Isosteviol reduces plasma glucose levels in the intravenous glucose tolerance test in Zucker diabetic fatty rats.

AIM: The aim of this study was to test the effect of isosteviol on blood glucose and insulin levels during the intravenous glucose tolerance test (IVGTT) in Wistar and Zucker diabetic fatty (ZDF) rats. METHODS: ZDF rats were divided into a control and three isosteviol treatment (1, 5 and 10 mg/kg) groups. Wistar rats were divided into a control group and an isosteviol treatment group (10 mg/kg). The rats were fasted for 12 h prior to infusion of isosteviol and glucose (1.0 g/kg). Blood samples were taken at 0, 5, 15, 30, 60, 90 and 120 min after the injection of glucose. Glucose concentrations were determined by the glucose oxidase method, and plasma insulin was analysed by radioimmunoassay. The area under the curve (AUC) of the net change in plasma glucose concentration was used to compare the isosteviol treatment and control groups. RESULTS: In ZDF rats, isosteviol at 5 and 10 mg/kg caused a significant (p < 0.05) reduction in the AUC of glucose during the IVGTT. However, isosteviol did not increase plasma insulin concentrations in ZDF rats. In Wistar rats, isosteviol did not significantly affect plasma glucose or insulin levels during the IVGTT. CONCLUSION: Isosteviol exerts an antihyperglycaemic effect during IVGTT in ZDF rats but not in Wistar rats. Isosteviol has no significant effect on plasma insulin concentrations. The glucose-lowering effect of isosteviol may be due to changes in the sensitivity of peripheral tissues to insulin.

Myrmecia pilosula (Jack Jumper) ant venom: identification of allergens and revised nomenclature.

BACKGROUND: The 'Jack Jumper Ant' (JJA; Myrmecia pilosula species complex) is the major cause of ant sting anaphylaxis in Australia. Our aims were to determine the allergenicity of previously described venom peptides in their native forms, identify additional allergens and if necessary, update nomenclature used to describe the allergens according to International Union of Immunological Societies criteria. METHODS: Various polyacrylamide gel electrophoresis methods were used to separate JJA venom. Gel resolved venom was Western-blotted and probed with individual sera taken from patients with a history of JJA sting anaphylaxis and immunoglobulin E radioallergosorbent test (IgE RAST) tracer uptakes of >1% to whole venom. RESULTS: Of 67 available sera, 54 had RAST uptakes >1%. Thirteen IgE binding bands were identified using these sera. Pilosulin 3, [Ile(5)]pilosulin 1, and pilosulin 4.1 were recognized by 42 (78%), 18 (33%) and nine (17%) of the 54 sera that were tested. Immunoglobulin E-binding proteins with estimated molecular masses of 6.6, 22.8, 25.6, 30.4, 32.1, 34.4 and 89.8 kDa were each recognized by three or more individual sera. Two of these (25.6 and 89.8 kDa) were recognized by 46% and 37% of sera, respectively. CONCLUSION: Nomenclature used to describe JJA venom allergens has been revised. Pilosulin 3 (Myr p 2) is the only major allergen, whilst [Ile(5)]pilosulin 1 (Myr p 1), and pilosulin 4.1 (Myr p 3) are minor allergens. There are an additional five IgE-binding proteins that require further characterization before they can be named as allergens. These findings provide a framework for standardizing venom extracts for diagnosis and immunotherapy.

The expression of apolipoprotein B epitopes is normal in LDL of diabetic and end-stage renal disease patients.

AIMS/HYPOTHESIS: When LDLs are exposed to glucose in vitro, glycation of apolipoprotein B100 (apoB) leads to a loss in its affinity for the LDL receptor and reproducible alterations in the immunoreactivity of specific apoB epitopes, including several epitopes close to the LDL receptor binding site. The aim of this work was to determine if similar immunological changes are observed in vivo in LDLs of diabetic and end-stage renal disease (ESRD) patients. SUBJECTS, MATERIALS AND METHODS: The immunoreactivity of LDLs isolated from 14 diabetic patients with normal renal function and 13 patients with ESRD was studied with a panel of 25 well-characterised anti-apoB monoclonal antibodies. RESULTS: Although diabetic and ESRD LDLs showed evidence of glycation modification, none of the changes in the apoB immunoreactivity induced by glucose in vitro was observed in vivo, including those for epitopes close to the LDL receptor binding domain. CONCLUSIONS/INTERPRETATION: These results suggest that in vivo glycation of LDLs is a complex process that is not mimicked by in vitro exposure of LDLs to high concentrations of glucose. This questions the clinical significance of the in vitro glycation studies used to understand the pathophysiological consequences of LDL glycation in diabetes and ESRD.

