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Haptoglobin (Hp) is the plasma protein that binds and clears cell-free hemoglobin (Hb), whereas apohemoglobin (apoHb, i.e., Hb devoid of heme) can bind heme. Therefore, the apoHb-Hp protein complex should facilitate holoHb-apoHb αβ-dimer exchange and apoHb-heme intercalation. Thus, we hypothesized that apoHb-Hp could facilitate both Hb and heme clearance, which, if not alleviated, could have severe microcirculatory consequences. In this study, we characterized apoHb-Hp and Hb/heme ligand interactions and assessed their in vivo consequences. Hb exchange and heme binding with the apoHb-Hp complex was studied with transfer assays using size-exclusion high-performance liquid chromatography coupled with UV-visible spectrophotometry. Exchange/transfer experiments were conducted in guinea pigs dosed with Hb or heme-albumin followed by a challenge with equimolar amounts of apoHb-Hp. Finally, systemic and microcirculatory parameters were studied in hamsters instrumented with a dorsal window chamber via intravital microscopy. In vitro and in vivo Hb exchange and heme transfer experiments demonstrated proof-of-concept Hb/heme ligand transfer to apoHb-Hp. Dosing with the apoHb-Hp complex reversed Hb- and heme-induced systemic hypertension and microvascular vasoconstriction, reduced microvascular blood flow, and diminished functional capillary density. Therefore, this study highlights the apoHb-Hp complex as a novel therapeutic strategy to attenuate the adverse systemic and microvascular responses to intravascular Hb and heme exposure. NEW & NOTEWORTHY This study highlights the apoHb-Hp complex as a novel therapeutic strategy to attenuate the adverse systemic and microvascular responses to intravascular Hb and heme exposure. In vitro and in vivo Hb exchange and heme transfer experiments demonstrated proof-of-concept Hb/heme ligand transfer to apoHb-Hp. The apoHb-Hp complex reverses Hb- and heme-induced systemic hypertension and microvascular vasoconstriction, preserves microvascular blood flow, and functional capillary density. In summary, the unique properties of the apoHb-Hp complex prevent adverse systemic and microvascular responses to Hb and heme-albumin exposure and introduce a novel therapeutic approach to facilitate simultaneous removal of extracellular Hb and heme.

Background: Babesia canis infection induces a marked acute phase response (APR) that might be associated with alteration in lipid and lipoprotein metabolism and disease prognosis., Hypothesis: Dogs with B. canis-induced APR develop dyslipidemia with altered lipoprotein concentration and morphology., Animals: Twenty-nine client-owned dogs with acute B. canis infection and 10 clinically healthy control dogs., Methods: Observational cross-sectional study. Serum amyloid A (SAA) was measured using ELISA. Cholesterol, phospholipids, and triglycerides were determined biochemically. Lipoproteins were separated using agarose gel electrophoresis. Lipoprotein diameter was assessed by polyacrylamide gradient gel electrophoresis; correlation with ApoA-1 (radioimmunoassay) and SAA was determined., Results: Dogs with B. canis infection had a marked APR (median SAA, 168.3 μg/mL; range, 98.1-716.2 μg/mL) compared with controls (3.2 μg/mL, 2.0-4.2 μg/mL) (P < .001). Dogs with B. canis infection had significantly lower median cholesterol (4.79 mmol/L, 1.89-7.64 mmol/L versus 6.15 mmol/L, 4.2-7.4 mmol/L) (P = .02), phospholipid (4.64 mmol/L, 2.6-6.6 mmol/L versus 5.72 mmol/L, 4.68-7.0 mmol/L) (P = .02), and α-lipoproteins (77.5%, 27.7%-93.5% versus 89.2%, 75.1%-93.5%) (P = .04), and higher ApoA-1 (1.36 U, 0.8-2.56 U versus 0.95 U, 0.73-1.54 U) concentrations (P = .02). Serum amyloid A correlated with high-density lipoproteins (HDLs) diameter (rho = .43; P = .03) and ApoA-1 (rho = .63, P < .001)., Conclusions and Clinical Importance: Major changes associated with B. canis-induced APR in dogs are related to concentration, composition, and morphology of HDL particles pointing to an altered reverse cholesterol transport. Parallel ApoA-1 and SAA concentration increase is a unique still unexplained pathophysiological finding., (© 2019 The Authors. Journal of Veterinary Internal Medicine published by Wiley Periodicals, Inc. on behalf of the American College of Veterinary Internal Medicine.)

Noninvasive tools that target tumor cells could improve the management of glioma. Cancer generally has a high demand for Fe(III), an essential nutrient for a variety of biochemical processes. We tested whether 68Ga-citrate, an Fe(III) biomimetic that binds to apo-transferrin in blood, detects glioma in preclinical models and patients using hybrid PET/MRI. Mouse PET/CT studies showed that 68Ga-citrate accumulates in subcutaneous U87MG xenografts in a transferrin receptor-dependent fashion within 4 hours after injection. Seventeen patients with WHO grade III or IV glioma received 3.7-10.2 mCi 68Ga-citrate and were imaged with PET/MR 123-307 minutes after injection to establish that the radiotracer can localize to human tumors. Multiple contrast-enhancing lesions were PET avid, and tumor to adjacent normal white matter ratios were consistently greater than 10:1. Several contrast-enhancing lesions were not PET avid. One minimally enhancing lesion and another tumor with significantly reduced enhancement following bevacizumab therapy were PET avid. Advanced MR imaging analysis of one patient with contrast-enhancing glioblastoma showed that metabolic hallmarks of viable tumor spatially overlaid with 68Ga-citrate accumulation. These early data underscore that high-grade glioma may be detectable with a radiotracer that targets Fe(III) transport.

Despite transferrin being the main circulating carrier of iron in body fluids, and iron overload conditions being known to worsen stroke outcome through reactive oxygen species (ROS)-induced damage, the contribution of blood transferrin saturation (TSAT) to stroke brain damage is unknown. The objective of this study was to obtain evidence on whether TSAT determines the impact of experimental ischemic stroke on brain damage and whether iron-free transferrin (apotransferrin, ATf)-induced reduction of TSAT is neuroprotective. We found that experimental ischemic stroke promoted an early extravasation of circulating iron-loaded transferrin (holotransferrin, HTf) to the ischemic brain parenchyma. In vitro, HTf was found to boost ROS production and to be harmful to primary neuronal cultures exposed to oxygen and glucose deprivation. In stroked rats, whereas increasing TSAT with exogenous HTf was detrimental, administration of exogenous ATf and the subsequent reduction of TSAT was neuroprotective. Mechanistically, ATf did not prevent extravasation of HTf to the brain parenchyma in rats exposed to ischemic stroke. However, ATf in vitro reduced NMDA-induced neuronal uptake of HTf and also both the NMDA-mediated lipid peroxidation derived 4-HNE and the resulting neuronal death without altering Ca 2+ -calcineurin signaling downstream the NMDA receptor. Removal of transferrin from the culture media or blockade of transferrin receptors reduced neuronal death. Together, our data establish that blood TSAT exerts a critical role in experimental stroke-induced brain damage. In addition, our findings suggest that the protective effect of ATf at the neuronal level resides in preventing NMDA-induced HTf uptake and ROS production, which in turn reduces neuronal damage., (Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.)

Cardiosiderosis is a leading cause of mortality in transfusion-dependent thalassemias. Plasma non-transferrin-bound iron and its redox-active component, labile plasma iron, are key sources of iron loading in cardiosiderosis. Risk factors were identified in 73 patients with or without cardiosiderosis. Soluble transferrin receptor-1 levels were significantly lower in patients with cardiosiderosis (odds ratio 21). This risk increased when transfusion-iron loading rates exceeded the erythroid transferrin uptake rate (derived from soluble transferrin receptor-1) by >0.21 mg/kg/day (odds ratio 48). Labile plasma iron was >3-fold higher when this uptake rate threshold was exceeded, but non-transferrin-bound iron and transferrin saturation were comparable. The risk of cardiosiderosis was decreased in patients with low liver iron, ferritin and labile plasma iron, or high bilirubin, reticulocyte counts or hepcidin. We hypothesized that high erythroid transferrin uptake rate decreases cardiosiderosis through increased erythroid re-generation of apotransferrin. To test this, iron uptake and intracellular reactive oxygen species were examined in HL-1 cardiomyocytes under conditions modeling transferrin effects on non-transferrin-bound iron speciation with ferric citrate. Intracellular iron and reactive oxygen species increased with ferric citrate concentrations especially when iron-to-citrate ratios exceeded 1:100, i.e. conditions favoring kinetically labile monoferric rather than oligomer species. Excess iron-binding equivalents of apotransferrin inhibited iron uptake and decreased both intracellular reactive oxygen species and labile plasma iron under conditions favoring monoferric species. In conclusion, high transferrin iron utilization, relative to the transfusion-iron load rate, decreases the risk of cardiosiderosis. A putative mechanism is the transient re-generation of apotransferrin by an active erythron, rapidly binding labile plasma iron-detectable ferric monocitrate species., (Copyright© 2017 Ferrata Storti Foundation.)

