Serum Fractions Research Articles

Organic consequences of ileal transposition in rats with diet-induced obesity

AbstractINTRODUCTION: The clinical management of metabolic syndrome - especially diabetes mellitus type 2 - is notoriously complex due to the progressive nature of this disease. At present, there is a need for a surgical procedure that is effective for the treatment of diabetes mellitus type 2, even in non-obese individuals. The isolated ileal transposition theory could lead to an effective alternative therapy. This intervention has not yet been performed in humans, and there are no reports of its use in an experimental model of diet-induced metabolic syndrome. OBJECTIVES: The objective of this study is to evaluate the physiological effects of ileal transposition in rats with diet-induced metabolic syndrome. The effects of this procedure on glucose and lipid metabolism will be assessed. METHODS: Forty 12-week-old male rats (albino Rattus norvegicus, Wistar, 2BAW, heterogeneous) will be divided into four groups of 10 animals each: the ileal transposition group (TG) comprising animals on a hypercaloric-hyperlipidic diet; the sham group (SG) containing animals that receive the same diet and undergo the sham surgery; control group 1 (CG1), which will receive a hypercaloric-hyperlipidic diet and will not undergo surgery; and control group 2 (CG2), which will consume standard feed and will not undergo surgery. The surgeries will be performed in 20-week-old animals. Blood samples for laboratory testing will be collected from 12-week-old animals on the day of surgery and after eight postoperative weeks, following determination of the weights of the animals and the administration of anesthesia. The levels of serum glucose, insulin, triglycerides, total cholesterol and fractions, glucagon-like peptide-1, C-peptide and glycated hemoglobin will be assessed in all of the animals. The insulin tolerance test will be performed using PRISMA software, and insulin resistance will be calculated by the HOMA-IR indirect test. On specific days, two 20-week-old rats will be separated and randomly distributed in TG and SG. These animals will be followed until the eighth postoperative week. Subsequently, they will be euthanized, and the retroperitoneal and periepididymal fat deposits will be collected and weighed using a precision scale. In addition, the pancreas, liver and intestinal segments will be sent for pathological and immunohistochemical studies.

Alteration upon Oral Ingestion of Monosodium Glutamate in Various Lipid and Lipoprotein Fractions in Serum of Adult Male Rat

Monosodium glutamate, a sodium salt of glutamic acid is commonly used as food additive in Chinese, Japanese and ready to serve foods all over the world as a flavor enhancer . Concomitantly, there is a tremendous increase in the incidences of coronary heart disease and atherosclerosis. So, the present study was conducted to elucidate the ef fect of oral ingestion of monosodium glutamate at dose levels of 4 and 8 mg/g body weight for 7-consecutive days to normal adult male rats by evaluating the changes in serum lipid and lipoprotein fractions, glucose and protein levels. A significant increase was observed in serum total lipids, phospholipids and free fatty acids in monosodium glutamate ingested rats with respect to normal healthy control animals whereas cholesterol levels were remained normal. Monosodium glutamate ingestion produced hyper glycemia by significantly increasing the glucose levels in 4 and 8mg/g. body weight ingested MSG rats and a nominal increase was seen in serum protein levels in MSG ingested rats. The oral ingestion of monosodium glutamate at dose levels of 4 mg g -1 body weight and above significantly increased the levels of low density lipoproteins, very low density lipoproteins whereas a significant decrease was observed in high density lipoproteins. All the above observations suggested that oral ingestion of monosodium glutamate at dose levels of 4mg/g body weight and above for 7 consecutive days produced hyperlipidemia, hyperlipoproteinemia and hyperglycemia and thereby could be responsible for the initiation of atherosclerosis.

Characterization of the human serum depletome by label‐free shotgun proteomics

While it is known that immunoaffinity depletion of abundant proteins in serum removes additional proteins beyond those targeted, there has been little characterization of the co-depleted proteins in the high abundant fraction, which we refer to here as the "depletome". We present evidence of co-depletion of non-targeted proteins in human serum using a top-20 immunodepletion column, as shown by label-free liquid chromatography mass spectrometry (LC-MS(E)) profiling. This led to identification of 147 proteins which were specific for this fraction and comprised proteins with functions predominantly in binding and transport of nucleotides, metal ions, carbohydrates and lipids. These results suggest that further studies on this commonly ignored serum fraction may provide new insights into clinical proteomics.

