Immunoaffinity Gel Research Articles

Isolation of melatonin by immunoaffinity chromatography

A single-step, highly specific and easy-to-use method was developed for isolation and purification of melatonin from complex biological matrices. Polyclonal antibodies highly specific against melatonin (with cross-reactivities with related compounds below 0.02%, except for 6-hydroxymelatonin) were raised, characterised by enzyme-linked immunosorbent assay (ELISA) and used for preparation of immunoaffinity gel. Melatonin recovery by the immunoaffinity method was ∼95%, allowing single-step processing of samples prior to electrospray HPLC–MS analysis (with detection limit 10 fmol). The method was successfully used for determining melatonin in human serum and turned out to be better than the non-specific solid-phase extraction published earlier.

Characterization of Lactoferrin (LF) from Colostral Whey Using Anti‐LF Antibody Immunoaffinity Chromatography

ABSTRACT: Lactoferrin (LF) in colostral whey was isolated by anti‐LF immunoglobulin in yolk (IgY)‐Sepharose 4B immunoaffinity chromatography, and parameters such as binding capacity (qm) and dissociation constant (Kd, × 10−6 M) of this immunoaffinity gel for LF were discussed. Purification folds for colostral whey I (from colostrum collected within 6 d of postpartum) and colostral whey II (from colostrum collected within 1 d of postpartum) by anti‐LF IgY‐immunoaffinity chromatography were 135.80 and 103.60, respectively. The recovery for LF in the same colostral whey sample by anti‐LF IgY‐immunoaffinity chromatography was 82 to 99 %. qm of anti‐LF IgY‐immunoaffinity gel for LF in colostral whey I and whey II were 0.372 and 0.272 mg LF/mL wet gel, respectively. Kd of anti‐LF IgY‐immunoaffinity gel for LF in colostral whey I was 1.594 × 10−6 M and II was 1.587 × 10−6 M.

Isolation of immunoglobulin in yolk (IgY) and rabbit serum immunoglobulin G (IgG) specific against bovine lactoferrin by immunoaffinity chromatography

Hens were intramuscularly immunized and rabbits were subcutaneously immunized once every two weeks for 6 weeks using bovine lactoferrin (LF) as antigen. Antibody titers of both yolk (IgY) and rabbit serum (IgG) were as high as 1.68×108 at the 6th and 8th weeks, respectively, after the initial immunization treatment. However, antibody titer against LF in yolk was 9.4×107 at 16 weeks. While antibody titer of rabbit serum declined sharply to 2.1×107 at the 12th week and to 2.6×106 at the 13th week after the initial immunization. The purification efficiency (specific activity of purified antibody against LF/specific activity of the corresponding antiserum or yolk against LF) of rabbit serum IgG purified by laboratory-prepared LF-Sepharose 4B immunoaffinity column (0.05 mg LF/ml wet gel) was about 2400, similar to that of IgY purified by LF-Sepharose 4B immunoaffinity column. Different amounts (0–15.0 mg) of IgY purified by LF-Sepharose 4B immunoaffinity chromatography were applied to the same column to determine the binding capacity (qm) and dissociation constant (Kd) of LF-Sepharose 4B immunoaffinity gel for IgY specific against LF. It was found that qm was 0.81 mg IgY/ml wet gel (1.620 mg IgY/mg LF) and Kd was 6.4×10−6 M as determined by Langmuir-type adsorption isotherms.

The atherogenic lipoprotein phenotype: small dense LDL and lipoprotein remnants in nephrotic range proteinuria