In the ILLUMINATE Trial, treatment with the cholesteryl ester transfer protein inhibitor torcetrapib resulted in a significant increase in both atherosclerotic cardiovascular disease events and total mortality which was not explained by changes in the routinely measured plasma lipids. To determine whether alterations in lipoproteins defined by their apoprotein content that are not estimated with conventional laboratory methods contributed to these unexpected events, we measured the apoB- and apoA-containing subclasses in a subgroup of ILLUMINATE participants. We find that torcetrapib treatment significantly increased the high-density lipoprotein subclasses LpA-I and LpA-I:A-II equally (p <0.0001) and the apoC-III content of high-density lipoprotein (p <0.001) without altering the apoB-containing subclasses. In conclusion, these findings provide further evidence that the untoward effects of torcetrapib were attributable to off-target effects and not related to disturbances in lipoprotein transport., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Background and Objective: Although salsalate administration consistently lowers plasma triglyceride concentrations in patients with type II diabetes, prediabetes, and/or insulin resistance, changes in low-density lipoprotein cholesterol (LDL-C) concentrations have been inconsistent; varying from no change to a significant increase. To evaluate the clinical relevance of this discordance in more detail, we directly measured LDL-C and obtained a comprehensive assessment of changes in lipid, lipoprotein, and apoprotein concentrations associated with salsalate use in insulin-resistant individuals, overweight or obese, but without diabetes, using vertical auto profile method., Methods: A single-blind, randomized, placebo-controlled study was performed in volunteers who were overweight or obese, without diabetes, and insulin resistant on the basis of their steady-state plasma glucose concentration during an insulin suppression test. Participants were randomized 2:1 to receive salsalate 3.5 g/d (n = 27) or placebo (n = 14) for 4 weeks. Comprehensive lipid, lipoprotein, and apoprotein analysis by vertical auto profile was obtained after an overnight fast, before and after study intervention., Results: There was no change in directly measured LDL-C concentration in salsalate-treated individuals. However, salsalate administration was associated with various changes considered to decrease atherogenicity; including decreases in triglyceride and total very low-density lipoprotein cholesterol (VLDL-C) concentrations, a shift from small denser LDL lipoproteins toward larger, more buoyant LDL particles, decreases in VLDL(1+2)-C and LDL(4)-C, and nonsignificant decreases in non-high-density lipoprotein cholesterol and apolipoprotein B. No significant changes occurred in the placebo-treated group., Conclusions: Atherogenicity of the lipid, lipoprotein, and apoprotein profile of insulin-resistant individuals who were overweight or obese improved significantly in association with salsalate treatment. The clinical importance of this finding awaits further study., (Copyright © 2015 National Lipid Association. Published by Elsevier Inc. All rights reserved.)

Objectives: Our aim was to assess the effects of the cholesteryl ester transfer protein (CETP) inhibitor anacetrapib and atorvastatin, both as monotherapy and in combination, on particle concentrations of low-density lipoproteins (LDL), very low-density lipoproteins (VLDL), and intermediate-density lipoproteins in dyslipidemic patients., Background: Although increases in high-density lipoproteins with CETP inhibition are well-documented, effects on atherogenic lipoprotein particle subclasses in dyslipidemic patients have not been extensively characterized., Methods: Ion mobility was performed on stored plasma samples collected from patients before and after treatment with anacetrapib alone (150 and 300 mg/d) or in combination with atorvastatin (20 mg/d) in a previously conducted 8-week phase IIb study., Results: Anacetrapib produced significant placebo-adjusted reductions of total LDL particles and all subfractions except for increases in very small LDL 4a and 4b. Atorvastatin reduced all LDL subfractions except LDL 4b. Results were generally additive for anacetrapib + atorvastatin. For patients treated with anacetrapib, the placebo-adjusted reduction in LDL 3a was attenuated and there was an increase in LDL 3b and 4a for those with low vs high triglyceride (TG) levels. For the atorvastatin alone vs placebo treatment comparison, there were small reductions in LDL 3a, 3b, and 4a for those with low vs high TG levels., Conclusions: Anacetrapib and atorvastatin produced similar reductions from baseline in total LDL particles, but did not have comparable effects on all LDL particle subfractions, and neither drug reduced the smallest LDL 4b particles. The clinical significance of these changes and the differential effects on very small LDL 4a in patients with higher vs lower TG remain to be determined (clinicaltrials.gov, NCT00325455)., (Copyright © 2015 National Lipid Association. Published by Elsevier Inc. All rights reserved.)

Hypertriglyceridemia is a common disorder encountered in daily medical practice. Since the causes of hypertriglyceridemia are various, we need to totally assess clinical history, physical findings, and laboratory examinations. In this section, we aim to show procedures of examination and diagnosis of hypertriglyceridemia. First, it is necessary to confirm a detection opportunity and conditions of blood sampling. Next, we move on to differentiate secondary hyperlipidemia and assess other arteriosclerosis risks. Finally, we diagnose primary hyperlipidemia by examining apoproteins, lipoproteins, and some metabolic enzymes of lipoproteins. To diagnose hypertriglyceridemia correctly, proficient clinical skills, understandings of pathophysiology, and knowledge of methods for respective examinations are needed.

Background and Aim: Because of the limitations of liver biopsy, reliable non-invasive serum biomarkers of liver fibrosis are needed. The aim of this study was to identify such markers by the use of serum proteomics in chronic hepatitis B (CHB)., Methods: Two-dimensional gel electrophoresis (2-DE) was used to identify differentially expressed protein spots in sera from 40 CHB patients [20 with mild fibrosis (S0-S1) and 20 with severe fibrosis (S3-S4)]. Mass spectrometry (MS) based multiple reaction monitoring (MRM) was used to quantify peptide ions of differential protein spots in another set of sera from 86 CHB patients with different liver fibrosis (S0-S4)., Results: Seven differentially expressed protein spots were found by 2-DE. Fourteen peptide ions of seven target protein spots were quantified by MS-based MRM. Summed peak areas ratio (SPAR) values of peptide ions from protein spot 1, 4 and 8, identified as apo serum transferrin, complement component C3c and transferrin, were significantly different from non-fibrosis (S0) to fibrosis stage 4. AUROCs of models established by peptide ions (protein spot 1, 4, 8) and model consisting of a combination of all ions were 0.848∼0.966 (S2-S4 versus S0-S1) and 0.785∼0.875 (S3-S4 versus S0-S2). Only the peptide ions model of transferrin had better sensitivity and specificity for predicting fibrosis stages than did aspartate aminotransferase-to-platelet ratio index (APRI), FIB-4 and Forn's index., Conclusions: Serum peptide ions of transferrin, detected by proteomic MRM, are new and promising biomarkers for staging liver fibrosis in CHB patients., (Copyright © 2013 Elsevier Masson SAS. All rights reserved.)

Plasma vitamin B-12 is the most commonly used biomarker of vitamin B-12 status, but the predictive value for low vitamin B-12 status is poor. The urinary methylmalonic acid (uMMA) concentration has potential as a functional biomarker of vitamin B-12 status, but the response to supplemental vitamin B-12 is uncertain. A study was conducted to investigate the responsiveness of uMMA to supplemental vitamin B-12 in comparison with other biomarkers of vitamin B-12 status [plasma vitamin B-12, serum holotranscobalamin (holoTC), plasma MMA] in elderly people with moderately poor vitamin B-12 status. A double-blind, placebo-controlled, randomized 8-wk intervention study was carried out using vitamin B-12 supplements (500 μg/d, 100 μg/d, and 10 μg/d cyanocobalamin) in 100 elderly people with a combined plasma vitamin B-12 <250 pmol/L and uMMA ratio (μmol MMA/mmol creatinine) >1.5. All biomarkers had a dose response to supplemental vitamin B-12. Improvements in plasma vitamin B-12 and serum holoTC were achieved at cobalamin supplements of 10 μg/d, but even 500 μg/d for 8 wk did not normalize plasma vitamin B-12 in 8% and serum holoTC in 12% of people. The response in uMMA was comparable with plasma MMA; 15-25% of people still showed evidence of metabolic deficiency after 500 μg/d cobalamin for 8 wk. There was a differential response in urinary and plasma MMA according to smoking behavior; the response was enhanced in ex-smokers compared with never-smokers. uMMA offers an alternative marker of metabolic vitamin-B12 status, obviating the need for blood sampling.

The transferrin (Tf) family of iron binding proteins includes important endogenous modulators of the immune function that may modulate autoimmune diseases. To define more clearly the role of apotransferrin (apoTf) in type 1 diabetes we determined the impact of this protein on type 1 diabetes as investigated in islet cells, animal models and patient sera. First, we demonstrated that recombinant apoTf counteracts the cytokine-induced death of murine pancreatic islet cells. Secondly, human apoTf administration favourably influences the course of type 1 diabetes in animal models, resulting in protection against disease development that was associated with reduction of insulitis and reduced levels of proinflammatory cytokines. Finally, we confirmed that patients with newly diagnosed type 1 diabetes manifest significantly lower apoTf serum levels compared to healthy controls and patients with long-lasting disease. In conclusion, our data suggest the apoTf pivotal role in the perpetuation of type 1 diabetes pathology., (© 2012 The Authors. Clinical and Experimental Immunology © 2012 British Society for Immunology.)