Stereoselective hepatic disposition of model diastereomeric acyl glucuronides.

Numerous studies have previously been conducted with the impulse-response isolated perfused rat liver (IR-IPRL) to establish the role of both physiological and physicochemical factors in determining solutes' pattern of hepatic disposition, however the impact of optical isomerism on hepatic disposition has hardly been studied using this methodology. In this study, the IR-IPRL was used to assess the extent of stereoselectivity exhibited by the kinetic processes involved in the hepatic disposition of the diastereomeric acyl glucuronides of (R)- and (S)-2-phenylpropionic acid (i.e. (R)- and (S)-PPAG). Moment and model-dependent (distributed model and axial dispersion model) analyses were conducted of the hepatic outflow profiles generated upon bolus administration of (R)-(14)C-PPAG or (S)-(14)C-PPAG and 3H-Sucrose (used as a marker of the hepatic vascular space) into the portal inflow of isolated perfused livers of male Sprague-Dawley rats (n = 4). Significant differences between (R)- and (S)-PPAG were apparent in the pharmacokinetic parameters defining the total hepatic disposition of the two diastereomers, the most marked being the hepatic availabilities, where the value for (S)-PPAG (0.721 +/- 0.059) was significantly lower than that of (R)-PPAG (0.909 +/- 0.042). The distributed and axial dispersion model analyses suggested that the more extensive hepatic extraction of (S)-PPAG was (at least in part) due to the higher sinusoidal membrane permeability-surface area product (PS UPT) of the diastereomer, and this has been considered in light of the emerging evidence regarding the role of hepatocellular membrane transport mechanisms. Furthermore, given the potential immunogenicity of acyl glucuronides (through covalent binding to plasma and intracellular proteins), the results of this study suggest that diastereomeric glucuronides may exhibit differing toxicity due to differences in their access to intracellular proteins.

The quantification of paracetamol, paracetamol glucuronide and paracetamol sulphate in plasma and urine using a single high-performance liquid chromatography assay.

A range of analytical methods exist for the determination of paracetamol in biological fluids. However, to understand the fate of paracetamol and the effect of other drugs on its disposition in vivo, the major metabolites require quantification in urine and plasma. A method to simultaneously quantify paracetamol, paracetamol glucuronide (PG) and paracetamol sulphate (PS) in plasma and urine with superior sensitivity is therefore desired, especially if the volume of plasma available is low. A simple isocratic reverse phase high-performance liquid chromatography (HPLC) assay with spectrophotometric detection has been developed. The method, requiring only 100 microl of plasma and 50 microl of urine, utilizes a reversed-phase C18 column, a wavelength of 254 nm for detection and a mobile phase composed of potassium dihydrogen orthophosphate (0.1 M)-isopropanol-tetrahydrofuran (THF) (100:1.5:0.1, v/v/v) adjusted to pH 3.7 with phosphoric acid. The method is sensitive and linear in plasma within a concentration range from 0.4 to 200 microM for paracetamol, PG and PS. For PG and PS in urine, the method is sensitive and linear within a concentration range from 100 to 20,000 microM. Over these ranges, accuracy and precision were less than 12%. The assay has been used to measure concentrations of paracetamol and the two metabolites in plasma collected by finger-prick sampling and of the metabolites in urine from healthy volunteers administered a single oral dose of 1000 mg of paracetamol.

Cerebral and lung kinetics of morphine in conscious sheep after short intravenous infusions.