The dyslipidaemia in nephrotic-range proteinuria is believed to contribute to the increased atherogenesis associated with the condition. Excess small dense low density lipoprotein (LDLIII) contributes to this risk. Lipoprotein remnants (RLP) may also be implicated but have not been studied in this population. We measured the plasma concentration of low density lipoprotein (LDL) subfractions (by density gradient ultracentrifugation), RLP (by immunoaffinity gel), very low density lipoprotein (VLDL) subfractions, post heparin lipases and cholesteryl ester transfer protein (CETP) activity in 27 patients with glomerular disease and albuminuria >2.0g. These were compared with 27 age and sex matched controls. Proteinuric patients had increased LDLIII concentration (patients 182 (84:267) vs. controls 31 (27:62); P<0.0001) with reduced lighter LDLI (36 (24:43) vs 69 (46:101); P<0.0005) and LDLII (124 (79:220) vs 178 (129:236); P<0.04, all mg/dl, median+interquartile range). RLP-cholesterol (RLP-C) and triglyceride (RLP-TG) were increased in proteinuric patients (RLP-C 18.9 (11.0:26.9) vs 7.7 (6.0:8.8); P<0.0001, RLP-TG 35.8 (11.8:54.7) vs. 7.2 (4.3:10.0); P<0.0001, all mg/dl). Increased LDLIII and RLP were independent of renal function. VLDL 1 and VLDL 2 concentrations were increased by 258 and 260% (both P<0.0001). CETP activity was increased by 46% ( P<0.005). Lipoprotein and hepatic lipase activities did not differ from control values. LDLIII concentration ( r 2=45.7%, P<0.001), RLP-C ( r 2=85.2%, P<0.001) and RLP-TG ( r 2=87.5%, P<0.001) all correlated positively with plasma triglyceride. Moreover, increased LDLIII was associated with both RLP-C ( r 2=31.3%, P<0.002) and RLP-TG ( r 2=33.6%, P<0.002). Excess LDLIII and RLP are present in nephrotic-range proteinuria and add to the spectrum of cardiovascular risk factors present in proteinuric patients. Increases in LDLIII and RLP are closely related to plasma triglyceride. The association between excess RLP and LDLIII suggests that RLP contribute to the increased atherogenicity attributed to the atherogenic lipoprotein phenotype.

Impaired glucose tolerance without hypertriglyceridemia does not enhance postprandial lipemia.

Postprandial lipemia has been thought to be one of risk factors for coronary heart disease, and enhances in potential patients for atherosclerotic disease. Patients with impaired glucose tolerance (IGT) often show hypertriglyceride, which is caused by enhanced portprandial lipemia. Therefore, postprandial lipemia in patients with IGT and without hypertriglyceridemia has not been cleared. We have examined the levels of plasma triglyceride and chylomicron remnants after a high fat meal load (1250 kcal, 40% fat and 420 mg cholesterol) in 13 normotriglyceridemic subjects with IGT and 10 controls with normal glucose tolerance (NGT). Chylomicron remnants were evaluated as remnant-like particles (RLP) that were not bound to an immunoaffinity gel mixture containing apo A-I and apo B-100 monoclonal antibody. RLP cholesterol levels 4 hours after the fat load were significantly lower in IGT subjects than in NGT subjects. Increase of RLP cholesterol after the fat meal load only significantly correlated with increase of insulin during the first 30 min after a 75 g oral glucose tolerance test, but not fasting lipid, insulinogenic index and HOMA-R (homeostasis model) in all subjects. These results suggest that postprandial response does not enhance in IGT subjects, and may associate with early-phase insulin secretion and without insulin resistance in normotriglyceridemic men with IGT or NGT.

Remnant-like particle cholesterol and triglyceride levels of hypertriglyceridemic patients in the fed and fasted state

Potentially atherogenic triglyceride-rich lipoprotein (TRL) remnants can be isolated and quantitated as remnant-like particles (RLP), using an immunoaffinity gel containing specific anti-human apolipoprotein A-I (apoA-I) and apoB-100 monoclonal antibodies. The aim of the present study was to determine the relationship between postprandial changes in RLP levels and changes in total serum triglyceride (TG) in patients with different forms of hypertriglyceridemia (HTG). Three groups of patients were selected, having similarly elevated serum TG levels: a) HTG with TRL remnant accumulation (i.e., type III patients, n = 15, TG: 3.8 +/- 0.2 mm), b) HTG with increased LDL (i.e., type IIb patients, n = 15, TG: 3.7 +/- 0.2 mm), and c) HTG without evidence of remnant or LDL accumulation (i.e., type IV patients, n = 15, TG: 3.9 +/- 0.3 mm). Ingestion of a 45-g fat meal caused a significant increase in serum TG (30;-50%) in all patients. Mean serum TG levels of the three groups were not significantly different at 4 or 6 h after the meal. RLP cholesterol (C) and TG levels increased after the meal in all patients, but these postprandial increases were also not significantly different among groups. Type III patients had significantly higher (P < 0.01) levels of RLP-C and RLP-apoE in the fasted and fed state, and also had significantly higher RLP-C-to-serum TG ratios (P < 0.001) compared with the other groups. These results indicate that 1) RLP-C and RLP-TG levels are significantly increased in the fed versus fasted state in patients with elevated fasting TG levels; 2) patients with different forms of HTG, but similar TG levels, have similar postprandial increases in RLP-C and RLP-TG; and 3) type III patients have significantly elevated levels of RLP-C and RLP-apoE in both the fed and fasted state.

