Cell-free, high-density lipoprotein-specific phospholipid efflux assay predicts incident cardiovascular disease.

BACKGROUNDCellular cholesterol efflux capacity (CEC) is a better predictor of cardiovascular disease (CVD) events than HDL-cholesterol (HDL-C) but is not suitable as a routine clinical assay.METHODSWe developed an HDL-specific phospholipid efflux (HDL-SPE) assay to assess HDL functionality based on whole plasma HDL apolipoprotein-mediated solubilization of fluorescent phosphatidylethanolamine from artificial lipid donor particles. We first assessed the association of HDL-SPE with prevalent coronary artery disease (CAD): study I included NIH severe-CAD (n = 50) and non-CAD (n = 50) participants, who were frequency matched for sex, BMI, type 2 diabetes mellitus, and smoking; study II included Japanese CAD (n = 70) and non-CAD (n = 154) participants. We also examined the association of HDL-SPE with incident CVD events in the Prevention of Renal and Vascular End-stage Disease (PREVEND) study comparing 340 patients with 340 controls individually matched for age, sex, smoking, and HDL-C levels.RESULTSReceiver operating characteristic curves revealed stronger associations of HDL-SPE with prevalent CAD. The AUCs in study I were as follows: HDL-SPE, 0.68; apolipoprotein A-I (apoA-I), 0.62; HDL-C, 0.63; and CEC, 0.52. The AUCs in study II were as follows: HDL-SPE, 0.83; apoA-I, 0.64; and HDL-C, 0.53. Also longitudinally, HDL-SPE was significantly associated with incident CVD events independent of traditional risk factors with ORs below 0.2 per SD increment in the PREVEND study (P < 0.001).CONCLUSIONHDL-SPE could serve as a routine clinical assay for improving CVD risk assessment and drug discovery.TRIAL REGISTRATIONClinicalTrials.gov NCT01621594.FUNDINGNHLBI Intramural Research Program, NIH (HL006095-06).

Effects of a yoga-based stress reduction intervention on stress, psychological outcomes and cardiometabolic biomarkers in cancer caregivers: A randomized controlled trial.

Caregiving stress is a risk factor for cardiometabolic disease. Therefore, integrating cardiometabolic biomarkers into caregiving research provides a more comprehensive assessment of an individual's health and response to an intervention. The objective of this study was to examine the effects of a yoga-based stress reduction intervention on stress, psychological outcomes, and cardiometabolic biomarkers in cancer caregivers. This prospective randomized controlled trial enrolled family caregivers of adult patients who underwent an allogeneic HSCT at the National Institutes of Health (NIH) Clinical Center. All subjects received usual care education. Participants in the intervention group received an Iyengar yoga intervention self-administered over six weeks using an audio recording file. The primary outcome was perceived stress (measured using the NIH toolbox Perceived Stress). The secondary outcomes were psychological factors (depression and anxiety measured using PROMIS® Depression and Anxiety), and cardiometabolic biomarkers measured by nuclear magnetic resonance spectroscopy. A total of 50 family caregivers (mean [SD] age, 44.9 [15.2] years; 42 [84.0%] women) were randomized, 25 to the intervention group and 25 to the control group. No group differences were noted in stress, depression, and anxiety. Significant interaction effects between group and time were found in large TRL-P (F(1,43) = 10.16, p = 0.003) and LP-IR (F(1,42) = 4.28, p = 0.045). Post-hoc analyses revealed that the levels of large TRL-P (mean difference = 1.68, CI = [0.86, 2.51], p< .001) and LP-IR (mean difference = 5.67, CI = [1.15, 10.18], p = 0.015) significantly increased over time in the control group but while remained stable in the intervention group (mean difference = -0.15, CI = [-0.96, 0.66], p = 0.718; mean difference = -0.81, CI = [-5.22, 3.61], p = 0.714, respectively). Even when perceptions of psychological distress remain unchanged, incorporating gentle yoga poses and breathing exercises may reduce the risk of cardiometabolic disease in caregivers by inhibiting the development of insulin resistance. Standard lipids of cardiometabolic risk do not appear to be robust enough to detect short-term early changes of cardiometabolic risk in caregivers. Trial registration: ClinicalTrials.gov Identifier: NCT02257853.

Volanesorsen, an antisense oligonucleotide to apolipoprotein C-III, increases lipoprotein lipase activity and lowers triglycerides in partial lipodystrophy.