BACKGROUND: The analgesic effects of morphine are delayed relative to its concentration in blood. The rate of equilibration of morphine between blood and brain may contribute to this delay, but the kinetics of this process have not been modelled. This was determined in conscious instrumented sheep. The lung kinetics of morphine were also determined given their importance in defining systemic kinetics after i.v. bolus administration. METHODS: Sheep were given short i.v. infusions of morphine (30 mg over 4 min). Cerebral kinetics were inferred from arterio-sagittal sinus concentration gradients and cerebral blood flow, and lung kinetics from the pulmonary artery-aortic gradient and cardiac output. These data were fitted to flow- and membrane-limited models of the kinetics in each organ. RESULTS: Morphine had minimal cardiovascular effects, did not alter cerebral blood flow and caused insignificant respiratory depression. Lung kinetics were best described by a single distribution volume (2036 ml) with a first-order loss (1370 ml min(-1)), which was attributed to deep distribution. The cerebral kinetics of morphine were characterized by a significant permeability barrier. Permeability across the barrier (7.44 ml min(-1)) was estimated with good precision, and was approximately one-fifth of the nominal cerebral blood flow. The distribution volume of morphine in the brain was estimated with less precision, but was described by a brain:blood partition coefficient of approximately 1.4. The time required for 50% equilibration between brain and blood concentrations was approximately 10.3 min. CONCLUSION: The cerebral equilibration of morphine was relatively slow, and was characterized by significant membrane limitation.

Effect of ketoprofen and its enantiomers on the renal disposition of methotrexate in the isolated perfused rat kidney.

Non-steroidal anti-inflammatory drugs (NSAIDs) have been shown to inhibit the renal tubular secretion of methotrexate. However, the relative contribution of the active S- and inactive R-enantiomers is unknown. This study examined the effect of racemic ketoprofen and its enantiomers on the renal disposition of methotrexate in the isolated perfused rat kidney (IPK). Nineteen kidneys were divided between a control and three treatment groups. Controls were perfused with methotrexate alone (25 micrograms mL-1, n = 5) over three 30-min periods. Treatment groups were perfused with methotrexate (25 micrograms m-1) for the first period, followed by a second period of methotrexate (25 micrograms mL-1) plus R- (n = 5), S- (n = 5) or RS-ketoprofen (n = 4) at 25 micrograms mL-1, and a third period of methotrexate (25 micrograms mL-1) plus R-, S- or RS-ketoprofen (50 micrograms mL-1). Perfusate and urine were collected over 10-min intervals. Methotrexate was measured by HPLC and its binding in perfusate by ultrafiltration. The clearance ratio (CR) for methotrexate was obtained by dividing the renal clearance by the product of its fraction unbound and the glomerular filtration rate. During control experiments, there was no significant change in the CR over 90 min. R-, S- and RS-ketoprofen at 50 micrograms mL-1 reduced the CR of methotrexate significantly, but there was no difference between the three groups. While the enantiomers of ketoprofen reduced the renal excretion of methotrexate, the interaction was not enantioselective.

A simple method for the assay of colistin in human plasma, using pre-column derivatization with 9-fluorenylmethyl chloroformate in solid-phase extraction cartridges and reversed-phase high-performance liquid chromatography.

A simple, selective and sensitive high-performance liquid chromatographic (HPLC) method is described for the determination of colistin in human plasma. Derivatization with 9-fluorenylmethyl chloroformate was performed in the same solid-phase extraction C18 cartridge used for sample pre-treatment, followed by reversed-phase HPLC with fluorimetric detection. Quantification was achieved using the ratio of the summed peak areas of colistin A and B derivatives to that of the derivative of netilmicin (internal standard). Linear calibration curves were obtained within the concentrations of colistin sulfate from 0.10 to 4.0 mg/l in plasma. Accuracy was within 10% and reproducibility (RSD) was less than 10%.

Low- and high-density lipoprotein metabolism in HepG2 cells expressing various levels of apolipoprotein E.

