Normal Rabbit Plasma Research Articles

Lipemia in the Plasma Sample Affects Fentanyl Measurements by Means of HPLC-MS2 after Liquid-Liquid Extraction.

Examination of fentanyl levels is frequently performed in certain scientific evaluations and forensic toxicology. It often involves the collection of very variable blood samples, including lipemic plasma or serum. To date, many works have reported the methods for fentanyl detection, but none of them have provided information about the impact on the assay performance caused by an excessive amount of lipids. This aspect may be, however, very important for highly lipophilic drugs like fentanyl. To address this issue, we developed the liquid chromatography method with mass spectrometry detection and utilized it to investigate the impact of lipids presence in rabbit plasma on the analytical method performance and validation. The validation procedure, conducted for normal plasma and lipemic plasma separately, resulted in good selectivity, sensitivity and linearity. The limits of detection and quantification were comparable between the two matrices, being slightly lower in normal plasma (0.005 and 0.015 µg/L) than in lipemic plasma (0.008 and 0.020 µg/L). Liquid–liquid extraction provided a low matrix effect regardless of the lipid levels in the samples (<10%), but pronounced differences were found in the recovery and accuracy. In the normal plasma, this parameter was stable and high (around 100%), but in the lipemic matrix, much more variable and less efficient results were obtained. Nevertheless, this difference had no impact on repeatability and reproducibility. In the present work, we provided reliable, convenient and sensitive method for fentanyl detection in the normal and lipemic rabbit plasma. However, construction of two separate validation curves was necessary to provide adequate results since the liquid-liquid extraction was utilized. Therefore, special attention should be paid during fentanyl quantification that involves lipemic plasma samples purified by this technique.

New Technique use to differentiate between Pseudo and true coagulase Activity of Staphylococcus aureus isolated from Different infected Sites in Patients from Baghdad Hospitals

A total of 300 strains of Staphylococci isolated from various pathological sites of infection from different hospitals; these specimens derived from cases ofhuman infections were tested for staphylocoagulase activity.&#x0D; Test systems employing normal citrated rabbit plasma and the same substrate supplemented with inhibitors of thrombin and proteolytic enzymes (but notinfluencing the staphylocoagulase activity) were used for testing suspensions of bacteria and cell-free culture supernatants. A total of 200 strains clotted normal rabbit plasma; however addition of Trasylol and heparine resulted in positive results in 100 strains, whereas plasma supplemented with Trasylol and hirudin was coagulated definitely by only 70 strains.It is postulated that proteolytic enzymes of staphylococci interfere with staphylocoagulase-induced clotting and may simulate coagulase-positve activity of staphylococci.To avoid such false results, attest system for detection of sse shtaphylocoagulaould include proteolytic enzyme inhibitors The purpose of this investigation was to verify this hypothesis by testing a collection of staphylococci strains from different sources of infections against rabbit plasma in atypical coagulase test versus the same supplemented with specific inhibitors of proteases and thrombin depending on the sever sites of infections.

Comparison of Colchicine, Dapsone, Triamcinolone, and Diphenhydramine Therapy for the Treatment of Brown Recluse Spider Envenomation

To compare the efficacy of dapsone, diphenhydramine, colchicine, and intralesional triamcinolone in the treatment of brown spider bites. We used a purified venom that reproducibly produces a large eschar. To mimic real-life circumstances, all agents were administered following a 2-hour delay after envenomation. The animals were evaluated for the presence of coagulopathy to determine if the incidence of systemic findings correlated with the type of treatment. In a research laboratory, 60 New Zealand white rabbits each received an intradermal injection of 20 microg of purified Loxosceles reclusa venom. The rabbits were divided into 5 groups of 12; a control group and 4 groups treated with a drug (either colchicine, triamcinolone, diphenhydramine, or dapsone). Measured end points included maximum eschar size as well as histologic grading of depth, inflammation, and thrombosis. Treatment with colchicine, triamcinolone, diphenhydramine, or dapsone. Maximum eschar size as well as histologic grading of depth, inflammation, and thrombosis. There was no significant difference with respect to eschar size (1-way analysis of variance, P = .003). There was no significant difference between any treatment with respect to presence or absence of ulcer, necrosis, large vessel vasculitis, or small vessel vasculitis. The only outcome of significance was that triamcinolone offered protection from thrombosis (chi2 likelihood ratio, P = .04). We also noted evidence of coagulopathy in all of the envenomated animals. The rabbits had grossly elevated activated partial thromboplastin time results, which were corrected with 1:1 mixing with normal rabbit plasma, suggesting an acquired factor deficiency. We did not detect an individual factor deficiency or a lupus anticoagulant. In a rabbit model, none of the agents tested (dapsone, diphenhydramine, colchicine, and intralesional triamcinolone) had an effect on eschar size. Triamcinolone appeared to offer some protection against histologic evidence of thrombosis, but this protection did not translate into a difference in clinical outcome. All animals developed evidence of coagulopathy, regardless of treatment. The coagulopathy could be corrected by fresh rabbit plasma, suggesting an acquired factor deficiency.