BACKGROUND: Partial lipodystrophy (PL) syndromes involve deficiency of adipose tissue, causing severe insulin resistance and hypertriglyceridemia. Apolipoprotein C-III (apoC-III) is elevated in PL and is thought to contribute to hypertriglyceridemia by inhibiting lipoprotein lipase (LPL). OBJECTIVE: We hypothesized that volanesorsen, an antisense oligonucleotide to apoC-III, would decrease apoC-III, increase LPL activity, and lower triglycerides in PL. METHODS: Five adults with PL enrolled in a 16-week placebo-controlled, randomized, double blind study of volanesorsen, 300 mg weekly, followed by 1-year open label extension. RESULTS: Within-subject effects of volanesorsen before and after 16 weeks of active drug are reported due to small sample size. From week 0 to 16, apoC-III decreased from median (25th, 75th %ile) 380 (246, 600) to 75 (26, 232) ng/mL, and triglycerides decreased from 503 (330, 1040) to 116 (86, 355) mg/dL while activation of LPL by subjects' serum increased from 21 (20, 25) to 36 (29, 42) nEq/mL*min. Although, A1c did not change, peripheral and hepatic insulin sensitivity (glucose disposal and suppression of glucose production during hyperinsulinemic clamp) increased and palmitate turnover decreased. After 32-52 weeks of volanesorsen, liver fat decreased. Common adverse events included injection site reactions and decreased platelets. CONCLUSIONS: In PL, volanesorsen decreased apoC-III and triglycerides, in part through an LPL dependent mechanism, and may improve insulin resistance and hepatic steatosis.

A Simple Fluorescent Cholesterol Labeling Method to Cryoprotect and Detect Plasma Lipoprotein-X.

Lipoprotein-X (LpX) are abnormal nephrotoxic lipoprotein particles enriched in free cholesterol and phospholipids. LpX with distinctive lipid compositions are formed in patients afflicted with either familial LCAT deficiency (FLD) or biliary cholestasis. LpX is difficult to detect by standard lipid stains due to the absence of a neutral lipid core and because it is unstable upon storage, particularly when frozen. We have recently reported that free cholesterol-specific filipin staining after agarose gel electrophoresis sensitively detects LpX in fresh human plasma. Herein, we describe an even more simplified qualitative method to detect LpX in both fresh and frozen-thawed human FLD or cholestatic plasma. Fluorescent cholesterol complexed to fatty-acid-free BSA was used to label LpX and was added together with trehalose in order to cryopreserve plasma LpX. The fluorescent cholesterol bound to LpX was observed with high sensitivity after separation from other lipoproteins by agarose gel electrophoresis. This methodology can be readily developed into a simple assay for the clinical diagnosis of FLD and biliary liver disease and to monitor the efficacy of treatments intended to reduce plasma LpX in these disease states.

Caregiving stress and burden associated with cardiometabolic risk in family caregivers of individuals with cancer.

Chronic stress is a well-established risk factor for cardiometabolic disease. Caregiving for individuals with cancer is perceived as a chronic stressor yet research on the risk for cardiometabolic disease in this population, opposed to the elderly and those with Alzheimer's disease, is limited. Additionally, few studies have explored the early physiological changes that occur in family caregivers suggesting an elevated risk for illness. This cross-sectional study was designed to examine levels of cardiometabolic risk biomarkers and their correlates in caregivers of patients with colorectal cancer. Caregivers completed questionnaires that measure exposures to stress and vulnerability factors, psychological distress, and health habits as potential correlates. Traditional lipid and nontraditional lipoprotein particle biomarkers (e.g. concentration and size for all lipoprotein classes) were assayed from blood serum. Caregivers (N = 83, mean age = 49.8, 73% female) displayed levels of cardiometabolic biomarkers that suggest an elevated risk for cardiometabolic disease. Caregivers who were Hispanic, married, highly educated, employed, reported more hours spent caregiving daily, experienced higher caregiver burden associated with the lack of family support and impact on schedule, and psychological distress, demonstrated an elevated risk for cardiometabolic disease; primarily determined by nontraditional lipid biomarkers - large TRL-P, LDL-P, small HDL-P, large HDL-P, TRL-Z, LDL-Z and HDL-Z. These findings suggest that traditional lipid biomarkers may not be robust enough to detect early physiological changes associated with cardiometabolic disease risk in family caregivers. Moreover, findings reiterate the importance of assessing caregiver burden and providing evidence-based interventions to manage caregiving stress with the potential to improve caregivers' cardiometabolic health.