Remnant Lipoproteins Induce Proatherothrombogenic Molecules in Endothelial Cells Through a Redox-Sensitive Mechanism

Triglyceride-rich lipoproteins (TGLs) are atherogenic. However, their cellular mechanisms remain largely unexplained. This study examined the effects of isolated remnant-like lipoprotein particles (RLPs) on the expression of intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and tissue factor (TF), proatherothrombogenic molecules, in cultured human endothelial cells. RLPs were isolated from plasma of hypertriglyceridemic patients by use of the immunoaffinity gel mixture of anti-apoA-1 and anti-apoB-100 monoclonal antibodies. The incubation of cells with RLPs significantly upregulated mRNA and protein expression of these molecules. Total TGLs (d<1.006) and LDL had fewer or minimal effects on expression of these molecules compared with RLPs. RLPs increased intracellular oxidant levels, as assessed with an oxidant-sensitive probe. Combined incubation with alpha-tocopherol or N-acetylcysteine, both antioxidants, suppressed RLP-induced increase in expression of these molecules. In patients with higher plasma levels of RLPs, plasma levels of soluble forms of ICAM-1 and VCAM-1 were significantly higher than in patients with lower RLP levels. Treatment with alpha-tocopherol for 1 month decreased levels of the soluble adhesion molecules concomitantly with an increase in resistance of RLPs to oxidative modification in patients with high RLP levels. RLPs upregulated endothelial expression of ICAM-1, VCAM-1, and TF, proatherothrombogenic molecules, partly through a redox-sensitive mechanism. RLPs may have an important role in atherothrombotic complications in hypertriglyceridemic patients.

Membrane active lipids in remnant lipoproteins cause impairment of endothelium-dependent vasorelaxation.

We have recently found that remnant lipoproteins (RLPs) and their lipid fractions impair endothelium-dependent vasorelaxation (EDR). This study was aimed at clarifying mechanisms responsible for RLP-induced endothelial dysfunction in isolated rabbit aortas. RLPs were isolated from plasma in hyperlipidemic subjects by use of the immunoaffinity gel mixture of anti-ApoA1 and anti-ApoB100 monoclonal antibodies and ultracentrifugation. Organ chamber experiments showed that EDR impairment was restored by addition of reduced glutathione (GSH) or N-acetylcysteine, antioxidants, into the incubation buffer containing isolated rabbit aortas and RLPs (0.75 mg of triglyceride/mL). Furthermore, the incubation of isolated human red blood cells (RBCs) with RLP and its lipids converted the normal shape of RBCs to echinocytes, but coincubation with antioxidants suppressed the RLP-induced RBC transformation, suggesting that they exerted oxidative damage on RBC surface membranes. Studies with HPLC and the postcolumn chemiluminescence method showed that RLPs contain a substantial amount of phosphatidylcholine hydroperoxides. Peroxidized phosphatidylcholine also impaired EDR and had echinocytogenic action, both of which were suppressed by N-acetylcysteine. RLPs isolated from the plasma of patients under treatment with alpha-tocopherol, an antioxidant, had a lower level of phosphatidylcholine hydroperoxides (15% of the amount in nontreated patients), which was associated with a lack of the inhibitory action on EDR and with lesser effect on RBC transformation. Oxidative damage caused by lipid components in RLPs, especially peroxidized phospholipids, deteriorates cell surface membrane and may be at least partly responsible for RLP-induced impairment of EDR.