Design, Synthesis, ADME Properties, and Pharmacological Activities of β‐Alanyl‐D‐histidine (D‐Carnosine) Prodrugs with Improved Bioavailability

β-Alanyl-D-histidine (D-CAR, the enantiomer of the natural dipeptide carnosine) is a selective and potent sequestering agent of reactive carbonyl species (RCS) that is stable against carnosinase, but is poorly absorbed in the gastrointestinal tract. Herein we report a drug discovery approach aimed at increasing the oral bioavailability of D-CAR. In our study we designed, synthesized, and evaluated a series of novel lipophilic D-CAR prodrugs. The considered prodrugs can be divided into two categories: 1) derivatives with both terminal groups modified, in which the carboxyl terminus is always esterified while the amino terminus is protected by an amidic (N-acetyl derivatives) or a carbamate (ethyloxy or benzyloxy derivatives) function; 2) derivatives with only one terminus modified, which can be alkyl esters as well as amidic or carbamate derivatives. The prodrugs were designed considering their expected lipophilicity and their hydrolysis predicted by docking simulations on the most important human carboxylesterase (hCES1). The stability and metabolic profile of the prodrugs were studied by incubating them with rat and human serum and liver fractions. The octyl ester of D-CAR (compound 13) was chosen as a candidate for further pharmacological studies due to its rapid hydrolysis to the bioactive metabolite in vitro. Pharmacokinetic studies in rats confirmed the in vitro data and demonstrated that the oral bioavailability of D-CAR is increased 2.6-fold if given as an octyl ester relative to D-CAR. Compound 13 was then found to dose-dependently (at daily doses of 3 and 30 mg kg(-1) equivalent of D-CAR) decrease the development of hypertension and dyslipidemia, to restore renal functions of Zucker fa/fa obese rats, and to inhibit the carbonylation process (AGEs and pentosidine) as well as oxidative stress (urinary 8-epi-prostaglandin F2α and nitrotyrosine). A plausible mechanism underlying the protective effects of 13 is RCS sequestration, as evidenced by the significant increase in the level of adduct between CAR and 4-hydroxy-trans-2-nonenal (HNE, the main RCS generated by lipid oxidation) in the urine of treated animals.

Comparative Drug Exsorption in the Perfused Rat Intestine

The factors affecting drug exsorption into the gastrointestinal tract are uncertain. In this study, the intestinal clearance (CLi) of compounds which vary in their lipophilicity, serum protein binding, molecular weight and ionic charge at physiological pH, has been measured. Male Sprague-Dawley rats with ligated bile ducts were infused with the test compounds through the jugular vein. The small intestine was intubated and perfused with Tyrode solution at 20 mL h-1. The CLi of the compounds investigated (urea, polyethylene glycol, inulin, albumin, dextran, barbituric acid, salicylic acid, thiobarbital, thiopental, thioseconal, theophylline, S-disopyramide and quinidine) was determined under anaesthesia by dividing the rate of a component's appearance rate in the perfusate by its carotid arterial concentration. Serum protein binding of the compounds was determined by equilibrium dialysis. The n-octanol-water partition coefficients of the compounds were measured as indices of lipophilicity. The CLi values of dextran, albumin, inulin, polyethylene glycol and urea were 0.56, 1.03, 4.5, 4.8 and 12.0 mL h-1, respectively. The larger the molecular weight of a compound, the smaller its CLi. The molecular weight is apparently one of the major determinants of CLi. Thiobarbital, thiopental and thioseconal are compounds of similar structure with increasing lipophilicity and serum protein binding. The CLi of thiobarbital, thiopental and thioseconal was proportional to the unbound fraction in serum. The unbound clearance (CLui) of three thiobarbiturates were similar (approximately 11 mL h-1). The unbound fraction of drug in serum appears to be a factor determining their CLi. Barbituric acid and salicylic acid, two acidic compounds, showed a low CLi (less than 1 mL h-1).(ABSTRACT TRUNCATED AT 250 WORDS)

Interaction of cyclosporin A with human lipoproteins.