Background: Lipid abnormalities, particularly high serum concentration of lipoprotein(a) [Lp(a)], are one of the major risk factors for cardiovascular disease (CVD) in peritoneal dialysis (PD) patients. The present study was designed to investigate the effects of soy consumption on serum lipids and apoproteins, especially Lp(a), in PD patients. ♢, Methods: This study was a randomized clinical trial in which 40 PD patients (20 males, 20 females) were randomly assigned to either the soy or the control group. Patients in the soy group received 28 g/day textured soy flour (containing 14 g of soy protein) for 8 weeks, whereas patients in the control group received their usual diet, without any soy. At baseline and the end of week 8 of the study, 5 mL of blood was collected from each patient after a 12- to 14-hour fast and serum triglyceride, total cholesterol, low density lipoprotein-cholesterol (LDL-C), high density lipoprotein-cholesterol (HDL-C), apoprotein B100 (apo B100), apoprotein AI (apo AI), and Lp(a) were measured. ♢, Results: In the present study, serum Lp(a) concentrations were above the normal range in 86% of the PD patients. Mean serum Lp(a) concentration was reduced significantly, by 41%, in the soy group at the end of week 8 compared to baseline (p < 0.01); the reduction was also significant compared to the control group (p < 0.05). During the study, mean serum Lp(a) concentration did not change significantly in the control group. There were no significant differences between the two groups in mean changes in serum triglyceride, total cholesterol, HDL-C, LDL-C, apo B100, or apoAI. ♢, Conclusion: The results of our study indicate that soy consumption reduces serum Lp(a) concentration, which is a risk factor for cardiovascular disease in peritoneal dialysis patients.

The idea that copper may play a role in the pathogenesis of Alzheimer's disease is gaining momentum. Serum copper and ceruloplasmin were measured by both enzymatic (eCp) and immunologic (iCp) methods in 28 patients with Alzheimer's disease and 29 age-matched controls. ''Free copper'' was determined by subtracting copper accounted for in the eCp assay from total serum copper. Percentage free copper, that is the proportion of serum copper not bound to ceruloplasmin, was significantly elevated in patients with Alzheimer's compared to controls. There was significantly more ''defective'' ceruloplasmin, which is apoceruloplamin lacking its copper, in Alzheimer's disease than in normal controls. This abnormality may precede the clinical onset of the disease and help predict risk of disease onset. Increased exposure to environmental copper (eg, the spread of copper plumbing and the use of copper in supplements) and/or defective ceruloplasmin function may play a role in the current epidemic of Alzheimer's disease.

Transfusional iron overload associated with thalassemia leads to the appearance of non-transferrin-bound iron (NTBI) in blood that is toxic and causes morbidity and mortality via tissue damage. Hence, a highly sensitive and accurate assay of NTBI, with broad clinical application in both diagnosis and validation of treatment regimens for iron overload, is important. An assay based on iron chelation by a high-affinity siderophore, azotobactin, has been developed. The steps consist of blocking of native apotransferrin iron binding sites, mobilization of NTBI, ultrafiltration of all serum proteins, and finally the addition of the probe, which has a chromophore that fluoresces at 490 nm. Binding of Fe3+ to azotobactin quenches the fluorescence in a concentration-dependent manner. Measured NTBI levels in 63 sera ranged from 0.07 to 3.24 microM (0.375+/-0.028 microM [means+/-SEM]). It correlated well with serum iron and percentage transferrin saturation but not with serum ferritin. Pearson's correlation coefficients were found to be 0.6074 (P<0.0001) and 0.6102 (P<0.0001) for percentage transferrin saturation and total serum iron, respectively. The low values are due to the patients being under regular chelation therapy even prior to sampling, indicating that the method is sensitive to very low levels of NTBI, allowing a much lower detection limit than the available methods.

Human plasma contains small amounts of a low density lipoprotein in which apoprotein is misfolded. Originally identified and isolated by means of anion-exchange chromatography, this component was subsequently described as electronegative low density lipoprotein (LDL)(-), with increased concentrations associated with elevated cardiovascular disease risk. It has been recognized recently as the trigger of LDL amyloidogenesis, which produces aggregates similar to subendothelial droplets observed in vivo in early atherogenesis. Although LDL(-) has been produced in vitro through various manipulations, the mechanisms involved in its generation in vivo remain obscure. By using a more physiological model, we demonstrate spontaneous, sustained and noticeable production of LDL(-) during incubation of unprocessed human plasma at 37 degrees C. In addition to a higher fraction of amyloidogenic LDL(-), LDL purified from incubated plasma contains an increased level of lysophospholipids and free fatty acids; analysis of LDL lipids packing shows their loosening. As a result, during plasma incubation, lipid destabilization and protein misfolding take place, and aggregation-prone particles are generated. All these phenomena can be prevented by inhibiting calcium-dependent secretory phospholipases A2. Our plasma incubation model, without removal of reaction products, effectively shows a lipid-protein interplay in LDL, where lipid destabilization after lipolysis threatens the apoprotein's structure, which misfolds and becomes aggregation-prone.

The interactions of various insulin mimetic oxovanadium(IV) compounds with serum proteins were studied in model systems and in ex vivo samples. For the modeling study, an earlier in situ method was extended and applied to the formation of ternary complexes of apotransferrin (apoTf)-V(IV)O-maltol (mal) and 1,2-dimethyl-3-hydroxy-4(1H)-pyridinone (dhp). Both systems were evaluated via simultaneous CD and EPR measurements. Determination of the formation constants of the ternary complexes allowed the calculation of more accurate stability constants for the V(IV)O-apoTf parent complexes and establishment of a better model for drug speciation in serum. It was found that dhp and the synergistic carbonate are non-competitive binders. Based on the stability constants obtained for V(IV)O-apoTf complexes and estimated for V(IV)O-HSA (= human serum albumin), modeling calculations were performed on the distribution of V(IV)O among the components of blood serum. The results were confirmed by HPLC-ICP-MS (liquid chromatography-inductively coupled plasma spectroscopy-mass spectrometry) measurements. The ex vivo interactions of the V(IV)O complexes formed with mal, picolinic acid (pic) and dhp with serum protein standards and also with human serum samples were evaluated. The proteins were firstly separated by (HPLC), and the V content of each fraction was determined by ICP-MS. All the studied V(IV)O compounds displayed similar chromatographic profiles, associated almost exclusively with apotransferrin as predicted by the modeling calculations. Under physiological conditions, the interactions with HSA of all of the species under study were negligible. Therefore Tf seems to be the main V(IV)O transporter in the serum under in vitro conditions, and this association is practically independent of the chemical form in which V(IV)O is administered.

Bis(maltolato)oxovanadium(IV) (BMOV), and its ethylmaltol analog, bis(ethylmaltolato)oxovanadium(IV) (BEOV), are candidate insulin-enhancing agents for the treatment of type 2 diabetes mellitus; in mid-2008, BEOV advanced to phase II clinical testing. The interactions of BMOV and its inorganic congener, vanadyl sulfate (VOSO(4)), with human serum apo-transferrin (hTf) were investigated using differential scanning calorimetry (DSC). Addition of BMOV or VOSO(4) to apo-hTf resulted in an increase in thermal stability of both the C- and N-lobes of transferrin as a result of binding to either vanadyl compound. A series of DSC thermograms of hTf solutions containing different molar ratios of BMOV and VOSO(4) were used to determine binding constants; at 25 degrees C the binding constants of BMOV to the C- and N-lobes of apo-hTf were found to be 3 (+/-1)x10(5) and 1.8 (+/-0.7)x10(5)M(-1), respectively. The corresponding values for VOSO(4) were 1.7 (+/-0.3)x10(5) and 7 (+/-2)x10(4)M(-1). The results show that the vanadium species initially presented as either BMOV or VOSO(4) had similar affinities for human serum transferrin due to oxidation of solvated vanadyl(IV) prior to complexation to transferrin. Binding of metavanadate (VO(3)(-)) was confirmed by DSC and isothermal titration calorimetry (ITC) experiments of the interaction between sodium metavanadate (NaVO(3)) and hTf.

Objective: The menstrual cycle represents a continuous state of change in terms of female sex steroid environment. Progesterone is linked to increased fat storage while estrogen exerts anti-lipogenic effects. This study investigated variations in the potent lipogenic factor acylation-stimulating protein (ASP), and examined its association with hormonal and lipid profile alterations across the menstrual cycle., Methods and Design: Nineteen non-obese women with regular menstrual cycles were investigated in a longitudinal study during the follicular, ovulatory, and mid-luteal phases (ML) of the cycle. Fasting ASP, LH, FSH, progesterone, estradiol, insulin, lipid profile, and apoproteins were evaluated during different phases of the cycle., Results: ASP levels changed significantly throughout the menstrual cycle (K-related Friedman test: P=0.013). Interestingly, these changes coincide with variations in progesterone levels across the cycle as no significant change in the ASP levels was seen across the follicular phases of the cycle, followed by a significant increase in the ovulatory phase, which continued to elevate toward the ML. The ASP levels correlated positively with the progesterone levels normally elevated in the ML. No significant correlation was seen between ASP and estrogen or any other measured female hormone. Multiple regression analysis including all measured parameters and body mass index showed that progesterone was the only significant predictor of the ASP levels., Conclusion: Our findings suggest that during the menstrual cycle of normal women, the ASP levels coincidentally fluctuate with the progesterone levels, possibly reflecting cooperation between them in fat storage enhancement.