Accumulation of LOX-1 Ligand in Plasma and Atherosclerotic Lesions of Watanabe Heritable Hyperlipidemic Rabbits: Identification by a Novel Enzyme Immunoassay

LOX-1 (lectin-like oxidized LDL receptor) is a newly identified cell surface receptor for oxidized LDL mainly expressed in endothelial cells. Recombinant soluble LOX-1(LOX-Fc) was generated by fusing the extracellular domain of LOX-1 with the Fc portion of IgG. A novel sandwich enzyme immunoassay specific for LOX-1 ligand is designed, using LOX-Fc and anti-apoB antibody. This immunoassay was used to determine LOX-1 ligand activity in normal and Watanabe heritable hyperlipidemic (WHHL) rabbit plasma. LOX-Fc was further applied for staining of atherosclerotic lesions of WHHL rabbits. LOX-1 ligand levels were significantly elevated in the plasma of hyperlipidemic rabbits compared with controls. Furthermore, LOX-1 ligand activity was detected in the atherosclerotic lesions in situ. These results support the potential roles of LOX-1 interacting with its ligand in the pathogenesis of atherosclerosis, which is enhanced in hyperlipidemia.

Leakage of Protein into Lungs of Preterm Ventilated Rabbits Is Correlated with Activation of Clotting, Complement, and Polymorphonuclear Leukocytes in Plasma

We investigated whether leakage of protein in lungs of preterm ventilated rabbits of 28- and 29-d gestational age is correlated with activation of clotting, complement, and polymorphonuclear leukocytes (PMN) in plasma. We found signs of systemic activation of clotting, complement and PMN in ventilated 28-d gestational age rabbits, as indicated, respectively, by increased median plasma fibrin monomer concentrations (83 versus 40% of normal adult rabbit plasma in nonventilated 28-d gestational age rabbits, p < 0.01), decreased median plasma CH50 activity (112 versus 122 U/L in nonventilated 28-d gestational age rabbits, p < 0.05), and increased median plasma beta-glucuronidase concentrations (159 versus 97% of maximal activated adult rabbit plasma in nonventilated 28-d gestational age rabbits p < 0.05). We did not find signs of systemic activation in the ventilated 29-d gestational age group. Higher median total protein concentrations in alveolar wash of the ventilated 28-d gestational age rabbits (2.7 versus 1.3 mg/mL in the nonventilated rabbits. p < 0.01) indicated protein leakage into the lungs, and this protein leakage was more pronounced in the lungs of ventilated 28-d gestational age rabbits than in those of ventilated 29-d gestational age rabbits (2.1 mg/mL, p < 0.01). The total protein concentration in the alveolar wash of all 28-d gestational age rabbits was correlated with the concentration of fibrin monomers (p = 0.51, p = 0.035) and beta-glucuronidase (p = 0.61, p = 0.011), and the CH50 activity (p = -0.73, p = 0.002) in plasma. We conclude that leakage of protein in lungs of preterm ventilated rabbits of 28-d gestational age is correlated with activation of clotting, complement, and PMN in plasma. This activation process may contribute to lung injury by intravascular and intraalveolar deposition of fibrin and formation of proteinaceous edema.

