Higher Albumin Concentrations Research Articles

Haematological and clinicochemical blood profiles in slaughter pigs.

In a cohort study, 40 pig finishing herds were selected: twenty pig herds with a low and twenty pig herds with a high prevalence of several pathological lesions recorded at slaughter in a six-month period before the start of the study. Blood samples were taken from 20 pigs per herd at the end of the finishing period to investigate haematological and clinicochemical profiles. There was only a significant difference in serum albumin concentration between the low and high lesion prevalence groups. There were distinct differences in blood profiles between pig herds, but also between batches of pigs within a herd, housed in different compartments. Differences between castrated males and gilts were also demonstrated with respect to mean values of the blood variables haemoglobin, iron, copper, beta-globulin, eosinophils and segmented neutrophils. However, the differences were not of a biologically important magnitude. The mean values of the blood variables pepsinogen and lymphocytes differed significantly between pig herds from the two participating integration groups. Pigs with a higher albumin concentration, a lower gamma-globulin concentration, a higher copper concentration and a higher creatine kinase activity in serum showed a higher daily weight gain.

Arachidonate Transfer Between Platelets and Lipoproteins

Although platelets have specific bindingsites for LDL and HDL, it is doubtful whether lipoproteins modulate platelet functions via receptor-mediated processes. We investigated platelet-lipoprotein interaction during prolonged incubation with concentrations of LDL and HDL that saturate the bindingsites within a few minutes. When [3H]arachidonate-labeled human platelets were incubated for 4 h with lipoproteins, part of the 3H-radioactivity transferred to LDL and to a lesser extent to HDL. The transfer was temperature-sensitive, unaffected by modification of lysine in LDL or indomethacin treatment of the platelets, and almost irreversible. [3H]arachidonate transfer to lipoproteins could be mimicked by incubating platelets with a high concentration of fatty acid free albumin. This showed, that the loss of 3H-radioactivity reflected a decrease in endogenous arachidonate, leading to impaired aggregation, secretion and thromboxane B2 formation in platelets after stimulation with thrombin but not with arachidonate. Thus, the decrease in platelet functions seen after long incubation with HDL is caused by depletion of platelet arachidonate. Despite an even stronger arachidonate depletion by LDL, this lipoprotein initiated arachidonate metabolism and secretion independent of specific binding sites for LDL on the platelet. Surprisingly, the major part of the secretion was preserved when the formation of prostaglandin endoperoxides/thromboxane A2 was inhibited with indomethacin. These findings argue against a role for LDL and HDL receptors in the modulation of platelet functions and are more in favor of lipid exchange processes between platelets and lipoproteins.

Stereoselective binding of etodolac to human serum albumin.

The protein binding of etodolac enantiomers was studied in vitro by equilibrium dialysis in human serum albumin (HSA) of various concentrations varying from 1 to 40 g/liter, by addition of each enantiomer at increasing concentrations. In the 1 g/liter solution, at the lowest drug levels, the (R)-form is more bound than its antipode, the contrary being observed at the highest drug levels. For higher albumin concentrations, S was bound in a larger extent than R. Using the displacement of specific markers of HSA sites I and II, studied by spectrofluorimetry, it was suggested that R and S are both bound to site I, while only S is strongly bound to site II.

Coupled water transport by rat proximal tubule

Simultaneous microperfusion of proximal tubules and peritubular capillaries in kidneys of rats anesthetized with Inactin was used to examine water reabsorption by this epithelium. Osmolality of the luminal solution was varied with changes in NaCl concentration and by the addition of raffinose. Capillary perfusates contained either low (2 g/dl) or high (16 g/dl) concentrations of albumin. We used low-bicarbonate perfusates for both lumen and capillary so that we might apply the nonequilibrium thermodynamic model of transport for a single solute (NaCl) to interpret our observations. Linear regression with the volume flux equation Jv = -Lp delta II - Lp sigma delta C + Jav (where Jv is volume flux, Lp is hydraulic conductance, delta II is oncotic force, sigma is osmotic reflection coefficient, delta C is salt concentration difference, and Jav is the component of Jv not attributed to transepithelial hydrostatic or osmotic forces) revealed a tubule water permeability (Pf = 0.11 +/- 0.01 cm/s) and a sigma (0.74 +/- 0.08) in agreement with previous determinations. These transport parameters were unaffected by changes in peritubular protein. We also found that Jav was substantial, approximately three-fourths of the rate of isotonic transport under these perfusion conditions. Further, this component of water transport nearly doubled with the transition from low- to high-protein peritubular capillary perfusion. When expressed as a capacity for water reabsorption against an osmotic gradient, the salt concentration differences required to null volume flux were 13.2 +/- 2.4 and 29.4 +/- 4.0 mosmol/kgH2O under low and high peritubular protein. Our data suggest that this protein effect is, most likely, an increase in solute transport by the tubule epithelial cells.