The ultracentrifugal fractionation of human serum after previous incubation with cyclosporin A showed that, in healthy fasting individuals, 8% of cyclosporin A was found in the very low density lipoproteins (VLDL), 31% in the low density lipoproteins (LDL), 46% in the high density lipoproteins (HDL) and 15% in the non-lipoprotein protein fraction. In non-fasted, healthy and in non-fasted, lipaemic individuals, 7 and 6% of cyclosporin A was found in chylomicrons, 9 and 13% in VLDL, 28 and 30% in LDL, 39 and 37% in HDL, and 12 and 13% in the non-lipoprotein protein fraction, respectively. In patients receiving cyclosporin A the distribution varied from 12 to 19% in VLDL, 21 to 28% in LDL, 33 to 43% in HDL, and 13 to 20% in non-lipoprotein proteins. The interaction between cyclosporin A and isolated normal human lipoproteins was also studied by ultrafiltration. All lipoproteins exhibited a non-saturable, low affinity, high capacity uptake for cyclosporin A. Analysis of the uptake by phospholipid vesicles showed a similar uptake, suggesting that cyclosporin A dissolves in the lipophilic portion of the lipoprotein molecule rather than being associated with specific binding sites.

Open‐label, ascending dose, prospective cohort study evaluating the antiviral efficacy of Rosuvastatin therapy in serum and lipid fractions in patients with chronic hepatitis C

HMG CoA reductase inhibition suppresses in vitro HCV replication through depletion of cellular sterol proteins such as geranylgeraniol. Our aims were to prospectively evaluate the changes in serum and lipid fraction HCV RNA with Rosuvastatin in non-responder (NR) patients with CHC. A total of 11 patients with CHC genotype-1 received Rosuvastatin at 20 mg qd (weeks 0-4), 40 mg qd (weeks 5-12), with 4 week follow up. Lipid fractions were separated by a sucrose density gradient ultracentrifugation, HCV RNA determined at wks 0, 2, 4, 8, 12, 16 in serum, and in selected very low- (VLDF) to high-density (HDF) lipid fractions. A reduction in LDL and total cholesterol (TC) was not accompanied by significant decline in HCV RNA. At baseline, there was an inverse correlation between HDL and HCV RNA (ρ = -0.45, P = 0.036). At 20 mg, there was correlation between change (Δ) in TG and Δ HCV RNA (ρ = 0.75, P = 0.007), Δ ALT and Δ TC (ρ = -0.64, P = 0.03) and Δ LDL (ρ = -0.67, P = 0.02). At 40 mg, Δ TG maintained a positive correlation with Δ HCV RNA (ρ = 0.65, P = 0.03). There was a group difference for HCV RNA in relation to lipid fractions (P = 0.04) but not study time intervals (P = 0.17); mean log HCV RNA was greater in VLDF compared to HDF (5.81 ± 0.59 vs 5.06 ± 0.67, P = 0.0002) with no other differences to study time intervals (P = 0.099). Short-term Rosuvastatin monotherapy is not associated with significant changes in serum or lipid fraction HCV RNA in NR patients. HCV co-localizes with the lowest density lipid fractions in serum.

Effects of Fractions of Bovine Follicular Fluid and Fetal Bovine Serum as Supplements to Maturation Medium on In Vitro Development of In Vitro Fertilized Bovine Embryos

Abstract: The purpose of this study was to examine the effects of different fractions of bovine follicular fluid (bFF) and fetal bovine serum (FBS) as maturation medium supplements on development to the blastocyst stage of bovine oocytes matured and fertilized in vitro. Three bFF fractions obtained by ultracentrifugation were designated as the 1st, 2nd and 3rd fractions, and four FBS fractions, also obtained by ultracentrifugation, were designated as the 1st, 2nd, 3rd and 4th fractions. Cumulus-oocyte complexes (COCs) were divided into two groups: those with homogeneous ooplasm and those with heterogeneous ooplasm. COCs were cultured in basic maturation medium with a bFF fraction or FBS fraction. In oocytes with homogeneous ooplasm, the rates of development to the blastocyst stage with bFF-1st and bFF-2nd were significantly higher than that with bFF. In oocytes with heterogeneous ooplasm, the rate of development to the blastocyst stage with bFF-2nd was significantly higher than that with bFF. In oocytes w...