Chylomicron Metabolism in Normal, Cholesterol-fed, and Watanabe Heritable Hyperlipidemic Rabbits: SATURATION OF THE SEQUESTRATION STEP OF THE REMNANT CLEARANCE PATHWAY

The plasma clearance of radiolabeled chylomicrons was compared in normal, cholesterol-fed, and Watanabe heritable hyperlipidemic (WHHL) rabbits. Chylomicron clearance was rapid in normal rabbits but was significantly retarded in cholesterol-fed and WHHL rabbits. At 40 min after the injection of chylomicrons, 14-17% of the injected dose remained in the plasma of normal rabbits, whereas approximately 40-50% of the injected dose remained in the plasma of cholesterol-fed and WHHL rabbits. The differences were reflected in the reduced plasma clearance by the liver and bone marrow of the cholesterol-fed and WHHL rabbits. The hyperlipidemic rabbits expressed normal levels of low density lipoprotein (LDL) receptor-related protein/alpha 2-macroglobulin receptor in the liver. In contrast, the hepatic levels of LDL receptors were lower in hyperlipidemic rabbits; as expected, they were significantly lower in WHHL rabbits compared with normal and cholesterol-fed rabbits. Furthermore, it was demonstrated that lipoproteins accumulating in the plasma of the hyperlipidemic rabbits competed for and retarded the clearance of chylomicrons from the plasma. Competition was demonstrated by cross-circulation of normal and cholesterol-fed or normal and WHHL rabbits, in which the rapid influx of plasma containing the accumulated plasma lipoproteins from cholesterol-fed or WHHL rabbits was shown to impair the uptake of chylomicrons by the liver and bone marrow of normal rabbits. These observations were extended by infusing isolated lipoproteins into normal rabbits. The rabbit d < 1.02 g/ml (remnant) fraction and the canine cholesterol-rich high density lipoproteins (HDL) with apolipoprotein E (HDLc) inhibited chylomicron clearance, whereas human LDL and HDL from humans and rabbits did not. We conclude that the low LDL receptor activity in the cholesterol-fed and WHHL rabbits may contribute, at least in part, to the impaired clearance by decreasing remnant uptake and causing the accumulation of chylomicron and/or very low density lipoprotein remnants. The accumulated remnant lipoproteins then compete for and saturate the mechanism responsible for the initial rapid clearance of chylomicrons from the plasma. We speculate that saturation of the initial rapid clearance may occur at the sequestration step, which involves the binding of remnants to heparan sulfate proteoglycans in the space of Disse.

Regulation of apolipoprotein B secretion in hepatocytes from Watanabe heritable hyperlipidemic rabbit, an animal model for familial hypercholesterolemia

Regulation of apolipoprotein B-100 (apo B) secretion in the liver in familial hypercholesterolemia (FH) remains largely unknown. In a previous study, we developed a rabbit hepatocyte culture system and investigated a cellular mechanism which regulates apo B secretion from hepatocytes in response to cellular cholesteryl ester contents [18]. Using this system, in the present study, we investigated regulation of apo B secretion in hepatocytes from the Watanabe heritable hyperlipidemic (WHHL) rabbit, an animal model for FH. Incubation with low density lipoproteins (LDL) at concentrations of 50 or 200 μg/ml, which increased both cellular cholesteryl ester and apo B secretion significantly in normal rabbit hepatocytes, did not cause such increases in WHHL rabbit hepatocytes. However, when WHHL rabbit hepatocytes were incubated with LDL at a concentration of 500 μg/ml, a significant increase in cellular cholesteryl ester and apo B secretion was observed. The effect of the increase in cellular level of cholesteryl ester upon apo B secretion in WHHL rabbit hepatocytes was compatible with that in normal rabbit hepatocytes. Indeed, when WHHL rabbit hepatocytes were incubated with LDL at 1680 μg/ml, a concentration comparable to that of WHHL rabbit plasma, the amount of LDL degradation, cellular cholesteryl ester level, and level of apo B secretion were the same as those in normal rabbit hepatocytes that were incubated with LDL at 160 μg/ml, a concentration comparable to that of normal rabbit plasma. In summary, our present study suggests that, at a steady state with such a high plasma concentration of LDL, the hepatic cholesterol content in WHHL rabbits could be set at the same level as in normal rabbits. It was also shown that cellular cholesteryl ester contents had the same regulatory effect on apo B secretion in WHHL rabbit hepatocytes as in normal rabbit hepatocytes. Therefore, we conclude that in the presence of the genetic defect of the LDL receptor, plasma cholesterol in WHHL rabbit is maintained at a concentration such that apo B secretion is similar to that in normal rabbit.