Elevated serum retinol-binding protein (RBP) concentration has been associated with obesity and insulin resistance, but accompanying retinol values have not been reported. Assessment of retinol is required to discriminate between apo-RBP, which may act as an adipokine, and holo-RBP, which transports vitamin A. The relations between serum RBP, retinol, retinyl esters, BMI, and measures of insulin resistance were determined in obese adults. Fasting blood (> or =8 h) was collected from obese men and women (n = 76) and blood chemistries were obtained. Retinol and retinyl esters were quantified by HPLC and RBP by ELISA. RBP and retinol were determined in age and sex-matched, nonobese individuals (n = 41) for comparison. Serum apo-RBP was two-fold higher in obese (0.90 +/- 0.62 microM) than nonobese subjects (0.44 +/- 0.56 microM) (P < 0.001). The retinol to RBP ratio (retinol:RBP) was significantly lower in obese (0.73 +/- 0.13) than nonobese subjects (0.90 +/- 0.22) (P < 0.001) and RBP was strongly associated with retinol in both groups (r = 0.71 and 0.90, respectively, P < 0.0001). In obese subjects, RBP was associated with insulin (r = 0.26, P < 0.05), homeostatic model assessment of insulin resistance (r = 0.29, P < 0.05), and quantitative insulin sensitivity check index (r = -0.27, P < 0.05). RBP was associated with BMI only when obese and nonobese subjects were combined (r = 0.25, P < 0.01). Elevated serum RBP, derived in part from apo-RBP, was more strongly associated with retinol than with BMI or measures of insulin resistance in obese adults. Investigations into the role of RBP in obesity and insulin resistance should include retinol to facilitate the measurement of apo-RBP and retinol:RBP. When evaluating the therapeutic potential of lowering serum RBP, consideration of the consequences of vitamin A metabolism is paramount.

Background: The levels of retinol-binding protein 4 (RBP4) - the carrier protein for Vitamin A in plasma - are tightly regulated under healthy circumstances. The kidney, the main site of RBP4 catabolism, contributes to an elevation of RBP4 levels during chronic kidney disease (CKD) whereas during chronic liver disease (CLD) RBP4 levels decrease. Little is known about RBP4 isoforms including apo-RBP4, holo-RBP4 as well as RBP4 truncated at the C-terminus (RBP4-L and RBP4-LL) except that RBP4 isoforms have been reported to be increased in hemodialysis patients. Since it is not known whether CLD influence RBP4 isoforms, we investigated RBP4 levels, apo- and holo-RBP4 as well as RBP4-L and RBP4-LL in plasma of 36 patients suffering from CKD, in 55 CLD patients and in 50 control subjects. RBP4 was determined by ELISA and apo- and holo-RBP4 by native polyacrylamide gel electrophoresis (PAGE). RBP4-L and RBP4-LL were analyzed after immunoprecipitation by mass spectrometry (MALDI-TOF-MS)., Results: RBP4 isoforms and levels were highly increased in CKD patients compared to controls (P < 0.05) whereas in CLD patients RBP4 isoforms were not different from controls. In addition, in hepatic dysfunction RBP4 levels were decreased whereas the amount of isoforms was not affected., Conclusion: The occurrence of RBP4 isoforms is not influenced by liver function but seems to be strongly related to kidney function and may therefore be important in investigating kidney function and related disorders.

Objectives: The aims of the present study were to determine if metabolic abnormalities and cytokine derangements are modified in HIV-1-infected patients after 12 months on treatment interruption (TI)., Design: The design of this study was prospective randomized study., Methods: Longitudinal multicenter study in HIV-1-infected patients with a 12-month follow-up. Patients on stable highly active antiretroviral therapy, with CD4 count >600/microL and HIV RNA <50 copies/mL for at least 6 months, were randomized to interrupt therapy or continue ongoing highly active antiretroviral therapy. Lipids (total cholesterol, triglycerides), apoproteins (A1, B, and E), and adipocytokines (leptin, adiponectin, plasminogen activator inhibitor-1, monocyte chemotactic protein-1, Interleukin-6, Interleukin-8, and tumor necrosis factor-alpha) were measured at baseline and at month 12. Multiplex suspension bead array immunoassay was performed using the Luminex 100 analyzer to identify protein expression in plasma., Results: Patients who underwent TI (n = 19) had a significant decrease in median cholesterol levels (P < 0.001), while median triglyceride levels remained unchanged. There was a significant decrease in Apo-A1 levels (P = 0.048) and Apo-B levels (P < 0.001) and an increase in tumor necrosis factor-alpha levels (P = 0.034). Given the greater decrease in Apo-B, the ratio Apo-A1/Apo-B increased after 12 months of TI (from 3.4 to 5.1, P = 0.008). We did not find significant variations in leptin or adiponectin levels. In patients who continued on highly active antiretroviral therapy (n = 18), there were no significant changes in any of the measured parameters., Conclusion: The lipid profile and apoproteins levels change toward a less atherogenic profile after TI, arguing against a lipid-mediated mechanism to explain the increased cardiovascular risk in patients who interrupt treatment.

During iron acquisition by the cell, complete homodimeric transferrin receptor 1 in an unknown state (R1) binds iron-loaded human serum apotransferrin in an unknown state (T) and allows its internalization in the cytoplasm. T also forms complexes with metals other than iron. Are these metals incorporated by the iron acquisition pathway and how can other proteins interact with R1? We report here a four-step mechanism for cobalt(III) transfer from CoNtaCO(3)(2-) to T and analyze the interaction of cobalt-loaded transferrin with R1. The first step in cobalt uptake by T is a fast transfer of Co(3+) and CO(3)(2-) from CoNtaCO(3)(2-) to the metal-binding site in the C-lobe of T: direct rate constant, k(1)=(1.1+/-0.1) x 10(6) M(-1) s(-1); reverse rate constant, k(-1)=(1.9+/-0.6) x 10(6) M(-1) s(-1); and equilibrium constant, K=1.7+/-0.7. This step is followed by a proton-assisted conformational change of the C-lobe: direct rate constant, k(2)=(3+/-0.3) x 10(6) M(-1) s(-1); reverse rate constant, k(-2)=(1.6+/-0.3) x 10(-2) s(-1); and equilibrium constant, K(2a)=5.3+/-1.5 nM. The two final steps are slow changes in the conformation of the protein (0.5 h and 72 h), which allow it to achieve its final thermodynamic state and also to acquire second cobalt. The cobalt-saturated transferrin in an unknown state (TCo(2)) interacts with R1 in two different steps. The first is an ultra-fast interaction of the C-lobe of TCo(2) with the helical domain of R1: direct rate constant, k(3)=(4.4+/-0.6)x10(10) M(-1) s(-1); reverse rate constant, k(-3)=(3.6+/-0.6) x 10(4) s(-1); and dissociation constant, K(1d)=0.82+/-0.25 muM. The second is a very slow interaction of the N-lobe of TCo(2) with the protease-like domain of R1. This increases the stability of the protein-protein adduct by 30-fold with an average overall dissociation constant K(d)=25+/-10 nM. The main trigger in the R1-mediated iron acquisition is the ultra-fast interaction of the metal-loaded C-lobe of T with R1. This step is much faster than endocytosis, which in turn is much faster than the interaction of the N-lobe of T with the protease-like domain. This can explain why other metal-loaded transferrins or a protein such as HFE-with a lower affinity for R1 than iron-saturated transferrin but with, however, similar or higher affinities for the helical domain than the C-lobe-competes with iron-saturated transferrin in an unknown state towards interaction with R1.

Background: In blood plasma, copper is transported in form of ceruloplasmin. Ceruloplasmin is not only a simple transport protein, it is a multifunctional protein with other various physiological functions. At least a part of these functions is connected with ceruloplasmin's enzymatic activity., Aim of the Study: The effect of alcoholic fatty liver on the metabolism of copper and ceruloplasmin was studied., Material and Methods: Patients suffering from alcoholic liver steatosis were enrolled in the study. The serum levels of copper, apoceruloplasmin and oxidase activity of ceruloplasmin were determined., Results: The copper level in patients with liver steatosis was moderately decreased in comparison to healthy controls. The difference in apoceruloplasmin levels between patients with liver steatosis and healthy controls was not significant, but the specific activity of ceruloplasmin in patients was significantly decreased in comparison to controls (0.59 vs. 0.82, p<0.001). The decreased specific activity of ceruloplasmin in spite of normal levels of apoceruloplasmin in patients with alcoholic liver steatosis suggested some problems in copper metabolism in these patients., Conclusions: The results of our study showed disturbances in copper and ceruloplasmin metabolism in patients with alcoholic liver steatosis. We can conclude that the determination of blood plasma ceruloplasmin level on the basis of its enzymatic activity is better than a simple determination of apoceruloplasmin (Tab. 2, Ref. 14).