Lipoprotein particle alterations due to androgen therapy in individuals with dyskeratosis congenita.

BACKGROUND: Dyskeratosis congenita (DC) is a telomere biology disorder associated with high rates of bone marrow failure (BMF) and other medical complications. Oral androgens are successfully used to treat BMF in DC but often have significant side effects, including elevation of serum lipids. This study sought to determine the extent to which oral androgen therapy altered lipid and lipoprotein levels. METHODS: Nuclear magnetic resonance (NMR) was used to evaluate serum lipid profiles, and lipoprotein particle number and size in nine androgen-treated individuals with DC, 45 untreated individuals with DC, 72 unaffected relatives of DC patients, and 19 untreated individuals with a different inherited BMF syndrome, Fanconi anaemia (FA). FINDINGS: Androgen-treated individuals with DC had significantly decreased serum HDL cholesterol, HDL particle number and HDL particle size (p < 0·001, p < 0·001 and p < 0·001, respectively); significantly increased serum LDL cholesterol and LDL particle number (p < 0·001, p < 0·001, respectively), decreased apoA-I and increased apoB (p < 0⋅001, p < 0⋅05 respectively) when compared with untreated individuals with DC. There were no significant lipid profile differences between untreated DC and untreated FA participants; or between untreated DC participants and their unaffected relatives. Branched chain amino acids and lipoprotein insulin resistance were not significantly different with androgen treatment. GlycA, an inflammatory acute phase reactant, was significantly increased with androgen treatment (p < 0⋅001). INTERPRETATION: Androgen treatment in DC creates an atherogenic lipoprotein profile, raising concern for the potential of elevated cardiovascular disease risk. Clinical guidelines for individuals on androgens for DC-related BMF should include cardiovascular disease monitoring. These findings could be relevant in individuals treated with androgen for other indications. FUNDING: Intramural research programs of the Division of Cancer Epidemiology and Genetics of the National Cancer Institute and National Heart, Lung, and Blood Institute.

A new phenotypic classification system for dyslipidemias based on the standard lipid panel.

BACKGROUND: Dyslipoproteinemias can be classified by their distinct lipoprotein patterns, which helps determine atherosclerotic cardiovascular disease (ASCVD) risk and directs lipid management but this has required advanced laboratory testing. OBJECTIVE: To develop a new algorithm for classifying lipoprotein disorders that only relies on the standard lipid panel. METHODS: Lipid thresholds for defining the different lipoprotein phenotypes were derived for Non-High-Density Lipoprotein-Cholesterol (NonHDL-C) and Triglycerides (TG) to be concordant when possible with the current US Multi-Society guidelines for blood cholesterol management. RESULTS: The new classification method categorizes patients into all the classical Fredrickson-like phenotypes except for Type III dysbetalipoproteinemia. In addition, a new hypolipidemic phenotype (Type VI) due to genetic mutations in apoB-metabolism is described. The validity of the new algorithm was confirmed by lipid analysis by NMR (N = 11,365) and by concordance with classification by agarose gel electrophoresis/beta-quantification (N = 5504). Furthermore, based on the Atherosclerosis Risk in Communities (ARIC) cohort (N = 14,742), the lipoprotein phenotypes differ in their association with ASCVD (TypeV>IIb > IVb > IIa > IVa > normolipidemic) and can be used prognostically as risk enhancer conditions in the management of patients. CONCLUSIONS: We describe a clinically useful lipoprotein phenotyping system that is only dependent upon the standard lipid panel. It, therefore, can be easily implemented for increasing compliance with current guidelines and for improving the care of patients at risk for ASCVD.

Apolipoprotein CIII and Angiopoietin-like Protein 8 are Elevated in Lipodystrophy and Decrease after Metreleptin.