Role of plasminogen activator inhibitor-1 in promoting fibrin deposition in rabbits infused with ancrod or thrombin

The role of defective fibrinolysis caused by elevated activity of plasminogen activator inhibitor-1 (PAI-1) in promoting fibrin deposition in vivo has not been well established. The present study compared the efficacy of thrombin or ancrod, a venom-derived enzyme that clots fibrinogen, to induce fibrin formation in rabbits with elevated PAI-1 levels. One set of male New Zealand rabbits received intravenous endotoxin to increase endogenous PAI-1 activity followed by a 1-hour infusion of ancrod or thrombin; another set of normal rabbits received intravenous human recombinant PAI-1 (rPAI-1) during an infusion of ancrod or thrombin. Thirty minutes after the end of the infusion, renal fibrin deposition was assessed by histopathology. Animals receiving endotoxin, rPAI-1, ancrod, or thrombin alone did not develop renal thrombi. All endotoxin-treated rabbits developed fibrin deposition when infused with ancrod (n = 4) or thrombin (n = 6). Fibrin deposition occurred in 7 of 7 rabbits receiving both rPAI-1 and ancrod and in only 1 of 6 receiving rPAI-1 and thrombin (P < .01). In vitro, thrombin but not ancrod was inactivated by normal rabbit plasma and by purified antithrombin III or thrombomodulin. The data indicate that elevated levels of PAI-1 promote fibrin deposition in rabbits infused with ancrod but not with thrombin. In endotoxin-treated rabbits, fibrin deposition that occurs with thrombin infusion may be caused by decreased inhibition of procoagulant activity and not increased PAI-1 activity.

Role of Plasminogen Activator Inhibitor-1 in Promoting Fibrin Deposition in Rabbits Infused With Ancrod or Thrombin

AbstractThe role of defective fibrinolysis caused by elevated activity of plasminogen activator inhibitor-1 (PAI-1) in promoting fibrin deposition in vivo has not been well established. The present study compared the efficacy of thrombin or ancrod, a venom-derived enzyme that clots fibrinogen, to induce fibrin formation in rabbits with elevated PAI-1 levels. One set of male New Zealand rabbits received intravenous endotoxin to increase endogenous PAI-1 activity followed by a 1-hour infusion of ancrod or thrombin; another set of normal rabbits received intravenous human recombinant PAI-1 (rPAI-1) during an infusion of ancrod or thrombin. Thirty minutes after the end of the infusion, renal fibrin deposition was assessed by histopathology. Animals receiving endotoxin, rPAI-1, ancrod, or thrombin alone did not develop renal thrombi. All endotoxin-treated rabbits de-developed fibrin deposition when infused with ancrod (n = 4) or thrombin (n = 6). Fibrin deposition occurred in 7 of 7 rabbits receiving both rPAI-1 and ancrod and in only 1 of 6 receiving rPAI-1 and thrombin (P < .01). In vitro, thrombin but not ancrod was inactivated by normal rabbit plasma and by purified antithrombin III or thrombomodulin. The data indicate that elevated levels of PAI-1 promote fibrin deposition in rabbits infused with ancrod but not with thrombin. In endotoxin-treated rabbits, fibrin deposition that occurs with thrombin infusion may be caused by decreased inhibition of procoagulant activity and not increased PAI-1 activity.This is a US government work. There are no restrictions on its use.

L-Carnitine Administration and Withdrawal Affect Plasma and Hepatic Carnitine Concentrations, Plasma Lipid and Lipoprotein Composition, and In Vitro Hepatic Lipogenesis from Labeled Mevalonate and Oleate in Normal Rabbits