Objectives: To investigate the effect of viral hepatitis on serum levels of apo- and lipoproteins in different forms of liver diseases., Materials and Methods: One hundred and thirty adult patients and 100 healthy age- and gender-matched control individuals participated in this study. Patients were grouped according to four types of liver disease: acute viral hepatitis, chronic active hepatitis (CAH), cirrhosis of the liver and fulminant hepatic failure., Results: Hepatitis B virus, C virus and E virus (HEV) infections were recorded in different combinations in these patients, but viral infections of hepatitis A and D were not seen in any of the patient groups. The results of lipo- and apoprotein analysis showed different patterns. The low-density lipoprotein value was high in the CAH group. In the other three groups, low-density lipoprotein level was comparable to the control value. The high-density lipoprotein level (p = 0.02) was significantly low in all groups except in the cirrhosis group. Apo-A was significantly reduced in the acute viral hepatitis and fulminant hepatic failure groups, whereas Apo-B level was low in the CAH and cirrhosis groups. The lipoprotein (a) level in these groups was low, compared to control., Conclusion: No apparent relationship was observed between etiological viruses and ensuing changes in lipid/lipoprotein profile..

We studied parameters of structural and oxidative modification of low density lipoproteins (LDL) in 80 men aged 35 - 65 years with coronary atherosclerosis verified at coronary angiography in comparison with 80 men of the same age without ischemic heart disease. Biochemical studies included determination of parameters of lipid blood composition by enzymatic methods, subfractional LDL profile by electrophoresis in 2 - 16% gradient polyacrylamide gel, degree of oxidative modification of lipid (lipid peroxidation products) and apoprotein (carbonyl groups) components of LDL by fluorometric and spectrophotometric methods. In men with coronary atherosclerosis we revealed elevated level of small dense LDL and substantially oxidized lipid and apoprotein components of these particles. Independent association of structural and oxidative modification was shown.

Objective: To investigate the role of serum cholesterol ester transfer protein (CETP) and the metabolism of various lipids including apoproteins in patients with type 2 diabetes., Materials and Methods: The relationships between serum concentrations of CETP and various lipids and apoproteins were investigated in 193 patients with type 2 diabetes mellitus and 68 age-matched healthy subjects. Serum CETP concentrations were measured by an enzyme-linked immunosorbent assay., Results: Serum CETP values were lower in diabetic patients than in healthy controls (p < 0.01). Female diabetic patients had significantly higher CETP concentrations than male patients. Serum CETP concentrations exhibited a significant positive correlation with serum concentrations of cholesterol (TC) and beta-lipoproteins in diabetic patients (r = 0.485, p = 0.013). Patients with relatively high serum concentrations of high-density lipoprotein cholesterol (HDL-C) tended to have much lower CETP concentrations than patients with lower HDL-C concentrations. Serum CETP concentrations showed significant positive correlations with those of apoproteins B (Apo B; r = 0.384, p = 0.024) and E (Apo E; r = 0.341, p = 0.035)., Conclusion: The data indicate that serum CETP is closely involved in the metabolism of TC, beta-lipoprotein, Apo B and Apo E in type 2 diabetic patients., (2007 S. Karger AG, Basel)

Background: To study the relationship between lipid profile and body mass index (BMI) in children after a 5-year follow-up., Method: A total of 281 children were evaluated at the ages of 6 and 11 years. Total cholesterol (TC), triglycerides (TG), high-density lipoprotein cholesterol (HDL-C), and apoproteins A1 (Apo A) and B100 (Apo B) were measured. Low-density lipoprotein cholesterol (LDL-C) was determined and the Apo B/Apo A, TC/HDL-C, LDL-C/HDL-C indexes, and the atherogenic index were calculated. BMI was also calculated (BMI 5 kg/m2). Evolution parameters were calculated (EVO 5 value 11 years - value 6 years). Associations between BMI and lipid profile were studied., Results: The prevalence of obesity (according to the criteria of the International Obesity Task Force) was 4.98 % (6 years) and 16,72 % (11 years). In children who were in the fourth BMI quartile at the age of 11 years, LDL-C/HDL-C and TC/HDL-C levels were significantly higher and than those in children in the first quartile but HDL-C and Apo A levels were lower. A significant positive correlation was found between the evolution of BMI and the four indexes studied and TG, but this correlation was negative for HDL-C and Apo A. The evolution of the indexes was positive in 11-year-old obese children and negative in nonobese children., Conclusions: Lipid profile was worse in 11-year-old children in the fourth BMI quartile than in the remaining children. Obese children had higher values of the indexes studied, supporting the importance of obesity as a cardiovascular risk factor.

The present study evaluated the effects of moderate intakes of myristic acid (MA), at 1.2% and 1.8% of total energy (TE), associated with a 0.9% TE intake of alpha-linolenic acid (ALA) on lipid and fatty acid profiles and red blood cell membrane fluidity. Twenty-nine monks without dyslipidaemia were enrolled in a 1-year nutritional study in which two experimental diets were tested for 3 months each: diet 1, MA 1.2 % and ALA 0.9%; diet 2, MA 1.8% and ALA 0.9%. A control diet (MA 1.2%, ALA 0.4%) was given 3 months before diets 1 and 2. Thus, two different levels of MA (1.2%, 1.8%) and ALA (0.4%, 0.9%) were tested. Intakes of other fatty acids were at recommended levels. Samples were obtained on completion of all three diets. For fluidity analysis, the red blood cells were labelled with 16-doxylstearate and the probe incorporated the membrane where relaxation-correlation time was calculated. Diet 1 was associated with a decrease in total cholesterol, in LDL-cholesterol, in triacylglycerols and in the ratio of total to HDL-cholesterol; ALA and EPA levels were increased in both phospholipids and cholesterol esters. Diet 2 was associated with a decrease in triacylglycerols and in the ratios of total to HDL-cholesterol and of triacylglycerols to HDL-cholesterol, and with an increase in HDL-cholesterol; EPA levels were decreased in phospholipids and cholesterol esters. Red blood cell membrane fluidity was increased in both diets (P<0.0001), but the higher increase was obtained with diet 1, mainly in the oldest subjects. Intakes of myristic acid (1.2%TE) and ALA (0.9%TE), both mainly in the sn-2 position, were associated with favourable lipid and n-3 long-chain fatty acid profiles. These beneficial effects coexisted with particularly high membrane fluidity, especially among the oldest subjects.

Clinical evidence indicates that a low level of high-density lipoprotein cholesterol (HDL-C) is a major risk of atherosclerosis. Raising HDL-C reduces this risk significantly, making HDL-C levels an important target of treatment for dyslipidaemia, especially in pre-existent atherosclerosis. HDL-C is protective against atherosclerosis, largely due to its function of reverse cholesterol transport. Additionally, some important roles include fibrinolysis, antioxidant functions, and reduction of platelet aggregability. A number of agents potentially modify HDL favourably. Niacin is the most potent HDL-C raising agent currently available in clinical practice, followed by fibrates. CETP inhibitors show greater HDL-C rising, but are still used in trial settings only. HDL mimetic agents are another group of agents that offer much promise. Clinical outcome data are awaited for these newer therapeutic agents.

ApoCI (apolipoprotein CI) is a potent inhibitor of plasma CETP [CE (cholesteryl ester) transfer protein]. The relevance of apoCI overexpression as a method for CETP blockade in vivo was addressed in the present study in CETPTg/apoCITg mice (mice expressing both human CETP and apoCI). Despite a significant reduction in specific CETP activity in CETPTg/apoCITg mice compared with CETPTg mice [transgenic mouse to human CETP; 46.8+/-11.1 versus 101.8+/-25.7 pmol x h(-1).(mug of plasma CETP)(-1) respectively; P<0.05], apoCI overexpression increased both the CETP mass concentration (3-fold increase; P<0.05) and the hepatic CETP mRNA level (4-fold increase, P<0.005), leading to an increase in total plasma CE transfer activity (by 39%, P<0.05). The ratio of apoB-containing lipoprotein to HDL (high-density lipoprotein) CE was 10-fold higher in CETPTg/apoCITg mice than in apoCITg mice (P<0.0005). It is proposed that the increased CETP expression in CETPTg/apoCITg mice is a direct consequence of liver X receptor activation in response to the accumulation of cholesterol-rich apoB-containing lipoproteins. In support of the latter view, hepatic mRNA levels of other liver X receptor-responsive genes [ABCG5 (ATP-binding cassette transporter GS) and SREBP-1c (sterol-regulatory-binding protein-1c)] were higher in CETPTg/apoCITg mice compared with CETPTg mice. In conclusion, overexpression of apoCI, while producing a significant inhibitory effect on specific CETP activity, does not represent a suitable method for decreasing total CE transfer activity in CETPTg/apoCITg mice, owing to an hyperlipidaemia-mediated effect on CETP gene expression.