Detection of serum α-fetoprotein in dogs with hepatic tumors

Summary Serum α-fetoprotein (afp) concentration was detected by use of 2 commercially available kits containing antibodies to human afp—a radioimmunoassay and an enzymetric test. Using neonatal canine serum (a source high in afp), it was determined that reagents from both kits were able to bind to canine afp, but a significant difference was detected in afp concentration. The enzymetric test was superior in detecting canine afp. Sera from dogs were classified into 6 groups: from dogs with primary hepatic tumors only (group 1); from dogs with primary hepatic tumors and other tumors (group 2); from dogs with normal liver but with other types of neoplasia (group 3); from dogs with nonneoplastic hepatic disease and tumors originating in other organs (group 4); from dogs with nonneoplastic hepatic disease only (group 5); and from clinically normal dogs (group 6). Serum biochemical determinations (alkaline phosphatase, alanine transaminase, albumin, total protein, total bilirubin, and serum bile acids) and values from the 2 afp assays were obtained for all dogs. Serum afp concentration detected by the enzymetric test was significantly higher in dogs with hepatocellular carcinoma and cholangiocarcinoma. Values > 250 ng/ml were detected in 5 of 9 dogs with cholangiocarcinoma and in 3 of 4 dogs with hepatocellular carcinoma. High serum afp concentration also was indicative of liver involvement in 2 of 3 dogs with primary hepatic lymphosarcoma; 2 dogs had values > 225 ng/ml. Serum afp concentration in dogs with other types of hepatic tumors was < 250 ng/ml, and serum afp concentration could not be correlated with such tumors. Discriminatory analysis was done to determine which serum biochemical values best aided differentiation of hepatic neoplasia from nonneoplastic hepatic disease. Along with high serum afp concentration, high serum alkaline phosphatase activity and low albumin concentration were the most important analytes. If hepatic tumor is suspected, we recommend use of the enzymetric test to determine afp concentration, in addition to use of serum biochemical values, clinical findings, and radiography, to aid in the diagnosis.

Effects of albumin on ionized calcium in vitro

Human serum albumin is used to treat hypoproteinemia in neonates or used as a volume expander. We tested the hypothesis that the addition of human serum albumin to neonatal serum decreases the serum ionized calcium (iCa) concentration. Concentrated human serum albumin was added to 12 placental and cord serum samples to reach seven incremental concentrations from 0 to 20.0 g/L. Serum iCa concentration decreased significantly with the addition of serum albumin. From multiple regression analysis, the effect of albumin addition on serum iCa concentration had more marked effects at high baseline albumin or low baseline Ca concentrations. From this in vitro study, we speculate that fast infusion of albumin in human neonates has the potential for acutely lowering serum iCa concentration.

Uptake of bilirubin glucuronides by isolated rat hepatocytes

The uptake of bilirubin diglucuronide (BDG) into isolated rat hepatocytes was investigated in order to characterize the mechanism by which bile pigments are transported by the liver. The BDG uptake by hepatocytes was saturable. The uptake was inhibited by bilirubin, sulfobromophthalein, and bilirubin monoglucuronide, but not by taurocholate. The uptake was not affected by replacement of sodium with other cations except for choline. Only when sodium was replaced with choline, was significant decrease in uptake observed. When chloride was replaced with nitrate, BDG uptake decreased, but it was not changed by replacement with sulfate. Metabolic inhibitors did not affect BDG uptake significantly. Thus bile pigments share a common sodium-independent and electrogenic potential-dependent transporter in liver cell membranes. A high concentration of albumin interferes with BDG uptake.