Carnitine was administered to normal rabbits to investigate the possible effects of pharmacologic doses on various aspects of normal lipid and lipoprotein metabolism. Carnitine concentrations were measured in the plasma and liver of normal rabbits that received L-carnitine orally [40 mg/(kg·d)] for 21 d and after withdrawal from the carnitine supplement for 21 d. Plasma lipids, plasma lipoprotein composition and in vitro hepatic lipid biosynthesis from [2-14C]mevalonate and [1-14C]oleate were also measured. Threefold elevations in plasma carnitine with carnitine treatment were essentially reversed after 48 h of carnitine withdrawal, but elevated hepatic carnitine accumulation (twofold) persisted for 21 d, suggesting that the accumulated carnitine constituted a pool that is only slowly miscible with plasma. The rabbits withdrawn from L-carnitine for 21 d experienced a 35% decrease in plasma cholesterol, a 50% decrease in VLDL cholesterol, and an increase in the protein content of HDL and of intermediate density lipoprotein + LDL. Additionally, the proportion of [14C]oleate incorporated into hepatic phospholipids increased 35% at the expense of triglyceride and the ratio of hepatic [14C]cholesterol to [14C]squalene derived from [14C]mevalonate increased over twofold following carnitine withdrawal. These studies provide evidence that normal lipid homeostasis can be altered by supplemental carnitine and that the perturbations are reflected by changes in plasma lipids and lipoproteins and in the proportions of the hepatic lipids synthesized.

Changes in plasma thrombomodulin antigen in rabbit developing endotoxin-induced disseminated intravascular coagulation and the effect of heparin

Soluble thrombomodulin (TM) antigen level was 1.64 ± 0.64 μg/ml (n=18, mean ± S.D.) in plasma of normal male rabbits as measured by enzyme immunoassay, and the antigen consisted of subspecies of 94, 83 and 51 kd. When disseminated intravascular coagulation (DIC) was induced by intravenous infusion of endotoxin into rabbits, the TM antigen level in plasma was elevated to about 1.5 times of the control value, and an increase in the 83 kd subspecies as well as the appearance of new subspecies of 76 and 48 kd was observed concomitantly with disappearance of the 94 kd subspecies in plasma. Elevation of the antigen level and disappearance of the 94 kd subspecies caused by infusion of endotoxin were reduced by simultaneous infusion of heparin. Addition of leukocytes stimulated with endotoxin plus FMLP to cultured endothelial cells induced release of TM antigen the medium accompanying cell injury as measured by 51Cr release, which was prevented by treatment with heparin. It was suggested that the increase in plasma TM antigen level in parallel with the generation of DIC reflected endothelial injury of rabbits, and that the elevation of TM antigen and the endothelial cell injury were prevented by heparin treatment.

Effect of Plasminogen Activator Inhibitor-1 on Tissue-Type Plasminogen Activator-Induced Fibrinolysis

The effect of fibrin on the interaction of human recombinant single-chain tissue plasminogen activator (t-PA) and plasminogen activator inhibitor-1 (PAI-1) was studied in normal rabbit plasma and in plasma with high levels of native PAI-1. t-PA was added to diluted plasma containing calcium (10 mM) and 125I-fibrinogen at 37 degrees C. Clotting was initiated with human thrombin, and lysis was monitored both turbidimetrically and by release of 125I-fibrin degradation products (fdp). The activity of t-PA (50 IU/ml) was rapidly reduced to 15% of the initial value in plasma containing PAI-1 (23 AU/ml). When thrombin and t-PA were added simultaneously to the plasma, more than 70% of the activity was retained through incorporation of t-PA into the fibrin clot. t-PA-induced fibrinolysis in PAI-1 enriched plasma was further delayed when the temperature was reduced from 37 to 25 degrees C. Turbidimetric and 125I-fdp release data provided complementary information. The former technique traced fiber dissolution, while the latter reflected network integrity. These results indicate that t-PA-induced fibrinolysis in PAI-1 enriched plasma is modulated by the presence of fibrin and by temperature.

Removal of apoprotein-B-containing lipoproteins by plasmapheresis using immobilized phosphorylcholine-binding protein affinity adsorbent