Background: Current reports claim that small and dense LDL particles are more atherogenic than larger LDL particles. There are many studies presenting LDL subclass distribution in adults, but there is not enough data regarding children in the literature on this problem. The aim of our study was to examine LDL subclass distribution in healthy children in the Republic of Macedonia., Materials/methods: Plasma LDL subclasses in 100 children aged 9-18 years were analyzed using non-denaturing polyacrilamide gradient (3-31%) gel electrophoresis. Conventional plasma lipid and apoprotein parameters thought to be related to LDL size were determined as well., Results: The results obtained showed the prevalence of large LDL subclasses (phenotype A) in 89% of the children, whereas small LDL subclasses (phenotype B) were observed in 11%. The mean LDL size was 26.37 +/- 0.68 nm, and there was no difference between gender groups. No association was noted between LDL size and plasma lipid and apoprotein levels, age, or BMI., Conclusions: LDL size and distribution is not gender- or age-dependent, or influenced by plasma lipid and apoprotein concentrations in childhood. This suggests that analysis of LDL subclass phenotype may provide better information on the risk of atherosclerosis development in adulthood.

The kinetics and thermodynamics of Bi(III) exchange between bismuth mononitrilotriacetate (BiL) and human serum transferrin as well as those of the interaction between bismuth-loaded transferrin and transferrin receptor 1 (TFR) were investigated at pH 7.4-8.9. Bismuth is rapidly exchanged between BiL and the C-site of human serum apotransferrin in interaction with bicarbonate to yield an intermediate complex with an effective equilibrium constant K(1) of 6 +/- 4, a direct second-order rate constant k(1) of (2.45 +/- 0.20) x 10(5) M(-1) s(-1), and a reverse second-order rate constant k(-1) of (1.5 +/- 0.5) x 10(6) M(-1) s(-1). The intermediate complex loses a single proton with a proton dissociation constant K(1a) of 2.4 +/- 1 nM to yield a first kinetic product. This product then undergoes a modification in its conformation followed by two proton losses with a first-order rate constant k(2) = 25 +/- 1.5 s(-1) to produce a second kinetic intermediate, which in turn undergoes a last modification in the conformation to yield the bismuth-saturated transferrin in its final state. This last process rate-controls Bi(III) uptake by the N-site of the protein and is independent of the experimental parameters with a constant reciprocal relaxation time tau(3)(-1) of (3 +/- 1) x 10(-2) s(-1). The mechanism of bismuth uptake differs from that of iron and probably does not involve the same transition in conformation from open to closed upon iron uptake. The interaction of bismuth-loaded transferrin with TFR occurs in a single very fast kinetic step with a dissociation constant K(d) of 4 +/- 0.4 microM, a second-order rate constant k(d) of (2.2 +/- 1.5) x 10(8) M(-1) s(-1), and a first-order rate constant k(-d) of 900 +/- 400 s(-1). This mechanism is different from that observed with the ferric holotransferrin and implies that the interaction between TFR and bismuth-loaded transferrin probably takes place on the helical domain of the receptor which is specific for the C-site of transferrin and HFE. The relevance of bismuth incorporation by the transferrin receptor-mediated iron acquisition pathway is discussed.

Aim: To explore the relationship between retinal vessel atherosclerosis and serum concentrations of total cholesterol, triglycerides, low density lipoprotein (LDL) cholesterol, high density lipoprotein (HDL) cholesterol, HDL(2) cholesterol, HDL(3) cholesterol, apoprotein B, apoprotein A-I, and apoprotein A-II., Methods: Lipids and lipoproteins were measured in 101 male patients aged between 26 and 69 years (median, 48 years), and the degree of their retinal vessels atherosclerosis (stage 1-4 according to Scheie) was determined. Diabetics, alcoholics, hypertensive, extremely obese patients, and patients with thyroid, liver, or kidney diseases were excluded from the analysis. The results were compared with those in 47 apparently healthy men aged between 22 and 65 years (median, 47 years) with no retinal vessel changes. Direct ophthalmoscopy for the fundus examination was carried out by a single ophthalmologist unaware of the patients' group., Results: Retinal vessel atherosclerosis strongly correlated with total serum cholesterol, LDL cholesterol, triglycerides, and apoprotein B concentrations. The higher these lipid and apoprotein values, the more advanced the stage of the disease. No significant association between HDL cholesterol as well as HDL(2) and HDL(3) cholesterol and atherosclerosis of retinal arteries could be proved but an inverse association, although a very weak one, between the low serum concentration of HDL cholesterol and the stage of the retinal artery atherosclerosis was found., Conclusion: Changes in lipoproteins and apoproteins associated with atherosclerosis of the retinal arteries correspond well to those associated with coronary artery atherosclerosis.

Objective: This double-blind, 15-month pilot study was designed to investigate the effect of soy protein isolate with varying concentrations of isoflavones on early postmenopausal bone loss and lipids., Design: A total of 65 women, with a mean age of 55 years and 7.5 years since menopause, were randomized to one of three groups; soy protein with 96 mg isoflavones/day, soy with 52 mg isoflavones/day, or soy without isoflavones (< 4 mg isoflavones/day). Soy was given for 9 months and then discontinued; participants were followed for an additional 6 months. Bone mineral density (BMD) and blood lipids were measured during this time., Results: Measurement of serum isoflavones at 3 months showed dose-related increases in the three groups. There was no significant effect of the soy supplements on BMD of the spine or femoral neck in any of the three groups. BMD increased significantly in the trochanter at 9 months (P = 0.0219) and at 15 months (P < 0.05) in the group given isoflavone-free soy compared with the other two groups. There was no significant effect of soy on lipid metabolism at the end of the intervention., Conclusion: The present study did not find a significant positive effect of soy protein isolate supplemented with isoflavones on BMD and the serum lipid profile in early postmenopausal women.

Twenty-nine women aged 35 years old or more, using triphasic combined oral contraceptive (COC) were evaluated during six cycles for the following parameters: total cholesterol, low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C) and its subfraction HDL2, triglycerides, apoproteins A and B, Castelli risk index I and II (cholesterol/HDL-C and LDL-C/HDL-C) and apoprotein ratio (apoprotein B/apoprotein A). The same laboratory measurements were done in a control group of 49 non-COC-user women. The results showed that there were no differences on most of the studied parameters between user and nonuser women. There was a significant reduction of HDL-C and HDL2-C, although within the normal range. In addition, it was observed a significant increment of triglycerides and apoprotein B at 6 months of follow-up only in user group (p < 0.05), although within the normal range. It is concluded that the use of levonorgestrel triphasic COC appeared to have no additional adverse impact when used by women aged over 35 years. Further studies are needed to obtain conclusive data.

The number of dual bonds (DB) was determined by the ozonization method in the lipid fraction of blood serm (BS) in healthy subjects and in patients with hyperlipidemia (HLP). Single-direction changes were detected in the DB quantity and in the lipid level in case of HLP; besides, a highly reliable positive correlation was established between the DB concentration and the basic BS lipid classes. The results suggest that an increasing DB quantity in HLP is preconditioned by a growing lipid contents and show their tense relation with components of the lipid transport system (LTS). The determination of the DB concentration, as of an index of the total unsaturation of the BS lipid fraction, provides for obtaining extra data useful in describing the LTS in different-type impairments of lipid metabolism.

Platelet-activating factor acetylhydrolase (PAF-AH) is a phospholipase A2 associated with lipoproteins that hydrolyzes platelet-activating factor (PAF) and oxidized phospholipids. We have developed an ELISA for PAF-AH that is more sensitive than previous methods, and have quantified HDL-associated and non-HDL-associated PAF-AH in healthy, hyperlipidemic, and diabetic subjects. In healthy subjects, plasma total PAF-AH concentration was positively correlated with PAF-AH activity and with plasma total cholesterol, triacylglycerol, LDL cholesterol and apolipoprotein B (apoB) concentrations (all P < 0.01). HDL-associated PAF-AH concentration was correlated positively with plasma apoA-I and HDL cholesterol. Subjects with hyperlipidemia (n = 73) and diabetes mellitus (n = 87) had higher HDL-associated PAF-AH concentrations than did controls (P < 0.01). Non-HDL-associated PAF-AH concentration was lower in diabetic subjects than in controls (P < 0.01). Both hyperlipidemic and diabetic subjects had lower ratios of PAF-AH to apoB (P < 0.01) and higher ratios of PAF-AH to apoA-I (P < 0.01) than did controls. Our results show that the distribution of PAF-AH mass between HDLs and LDLs is determined partly by the concentrations of the lipoproteins and partly by the mass of enzyme per lipoprotein particle, which is disturbed in hyperlipidemia and diabetes mellitus.

Protein ion charge state distributions in electrospray ionization mass spectra have a potential to provide a wealth of information on protein dynamics, because they contain contributions from all protein conformers present in solution. Such ionic contributions often overlap, limiting the amount of useful information that can be extracted from the spectra. This difficulty is overcome in the present work by using a chemometric approach, which allows spectral deconvolution to be carried out and information on individual protein conformers to be extracted. Experiments are carried out by acquiring a series of spectra over a range of near-native and denaturing conditions to ensure significant changes in the conformers' populations. A total number of protein conformers sampled in the course of the experiments is determined by subjecting the set of collected spectra to singular value decomposition. Ionic contributions of each conformer to the total signal are then determined using a supervised optimization routine. Validation of the method has been carried out by monitoring acid- and alcohol-induced equilibrium states of well-characterized model proteins, chymotrypsin inhibitor 2 (two states), ubiquitin (three states) and apo-myoglobin (four states). For each of the model proteins, a new chemometric procedure yielded a picture of protein dynamics that was in excellent agreement with their documented behavior (as studied with other biophysical tools). The new method appears to be well-suited for monitoring protein dynamics in highly heterogeneous systems consisting of multiple proteins sampling a range of conformational states.