CONTEXT: Lipodystrophy syndromes cause hypertriglyceridemia that improves with leptin treatment using metreleptin. Mechanisms causing hypertriglyceridemia and improvements after metreleptin are incompletely understood. OBJECTIVE: Determine relationship of circulating lipoprotein lipase (LPL) modulators with hypertriglyceridemia in healthy controls and in patients with lipodystrophy before and after metreleptin. METHODS: Cross-sectional comparison of patients with lipodystrophy (generalized lipodystrophy n = 3; partial lipodystrophy n = 11) vs age/sex-matched healthy controls (n = 28), and longitudinal analyses in patients before and after 2 weeks and 6 months of metreleptin. The study was carried out at the National Institutes of Health, Bethesda, Maryland. Outcomes were LPL stimulators apolipoprotein (apo) C-II and apoA-V and inhibitors apoC-III and angiopoietin-like proteins (ANGPTLs) 3, 4, and 8; ex vivo activation of LPL by plasma. RESULTS: Patients with lipodystrophy were hypertriglyceridemic and had higher levels of all LPL stimulators and inhibitors vs controls except for ANGPTL4, with >300-fold higher ANGPTL8, 4-fold higher apoC-III, 3.5-fold higher apoC-II, 1.9-fold higher apoA-V, 1.6-fold higher ANGPTL3 (P < .05 for all). At baseline, all LPL modulators except ANGPLT4 positively correlated with triglycerides. Metreleptin decreased apoC-II and apoC-III after 2 weeks and 6 months, and decreased ANGPTL8 after 6 months (P < 0.05 for all). Plasma from patients with lipodystrophy caused higher ex vivo LPL activation vs hypertriglyceridemic control plasma (P < .0001), which did not change after metreleptin. CONCLUSION: Elevations in LPL inhibitors apoC-III and ANGPTL8 may contribute to hypertriglyceridemia in lipodystrophy, and may mediate reductions in circulating and hepatic triglycerides after metreleptin. These therefore are strong candidates for therapies to lower triglycerides in these patients.

Vitamin E treatment in NAFLD patients demonstrates that oxidative stress drives steatosis through upregulation of de-novo lipogenesis.

Oxidative stress (OS) in non-alcoholic fatty liver disease (NAFLD) promotes liver injury and inflammation. Treatment with vitamin E (α-tocopherol, αT), a lipid-soluble antioxidant, improves liver injury but also decreases steatosis, thought to be upstream of OS, through an unknown mechanism. To elucidate the mechanism, we combined a mechanistic human trial interrogating pathways of intrahepatic triglyceride (IHTG) accumulation and in vitro experiments. 50% of NAFLD patients (n = 20) treated with αT (200-800 IU/d) for 24 weeks had a ≥ 25% relative decrease in IHTG by magnetic resonance spectroscopy. Paired liver biopsies at baseline and week 4 of treatment revealed a decrease in markers of hepatic de novo lipogenesis (DNL) that strongly predicted week 24 response. In vitro, using HepG2 cells and primary human hepatocytes, αT inhibited glucose-induced DNL by decreasing SREBP-1 processing and lipogenic gene expression. This mechanism is dependent on the antioxidant capacity of αT, as redox-silenced methoxy-αT is unable to inhibit DNL in vitro. OS by itself was sufficient to increase S2P expression in vitro, and S2P is upregulated in NAFLD livers. In summary, we utilized αT to demonstrate a vicious cycle in which NAFLD generates OS, which feeds back to augment DNL and increases steatosis. Clinicaltrials.gov: NCT01792115.

Leptin decreases de novo lipogenesis in patients with lipodystrophy.

De novo lipogenesis (DNL) plays a role in the development of hepatic steatosis. In humans with lipodystrophy, reduced adipose tissue causes lower plasma leptin, insulin resistance, dyslipidemia, and ectopic triglyceride (TG) accumulation. We hypothesized that recombinant leptin (metreleptin) for 6 months in 11 patients with lipodystrophy would reduce DNL by decreasing insulin resistance and glycemia, thus reducing circulating TG and hepatic TG. The percentage of TG in TG-rich lipoprotein particle (TRLP-TG) derived from DNL (%DNL) was measured by deuterium incorporation from body water into palmitate. At baseline, DNL was elevated, similar to levels previously shown in obesity-associated nonalcoholic fatty liver disease (NAFLD). After metreleptin, DNL decreased into the normal range. Similarly, absolute DNL (TRLP-TG × %DNL) decreased by 88% to near-normal levels. Metreleptin improved peripheral insulin sensitivity (hyperinsulinemic-euglycemic clamp) and lowered hemoglobin A1c and hepatic TG. Both before and after metreleptin, DNL positively correlated with insulin resistance, insulin doses, and hepatic TG, supporting the hypothesis that hyperinsulinemia stimulates DNL and that elevated DNL is integral to the pathogenesis of lipodystrophy-associated NAFLD. These data suggest that leptin-mediated improvement in insulin sensitivity increases clearance of blood glucose by peripheral tissues, reduces hepatic carbohydrate flux, and lowers insulinemia, resulting in DNL reductions and improvements in hepatic steatosis and dyslipidemia.