99 Phosphorylated Signaling Proteins as Biomarkers of Antibody-Mediated Heart Allograft Rejection

Purpose Ligation of class I molecules on endothelial cells (EC) by anti-HLA Ab stimulates phosphorylation of p70 S6K, S6RP, ERK1/2 and Akt that in turn mediate cell survival and proliferation. We postulate that the capacity of Ab to mediate these signals is dependent upon the HLA Ab titer and level of HLA antigen expression on the endothelium. Methods and Materials Purified IgG fractions of heart recipient sera containing HLA Ab were tested for their ability to induce phosphorylation of S6RP, Akt, S6K and ERK in primary EC bearing relevant HLA antigens. Phosphorylation was measured by Western blot and normalized to EC treated with IgG containing no HLA Ab. The Ab binding capacity (ABC) of IgG fractions to EC was measured by flow-cytometry. Phosphorylation of paraffin embedded endomyocardial biopsies was determined by immunohistochemical staining. Results DSA with ABC values >=14000 triggered higher levels of phosphorylation of S6K (T421/S424), S6RP (S235/6), and ERK1/2 (T202/Y204). In contrast, DSA with low ABC values = Conclusions These results provide mechanistic insights into how DSA contribute to the process of AMR. High levels of DSA activate cell proliferation pathways which may play a role in the development of transplant vasculopathy. Low levels of DSA may play a role in accommodation by activating cell survival pathways. The study also emphasizes the value of immunostaining of phosphorylated kinases, such as S6K, as biomarkers of AMR.

315 Novel Use of a Serum Fractionation Centrifuge Tubes in Measuring Inorganic Pyrophosphate in Dialysis Patients

315 Novel Use of a Serum Fractionation Centrifuge Tubes in Measuring Inorganic Pyrophosphate in Dialysis Patients

Characterization of associated proteins and phospholipids in natural rubber latex

Non-rubber components present in natural rubber (NR) latex, such as proteins and phospholipids, are presumed to be distributed in the serum fraction as well as surrounding the rubber particle surface. The phospholipid-protein layers covering the rubber particle surface are especially interesting due to their ability to enhance the colloidal stability of NR latex. In this study, we have characterized the components surrounding the NR particle surface and investigated their role in the colloidal stability of NR particles. Proteins from the cream fraction were proteolytically removed from the NR latex and compare to those from the serum fractions using SDS-polyacrylamide gel electrophoresis revealing that both fractions contained similar proteins in certain molecular weights such as 14.5, 25 and 27 kDa. Phospholipids removed from latex by treatment with NaOH were analyzed using (1)H-NMR spectroscopy and several major signals were assignable to -(CH(2))(n)-, -CH(2)OP, -CH(2)OC═O and -OCH(2)CH(2)NH-. These signals are important evidence that indicates phospholipids associate with the rubber chain. The colloidal behavior of rubber lattices before and after removal of protein-lipid membrane was evaluated by zeta potential analysis and scanning electron microscope (SEM). The lowest zeta potential value of NR particles was observed at pH 10, consequently leading to the highest stability of rubber particles. Additionally, SEM micrographs clearly displayed a gray ring near the particle surface corresponding to the protein-lipid membrane layer.