Production of a polyclonal antibody to Phellinus pini and examination of its potential use in diagnostic assays

SummaryIn order to differentiate among Phellinus pini, Inonotus tomentosus and Inonotus circinatus a polyclonal antibody was raised to a N‐terminal part of 25‐kDa P. pini‐specific protein. The specificity of the polyclonal antibody produced against a synthetic N‐terminal peptide of this protein was investigated for diagnostic purposes using Western immunoblot, indirect enzyme‐linked immunosorbent assay (ELISA), and inhibition ELISA techniques. The N‐terminal synthetic peptide, used as the immunogen, was found to be more than 80% pure by reverse‐phase high‐performance liquid chromatography. Following immunization, antisera were collected at three different time intervals. The antibody molecules were purified from the crude antisera using immunoaffinity gel chromatography. Following one‐dimensional sodium dodecylsulfate‐polyacrylamide gel electrophoresis, Western immunoblot analysis showed that the P. pini I polyclonal‐antibody detected the immunogen, the 25‐kDa protein, in all but one of the P. pini isolates examined, but in none of the isolates of the nontarget species I. tomentosus and I. circinatus. Nevertheless, cross‐reactivity was a problem because the P. pini I polyclonal‐antibody also recognized bands at other molecular weights in nearly all of the isolates of the other species tested. With the indirect ELISA the P. pini isolates tended to have higher affinity for the polyclonal antibody than the nontarget species, but some cross‐reactivity did occur. Inhibition ELISAs, performed over a range of soluble antigen concentrations (1.56–400 ng/100 μl), failed to show a clear distinction between P. pini and the two Inonotus spp. The low level of cross‐reactivity observed for I. tomentosus isolate 52 (9%) was also apparent in the indirect ELISA analysis. All three assays indicated that P. pini isolate 41 was the most antigenic. Despite cross‐reactivity, the antibody is useful in Western immunoblots for the diagnosis of most P. pini isolates.

Characterization of remnant-like lipoprotein particles isolated by immunoaffinity gel from subjects with LDL subclass phenotypes A and B

Characterization of remnant-like lipoprotein particles isolated by immunoaffinity gel from subjects with LDL subclass phenotypes A and B

Measurement of a Malondialdehyde‐DNA Adduct

Determining the levels of various DNA adducts has become an essential tool in understanding the toxicology of carcinogens. Direct measurement of DNA adduct levels, the true biologically effective dose of a mutagen, can be correlated with biological outcomes or used to probe mechanisms of adduct formation. Each adduct to be measured requires a specific assay. Malondialdehyde is a carcinogenic and mutagenic electrophile that is endogenously produced during peroxidation of polyunsaturated fatty acids. Its reaction with deoxyguanosine produces a fluorescent exocyclic pyrimidopurinone that can be detected by gas chromatographic/negative chemical ionization-electron capture mass spectroscopy. Methods for preparing an immunoaffinity gel and for HPLC quantification of nucleosides are also included.

Characterization of remnant-like particles isolated by immunoaffinity gel from the plasma of type III and type IV hyperlipoproteinemic patients

Previous studies have investigated the potential atherogenicity and thrombogenicity of triglyceride-rich lipoprotein (TRL) remnants by isolating them from plasma within a remnant-like particle (RLP) fraction, using an immunoaffinity gel containing specific anti-apoB-100 and anti-apoA-I antibodies. In order to characterize lipoproteins in this RLP fraction and to determine to what extent their composition varies from one individual to another, we have used automated gel filtration chromatography to determine the size heterogeneity of RLP isolated from normolipidemic control subjects (n = 8), and from type III (n = 6) and type IV (n = 9) hyperlipoproteinemic patients, who by selection had similarly elevated levels of plasma triglyceride (406 ± 43 and 397 ± 35 mg/dl, respectively). Plasma RLP triglyceride, cholesterol, apoB, apoC-III, and apoE concentrations were elevated 2- to 6-fold (P < 0.05) in hyperlipoproteinemic patients compared to controls. RLP fractions of type III patients were enriched in cholesterol and apoE compared to those of type IV patients, and RLP of type IV patients were enriched in triglyceride and apoC-III relative to those of normolipidemic subjects. In normolipidemic subjects, the majority of RLP had a size similar to LDL or HDL. The RLP of hyperlipoproteinemic patients were, however, larger and were similar in size to TRL, or were intermediate in size (i.e., ISL) between that of TRL and LDL. Compared to controls, ISL in the RLP fraction of type III patients were enriched in apoE relative to apoC-III, whereas in type IV patients they were enriched in apoC-III relative to apoE. These results demonstrate that: 1) RLP are heterogeneous in size and composition in both normolipidemic and hypertriglyceridemic subjects, and 2) the apoE and apoC-III composition of RLP is different in type III compared to type IV hyperlipoproteinemic patients.—Marcoux, C., M. Tremblay, K. Nakajima, J. Davignon, and J. S. Cohn. Characterization of remnant-like particles isolated by immunoaffinity gel from the plasma of type III and type IV hyperlipoproteinemic patients. J. Lipid Res. 1999. 40: 636–647.