A New Equation for Calculation of Low-Density Lipoprotein Cholesterol in Patients With Normolipidemia and/or Hypertriglyceridemia.

Importance: Low-density lipoprotein cholesterol (LDL-C), a key cardiovascular disease marker, is often estimated by the Friedewald or Martin equation, but calculating LDL-C is less accurate in patients with a low LDL-C level or hypertriglyceridemia (triglyceride [TG] levels ≥400 mg/dL). Objective: To design a more accurate LDL-C equation for patients with a low LDL-C level and/or hypertriglyceridemia. Design, Setting, and Participants: Data on LDL-C levels and other lipid measures from 8656 patients seen at the National Institutes of Health Clinical Center between January 1, 1976, and June 2, 1999, were analyzed by the ß-quantification reference method (18 715 LDL-C test results) and were randomly divided into equally sized training and validation data sets. Using TG and non-high-density lipoprotein cholesterol as independent variables, multiple least squares regression was used to develop an equation for very low-density lipoprotein cholesterol, which was then used in a second equation for LDL-C. Equations were tested against the internal validation data set and multiple external data sets of either ß-quantification LDL-C results (n = 28 891) or direct LDL-C test results (n = 252 888). Statistical analysis was performed from August 7, 2018, to July 18, 2019. Main Outcomes and Measures: Concordance between calculated and measured LDL-C levels by ß-quantification, as assessed by various measures of test accuracy (correlation coefficient [R2], root mean square error [RMSE], mean absolute difference [MAD]), and percentage of patients misclassified at LDL-C treatment thresholds of 70, 100, and 190 mg/dL. Results: Compared with ß-quantification, the new equation was more accurate than other LDL-C equations (slope, 0.964; RMSE = 15.2 mg/dL; R2 = 0.9648; vs Friedewald equation: slope, 1.056; RMSE = 32 mg/dL; R2 = 0.8808; vs Martin equation: slope, 0.945; RMSE = 25.7 mg/dL; R2 = 0.9022), particularly for patients with hypertriglyceridemia (MAD = 24.9 mg/dL; vs Friedewald equation: MAD = 56.4 mg/dL; vs Martin equation: MAD = 44.8 mg/dL). The new equation calculates the LDL-C level in patients with TG levels up to 800 mg/dL as accurately as the Friedewald equation does for TG levels less than 400 mg/dL and was associated with 35% fewer misclassifications when patients with hypertriglyceridemia (TG levels, 400-800 mg/dL) were categorized into different LDL-C treatment groups. Conclusions and Relevance: The new equation can be readily implemented by clinical laboratories with no additional costs compared with the standard lipid panel. It will allow for more accurate calculation of LDL-C level in patients with low LDL-C levels and/or hypertriglyceridemia (TG levels, ≤800 mg/dL) and thus should improve the use of LDL-C level in cardiovascular disease risk management.

Supplementation with saury oil, a fish oil high in omega-11 monounsaturated fatty acids, improves plasma lipids in healthy subjects.

BACKGROUND: Fish oil enriched in omega-11 long-chain monounsaturated fatty acids (LCMUFAs; C20:1 and C22:1 isomers combined) have shown lipid-lowering and atheroprotective effects in animal models. OBJECTIVE: To perform a first-in-human trial of LCMUFA-rich saury fish oil supplementation to test its safety and possible effect on plasma lipids. METHODS: A double-blind, randomized, crossover clinical trial was carried out in 30 healthy normolipidemic adults (BMI <25 kg/m2; mean TG, 84 mg/dL). Treatment periods of 8 weeks were separated by an 8-week washout period. Subjects were randomized to receive either 12 g of saury oil (3.5 g of LCMUFA and 3.4 g of omega-3 FAs) or identical capsules with control oil (a mixture of sardine and olive oil; 4.9 g of shorter-chain MUFA oleate and 3 g of omega-3 FAs). RESULTS: Saury oil supplementation was safe and resulted in LDL particle counts 12% lower than control oil (P < .001). Saury oil also had a minor effect on increasing HDL particle size (9.8 nm vs 9.7 nm; P < .05) based on a linear mixed effect model. In contrast, control oil, but not saury oil, increased LDL-C by 7.5% compared with baseline (P < .05). Saury oil had similar effects compared with control oil on lowering plasma TG levels, VLDL, and TG-rich lipoprotein particle counts (by ∼16%, 25%, and 35%, respectively; P < .05), and increasing HDL-C and cholesterol efflux capacity (by ∼6% and 8%, respectively; P < .05) compared with baseline. CONCLUSION: Saury oil supplementation is well tolerated and has beneficial effects on several cardiovascular parameters, such as LDL particle counts, HDL particle size, and plasma TG levels.

