyslipidaemia.

Treatment with a dietary fat substitute decreased Arochlor 1254 contamination in an obese diabetic male.

A case manifesting symptoms due to organochlorine toxicity was treated with the fat substitute olestra in his diet. Before treatment, the patient was obese, with severe type 2 diabetes mellitus and mixed hyperlipidemia, chloracne, frequent headaches, and numbness and paraesthesias of his trunk and lower limbs. Earlier attempts at weight loss had been unsuccessful due to worsening of his symptoms. After inclusion of olestra in his diet for 2 years, weight loss was successful without aggravation of his symptoms, and the patient reverted to normoglycemia and normolipidemia. Olestra may have assisted weight loss and amelioration of his diabetes by increasing fecal elimination of organochlorines, rather than by preventing the partitioning of these pollutants into tissues, where they have been reported to exert antimetabolic effects on substrate oxidation.

Obesity and post-prandial lipid metabolism. Feast or famine?

Both in Western countries and in third world countries there is an increasing incidence of obesity. Obesity per se or insulin resistance associated with obesity may increase cardiovascular risk factors including dyslipidemia, hypertension and Type 2 diabetes. Over the past decade the understanding has increased of specific mediators in the hypothalamus that are involved in regulating food intake and body weight. In obese humans fasting plasma lipids can be normal but postprandial lipid metabolism is abnormal with an accumulation of triglyceride-rich remnant lipoproteins. In viscerally obese men chylomicron remnant catabolism was markedly decreased when compared with lean individuals. The decreased clearance of chylomicron remnants in viscerally obese subjects may be explained by competition between chylomicron remnants and the increased hepatic production of VLDL for clearance by low density lipoprotein receptors. Increased food intake in rodent models of obesity was shown to be associated with a delay in the catabolism of remnant lipoprotein particles. Prevention of hyperphagia was found to correct the impairment in the metabolism of remnant lipoproteins. Under fasting and food restricted conditions the improvement of remnant metabolism was associated with an increased oxidation of remnant lipids as determined by a novel stable isotope breath test. Anti-obesity and lipid lowering drugs have been used for the treatment of obesity. Inhibitors of cholesterol synthesis inhibitors (statins) have been shown to be effective in treating dyslipidemia. Inhibition of cholesterol synthesis with Atorvastatin was shown to improve chylomicron metabolism by increasing chylomicron remnant catabolism in obese subjects as assessed by the newly developed stable isotope breath test.

Relationships between cholesterol homoeostasis and triacylglycerol-rich lipoprotein remnant metabolism in the metabolic syndrome.

The dysmetabolic syndrome of insulin resistance and visceral obesity is characterized by elevated plasma concentration of triacylglycerol-rich lipoprotein (TRL) remnants that may be related to increased cardiovascular risk. Perturbed hepato-intestinal cholesterol metabolism may play a contributory role in this abnormality. We therefore investigated the association between plasma markers of cholesterol absorption and synthesis with TRL remnant metabolism in 35 men with the metabolic syndrome (MS). Plasma campesterol:cholesterol and lathosterol:cholesterol ratios were measured as estimates of cholesterol absorption and synthesis respectively. Remnant metabolism was assessed by measuring remnant-like particle-cholesterol (RLP-C), apolipoprotein (apo)B-48 and the fractional catabolic rate (FCR) of a labelled remnant-like emulsion. Compared with controls, subjects with the MS had significantly lower plasma campesterol:cholesterol ratio, but higher lathosterol:cholesterol ratio ( P <0.05). Plasma RLP-C and apoB-48 concentrations were also higher ( P <0.01) and the remnant-like emulsion FCR was lower ( P <0.05). The plasma campesterol:cholesterol ratio was inversely correlated ( P <0.05) with plasma triacylglycerols ( r =-0.346), RLP-C ( r =-0.443), apoB-48 ( r =-0.427) and plasma lathosterol:cholesterol ratio ( r =-0.366); the campesterol:cholesterol ratio was also positively correlated with the remnant-like emulsion FCR ( r =0.398, P <0.05). In multiple regression analysis, the significant correlations between plasma campesterol:cholesterol ratio and plasma triacylglycerols, RLP-C, apoB-48 and FCR of the remnant-like emulsion were independent of age, dietary energy and plasma lathosterol. Our findings suggest that in subjects with the MS alterations in cholesterol absorption and synthesis may be closely linked with the kinetic defects in TRL metabolism.