To determine the importance of hepatic apolipoprotein (apo) E in lipoprotein metabolism, HepG2 cells were transfected with a constitutive expression vector (pRc/CMV) containing either the complete or the first 474 base pairs of the human apoE cDNA inserted in an antisense orientation, for apoE gene inactivation, or the full-length human apoE cDNA inserted in a sense orientation for overexpression of apoE. Stable transformants were obtained that expressed 15, 24, 226, and 287% the apoE level of control HepG2 cells. The metabolism of low-density lipoprotein (LDL) and high-density lipoprotein-3 (HDL(3)), two lipoprotein classes following both holoparticle and cholesteryl esters (CE)-selective uptake pathways, was compared between all these cells. LDL-protein degradation, an indicator of the holoparticle uptake, was greater in low apoE expressing cells than in control or high expressing cells, while HDL(3)-protein degradation paralleled the apoE levels of the cells (r(2) = 0.989). LDL- and HDL(3)-protein association was higher in low apoE expressing cells compared to control cells. In opposition, LDL- and HDL(3)-CE association was not different from control cells in low apoE expressing cells but rose in high apoE expressing cells. In consequence, the CE-selective uptake (CE/protein association ratio) was positively correlated with the level of apoE expression in all cells for both LDL (r(2) = 0.977) and HDL(3) (r(2) = 0.998). We also show that, although in normal and low apoE expressor cells, 92% of LDL- and 80% HDL(3)-CE hydrolysis is sensitive to chloroquine suggesting a pathway linked to lysosomes for both lipoproteins, cells overexpressing apoE lost 60% of chloroquine-sensitive HDL(3)-CE hydrolysis without affecting that of LDL-CE. Thus, the level of apoE expression in HepG2 cells determines the fate of LDL and HDL(3).

Well-defined regions of apolipoprotein B-100 undergo conformational change during its intravascular metabolism.

Apolipoprotein B (apoB)-100-containing lipoproteins are secreted from the liver as large triglyceride-rich very low density lipoproteins (VLDLs) into the circulation, where they are transformed, through the action of lipases and plasma lipid transfer proteins, into smaller, less buoyant, cholesteryl ester-rich low density lipoproteins (LDLs). As a consequence of this intravascular metabolism, apoB-containing lipoproteins are heterogeneous in size, in hydrated density, in surface charge, and in lipid and apolipoprotein composition. To identify specific regions of apoB that may undergo conformational changes during the intravascular transformation of VLDLs into LDLs, we have used a panel of 29 well-characterized anti-apoB monoclonal antibodies to determine whether individual apoB epitopes are differentially expressed in VLDL, intermediate density lipoprotein (IDL), and LDL subfractions isolated from 6 normolipidemic subjects. When analyzed in a solid-phase radioimmunoassay, the expression of most epitopes was remarkably similar in VLDLs, IDLs, and LDLs. Two epitopes that are close to the apoB LDL receptor-binding site show an increased expression in large (1.019 to 1.028 g/mL), medium (1.028 to 1.041 g/mL), and small (1.041 to 1.063 g/mL) LDLs compared with VLDLs and IDLs, and 2 epitopes situated between apoB residues 4342 and 4536 are significantly more immunoreactive in small and medium-sized LDLs compared with VLDLs, IDLs, and large LDLs. Therefore, as VLDL is converted to LDL, conformational changes identified by monoclonal antibodies occur at precise points in the metabolic cascade and are limited to well-defined regions of apoB structure. These conformational changes may correspond to alterations in apoB functional activities.

Hepatic disposition of fexofenadine: influence of the transport inhibitors erythromycin and dibromosulphothalein.

PURPOSE: To examine the disposition of fexofenadine in the isolated perfused rat liver and the influence of erythromycin and dibromosulphthalein (DBSP) on the hepatic uptake and biliary excretion of fexofenadine. METHODS: Livers from four groups of rats were perfused in a recirculatory manner with fexofenadine HCl added as a bolus (125, 250, 500, or 1000 microg) to perfusate. Livers from another three groups of rats were perfused with 250 microg of fexofenadine HCl. With one group as control, erythromycin (4.0 microg/ml) or DBSP (136 microg/ml) was added to the perfusate of the other groups. In all experiments, perfusate and bile were collected for 60 min; in addition, livers from the second experiment were retained for assay. Fexofenadine was determined in perfusate, bile, and homogenized liver by HPLC. RESULTS: The area under the curve (AUC) of fexofenadine was linearly related to concentration. It was unchanged from control (12,800 +/- 200 ng x h/ml) by erythromycin (14,400 +/- 2000 ng x h/ml), but was increased 95% by DBSP (25,000 +/- 2600 ng x h/ml, P <0.001). The ratios of the concentrations of fexofenadine in liver/perfusate were decreased significantly by DBSP; those for bile/liver were increased by erythromycin. CONCLUSIONS: Erythromycin reduced the canalicular transport of fexofenadine into bile, whereas DBSP reduced uptake across the sinusoidal membrane.