Vitamin E sequestration by liver fat in humans.

BACKGROUNDWe hypothesized that obesity-associated hepatosteatosis is a pathophysiological chemical depot for fat-soluble vitamins and altered normal physiology. Using α-tocopherol (vitamin E) as a model vitamin, pharmacokinetics and kinetics principles were used to determine whether excess liver fat sequestered α-tocopherol in women with obesity-associated hepatosteatosis versus healthy controls.METHODSCustom-synthesized deuterated α-tocopherols (d3- and d6-α-tocopherols) were administered to hospitalized healthy women and women with hepatosteatosis under investigational new drug guidelines. Fluorescently labeled α-tocopherol was custom-synthesized for cell studies.RESULTSIn healthy subjects, 85% of intravenous d6-α-tocopherol disappeared from the circulation within 20 minutes but reappeared within minutes and peaked at 3-4 hours; d3- and d6-α-tocopherols localized to lipoproteins. Lipoprotein redistribution occurred only in vivo within 1 hour, indicating a key role of the liver in uptake and re-release. Compared with healthy subjects who received 2 mg, subjects with hepatosteatosis had similar d6-α-tocopherol entry rates into liver but reduced initial release rates (P < 0.001). Similarly, pharmacokinetics parameters were reduced in hepatosteatosis subjects, indicating reduced hepatic d6-α-tocopherol output. Reductions in kinetics and pharmacokinetics parameters in hepatosteatosis subjects who received 2 mg were echoed by similar reductions in healthy subjects when comparing 5- and 2-mg doses. In vitro, fluorescent-labeled α-tocopherol localized to lipid in fat-loaded hepatocytes, indicating sequestration.CONCLUSIONSThe unique role of the liver in vitamin E physiology is dysregulated by excess liver fat. Obesity-associated hepatosteatosis may produce unrecognized hepatic vitamin E sequestration, which might subsequently drive liver disease. Our findings raise the possibility that hepatosteatosis may similarly alter hepatic physiology of other fat-soluble vitamins.TRIAL REGISTRATIONClinicalTrials.gov, NCT00862433.FUNDINGNational Institute of Diabetes and Digestive and Kidney Diseases and NIH grants DK053213-13, DK067494, and DK081761.

Advanced Lipoprotein Analysis Shows Atherogenic Lipid Profile That Improves After Metreleptin in Patients with Lipodystrophy.

CONTEXT: Patients with lipodystrophy have dyslipidemia and insulin resistance. Leptin treatment with metreleptin in lipodystrophy decreases insulin resistance and lowers triglycerides without changing high-density lipoprotein. Detailed measurement of lipoprotein particles with nuclear magnetic resonance (NMR) spectroscopy can offer insights into cardiovascular disease (CVD) risk and lipid metabolism beyond a standard lipid panel. We hypothesized that patients with lipodystrophy would have a more atherogenic lipid profile than controls at baseline, which would be ameliorated with metreleptin treatment. OBJECTIVE: To characterize the lipoprotein profile in patients with lipodystrophy compared with controls and to evaluate effects of metreleptin treatment. DESIGN SETTING PATIENTS AND INTERVENTION: Patients with lipodystrophy (N = 17) were studied before and after metreleptin for 2 weeks and 6 months and compared with 51 insulin-sensitive sex-matched controls. MAIN OUTCOME MEASURES: Lipoprotein profiles were measured by NMR with the LP4 deconvolution algorithm, which reports triglyceride-rich lipoprotein particles (TRLPs), high-density lipoprotein particles (HDLPs), and low-density lipoprotein particles (LDLPs). RESULTS: Patients with lipodystrophy had elevated large TRLPs and smaller HDLPs and LDLPs compared with controls. Five patients with lipodystrophy had chylomicrons, compared with zero controls. Metreleptin decreased the size and concentration of TRLPs, eliminated chylomicrons in all but one patient, decreased LDLPs, and increased LDLP size. Metreleptin treatment did not have major effects on HDLPs. CONCLUSIONS: Patients with lipodystrophy had an atherogenic lipoprotein profile at baseline consistent with elevated CVD risk, which improved after metreleptin treatment. The presence of fasting chylomicrons in a subset of patients with lipodystrophy suggests saturation of chylomicron clearance by lipoprotein lipase.