Rat serum phosphorylcholine binding protein was earlier shown to bind lipoproteins containing apoproteins B and E from human very low and low density lipoproteins. The present studies were undertaken to show the effectiveness of rat serum phosphorylcholine-binding protein immobilized on Sepharose affinity column to remove apoprotein-B-containing lipoproteins from normal and hypercholesterolemic rabbit plasma, when used in a plasmapheresis system. The maximum in vitro binding of very low and low density lipoproteins from hypercholesterolemic rabbit plasma to the affinity adsorbent was Ca2+ dependent, and the cholesterol bound to the column at the optimum calcium concentration (2.5 mM) was 21% of the total plasma cholesterol applied. The in vivo binding of total cholesterol from normal and hypercholesterolemic rabbit plasma during plasmapheresis ranged from 0.22 to 7.7%. Total mass of cholesterol bound ranged from 3.86 and 27.52 mg at plasma cholesterol concentrations 13.8 and 282 mg/dL, respectively. Most (greater than 95%) of the bound cholesterol was associated with very low and low density lipoproteins. These studies show the ability of immobilized rat serum phosphorylcholine-binding protein to lower the atherogenic apoprotein-B-containing lipoproteins from plasma of hypercholesterolemic rabbits.

The role of the low density lipoprotein receptor for alpha-tocopherol delivery to tissues.

To study the role of the LDL receptor pathway for the maintenance of alpha-tocopherol concentrations in tissues of intact animals, we have compared vitamin E levels in plasma and tissues of normal rabbits and WHHL rabbits. WHHL rabbits are deficient in LDL receptor activity. For WHHL rabbits, alpha-tocopherol plasma concentrations were elevated to 10 times normal levels. When plasma lipoprotein profiles were analyzed by density gradient centrifugation, concentrations of VLDL, IDL, and LDL were increased in WHHL rabbits, and plasma alpha-tocopherol was only recovered in these fractions. In normal rabbits, plasma alpha-tocopherol was confined mainly to the HDL fraction; levels associated with LDL were markedly lower. Despite LDL receptor deficiency, alpha-tocopherol concentrations in various tissues of WHHL rabbits were not found to be reduced, with the exception of the adrenal. Vitamin E levels in muscle and kidney of WHHL rabbits exceeded those of normal animals. Our results demonstrate the importance of the LDL receptor pathway for vitamin E clearance in the normal rabbit, although at high LDL concentrations, alternative mechanisms may become more efficient for the delivery of vitamin E to tissues.

Bile acid synthesis by cultured rabbit hepatocytes: stimulation by three lipoprotein fractions

Bile acid and cholesterol synthesis were measured in monolayer cultures of rabbit hepatocytes maintained in a defined culture medium. In the absence of lipoproteins, bile acid synthesis and secretion were correlated with cholesterol synthesis and were increased 245% by mevalonolactone (10 mM) and inhibited 45% by lovastatin (50 μg/ml) over 24 h. When included in the culture medium, normal rabbit plasma low-density and high-density lipoproteins increased bile acid synthesis and secretion by up to 140% of values obtained without lipoproteins in hepatocytes from normal or cholestyramine-fed rabbits. Three cholesterol-rich lipoprotein fractions (β-very low density, low density and high density) also were isolated from rabbits fed 1% cholesterol for 14 days. When added to rabbit hepatocyte cultures, each fraction markedly increased hepatocellular cholesterol content, stimulated bile acid synthesis and secretion in a dose-dependent manner, and inhibited cholesterol synthesis from radioactive acetate. These data indicate that three different lipoprotein fractions can provide cholesterol for uptake and subsequent breakdown to bile acids by cultured rabbit hepatocytes.

Dependence of fibrinolytic activity on the concentration of free rather than total tissue-type plasminogen activator in plasma after pharmacologic administration.

To identify factors responsible for the decline of plasma tissue-type plasminogen activator (t-PA)-specific activity that we have observed after infusions of the activator and to define the potential usefulness of selected variants of t-PA in obviating them in patients with infarction, serial plasma samples from patients (n = 4) and rabbits (n = 15) given t-PA were assayed for total t-PA antigen, t-PA activity, and free as opposed to type-1 plasminogen activator inhibitor (PAI-1)--complexed t-PA. In patients, attenuation of t-PA specific activity after infusions was evident with concentrations of total t-PA antigen that were as much as sevenfold greater than pretreatment values (62 compared with 9 ng/ml). Attenuation of t-PA activity corresponded with the disappearance of free t-PA from plasma and was associated with persistence of complexes of t-PA with PAI-1. In normal rabbits (n = 4) given wild-type t-PA by bolus injection, PAI-1 activity was 4 +/- 1 arbitrary units/ml. Attenuation of t-PA activity was not evident until 60 minutes after injection at a time when total plasma t-PA antigen concentration was as low as 13 +/- 8 ng/ml. Under these conditions, plasma t-PA was composed predominantly of free t-PA. In rabbits (n = 5) given lipopolysaccharide to increase plasma PAI-1 activity to 193 +/- 84 arbitrary units/ml, the specific activity of t-PA was attenuated as early as 15 minutes after injection at a time when total t-PA antigen concentration was as high as 164 +/- 79 ng/ml. As was the case with samples from patients, attenuation was associated with the disappearance of free t-PA and the persistence of complexes of t-PA with PAI-1. A genetically engineered variant of t-PA with comparable specific activity and a comparable rate constant of association with PAI-1 but designed to persist in the circulation manifested prolonged clearance from plasma of normal rabbits (n = 3) (t1/2 = 24.6 +/- 1.6 minutes compared with an alpha phase t1/2 of 1.9 minutes for wild-type t-PA). The variant lacked the epidermal growth factor and kringle one domains and contained a duplicated kringle two domain.(ABSTRACT TRUNCATED AT 400 WORDS)