Studying low-density lipoprotein-monoclonal antibody complexes using dynamic laser light scattering and analytical ultracentrifugation.

Monoclonal antibody complexes have proven very useful in the study of low-density lipoproteins (LDLs). Thus, complexes composed of two different monoclonal antibodies, selected from a panel of 11 different antibodies, and LDL have been employed to map apolipoprotein B (apoB) on the surface of the LDL. In this way, apoB was found to surround the LDL as a ribbon with a bow [Chatterton, J. E., et al. (1995) J. Lipid Res. 36, 2027-2037]. Moreover, monoclonal MB19, which recognizes a polymorphic site, has been employed to quantitate the two different allelic forms of apoB found on LDL in human sera, and in this way, we assessed the effect of most of the known common polymorphisms of this protein as well as detected the depletion of the normal allele product in two forms of familial defective apoB-100 [Chatterton, J. E., et al. (1995) Biochemistry 34, 9571-9580; Pullinger, C. R., et al. (1995) J. Clin. Invest. 95, 1225-1234]. In this paper, these studies have been extended by examining by dynamic light scattering and sedimentation velocity techniques the complexes formed with only one antibody, and complexes formed using two antibodies. Our data show that the largest complex formed with a single monoclonal antibody was that of an LDL dimer; no larger, nonspecific complexes were present. With two antibodies, a variety of complexes were seen. Thus, monoclonal antibodies MB47 and 4G3, which bound about 55 degrees apart, formed a very stable dimer. Monoclonal antibodies MB47 and 2D8, which bound 136 degrees apart, formed a very stable tetramer, with four LDLs held together in probably a circular structure with four monoclonal antibodies. Finally, monoclonal antibodies 2D8 and 1D1, which bound 86 degrees apart, probably formed a less stable LDL tetramer, held together by three to four monoclonal antibodies. A rationale for these structures is discussed, as well as the biological relevance of these complexes.

Differences in apolipoprotein and lipid composition between human chylomicron remnants and very low density lipoproteins isolated from fasting and postprandial plasma.

Triglyceride-rich lipoproteins (TRLs) that are modified during alimentary lipemia and their remnants are indicated to play an important role in the development of atherosclerosis. Although recent studies in transgenic and gene knock-out animal models have shed new light on the function of different apolipoproteins (apos) in the metabolism of TRLs and on their respective role in atherogenesis in these models, little is known about the compositional properties of human chylomicron remnants and very low density lipoprotein (VLDL). To address this issue, apos E, C-I, C-II, and C-III and lipids (triglycerides, phospholipids and cholesterol) were measured in Svedberg flotation rate (Sf) 60-400 and Sf 20-60 subfractions of VLDL and chylomicron remnants isolated from fasting and postprandial plasma samples in ten normotriglyceridemic men. VLDL was separated from chylomicron remnants by immunoaffinity chromatography using monoclonal antibodies (4G3 and 5E11) recognizing apoB-100 but not apoB-48 epitopes. The triglyceride, cholesterol and apoC-II contents of large (Sf 60-400) chylomicron remnants were significantly higher compared with large VLDL particles, while the small (Sf 20-60) chylomicron remnants contained significantly more apoC-II molecules but fewer apoC-I molecules than small VLDL. Whereas the apoC-III contents of large chylomicrons decreased, the apoC-III contents of large VLDL increased postprandially. The cholesterol to triglyceride ratio of large VLDL particles increased transiently by 50% in response to the oral fat load, whereas the cholesterol to triglyceride ratio of large chylomicron remnant particles and small TRL remnants increased 50-100% throughout the entire postprandial period. The specific alterations of the apolipoprotein and lipid composition of chylomicron remnants and VLDL particles observed during alimentary lipemia are likely to target these lipoprotein species differently to metabolic routes and to confer both endogenous and exogenous remnant lipoprotein roles in atherogenesis.