Background and Aim: To evaluate the role of lipoprotein abnormalities as risk factors for macroangiopathy in Type 2 diabetes., Methods and Results: This prospective nine-year follow-up study involved 113 Type 2 diabetic patients (50 men and 63 women, mean age 66.9 +/- 9.9 years), 37 of whom had clinical signs of coronary heart disease (CHD) and cerebrovascular disease (CVD) at baseline. During the follow-up, 32 patients died: 17 of CHD, five of CVD, and 10 of non-vascular causes. The patients who died because of vascular disease were more frequently smokers, and had baseline symptoms of vascular disease; they were also significantly different from the other patients insofar as they were older, and had higher fasting plasma glucose levels, lower fasting C-peptide levels, and lower apoprotein (apo) AII, apo CII, apo CIII and apo E levels. Univariate analysis showed that baseline symptoms of vascular disease, current smoking, age, high fasting plasma glucose levels, low fasting C-peptide levels, and low apo AII, apo CII, apo CIII and apo E levels [but not cholesterol, triglyceride, high-density lipoprotein (HDL)-cholesterol or qualitative low-density lipoprotein or HDL abnormalities] were associated with cardiovascular mortality. Multivariate analysis showed that only age, smoking, glycated hemoglobin (HbA1c) and fasting C-peptide levels were significant independent determinants of macrovascular death., Conclusions: In Type 2 normolipidemic diabetic patients, only age, smoking, HbA1c and fasting C-peptide levels are independent vascular risk factors. The differences in apo concentrations between patients with and without vascular disease may reflect qualitative abnormalities in plasma lipoproteins related to vascular disease.

Both renal failure and type 2 diabetes may contribute synergistically to the dyslipemia of diabetic renal failure with the development of atherosclerosis as the possible consequence. It has not yet been conclusively evaluated whether diabetic patients with end-stage renal failure under maintenance hemodialysis (HD) show accentuated alterations in plasma lipids and lipoproteins in comparison to nondiabetics under HD. These abnormalities would involve hepatic lipase activity and the regulation of triglyceride-rich lipoprotein metabolism. The purpose of the present study was to evaluate whether type 2 diabetic patients undergoing HD exhibited a lipid-lipoprotein profile different from that of nondiabetic hemodialyzed patients. We compared plasma lipids, apoprotein (apo) A-I and B, and lipoprotein parameters among 3 groups: 25 type 2 diabetics, 25 nondiabetics, both undergoing HD, and 20 healthy control subjects. Intermediate-density lipoprotein (IDL) and low-density lipoprotein (LDL) were isolated by sequential ultracentrifugation. Hepatic lipase activity was measured in postheparin plasma. Both groups of HD patients showed higher triglyceride and IDL cholesterol (P <.001), and lower high-density lipoprotein (HDL) cholesterol (P <.01) and apo A-I (P <.001) levels compared to the control group, even after adjustment for age and body mass index (BMI). However, no differences were found in lipid, lipoprotein, and apoprotein concentrations between diabetic and nondiabetic HD patients, except for high LDL triglyceride content of diabetic HD patients (P <.01). Nondiabetics undergoing HD also presented higher LDL triglyceride levels than controls (P <.05). LDL triglyceride correlated with plasma triglycerides (r = 0.51, P <.001). A lower LDL cholesterol/apo B ratio was found in each group of HD patients in comparison to controls (P <.02). Comparing the diabetic and nondiabetic patients, hepatic lipase activity remained unchanged, but significantly lower than control subjects (P <.001). Hepatic lipase correlated with log-triglyceride (r = -0.31, P <.01), IDL cholesterol (r = -0.41, P <.001), and LDL triglyceride (r = -0.32, P <.01). In conclusion, both diabetic and nondiabetic HD patients shared unfavorable alterations in lipid-lipoprotein profile not different between them but different from a healthy control group. The only difference between the groups of HD patients was a significant LDL triglyceride enrichment, which correlated negatively with hepatic lipase activity. Lipoprotein abnormalities in HD patients would enhance their risk for the development of atherosclerosis., (Copyright 2003, Elsevier Science (USA). All rights reserved.)

Aim: To study specific features of lipid plasm profile in patients with metabolic syndrome (MS) and manifest disorder of carbohydrate metabolism (CM) as well as their relationships with clinical symptoms of MS., Materials and Methods: Contents of lipids and apolipoproteins of plasm was measured on an empty stomach in 30 patients (18 females and 12 males) with MS and manifest disorder of CM (diabetes mellitus type 2 at the stage of good and satisfactory compensation of CM without terminal complications)., Results: The patients were found to have a subnormal level of HDLP cholesterol, high levels of total cholesterol, LDLP cholesterol and LP alpha. Hypertriglyceridemia occurred only in 31.4% patients (TG < 200 mg/dl). Mean TG level was 173.69 +/- 21.52 mg/dl. TG and diastolic arterial pressure, fasting glycemia and waist circumference significantly correlated. If the WC/TC was 1.0 and higher, the antiatherogenic plasm factor--HDLP cholesterol--significantly lowered. Positive correlation existed between TG and total cholesterol, LDLP cholesterol, apoB, LDLP cholesterol/HDLP cholesterol, apoB/apoA1, while negative correlation was seen between TG and HDLP cholesterol levels. Patients with hypertriglyceridemia had significantly higher levels of total cholesterol, LDLP cholesterol, apoB, apoB/apoA1., Conclusion: Plasm levels of TG most significantly reflect severity of basic clinical manifestations of MS: abdominal obesity, arterial hypertension, CM compensation. The presence of hypertriglyceridemia in MS patients can be considered as an indicator of high atherogenic potential of plasm.

Objective: Cardiovascular disease is the major cause of excessive mortality in patients with rheumatoid arthritis (RA). We determined whether endothelial dysfunction characterizes patients with newly diagnosed RA (n=10) compared with normal subjects (control group, n=33) and whether it is reversible with 6 months of anti-inflammatory therapy., Methods and Results: Endothelial function was determined by measuring vasodilatory responses to intrabrachial artery infusions of acetylcholine (ACh at 7.5 and 15 microg/min, low and high dose, respectively), an endothelium-dependent vasodilator, and to sodium nitroprusside (SNP, 3 and 10 micro g/min), an endothelium-independent vasodilator. Before treatment, blood flow responses (fold increase in flow) to low-dose SNP were 30% lower in the RA versus the control group (4.1+/-0.4-fold versus 5.9+/-0.5-fold, respectively), and responses to high-dose SNP were 34% lower in the RA group versus the control group (5.1+/-0.6-fold versus 7.7+/-0.7-fold, respectively; P<0.001). The responses to low-dose ACh were 50% lower in the RA group versus the control group (3.0+/-0.5-fold versus 6.6+/-0.7-fold, respectively), and responses to high-dose ACh were 37% lower in the RA group versus the control group (5.0+/-0.4-fold versus 7.9+/-0.8-fold, respectively; P<0.001). After therapy, clinical and laboratory markers of inflammation had significantly decreased. Blood flow responses to ACh increased significantly (P=0.02)., Conclusions: We conclude that newly diagnosed patients with RA have vascular dysfunction, which is reversible with successful therapy. Therefore, early suppression of inflammatory activity may reduce long-term vascular damage.

Background: Postmenopausal women are at increased risk of cardiovascular disease. Exogenous estrogens may potentially lower the risk of cardiovascular disease through beneficial changes in lipoprotein profile and hemostasis. The effect on the vascular system of combined estrogen/progestagen hormone replacement therapy is less clear., Material/methods: A group of 44 peri- and postmenopausal women was studied during treatment with Femoston, a sequentially combined hormone replacement therapy consisting of 17-beta-estradiol and dydrogesterone. Comparative analyses were made between the estrogen-only phase and the combined phase of the hormone replacement therapy. Comparisons were also made against the different phases of the spontaneous menstruation cycle in 10 pre- and perimenopausal controls., Results: Femoston had a neutral effect on blood coagulation factors and had no effect on plasma glucose concentrations. With Femoston, there was a positive change in the atherogenic index and the total cholesterol/HDL cholesterol ratio., Conclusions: The findings from the present study provide evidence for the efficacy of Femoston hormone replacement therapy in terms of control of hemostasis and inducement of a potentially beneficial lipoprotein profile, which is important for reducing the risk of vascular pathology in peri- and postmenopausal women.