Anti-GVHD Effect of Antithymocyte Globulin is Mediated by Antibodies Binding to T Cells and Regulatory NK Cells, and Less So or Not at All by Antibodies Binding to Other Mononuclear Cell Subsets

Abstract 2346 Introduction: Polyclonal rabbit-anti-human T cell globulin may decrease the likelihood of graft-vs-host disease (GVHD) without increasing the likelihood of relapse. We have recently shown that high levels of antithymocyte globulin (ATG) capable of binding to total lymphocytes are associated with a low likelihood of acute GVHD grade 2–4 (aGVHD) as well as chronic GVHD needing systemic therapy (cGVHD) but not increased likelihood of relapse (Podgorny PJ et al, BBMT 16:915, 2010). ATG is polyclonal, composed of antibodies for antigens expressed on multiple cell subsets, including T cells, B cells, NK cells, monocytes and dendritic cells. These cell subsets may play a role in the pathogenesis of GVHD. The anti-GVHD effect of ATG may be mediated through killing/inhibition of one or several of these cell subsets (eg, T cells) or their subsets (eg, naive T cells as based on mouse experiments naive T cells are thought to play a major role in the pathogenesis of GVHD). To better understand the mechanism of action of ATG on GVHD, we set out to determine levels of which ATG fraction (capable of binding to which cell subset) are associated with subsequent development of GVHD. Patients and Methods: A total of 121 patients were studied, whose myeloablative conditioning included 4.5 mg/kg ATG (Thymoglobulin). Serum was collected on day 7. Using flow cytometry, levels of the following ATG fractions were determined: capable of binding to 1. naive B cells, 2. memory B cells, 3. naive CD4 T cells, 4. central memory (CM) CD4 T cells, 5. effector memory (EM) CD4 T cells, 6. naive CD8 T cells, 7. CM CD8 T cells, 8. EM CD8 T cells not expressing CD45RA (EMRA-), 9. EM CD8 T cells expressing CD45RA (EMRA+), 10. cytolytic (CD16+CD56+) NK cells, 11. regulatory (CD16-CD56high) NK cells, 12. CD16 + CD56 − NK cells, 13. monocytes and 14. dendritic cells/dendritic cell precursors (DCs). For each ATG fraction, levels in patients with versus without aGVHD or cGVHD were compared using Mann-Whitney-Wilcoxon test. For each fraction for which the levels appeared to be significantly different (p Results: In univariate analyses, patients developing aGVHD had significantly lower levels of the following ATG fractions: binding to naive CD4 T cells, EM CD4 T cells, naive CD8 T cells and regulatory NK cells. Patients developing cGVHD had significantly lower levels of the following ATG fractions: capable of binding to naive CD4 T cells, CM CD4 T cells, EM CD4 T cells, naive CD8 T cells and regulatory NK cells. Patients who did vs did not develop relapse had similar levels of all ATG fractions. In multivariate analyses, high levels of the following ATG fractions were significantly associated with a low likelihood of aGVHD: capable of binding to naive CD4 T cells (relative risk=.33, p=.001), EM CD4 T cells (RR=.30, p Conclusion: For both aGVHD and cGVHD, the anti-GVHD effect with relapse-neutral effect of ATG appears to be mediated by antibodies to antigens expressed on naive T cells (both CD4 and CD8), EM CD4 T cells and regulatory NK cells, and to a lesser degree or not at all by antibodies binding to antigens expressed on B cells, cytolytic NK cells, monocytes or DCs. This is the first step towards identifying the antibody(ies) within ATG important for the anti-GVHD effect without impacting relapse. If such antibody(ies) is (are) found in the future, it should be explored whether such antibody(ies) alone or ATG enriched for such antibody(ies) could further decrease GVHD without impacting relapse. Disclosures: No relevant conflicts of interest to declare.