Evidence for lipid-dependent structural changes in specific domains of apolipoprotein B100.

The structural organization and stability of apoB100 in complexes containing triglyceride (TG) and phospholipid have been examined. LDL was delipidated to form aqueous soluble apoB100-TG complexes that retain approximately 70% of LDL TG, but contain no other lipids. The apoB100-TG complexes exhibited reduced amphipathic alpha-helical content (17%) and net negative charge (-2.9 mV) as compared to native LDL-apoB100 (49% and -6 mV, respectively). Of 28 anti-apoB monoclonal antibodies tested, 15 showed partial or full reactivity with apoB100-TG. The immunoreactive epitopes of apoB100-TG were restricted to those situated in either the amino terminal globular domain (4 of 6) or in regions of apoB100 that are predicted to be composed of amphipathic beta-strands (11 of 13). Incubation of the apoB100-TG complex with palmitoyloleoylphosphatidylcholine (POPC) spontaneously (< 10 min) formed homogeneous lipoproteins (20 nm) that contained approximately 300 molecules of POPC per particle (apoB100-PC). Phospholipidation of apoB100-TG complexes partially recovered the alpha-helical content (34%) and net negative charge (-4.9 mV) of the native LDL and restored resistance of apoB100 to denaturation by guanidine HCl (5.8 M). Addition of phospholipids to apoB100-TG also increased the immunoreactivity of specific epitopes that are located primarily in regions of apoB100 that are thought to be constituted of amphipathic beta-strands. The effects of TG and phospholipid on apoB100 conformation appear to be highly domain-specific. On the basis of these results, we propose that the beta-strands of apoB100 may represent a nonflexible lipid-associating backbone, while the amphipathic alpha-helical domains may represent flexible lipid-binding regions that allow the particle to accommodate varying amounts of lipid.

Influence of renal failure on the disposition of morphine, morphine-3-glucuronide and morphine-6-glucuronide in sheep during intravenous infusion with morphine.

The influence of experimentally induced renal failure on the disposition of morphine, morphine-3-glucuronide (M3G) and morphine-6-glucuronide (M6G) was examined in seven sheep infused intravenously with morphine for 6 hr. Between 5 and 6 hr, blood was collected from the aorta, pulmonary artery, hepatic, hepatic portal and renal veins and posterior vena cava. Additional samples from the aorta and urine were collected up to 144 hr. Morphine, M3G and M6G were determined in plasma and urine by high-performance liquid chromatography. Constant concentrations of morphine, but not of M3G and M6G, were achieved in plasma between 5 and 6 hr. Significant (P < .001) extraction of morphine by the liver (0.72 +/- 0.05) and kidney (0.42 +/- 0.15) occurred. Compared with sheep with normal kidneys (Milne et al., 1995), renal failure did not alter (P = .11) the mean total clearance of morphine (1.5 +/- 0.3 liters/min); clearance by the kidney was less (P < .001). However, a paired comparison using sheep common to this study and from the study when their kidneys were normal revealed a significant reduction in mean total clearance of 25%. The renal extraction of M3G and M6G and urinary recovery of the dose as summed morphine, M3G and M6G were reduced by renal failure. The kidney metabolized morphine to M3G. The data suggest that nonrenal elimination of M3G becomes more important during renal failure.

Comparison of the disposition of hepatically-generated morphine-3-glucuronide and morphine-6-glucuronide in isolated perfused liver from the guinea pig.

PURPOSE: Humans and guinea pigs metabolise morphine extensively, forming the isomers morphine-3-glucuronide (M3G) and morphine-6-glucuronide (M6G) in relatively similar ratios. Both metabolites are formed in the liver, and their greater polarity relative to the parent aglycone may limit their permeability across hepatic membranes. This study compared the disposition of hepatically-generated M3G and M6G in perfused livers isolated from guinea pigs. METHODS: Livers were perfused at 30 ml/min in a non-recirculating manner with Krebs bicarbonate buffer containing morphine (6 to 7 microM). Perfusing medium, venous perfusate and bile were collected at regular intervals and concentrations of morphine, M3G and M6G determined by reversed-phase HPLC. RESULTS: Concentrations of morphine, M3G and M6G in perfusate and the rates of biliary excretion of M3G and M6G were consistent between 20 and 50 min of perfusion. The mean (+/-s.d.) ratio for the rate of formation of M3G relative to M6G was 3.7 +/- 1.5. A mean 33 +/- 3% of morphine extracted by the liver was recovered as summed M3G and M6G. Of the M3G and M6G formed during a single passage, 19 +/- 11% and 9 +/- 9%, respectively, was excreted into bile; the values were significantly different (P = 0.002). CONCLUSIONS: A greater fraction of hepatically-generated M3G excreted into bile compared to that for M6G reflects differences in their relative transport across sinusoidal and canalicular membranes of hepatocytes, possibly via carrier-mediated systems.