Randomized controlled trial of the effect of n-3 fatty acid supplementation on the metabolism of apolipoprotein B-100 and chylomicron remnants in men with visceral obesity.

BACKGROUND: Lipid abnormalities may contribute to the increased risk of atherosclerosis and coronary disease in visceral obesity. Fish oils lower plasma triacylglycerols, but the underlying mechanisms are not fully understood. OBJECTIVE: We studied the effect of fish oils on the metabolism of apolipoprotein B-100 (apo B) and chylomicron remnants in obese men. DESIGN: Twenty-four dyslipidemic, viscerally obese men were randomly assigned to receive either fish oil capsules (4 g/d, consisting of 45% eicosapentaenoic acid and 39% docosahexaenoic acid as ethyl esters) or matching placebo (corn oil, 4 g/d) for 6 wk. VLDL, intermediate-density lipoprotein (IDL), and LDL apo B kinetics were assessed by following apo B isotopic enrichment with the use of gas chromatography-mass spectrometry after an intravenous bolus injection of trideuterated leucine. Chylomicron remnant catabolism was measured with the use of an intravenous injection of a chylomicron remnant-like emulsion containing cholesteryl [(13)C]oleate, and isotopic enrichment of (13)CO(2) in breath was measured with isotope ratio mass spectrometry. Kinetic values were derived with multicompartmental models. RESULTS: Fish oil supplementation significantly (P < 0.05) lowered plasma concentrations of triacylglycerols (-18%) and VLDL apo B (-20%) and the hepatic secretion of VLDL apo B (-29%) compared with placebo. The percentage of conversions of VLDL apo B to IDL apo B, VLDL apo B to LDL apo B, and IDL apo B to LDL apo B also increased significantly (P < 0.05): 71%, 93%, and 11%, respectively. Fish oils did not significantly alter the fractional catabolic rates of apo B in VLDL, IDL, or LDL or alter the catabolism of the chylomicron remnant-like emulsion. CONCLUSION: Fish oils effectively lower the plasma concentration of triacylglycerols, chiefly by decreasing VLDL apo B production but not by altering the catabolism of apo B-containing lipoprotein or chylomicron remnants.

Apolipoprotein B-100 kinetics in visceral obesity: associations with plasma apolipoprotein C-III concentration.

Obesity is strongly associated with dyslipidemia, which may account for the associated increased risk of atherosclerosis and coronary disease. We aimed to test the hypothesis that kinetics of hepatic apolipoprotein B-100 (apoB) metabolism are disturbed in men with visceral obesity and to examine whether these kinetic defects are associated with elevated plasma concentration of apolipoprotein C-III (apoC-III). Very-low-density lipoprotein (VLDL), intermediate-density lipoprotein (IDL), and low-density lipoprotein (LDL) apoB kinetics were measured in 48 viscerally obese men and 10 age-matched normolipidemic lean men using an intravenous bolus injection of d(3)-leucine. ApoB isotopic enrichment was measured using gas chromatography-mass spectrometry (GCMS). Kinetic parameters were derived using a multicompartmental model (Simulation, Analysis, and Modeling Software II [SAAM-II]). Compared with controls, obese subjects had significantly elevated plasma concentrations of plasma triglycerides, cholesterol, LDL-cholesterol, VLDL-apoB, IDL-apoB, LDL-apoB, apoC-III, insulin, and lathosterol (P <.01). VLDL-apoB secretion rate was significantly higher (P =.034) in obese than control subjects; the fractional catabolic rates (FCRs) of IDL-apoB and LDL-apoB (P <.01) and percent conversion of VLDL-apoB to LDL-apoB (P <.02) were also significantly lower in obese subjects. However, the decreased VLDL-apoB FCR was not significantly different from the lean group. In the obese group, plasma concentration of apoC-III was significantly and positively associated with VLDL-apoB secretion rate and inversely with VLDL-apoB FCR and percent conversion of VLDL to LDL. In multiple regression analysis, plasma apoC-III concentration was independently and significantly correlated with the secretion rate of VLDL-apoB (regression coefficient [SE] 0.511 [0.03], P =.001) and with the percent conversion of VLDL-apoB to LDL-apoB (-0.408 [0.01], P =.004). Our findings suggest that plasma lipid and lipoprotein abnormalities in visceral obesity may be due to a combination of overproduction of VLDL-apoB particles and decreased catabolism of apoB containing particles. Elevated plasma apoC-III concentration is also a feature of dyslipidemia in obesity that contributes to the kinetic defects in apoB metabolism.