Receptor-mediated endocytosis of albumin in cultured opossum kidney cells: a model for proximal tubular protein reabsorption.

The accumulation of fluorescein (FITC)-labelled bovine albumin was measured against the extracellular-fluid-phase marker FITC-inulin within confluent monolayers of the opossum kidney cell line OK. Fluorescence and electron microscopic pictures show that FITC-albumin is taken up by endocytosis and appears in a vesicular intracellular distribution. The uptake of FITC-albumin was quantified by measuring the cell-adherent fluorescence fluorimetrically. FITC-albumin uptake shows a time- and concentration-dependent saturation kinetics in contrast to the non-saturable FITC-inulin uptake, and exceeds the latter more than tenfold at low concentrations. Half-maximum saturation occurs at 20-30 mg/l. Initial FITC-albumin uptake/mg protein is stimulated by cell maturation, being six-to sevenfold higher in the confluent than in the subconfluent state, while FITC-inulin uptake is unchanged. Both an elevation of ambient osmolality to 600-750 mOsm/kg and disruption of the cytoskeleton by cytochalasin B (0.1 mmol/l) reduce initial FITC-albumin uptake by 50%-60% in a non-additive fashion. Albumin endocytosis is reduced both in acidic (pH 5.4) and alkaline (pH 8.4) medium, but does not depend on extracellular sodium, calcium or chloride. High concentrations of fetal calf serum or unlabelled albumin reduce FITC-albumin endocytosis dose-dependently. The present study is the first to investigate both the protein uptake and the fluid-phase endocytosis in a cultured proximal tubular cell line, using these cells as a model system-for proximal tubular protein reabsorption.

Blood compatibility of PEO grafted polyurethane and HEMA/styrene block copolymer surfaces

HEMA/styrene (HEMA/STY) block copolymers and poly(ethylene oxide) 4,000 M.W. (PEO4K) grafted Biomer (B-PEO4K) surfaces have been synthesized, characterized, and evaluated as blood-contacting materials. These surfaces have demonstrated improved blood compatibility, compared to Biomer, in in vitro and ex vivo experiments. Biomer vascular grafts (6 mm I.D. 7 cm in length) were fabricated by a dip coating process. The luminal surface was modified either with PEO grafting, HEMA/STY coating, or Biomer coating (control). These surface-modified grafts were implanted in the abdominal aortas of dogs and evaluated for graft patency and protein adsorption. Surface protein layer thickness was measured by transmission electron microscopy (TEM). B-PEO4K and Biomer showed thick multilayers of adsorbed proteins (1000-2000 A) after 3 weeks to 1 month implantation. In contrast, HEMA/STY only showed a monolayer protein thickness (less than 200 A), even after 3 months. Visualization of adsorbed plasma proteins (albumin, IgG, and fibrinogen) was performed with scanning electron microscopy (SEM)/TEM using an immunogold double antibody technique. The pattern of protein distribution showed high concentrations of fibrinogen and IgG, and less albumin adsorbed onto Biomer and B-PEO4K. In contrast, HEMA/STY showed a patchy protein distribution pattern with high concentrations of albumin and IgG, and relatively less fibrinogen. Adsorbed monolayer patterns showed improved compatibility over multilayered proteins. The Biomer and B-PEO4K grafts occluded within 1 month, while HEMA/STY grafts were patent for over 3 months. The thin and stable adsorbed protein layer on HEMA/STY surfaces may be associated with the microdomain structures of the surface, and will play an important role in long-term in vivo blood compatibility. This manuscript will evaluate the long-term in vivo performance of these polymers, analyze the extent of protein adsorption onto the surfaces, and correlate protein layer thickness to the thrombogenicity of the polymer surfaces.