Evolution of mammalian apolipoprotein A-I and conservation of antigenicity: correlation with primary and secondary structure.

We have evaluated the immunoreactivity of 20 monoclonal antibodies (mAbs) directed against human apolipoprotein (apo)A-I with a panel of high density lipoproteins (HDL) from 13 mammalian species. The pattern of cross-reactivity showed that 20 mAbs had different specificity. While not all mAbs recognized apoA-I from all of the different species, the antigenicity of some sequences was well conserved. Thus, mAb A05 cross-reacted with all species except guinea pig and rat. In contrast, the mAb 4H1, which recognized residues 2-8, required a specific proline in position 3, as no immunoreactivity was found in the species missing this amino acid. Furthermore, the presence of a threonine residue in place of serine (in position 6) in the cynomolgus monkey was associated with a 20-fold loss of immunoreactivity in radioimmunometric assay with 4H1. As most of the epitopes were found in CNBr fragments 2 and 3, we sequenced these regions in four species (horse, goat, sheep, and cat) and analyzed the alignment of most known sequences to evaluate their consensus. Except for the rat and the chicken, considerable identity was observed. This permitted us to deduce the involvement of the residues in some antigenic epitopes. In the middle of apoA-I, a conservative mutation Asp103-->Glu was found sufficient to eliminate all reactivity of this epitope for A11 (residues 99-105 ... 12l6-132) in five species (rabbit, cow, goat, sheep, and rat). The residues essential to the expression of two other epitopes overlapping with A11 were also characterized. Edmundson-wheel representation of 18-residue repeated sequences of the different apoA-I species (for the eight amphipatic helices of residues 46-63, 68-85, 101-118, 123-140, 143-160, 167-184, 189-206, and 222-239) showed that secondary structure of apoA-I was more conserved than the antigenic epitopes. The N-terminal region, residues 1 to about 98, is rich in both strictly preserved sequences and epitope expression in most of the species surveyed. This evolutionary conservation of the N-terminal domain suggests an important yet unknown function.

Alterations of VLDL composition during alimentary lipemia.

Apoliprotein (apo) B-100-containing very low density lipoprotein (VLDL) particles secreted from the liver accumulate in plasma during alimentary lipemia. To determine whether changes of VLDL composition occur in the postprandial state that may render these lipoproteins more atherogenic, apoE, C-I, C-II, and C-III, and lipids (triglycerides, phospholipids, and cholesterol) were measured in Svedberg flotation (Sf) 60-400 (large) and Sf 20-60 (small) VLDL before and after an oral fat load. Ten normotriglyceridemic (NTG) and three hypertriglyceridemic (HTG) healthy men were given a fat-rich mixed meal (1,000 kCal with 60.2 E% from fat). Triglyceride-rich lipoproteins were isolated by density gradient ultracentrifugation from plasma samples obtained before (fasting) and at 2-h intervals after the meal. VLDL was then separated from chylomicrons and their remnants by immunoaffinity chromatography using monoclonal antibodies 4G3 and 5E11, recognizing apoB-100, but not apoB-48 epitopes. Large and small VLDL isolated from the NTG group were enriched with apoE and C-I, and cholesterol, but depleted of apoC-II in the postprandial state, whereas the apoC-III, triglyceride, and phospholipid contents were essentially unchanged. The compositional changes of VLDL in HTG subjects were similar but more pronounced compared with NTG subjects. We conclude that postprandial lipemia in healthy men induces transient compositional alterations of VLDL that link these lipoprotein species to the formation of atherosclerosis.