Regulatory effects of HMG CoA reductase inhibitor and fish oils on apolipoprotein B-100 kinetics in insulin-resistant obese male subjects with dyslipidemia.

Hepatic accumulation of lipid substrates perturbs apolipoproteinB-100 (apoB) metabolism in insulin-resistant, obese subjects and may account for increased risk of cardiovascular disease. In a placebo-controlled trial, we examined the independent and combined effects of decreasing cholesterol synthesis with atorvastatin (40 mg/day) and triglyceride synthesis with fish oils (4 g/day) on apoB kinetics. The subjects were 48 viscerally obese, insulin-resistant men with dyslipidemia who were studied in a fasted state. We found that atorvastatin significantly decreased plasma apoB-containing lipoproteins (P < 0.001, main effect) through increases in the fractional catabolic rate (FCR) of VLDL-, IDL-, and LDL-apoB (P < 0.01). Fish oils significantly decreased plasma levels of triglycerides and VLDL-apoB (P < 0.001), decreased the VLDL-apoB secretion rate (P < 0.01), but increased the conversion of VLDL to LDL (P < 0.001). Compared with placebo, combined treatment with atorvastatin and fish oils decreased VLDL-apoB secretion (P < 0.03) and increased the FCR of apoB in each lipoprotein fraction (P < 0.03) and the percent conversion of VLDL to LDL (P < 0.05). None of the treatments altered insulin resistance. In conclusion, in visceral obesity, atorvastatin increased hepatic clearance of all apoB-containing lipoproteins, whereas fish oils decreased hepatic secretion of VLDL-apoB. The differential effects of atorvastatin and fish oils on apoB kinetics support their combined use in correcting defective apoB metabolism in obese, insulin-resistant subjects.

Mechanism of action of a 3-hydroxy-3-methylglutaryl coenzyme a reductase inhibitor on apolipoprotein B-100 kinetics in visceral obesity.

We examined the effect of atorvastatin, an inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase, on the kinetics of apolipoprotein B-100 (apoB) metabolism in 25 viscerally obese men in a placebo-controlled study. Very-low-density lipoprotein (VLDL), intermediate-density lipoprotein (IDL), and low-density lipoprotein (LDL) apoB kinetics were measured using an iv bolus injection of [(2)H(3)]leucine. ApoB isotopic enrichment was measured using gas chromatography-mass spectrometry. Kinetic parameters were derived by using a multicompartmental model (SAAM-II). Compared with the placebo group, atorvastatin treatment resulted in significant (P < 0.001) decreases in total cholesterol (-34%), triglyceride (-19%), LDL cholesterol (-42%), total apoB (-39%), and lathosterol (-86%); VLDL-apoB, IDL-apoB, and LDL-apoB pool sizes also fell significantly (P < 0.002) by -27%, -22%, and -41%, respectively. This was associated with an increase in the fractional catabolic rates of VLDL-apoB (+58%, P = 0.019), IDL-apoB (+40%, P = 0.049), and LDL-apoB (+111%, P = 0.001). However, atorvastatin did not significantly alter the production and conversion rates of apoB in all lipoproteins. We conclude that in obese subjects, atorvastatin decreases the plasma concentration of all apoB-containing lipoproteins chiefly by increasing their catabolism and not by decreasing their production or secretion. This may be owing to up-regulation of hepatic receptors as a consequence of inhibition of cholesterogenesis.

Effect of atorvastatin on chylomicron remnant metabolism in visceral obesity: a study employing a new stable isotope breath test.