Objective: Our purpose was to investigate iron status parameters in preeclampsia with a view to exploring their possible contribution to the etiology., Study Design: In prepared serum samples from 40 preeclamptic women and matched pregnant control subjects at the John Radcliffe Hospital, Oxford, a number of iron status parameters were measured. Statistical analysis was by the Wilcoxon signed rank test and linear regression., Results: Serum iron concentration, ferritin, and percent saturation of transferrin were significantly higher in the preeclamptic patients than in control subjects, whereas unsaturated iron-binding capacity and apotransferrin levels were significantly lower. No difference was found in hemopexin concentrations in the two groups. Gestational age at the time of sampling was correlated (positively) with only two parameters, total and unsaturated iron-binding capacity, but only in the preeclampsia group. Eighteen percent of preeclamptic subjects had percent transferrin saturation levels in the region associated with iron overload., Conclusion: Released iron species in preeclampsia may contribute to the etiology and will exacerbate lipid peroxidation and endothelial cell injury. Given the high prevalence of heterozygosity for hemochromatosis with the associated reduced ability to exclude ingested iron, it would seem inadvisable, in the absence of evidence of iron deficiency, to give iron supplements to pregnant women at high risk for preeclampsia.

Background: Cholesterol is the dietary component that has elicited the most public interest in conjunction with coronary heart disease. However, the impact of excess dietary cholesterol intake on plasma cholesterol levels cannot be accurately predicted; therefore, its role in disease progression is not straightforward. Individual response variation can be due to factors such as ethnicity, hormonal status, obesity and genetic predisposition., Objective: The purpose of this study was to evaluate the differences that occur within the plasma compartment of normolipidemic pre-menopausal women, classified based on their response to a high dietary cholesterol challenge., Design: We recruited 51 pre-menopausal women (29 Caucasian and 22 of Hispanic origin) aged 18 to 49 years with initial plasma cholesterol concentrations ranging from 3.62 to 5.17 mmol/L. Using a cross-over research design, women were randomly allocated to an egg (640 mg additional dietary cholesterol per day) or placebo group (0 mg additional dietary cholesterol per day) initially, and the two 30 day periods were separated by a three-week washout., Results: An initial evaluation of the ethnicity effects revealed elevations in both plasma LDL-C (p < 0.0001) and HDL-C (p < 0.001) concentrations in both Hispanics and Caucasians during the high dietary cholesterol period. However, these increases were not accompanied by a change in the LDL/HDL ratio. Subjects were then classified as hypo- (< 0.05 mmol/L increase in total plasma cholesterol per each additional 100 mg of dietary cholesterol consumed per day) or hyper-responders (> or =0.06 mmol/L increase in total blood cholesterol per each additional 100 mg of dietary cholesterol consumed per day), based on their reaction to the additional dietary cholesterol provided. Hypo-responders did not experience an increase in LDL-C or HDL-C during the egg period, while both lipoproteins were elevated in hyper-responders. However, the LDL/HDL ratio, an important parameter of coronary heart disease risk, was maintained for all subjects during the egg period independent of response. Furthermore, hyper-responders had higher concentrations of apo C-III (p < 0.001), apo B (p < 0.001) and cholesterol ester transfer protein (CETP) (p < 0.05) during this period., Conclusion: These data revealed that excess dietary cholesterol does not increase the risk of developing an atherogenic lipoprotein profile in pre-menopausal women, regardless of their response classification. Although the addition of 640 mg of cholesterol to the diet did result in an increase in plasma cholesterol in hyperresponders, the LDL/HDL ratio was maintained. This result, accompanied by increases in CETP activity, leads to the speculation that hyper-responders may process the excess cholesterol in the plasma compartment through an enhancement of the reverse cholesterol transport pathway. With this mechanism identified, further measurement of additional parameters is needed to verify this conclusion.

Purpose: To evaluate the accuracy of lipoprotein lipids and apoproteins in predicting coronary heart disease (CHD) in diabetic American Indians., Methods: This study included 2099 diabetic participants of the Strong Heart Study, which is a longitudinal study of cardiovascular disease (CVD) and its risk factors in American Indians in Arizona, Oklahoma, and South and North Dakota. Diabetic participants with incident CHD (N = 126) were selected as the case group, and those without CHD or any cardiovascular events were the control group (N = 1732). Previous vascular events such as stroke were the sole exclusion criterion (N = 241). Baseline measurements of lipoprotein lipids and apoproteins were used to predict CHD diagnosed at the 4-year follow-up examination by using Receiver-Operating Characteristic (ROC) curve analysis., Results: The ratio of high-density lipoprotein (HDL) to total cholesterol had the highest area under the ROC curve (0.69 +/- 0.02). The areas for the ratios of HDL to low-density lipoprotein (LDL) cholesterol (0.68 +/- 0.02), apo AI/B (0.66 +/- 0.02), and the single component of total cholesterol (0.64 +/- 0.03) and LDL cholesterol (0.63 +/- 0.05) were not significantly different from the area for HDL/total cholesterol. However, the areas for apo B (0.64 +/- 0.02), HDL cholesterol (0.62 +/- 0.03), triglycerides (0.58 +/- 0.03), and apo AI (0.57 +/- 0.05) were significantly lower than the area for HDL/total cholesterol. Logistic regression analysis indicates that only HDL and LDL cholesterol were significant independent lipoprotein lipid and apoprotein predictors for CHD. The other significant predictors in the model were study center, age, gender, and albuminuria. The ROC area for this model is 0.75., Conclusions: HDL and LDL cholesterol were the most important independent predictors for incident CHD in diabetic American Indians. The ratios of HDL/total cholesterol, HDL/LDL cholesterol, and apo AI/B had higher accuracy for predicting CHD. Although the values for all lipoprotein lipids and apoproteins and their ratios were not large enough to definitely predict CHD, they can be used as screening tools for CHD in diabetic American Indians.

The guideline for the reference value of serum lipid(total cholesterol, LDL-cholesterol, HDL-cholesterol and triglyceride) was proposed by the Japan Atherosclerosis Society in 1997. These values are utilized for the diagnosis and treatment of hyperlipidemia to protect patients from ischemic heart disease. The upper limit of reference value was not determined by the 95 percentile range from healthy subjects, but on the basis of clinical data on serum lipid in the Japanese. This way to determine the reference value will be suitable in case of lipid, because there is a tendency to increase cholesterol level in the Japanese for these thirty years. The standardization for each lipid assay is crucial. The CDC-established Reference Methods for measuring total cholesterol(TC), HDL-C. LDL-C, and triglyceride(TG) are used to set reference values for the serum pools used for LSP(Lipid Standardization Panel) standardization. In Japan, the routine methods for cholesterol and triglyceride have been proposed by the special committee in the Japan Society of Clinical Chemistry. In contrast, homogeneous assay for HDL- and LDL-C, which are used routinely at present, have still some problems in the point of accuracy. A new ELISA for Lp(a) has been developed using antibody not reacting to kringle IV type 2. Its standardized assay, thus, will be available sooner or later. With respect to other lipid-related test such as RLP(remnant-like particles) and oxidized LDL, it is necessary to accumulate more clinical data for the determination of reference value.

We investigated whether the apolipoprotein-E (apoE) phenotype and the basal activity of low-density lipoprotein (LDL) receptor, which were reported to be the major determinants for increase in plasma LDL levels by cholesterol ingestion, have the same role in Japanese subjects whose diet is low in fat and cholesterol. Cholesterol (750 mg/d) was added to the ordinary diet as a dried egg-yolk supplement for 4 wk to 110 subjects. Plasma levels of lipids, apolipoproteins, and cholesterol in lipoprotein subfractions were measured at the beginning and end of the test period. Phenotyping of apoE was determined by an isoelectric focusing-immunoblotting method, and LDL receptor activity in lymphocytes was determined by flow cytometry. Plasma levels of cholesterol in less-dense LDL (LDL(1)) and less-dense high-density lipoprotein (HDL(2)) were slightly but significantly increased, 3.4% and 4.1%, respectively, by cholesterol ingestion, but the increases were not statistically significant in any of E2, E3, and E4 groups. The distribution of the apoE phenotype was equivalent in all three LDL-cholesterol groups (no change, increase, and decrease by cholesterol ingestion). Plasma levels of LDL, LDL(1), and LDL(2) cholesterol were not significantly increased in the three groups of subjects with lymphocyte LDL-receptor activities (low, medium, and high). As with apoE phenotype, LDL-receptor activities were the same in all three LDL-cholesterol groups. In addition, there were no significant correlations between LDL-receptor activity and changes in plasma levels of lipids, apolipoproteins, and cholesterol in lipoprotein subfractions. Therefore, we concluded that cholesterol ingestion significantly increases plasma levels of less-dense LDL and HDL, but neither apoE phenotype nor basal LDL-receptor activity explain the variability in changes in plasma lipoprotein subfractions by cholesterol ingestion in Japanese subjects.

The brain efflux index method is used to examine the extent to which transferrin effluxes from brain to blood across the blood-brain barrier (BBB) following intracerebral injection. Whereas high-molecular-weight dextran is nearly 100% retained in brain for up to 90 min after intracerebral injection in the Par2 region of the parietal cortex of brain, there is rapid efflux of transferrin from brain to blood across the BBB. The efflux of apotransferrin is 3.5-fold faster than the efflux of holo-transferrin. The brain to blood efflux of apotransferrin is completely saturable by unlabeled transferrin, but is not inhibited by other plasma proteins. These studies provide evidence for reverse transcytosis of transferrin from brain to blood across the BBB. As circulating transferrin is known to undergo transcytosis across the BBB in the blood-to-brain direction, these studies support the model of bidirectional transcytosis of transferrin through the BBB in vivo.