Vitamin E absorption and kinetics in healthy women, as modulated by food and by fat, studied using 2 deuterium-labeled α-tocopherols in a 3-phase crossover design.

BACKGROUND: Determining the human vitamin E [α-tocopherol (α-T)] requirement is difficult, and novel approaches to assess α-T absorption and trafficking are needed. OBJECTIVE: We hypothesized that the dual-isotope technique, using 2 deuterium-labeled [intravenous (IV) d6- and oral d3-] α-T, would be effective in determining α-T fractional absorption. Further, defined liquid meal (DLM) fat or fasting would modulate α-T fractional absorption and lipoprotein transport. METHODS: A 3-phase cr ossover design was used. At 0 h, participants received IV d6-α-T and consumed d3-α-T with a 600-kcal DLM (40% or 0% fat) followed by controlled meals or by the 0% fat DLM, a 12-h fast, and then controlled meals. Blood samples and fecal samples were collected at intervals and analyzed by LC-MS. Pharmacokinetic parameters were calculated from plasma tracer concentrations and enrichments. Fractional absorption was calculated from d3- to d6-α-T areas under the curve, from a novel mathematical model, and from the balance method (oral d3-α-T minus fecal d3-α-T excreted). RESULTS: Estimated α-T fractional absorption during the 40% fat intervention was 55% ± 3% (mean ± SEM; n = 10), which was 9% less than during the 0% fat intervention (64% ± 3%, n = 10; P < 0.02). Fasting had no apparent effect (56% ± 3%, n = 7), except it slowed plasma oral d3-α-T appearance. Both balance data and model outcomes confirmed that the DLM fat did not potentiate d3-α-T absorption. During the IV emulsion clearance, HDL rapidly acquired d6-α-T (21 ± 2 nmol/L plasma per minute). During the first 8 h postdosing, triglyceride-rich lipoproteins (TRLs) were preferentially d3-α-T enriched relative to LDL or HDL, showing the TRL precursor role. CONCLUSIONS: Quantitatively, α-T absorption is not limited by fat absence or by fasting. However, α-T leaves the intestine by a process that is prolonged during fasting and potentiated by eating, suggesting that α-T absorption is highly dependent on chylomicron assembly processes. This trial was registered at clinicaltrials.gov as NCT00862433.

Plasma lipoprotein-X quantification on filipin-stained gels: monitoring recombinant LCAT treatment ex vivo.

Familial LCAT deficiency (FLD) patients accumulate lipoprotein-X (LP-X), an abnormal nephrotoxic lipoprotein enriched in free cholesterol (FC). The low neutral lipid content of LP-X limits the ability to detect it after separation by lipoprotein electrophoresis and staining with Sudan Black or other neutral lipid stains. A sensitive and accurate method for quantitating LP-X would be useful to examine the relationship between plasma LP-X and renal disease progression in FLD patients and could also serve as a biomarker for monitoring recombinant human LCAT (rhLCAT) therapy. Plasma lipoproteins were separated by agarose gel electrophoresis and cathodal migrating bands corresponding to LP-X were quantified after staining with filipin, which fluoresces with FC, but not with neutral lipids. rhLCAT was incubated with FLD plasma and lipoproteins and LP-X changes were analyzed by agarose gel electrophoresis. Filipin detects synthetic LP-X quantitatively (linearity 20-200 mg/dl FC; coefficient of variation <20%) and sensitively (lower limit of quantitation <1 mg/ml FC), enabling LP-X detection in FLD, cholestatic, and even fish-eye disease patients. rhLCAT incubation with FLD plasma ex vivo reduced LP-X dose dependently, generated HDL, and decreased lipoprotein FC content. Filipin staining after agarose gel electrophoresis sensitively detects LP-X in human plasma and accurately quantifies LP-X reduction after rhLCAT incubation ex vivo.

Development of a novel fluorescent activity assay for lecithin:cholesterol acyltransferase.