A purification method for apolipoprotein A-I and A-II

Apolipoproteins A-I and A-II were isolated from precipitates obtained by cold ethanol fractionation of human plasma. The starting material used in this report was precipitate B of the Kistler and Nitschmann method which corresponds approximately to fraction III of the Cohn and Oncley procedure. Through the use of urea, chloroform, and ethanol in appropriate concentrations, apolipoproteins A-I and A-II were isolated by a simple extraction technique avoiding time-consuming ultracentrifugation. Starting from 10 g of centrifuged precipitate B, approximately 100 mg of apolipoprotein A-I and 10 mg of apolipoprotein A-II were obtained. When incubated with normal human or rabbit plasma, both apolipoproteins were readily incorporated into high-density lipoproteins. Apolipoprotein A-I obtained by the cold ethanol method activated lecithin-cholesterol acyltransferase to the same extent as apolipoprotein A-I prepared by the classical flotation method. Apolipoprotein A-II had no such properties by itself, but was capable of potentiating lecithin-cholesterol acyltransferase activity of apolipoprotein A-I.

Determination of hyaluronic acid in rabbit plasma by HPLC with fluorometric postcolumn detection.

A sensitive high performance liquid chromatographic method with fluorometric postcolumn labeling using 2-cyanoacetamide was developed for determination of unsaturated oligosaccharides produced from hyaluronic acid (HA) by digestion with Streptomyces Hyaluronidase. The unsaturated tetra-and hexasaccharides produced enzymatically from HA were separated completely within 30 min by HPLC. The detection limit was 10 ng of HA. This method was applied to the determination of HA in normal rabbit plasma to verify the sensitivity and practicability.

Hyperresponsiveness of arterial rings induced by renal prehypertensive rabbit plasma.

Vascular rings from normal rabbit renal arteries, when bathed in processed plasma from 3-day renal artery stenosis (RAS) rabbits, had greater contractile responses to norepinephrine than did matched rings bathed in processed plasma from sham-operated rabbits. This vascular hyperresponsiveness of the rings produced by 3-day RAS plasma was abolished by the angiotensin II (ANG II) antagonist [Sar1, Ile8] ANG II but not by [Sar1, Ala8] ANG II at a dose that completely blocked the contractile responses of the rings to ANG II. The addition of [Sar1, Ala8]-ANG II to rings bathed in normal rabbit plasma did not alter the contractile responses to norepinephrine, indicating a lack of agonistic action by this ANG II analogue. These studies demonstrated that the ANG II receptors involved in the hormonally mediated vascular hyperresponsiveness in 3-day RAS rabbits are contained in the tissues that comprise these renal arterial rings.

Microdetermination of Unsaturated Disaccharides Produced from Chondroitin Sulfates in Rabbit Plasma by High Performance Liquid Chromatography with Fluorometric Detection

A highly sensitive high performance liquid chromatographic method with fluorometric postcolumn labeling using 2-cyanoacetamide was developed for determination of unsaturated disaccharides produced enzymatically from chondroitin sulfates (ChS). The calibration curves of the unsaturated disaccharides were linear over the range from 2 pmol to 5 nmol. This method was applied to the analysis of normal rabbit plasma for ChS to verify the sensitivity and practicality.