Elevated remnant-like lipoprotein particles in impaired glucose tolerance and type 2 diabetic patients.

Impaired glucose tolerance (IGT) in association with insulin resistance is considered to be a risk factor for atherosclerosis. Thus, patients with IGT may have abnormal lipid and lipoprotein profiles. The purpose of this study was to investigate presence of remnant-type hyperlipoproteinemia in patients with IGT. Serum levels of remnant-like lipoprotein particles (RLP) were measured in 541 subjects (362 men and 179 women, age 53 +/- 7.9 years) who visited our health center for routine medical examinations. We measured RLP cholesterol (RLP-C) and RLP triglycerides (RLP-TG) using immunoaffinity gel containing monoclonal anti-human apoproteins A-I (H-12) and B-100 (JI-H) antibodies. After a 75-g oral glucose tolerance test, subjects were divided into three groups: normal, IGT, and type 2 diabetic. After matching for sex, age, and body weight, serum RLP-C in normal, IGT, and diabetic groups were 4.2 +/- 1.7, 6.2 +/- 3.4, and 6.2 +/- 4.2 mg/dl, respectively. The corresponding RLP-TG values were 16.7 +/- 9.2, 28.0 +/- 19.1, and 29.0 +/- 27.2 mg/dl. We found that RLP-C and RLP-TG values were significantly higher in the IGT and diabetic groups compared with the normal group (P < 0.001). In the same order, total serum cholesterol levels were 206 +/- 29, 205 +/- 34, and 206 +/- 34 mg/dl and LDL cholesterol levels were 127 +/- 27, 124 +/- 34, and 123 +/- 34 mg/dl, showing no marked difference in these groups. However, serum levels of triglyceride were higher in the IGT and diabetes groups (155 +/- 76 and 151 +/- 81 mg/dl vs. 106 +/- 41 mg/dl; P < 0.0001). Further, the incidence of remnant hyperlipoproteinemia in normocholesterolemic subjects was up to four times higher in IGT and diabetic groups compared with the normal group. High serum RLP-C and RLP-TG levels in IGT and diabetic patients may represent an increased risk of atherosclerosis in these patients.

Plasma remnant-like particle lipid and apolipoprotein levels in normolipidemic and hyperlipidemic subjects