Background Lecithin:cholesterol acyltransferase (LCAT) is a plasma enzyme that esterifies cholesterol. Recombinant human LCAT (rhLCAT) is now being developed as an enzyme replacement therapy for familial LCAT deficiency and as a possible treatment for acute coronary syndrome. The current 'gold standard' assay for LCAT activity involves the use of radioisotopes, thus making it difficult for routine clinical use. Methods We have developed a novel and more convenient LCAT activity assay using fluorescence-labelled cholesterol (BODIPY-cholesterol), which is incorporated into proteoliposomes as a substrate instead of radiolabelled cholesterol. Results The apparent Km and Vmax were 31.5 µmol/L and 55.8 nmol/h/nmoL, rhLCAT, respectively, for the 3H-cholesterol method and 103.1 µmol/L and 13.4 nmol/h/nmol rhLCAT, respectively, for the BODIPY-cholesterol method. Although the two assays differed in their absolute units of LCAT activity, there was a good correlation between the two test assays ( r = 0.849, P < 1.6 × 10-7, y = 0.1378x + 1.106). The BODIPY-cholesterol assay had an intra-assay CV of 13.7%, which was superior to the intra-assay CV of 20.8% for the radioisotopic assay. The proteoliposome substrate made with BODIPY-cholesterol was stable to storage for at least 10 months. The reference range ( n = 20) for the fluorescent LCAT activity assay was 4.6-24.1 U/mL/h in healthy subjects. Conclusions In summary, a novel fluorescent LCAT activity assay that utilizes BODIPY-cholesterol as a substrate is described that yields comparable results to the radioisotopic method.

Lecithin:Cholesterol Acyltransferase Activation by Sulfhydryl-Reactive Small Molecules: Role of Cysteine-31.

Lecithin:cholesterol acyltransferase (LCAT) catalyzes plasma cholesteryl ester formation and is defective in familial lecithin:cholesterol acyltransferase deficiency (FLD), an autosomal recessive disorder characterized by low high-density lipoprotein, anemia, and renal disease. This study aimed to investigate the mechanism by which compound A [3-(5-(ethylthio)-1,3,4-thiadiazol-2-ylthio)pyrazine-2-carbonitrile], a small heterocyclic amine, activates LCAT. The effect of compound A on LCAT was tested in human plasma and with recombinant LCAT. Mass spectrometry and nuclear magnetic resonance were used to determine compound A adduct formation with LCAT. Molecular modeling was performed to gain insight into the effects of compound A on LCAT structure and activity. Compound A increased LCAT activity in a subset (three of nine) of LCAT mutations to levels comparable to FLD heterozygotes. The site-directed mutation LCAT-Cys31Gly prevented activation by compound A. Substitution of Cys31 with charged residues (Glu, Arg, and Lys) decreased LCAT activity, whereas bulky hydrophobic groups (Trp, Leu, Phe, and Met) increased activity up to 3-fold (P < 0.005). Mass spectrometry of a tryptic digestion of LCAT incubated with compound A revealed a +103.017 m/z adduct on Cys31, consistent with the addition of a single hydrophobic cyanopyrazine ring. Molecular modeling identified potential interactions of compound A near Cys31 and structural changes correlating with enhanced activity. Functional groups important for LCAT activation by compound A were identified by testing compound A derivatives. Finally, sulfhydryl-reactive ß-lactams were developed as a new class of LCAT activators. In conclusion, compound A activates LCAT, including some FLD mutations, by forming a hydrophobic adduct with Cys31, thus providing a mechanistic rationale for the design of future LCAT activators.

Cardiometabolic risk factors and health behaviors in family caregivers.

The purpose of this study was to compare components of cardiometabolic risk and health behaviors of 20 family caregivers of allogeneic hematopoietic stem cell transplant patients to those of age, gender, and race/ethnicity-matched controls. A prospective, repeated measures design was used to compare cardiometabolic risk and health behaviors in caregivers and controls at three time-points: pre-transplantation, discharge, and six weeks post-discharge. Measures included components of metabolic syndrome, Reynolds Risk Score, NMR serum lipoprotein particle analyses, and the Health-Promoting Lifestyle Profile II (HPLP-II). Mixed-model repeated measure analyses were used. There were no between or within group differences in LDL cholesterol, HDL cholesterol, and triglycerides. There was a significant interaction effect between time and role in large VLDL concentration (VLDL-P) (F (2, 76) = 4.36, p = .016), with the trajectory of large VLDL-P increasing over time in caregivers while remaining stable in controls. Within caregivers, VLDL particle size (VLDL-Z) was significantly larger at time-point three compared to time-points one (p = .015) and two (p = .048), and VLDL-Z was significantly larger in caregivers than in controls at time point three (p = .012). HPLP-II scores were lower in caregivers than controls at all time-points (p < .01). These findings suggest that caregiving may have a bigger impact on triglycerides than on other lipids, and it is through this pathway that caregivers may be at increased cardiometabolic risk. More sensitive measurement methods, such as NMR lipoprotein particle analyses, may be able to detect early changes in cardiometabolic risk.