Elevated plasma concentration of chylomicron remnants may be causally related to atherosclerosis in obesity. We examined the effect of atorvastatin on chylomicron remnant metabolism in 25 obese men with dyslipidaemia. A remnant-like emulsion labeled with cholesteryl [(13)C]oleate was injected intravenously into patients; the fractional catabolic rate (FCR) of the remnant-like emulsion was determined by measurement of (13)CO(2) in the breath and analyzed using compartmental modelling. Compared with placebo, atorvastatin significantly decreased the plasma concentrations of total cholesterol, triglycerides, LDL cholesterol, apolipoprotein B (apoB), and lathosterol (P < 0.001). ApoB-48 and remnant-like particle-cholesterol (RLP-C) both decreased significantly by 23% (P = 0.002) and 33% (P = 0.045), respectively. The FCR of the remnant-like emulsion increased significantly from 0.054 +/- 0.008 to 0.090 +/- 0.010 pools/h (P = 0.002). The decrease in RLP-C was associated with the decrease in plasma triglycerides (r = 0.750, P = 0.003). Furthermore, the change in FCR of remnant-like emulsions was inversely associated with the change in LDL-C (r = -0.575, P = 0.040), suggesting removal of LDL and chylomicron remnants by similar hepatic receptor pathways. We conclude that in obese subjects, inhibition of cholesterol synthesis with atorvastatin decreases the plasma concentrations of both LDL-C and triglyceride-rich remnants and that this may be partially due to an enhancement in hepatic clearance of these lipoproteins.

Food restriction normalizes chylomicron remnant metabolism in murine models of obesity as assessed by a novel stable isotope breath test.

Evidence is increasing that defective metabolism of postprandial remnants of triglyceride-rich lipoproteins contributes to atherogenesis. In obesity, postprandial lipemia is increased by mechanisms that are not currently established. In the present study, a recently developed (13)CO(2) breath test was used to assess the metabolism of chylomicron remnants (CR) in obese mice. Six murine obese models ob/ob, fat/fat, New Zealand Obese (NZO), db/db, gold thioglucose (GTG)-treated and agouti (A(y)) were studied. All obese mice were hyperphagic and their breath test metabolism was markedly impaired (P < 0.01) compared with control, nonobese mice. The breath test was also impaired (P < 0.01) in all obese mice except A(y) mice after 24-h food deprivation. However, after restriction to the food intake of paired control mice for 6 wk, the breath test in all obese mice improved to values of control, nonobese mice. The obese NZO, fat/fat and ob/ob mice had significant (P < 0.02) weight loss when food restricted, whereas A(y), GTG, and db/db mice did not. In all obese mice, plasma cholesterol levels decreased (P < 0.02) after the 6-wk period of food restriction. Plasma triglyceride levels significantly decreased (P < 0.02) in NZO, GTG and db/db mice, but not in other obese mice. Plasma glucose levels were significantly decreased (P < 0.02) after the 6-wk period in the obese mice except for the A(y) and NZO mice; levels were greater in food-restricted db/db mice. Although some of the obese models such as db/db were diabetic, our data suggest that the defective breath test was independent of diabetes because all obese and diabetic models responded similarly to food restriction. Impaired hepatic catabolism of CR was excluded as a cause of the abnormal breath tests. In summary, the impairment (P < 0.05) in remnant metabolism as assessed by the breath test in obese mice was corrected by food restriction, associated with improvements in plasma glucose, triglyceride and cholesterol levels.

Markers of triglyceride-rich lipoprotein remnant metabolism in visceral obesity.

BACKGROUND: Triglyceride-rich lipoprotein remnants are atherogenic, and this may be particularly important in visceral obesity. We investigated remnant metabolism in obese men by measuring remnant-like particle-cholesterol (RLP-C), apolipoprotein (apo) B-48, apoC-III, and the clearance of a labeled remnant-like emulsion. METHODS: Fasting RLP-C, apoB-48, and apoC-III concentrations were measured in 48 viscerally obese men and 10 lean controls. RLP-C was determined by immunoseparation assay, apoB-48 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and enhanced chemiluminescence, and apoC-III by immunoturbidimetric assay. The catabolism of chylomicron remnants was measured by intravenous injection of a remnant-like emulsion containing cholesteryl [(13)C]oleate, with isotopic enrichment of (13)CO(2) in breath determined by isotope-ratio mass spectrometry and a multicompartmental model to estimate fractional catabolic rate (FCR) of the emulsion. RESULTS: Compared with controls, obese men had significantly increased plasma concentrations of RLP-C, apoB-48, and apoC-III (P <0.001 for all). Plasma total apoB-100, non-HDL-cholesterol, LDL-cholesterol, triglycerides, and insulin resistance (HOMA score) were also significantly higher in the obese group (P <0.001 for all). Obese men had a significantly lower FCR of the remnant-like emulsion compared with controls (P = 0.020). CONCLUSIONS: Viscerally obese individuals have insulin resistance and increased plasma concentrations of triglyceride-rich lipoprotein remnants, which may be attributable to decreased catabolism of these particles.