Remnant-like particle (RLP) lipid and apolipoprotein (apo) levels were determined in the plasma of normolipidemic and hyperlipidemic subjects, in order to investigate the relationship between RLP levels and the concentration of other plasma lipoprotein parameters. Plasma RLP fractions were isolated with the use of an immunoaffinity gel (RLP-Cholesterol Jimro II, Japan Immunoresearch Lab.), containing specific anti-apoB-100 and anti-apoA-I antibodies. Four groups of human subjects were selected, who had either matching or significantly different levels of plasma triglyceride (TG) and low-density lipoprotein cholesterol (LDL-C): (1) normolipidemic control (NC) subjects ( n=10), (2) patients with elevated levels of LDL-C (type IIa, LDL-C (mean±S.E.), 4.65±0.09 mmol/l, n=10), (3) hypertriglyceridemic (HTG) patients with elevated LDL-C (type IIb, TG: 3.86±0.36; LDL-C: 4.67±0.21 mmol/l, n=10), and (4) HTG patients with normal LDL-C (type IV, TG: 3.71±0.39 mmol/l, n=10). NC subjects (RLP-C: 0.22±0.01; RLP-TG: 0.24±0.03 mmol/l) had RLP apoB, apoC-III and apoE levels of 3.2±0.3, 1.8±0.3, and 1.4±0.1 mg/dl, representing 3.2±0.4, 14.5±1.4 and 32.1±2.1% of total plasma levels, respectively. RLP lipid and apolipoprotein concentrations were significantly higher in HTG groups (type IIb and IV) compared to NTG groups (NC and type IIa) (e.g. RLP-C: 0.50±0.07 and 0.58±0.11 vs. 0.22±0.01 and 0.21±0.01 mmol/l, respectively ( P<0.01); RLP apoB: 8.4±1.6 and 8.2±0.9 vs. 3.2±0.3 and 3.4±0.2 mg/dl, respectively ( P<0.01)). No significant difference in RLP levels was observed between groups having different LDL levels, and thus no correlation existed between RLP-C and LDL-C levels ( r=0.24, n.s.). RLP-C and RLP apoB levels were, however, correlated with VLDL-C and VLDL apoB ( r=0.86, P<0.001 and r=0.70, P<0.001, respectively). These results demonstrate that elevated levels of both RLP lipids and apolipoproteins are characteristic of patients with increased levels of plasma triglyceride, and not patients with increased levels of LDL.

Remnants of chylomicron and very low density lipoprotein impair endothelium-dependent vasorelaxation

Remnants of chylomicron and very low density lipoprotein (VLDL) have been implicated as potentially atherogenic. Since endothelial dysfunction is an early event in atherosclerosis, we examined effects of the remnants on endothelium-dependent vasorelaxation. The remnant lipoproteins were isolated from postprandial plasma in hyperlipidemic subjects using the immunoaffinity gel mixture of anti apo A-1 and anti apo B-100 monoclonal antibodies and ultracentrifugation. Rabbit aortic strips suspended in the organ chambers were incubated for 2 h with the preparations of lipoproteins and lipids. After incubation, the strips were tested with vasodilators after precontraction with phenylephrine (1 μM). The remnant lipoproteins (750–1500 μg triglyceride/ml) but not VLDL fraction (up to 1500 μg triglyceride/ml) impaired vasorelaxation in responses to acetylcholine, substance P and A23187. Carbamylated or methylated remnant lipoproteins, chemically modified remnant lipoproteins, had comparable impairment of the vasorelaxation as unmodified remnant lipoproteins. Incubation with lipid extracts from the remnant lipoproteins also exerted an inhibitory effect on the vasorelaxation. Relaxation to sodium nitroprusside was fully preserved in all aortas exposed to the lipoprotein preparations. Thus, the remnant lipoproteins impair endothelium-dependent arterial relaxation at the concentrations observed in the plasma in patients with coronary artery disease (500–2000 μg triglyceride of remnant lipoprotein/ml). The impairment may be in apoprotein receptor-independent manner, and the lipids in the remnants seem to contribute to the inhibitory effect. The endothelial dysfunction caused by the remnant lipoproteins may play a role in the high prevalence of atherosclerotic coronary artery disease in postprandial hyperlipidemic patients.

Quantitative determination of several synthetic corticosteriods by gas chromatography-mass spectrometry after purification by immunoaffinity chromatography

A study was conducted to test a multiresidue analytical procedure for detecting and quantifying several corticosteroids on which the European Union imposes maximum residue limits (MRLs). Primary extracts from different matrices (liver, milk, urine, faeces) were first purified on C 18 cartridges. A new immunoaffinity clean-up step was included. The immunoaffinity gel was used to purify several corticosteroids simultaneously with enrichment of the corresponding fractions. The extracts were treated with an aqueous solution of pyridinium chlorochromate to fully oxidise all corticosteroids and to facilitate their extraction with dichloromethane. After evaporation, the final extract was reconstituted with toluene before injection into the GC-MS apparatus. The analysis was performed in the CI-negative ionisation mode using ammonia as the reactant gas. The estimated detection and quantification limits were, respectively, 0.25 and 0.5 ppb or lower. Overall, the method is reproducible to within 20%. Recovery is between 50 and 80% according to the corticosteroid.