S1P, dihydro-S1P and C24:1-ceramide levels in the HDL-containing fraction of serum inversely correlate with occurrence of ischemic heart disease

BackgroundThe lysosphingolipid sphingosine 1-phosphate (S1P) is carried in the blood in association with lipoproteins, predominantly high density lipoproteins (HDL). Emerging evidence suggests that many of the effects of HDL on cardiovascular function may be attributable to its S1P cargo.MethodsHere we have evaluated how levels of S1P and related sphingolipids in an HDL-containing fraction of human serum correlate with occurrence of ischemic heart disease (IHD). To accomplish this we used liquid chromatography-mass spectrometry to measure S1P levels in the HDL-containing fraction of serum (depleted of LDL and VLDL) from 204 subjects in the Copenhagen City Heart Study (CCHS). The study group consisted of individuals having high serum HDL cholesterol (HDL-C) (females:≥73.5 mg/dL; males:≥61.9 mg/dL) and verified IHD; subjects with high HDL-C and no IHD; individuals with low HDL-C (females:≤38.7 mg/dL; males:≤34.1 mg/dL) and IHD, and subjects with low HDL-C and no IHD.ResultsThe results show a highly significant inverse relationship between the level of S1P in the HDL-containing fraction of serum and the occurrence of IHD. Furthermore, an inverse relationship with IHD was also observed for two other sphingolipids, dihydro-S1P and C24:1-ceramide, in the HDL-containing fraction of serum. Additionally, we demonstrated that the amount of S1P on HDL correlates with the magnitude of HDL-induced endothelial cell barrier signaling.ConclusionsThese findings indicate that compositional differences of sphingolipids in the HDL-containing fraction of human serum are related to the occurrence of IHD, and may contribute to the putative protective role of HDL in IHD.

Mechanisms of the Plurality of Scorpaena Porcus L. Serum Albumin

The proteins, which bind albuminspecific dye Evans blue, are revealed in the low-molecular protein fraction of the blood serum from Scorpaena porcus L. and identified as serum albumin. They were represented by three bands in 2D-SDS-PAAG. MALDI-TOF-analysis revealed the fundamental similarity of the mass spec-trum of the fragments of tryptic cleavage of proteins with molecular weight 73 and 76 kDa. The role of du-plications and intragenic reconstructions in the creation of the plurality of scorpaena albumins is discussed.

Effect of extrusion-cooking of some grain species on piglet performance and blood lipid profile

The objective of the research was to determine the effect of extruded grain of wheat, barley, rice and naked oats on piglet performance till 56 day of age as well as the parameters of blood lipid profile. The study involved 400 piglets from 40 hybrid PIC line gilts mated to P-76 boars. The experiment comprised 4 treatment groups, each consisting of 100 weaned piglets penned in groups of 10 animals. Piglets from group I received diet with extruded wheat, group II – with barley, group III – with rice and group IV – naked oats. The grains were extruded at 14% moisture, temperature profile 120/160,180C and screw rotation speed 80 rpm. During the study, the production effects on the basis of individual body weight of animals on 4, 28 day of life (weaning) and 56 day as well as the feed intake for each litter were evaluated. The digestibility studies by indicator method were performed on 6 barrows from each group. Blood for analyses was collected twice from 10 piglets from a group on 28 and 56 days of age. According to the research results, extruded rice and oats proved to be more palatable components of diets for piglets than barley or wheat. Their dietary inclusion significantly increased the feed intake and assured a high health status of piglets at the rearing period. Dietary supplement of extruded rice significantly improved piglet performance manifested by higher daily weight gains and better feed conversion ratio. The piglets fed naked oats-based diet showed a significantly lower content of total cholesterol and triacyloglycerols as well as an elevated HDL cholesterol fraction in blood serum as compared to the animals receiving a wheat, barley or rice supplemented diet.