The in vitro effects of chylomicron remnant and very low density lipoprotein remnant on platelet aggregation in blood obtained from healthy persons

We investigated the in vitro influence of chylomicron (CM) remnant and very low density lipoprotein (VLDL) remnant on platelet aggregation in healthy persons. The separation of CM and VLDL remnants from serum was performed using an immunoaffinity gel mixture containing anti apo B-100 and anti apo A-1 antibodies coupled to Sepharose 4B. The preincubation with CM and VLDL remnants significantly enhanced the platelet aggregation in whole blood and in platelet rich plasma (PRP) induced by collagen. This effect was observed in whole blood with the addition of VLDL remnant at the concentration of 5 μg/ml, and in PRP with the addition of CM remnant at the concentration of 1 and 5 μg/ml, and with the addition of VLDL remnant at the concentration of 5 and 10 μg/ml. These results may indicate that increase in remnant lipoproteins may be a part of the reasons for atherosclerotic and thrombotic complications.

Analysis of Dexamethasone and Betamethasone in Bovine Urine by Purification with an “On-Line” Immunoaffinity Chromatography–High-Performance Liquid Chromatography System and Determination by Gas Chromatography–Mass Spectrometry

A method for the immunoaffinity extraction of dexamethasone and betamethasone in bovine urine, followed by high-pressure liquid chromatography (HPLC) fractionation and gas chromatography–mass spectrometry determination, is described. A commercial immunoaffinity gel, containing antibodies raised against dexamethasone, was used to prepare an immunoaffinity cartridge which was inserted in an automatic HPLC system for on-line extraction and purification. By injecting urine samples (spiked with flumethasone as internal standard) directly into the system, it was possible to collect purified fractions, containing the analytes of interest. The fractions were dried and derivatized to yield the tetra-trimethylsilyl derivatives of the three corticosteroids, which were analyzed by selected ion monitoring gas chromatography–mass spectrometry. The method allowed a very good purification of samples and reached a detection limit of 0.1 ng/ml for dexamethasone and 0.2 ng/ml for betamethasone. Several samples, coming from a steer treated with dexamethasone and from other bovines coming from breedings in northern Italy, were analyzed with the method described. Dexamethasone levels ranged from 0.12 to 146 ng/ml.

Remnant-like lipoproteins stimulate whole blood platelet aggregation in vitro

We have developed a simple, rapid assay method to measure remnant- like lipoproteins by using an immunoaffinity gel mixture of anti apo B-100 and apoA-1 antobodies to Sepharose 4B. Characterization of the unbound lipoproteins has shown that they represent chylomicron and VLDL remnant particles (RLP). Preincubation of whole blood with RLP resulted in the enhanced activation of aggregation with ADP and collagen. Such enhancement was not observed in the presence of lipoprotein deficient serum or alubumin preparation. The extent of enhancement was 2.78 times by 7.5 μM of ADP and 44 times by 0.5 μg/ml of collagen in the presence of RLP-prepalation 1 (RLP-1), respectively. In the presence of RLP-2, the enhancement was 5.37 times by 7.5 μM of ADP and 102 times by 0.5 μg/ml of collagen, respectively. On the other hand RLP slightly inhibited PRP aggregation by these agonists. Inhibitions were 19% by 7.5 μM of ADP and 18 % by 1.0 μg/of collagen in the presence of RLP-1, respectively. Incubation of whole blood with RLP did not result in the release of factors to stimulate platelets or ADP- or collagen-induced platelet aggregation in vitro. The extents of enhanced aggregation in whole blood or inhibition in PRP were not correlated with RLP-cholesterol nor RLP-protein concentrations of RLP preparations used. These results may indicate that RLP not only interact with platelets but with erythrocytes or leukocytes. Our findings support the hypothesis that the postprandial increase in remnant lipoproteins is an atherosclerotic risk factor and may be a part of the reasons of thrombotic complications by stimulating platelets in patients with remnant hyperlipoproteinemia.

Measurement of remnant-like particles in human serum with a mixed immunoaffinity gel

Measurement of remnant-like particles in human serum with a mixed immunoaffinity gel