Levels of protein fractions in blood serum of periparturient goats

The aim of the study was to compare contents of individual protein fractions determined by electrophoresis in blood serum of healthy periparturient goats. Eight clinically healthy white shorthaired goats were examined. Blood samples of these goats were taken from v. jugularis three weeks and two weeks before the anticipated parturition, on the parturition day, 7 days after the parturition, and 28 days after the parturition. Individual protein fractions, albumins, α1-, α2-, β1-, β2- and γ-globulins were identified by electrophoretic analysis of blood serum. Percentage shares of individual protein fractions were converted from total protein values to g·l-1values. The results indicated that individual serum protein fractions in periparturient goats showed significant dynamics of change. There were no significant differences between samples taken before the parturition and on the parturition day (except for the increase of β2 from 2.5 ± 0.3 g·l-1observed 3 weeks before the parturition to 3.2 ± 0.4 g·l-1measured 2 weeks before the parturition, and the decrease of γG levels from 9.5 ± 2.6 g·l-1observed three weeks before the parturition to 8.1 ± 1.7 g·l-1found on the parturition day), yet most indicators measured after the parturition were significantly higher (p< 0.05) than on the parturition day (values in g·l-1observed on the parturition day versus values measured 28 days after the parturition: total protein 60.6 ± 4.3 vs. 71.3 ± 2.4, albumins 37.4 ± 2.9 vs. 42.7 ± 1.0, α2 3.4 ± 0.5 vs. 4.1 ± 0.8, β1 5.6 ± 0.5 vs. 6.3 ± 0.9, γG 8.1 ± 1.7 vs. 12.3 ± 1.9). The results bring new knowledge on the values of individual fractions of serum proteins in healthy goats, which is necessary for diagnostic interpretation of pathological findings in animals and also for experimental studies.

Hypolipidemic and Antioxidant Potentials ofXylopia aethiopicaSeed Extract in Hypercholesterolemic Rats

A short-term study was carried out on Wistar strain rats to determine the effects of Xylopia aethiopica extract on serum and postmitochondrial fractions (PMFs) of visceral organs in experimental hypercholesterolemia. Animals received normal diet and were administered cholesterol orally by intubations at a dose of 40 mg/kg/0.3 mL, plant extracts at 250 mg/kg, and cholestyramine (Questran®, Bristol-Myers Squibb, Hounslow, United Kingdom) at 0.26 g/kg five times a week for 8 consecutive weeks. Thereafter the hypolipidemic effects were assessed by measuring total cholesterol, low-density lipoprotein-cholesterol (LDL-C), high-density lipoprotein-cholesterol, and triglycerides, whereas the extent of oxidative stress was assayed by measuring thiobarbituric acid-reactive substances and enzymatic antioxidants such as superoxide dismutase, catalase, and reduced glutathione (GSH) in serum and PMF of liver and kidney. We assayed two liver biomarkers-alanine aminotransferase and aspartate aminotransferase-for safety of X. aethiopica at the dose given in this experiment. Cholesterol feeding resulted in a significant increase (P < .05) in body weight of the hypercholesterolemic animals relative to control animals, and administration of X. aethiopica (250 mg/kg) caused a more than 60% reduction in body weight. Simultaneous treatment with X. aethiopica and Questran elicited 33.75% and 23.94% reductions, respectively, in serum cholesterol levels of hypercholesterolemic rats. In addition, the LDL-C level decreased significantly (P < .05) by 49.09% and 78.92% in serum and by 64.97% and 37.29% in the liver with cotreatment with the plant extract and Questran, respectively, compared to untreated hypercholesterolemic rats. X. aethiopica counteracted the decreases in enzymatic antioxidants, especially in GSH, where there was a greater than 300% increase compared with hypercholesterolemic animals. This study has shown that intake of X. aethiopica reduced the composition of lipids and produced a favorable lipid profile in the serum and PMF of visceral organs in experimental hypercholesterolemia.

Rapid fractionation of Al in human serum by the use of HiTrap desalting size exclusion column with ICP-MS detection

To understand the pathways of Al transport in human body it is necessary to identify and quantify Al species present in human serum. It has been demonstrated that about 90% of Al in human serum corresponds to high molecular mass species (HMM-Al) and that Al is bound exclusively to transferrin (Tf). The remaining Al exists in low molecular mass species (LMM-Al) bound predominantly to Al-citrate. In studies of the efficiencies of the chelation therapies and to perform kinetic studies of Al binding to Tf, rapid and reliable analytical procedures are required. Therefore, a new analytical procedure for the efficient, reliable and fast separation of proteins from LMM compounds in serum was developed by the use of a HiTrap desalting size exclusion column. Unspiked and spiked human serum that was left for six hours to reach approximately thermodynamic equilibrium were studied. The separation of HMM-Al from LMM-Al species was accomplished with tris-HCl buffer (pH 7.4) in 10 min. Al elution profile was followed by ICP-MS detection. In the first 5 min HMM-Al compounds were eluted, followed by the elution of LMM-Al species from 5 to 10 min. Results demonstrated that about 94% of Al was present in the HMM (protein) fraction. To prove the reliability of the fractionation procedure developed, the protein peak and LMM separated species were collected in 5 mL fractions, followed by the previously developed speciation procedures. Speciation of the protein peak by CIM® DEAE-ICP-MS verified that about 98% of Al was bound to Tf. In the LMM serum fraction Al-citrate was separated on the FPLC column coupled to ICP-MS. Speciation data confirmed the reliability of the developed fast fractionation procedure. It represents a promising tool for investigations of kinetics of Al binding to Tf and may also be applied in studies of the efficiencies of the chelation therapies in patients overloaded with Al.

Enhancement in MALDI‐TOF MS analysis of the low molecular weight human serum proteome

The combination in advances in MALDI-TOF MS instrumentation(1) and serum sample preparation techniques,(2-4) has lead to the emergence of MALDI serum protein expression profiling as a promising tool for biomarker discovery.(5) However, three factors still pose significant challenges for MALDI profiling of serum: the limited mass window of MALDI, serum protein complexity,(6) and analytical reproducibility.(7, 8) MALDI has a small mass preference due to the ionization efficiency of intact protein molecules and the focusing of flight of ions towards the detector. In linear mode, TOF analyzers have limited sensitivity for masses above 20 kDa. To address this, we previously reported the enhancement of MALDI broad mass range detection of protein signals.(9) Spanning a 100 kDa mass range, we improved the sensitivity of detection by an order of magnitude through the combined optimization of sample preparation, instrument parameters and data processing procedures. The complexity of the blood proteome is very high, with protein concentrations differing by up to ten orders of magnitude.(10) This large dynamic range exceeds current proteomic analytical capabilities; thus analysis of easily prepared subproteomes of serum or plasma is essential. Here, we shift our focus back to the enhancement of MALDI analysis in the low mass regime. The low molecular weight (LMW) subcomponent of serum promises to be a rich source of undiscovered biomarkers, as biological processes give rise to a plethora of proteolytic protein fragments.(11) Currently, there is no consensus on what constitutes the mass limits of this derivative proteome (also termed peptidome). However, “<15 kDa” is often cited in the literature based on a serum MALDI study using 20 MWCO filters.(12) To date, small native protein/peptide mass measurements have been mainly conducted in reflectron mode (< 4 kDa),(13) and linear mode up to 10 kDa.(14) However, no systematic comparison has been described for a combination of preparation steps: spotting, filtering and matrix choice, to optimize the performance. A rigorous reproducibility study for different preparation strategies is likewise missing, which prevents extension of the technique to clinical proteomics applications. The purpose of this work was to provide such a systematic comparison, using rigorous performance metrics to optimize sample preparation for LMW serum proteome profiling by MALDI-TOF MS analysis up to 20 kDa. We explored a combination of MALDI sample preparation and spotting methods. Procedures that gave the best results included: 1) MALDI spotting with a thin layer technique using sinapinic acid on a ground steel plate, and 2) centrifugal ultrafiltration with 50000 MWCO filters in the presence of 2% TFA, followed by desalting with C3 magnetic beads. Ultrafiltration has been utilized previously to improve MALDI profiling of serum.(2, 13, 15-17) However, our approach extensively improves MALDI peak intensities of higher MW peptides and small proteins, thus increasing biomarker coverage in the 3-20 kDa range. Reproducibility studies of a protein standard and serum samples gave excellent results, all of which suggests that this procedure can be a useful tool for proteome profiling of the LMW fraction of serum.

Fetuin-A (α2HS-Glycoprotein) Is a Major Serum Adhesive Protein That Mediates Growth Signaling in Breast Tumor Cells

Fetuin-A (α2HS-Glycoprotein) Is a Major Serum Adhesive Protein That Mediates Growth Signaling in Breast Tumor Cells