Glucagon Inhibits Insulin Activation of Glucose Transport in Rat Adipocytes Mainly through a Postbinding Process

Incubation of rat adipocytes with 1 microM glucagon plus adenosine deaminase (5 micrograms/ml) inhibited maximally insulin-stimulated 3-O-methyl-D-glucose (MeGlc) transport by approximately 70%, concomitant with 30% and 55% decreases in insulin binding and cellular ATP, respectively. In contrast, under conditions where cellular ATP levels are well preserved (i.e. high albumin concentration in the medium), the inhibition of transport was reduced to about 30%, but that of insulin binding was not. Because depletion of the cellular ATP level by more than 60% by metabolic inhibitors induced 40% or more inhibition of insulin-stimulated MeGlc transport, the greater inhibition of the transport with the low albumin concentration appears to be caused in part by the secondary effect of ATP loss. The relationship between the amount of cell-bound insulin and hormone-stimulated transport activity showed that glucagon does not modulate insulin action at the step of insulin binding to its receptors. Furthermore, glucagon suppressed insulin-stimulated MeGlc transport, mainly through an attenuation of the hormone-induced increase in maximum velocity. The data show that glucagon modulates the process of signal transduction of insulin action. However, the possibility that glucagon directly modulates the process of translocation or the intrinsic activity of the glucose transporters cannot be eliminated.

Interactions of glycolytic enzymes with erythrocyte membranes

Partition equilibrium experiments have been used to characterize the Interactions of erythrocyte ghosts with four glycolytic enzymes, namely aldolase, glyceraldehyde-3-phosphofructokinase and lactate dehydrogenase, in 5 m M sodium phosphate buffer (pH 7.4). For each of these retrameric enzymes a sing intrinsie association constant sufficed to describe its interaction with crythrocyte matrix sites, the membrane capacity for the first three enzymes coinciding with the band 3 protein content. For lactate dehydrogenase the erythrocyte membrane capacity was twice as great. The membrane interactions of aldolase and glyceraldehyde-3-phosphate dehydrogenase were mutually inhibitory, as were those involving either of these enzymes and lactate dehydrogenase. Although the binding of phosphofructokinase to erythrocyte membranes was inhibited by aldolase, there was a transient concentration range of aldolase for which its interaction with matrix sites was enhanced by the presence of phosphofructokinase. In the presence of a moderate concentration of bovine serum albumin (15 mg/ml) the binding of aldolase to erythrocyte ghosts was enhanced in accordance with the prediction of thermodynamic nonideality based on excluded volume. At higher concentrations of albumin, however, the measured association constant decreased due to very weak binding of the space-filling protein to either the enzyme or the erythrocyte membrane. The implications of these finding are discussed in relation to the likely subcellular distribution of glycolytic enzymes in the red blood cell.

Determination of copper(II) in a complex matrix after prereduction to copper(I) in a flow-injection system with an amperometric detector

A flow-injection system with a carbon paste detector is proposed for the determination of copper in complex media at +0.050 V vs. Ag/AgCl. Both the reduction current peak of Cu(II) and the oxidation peak of Cu(I) (obtained in the presence of hydroxylamine in the reagent stream) were proportional to the Cu(II) concentration in the original sample. The stabilization of Cu(I) in the hydroxylamine system provides a novel approach; the inherent repeatability of operation of flow-injection systems (timing, etc.) proved ideal for this utilization. Acidified Cu(II) samples containing additional metal ions [Fe(lI), Zn(II), Pb(II)] and relatively high concentrations of serum albumin were analysed. A chelating column retained heavy metal ions while allowing albumin to run to waste. The retained metals were subsequently eluted with nitric acid into a stream of sodium acetate or sodium acetate-hydroxyl- amine. Before reaching the detector, the pH of the sample plug was adjusted. With the hydroxylamine system, Fe(III) interference was minimized and the sensitivity and reproducibility were improved. The sample throughput was 25 h−1. National Bureau of Standards Standard Reference Material 909 Human Serum was used to test the method.

In vivo protein adsorption onto polymers: a transmission electron microscopic study.

This paper reports in vivo protein adsorption onto polymers, including Biomer, PEO grafted Biomer (B-PEO-4K), heparin immobilized Biomer with PEO spacers (B-PEO-4K-HEP), and HEMA-Styrene block copolymer (H-S). Vascular grafts (6 mm ID, 7 cm in length) were fabricated with Biomer, coated on their luminal surfaces with test polymers, and implanted into the abdominal aorta of dogs. After 3 weeks-1 month, the grafts were retrieved and processed for TEM and SEM. TEM measured the thickness of adsorbed protein layers stained with a OsO4 solution, and the distribution pattern of adsorbed proteins (albumin, IgG and fibrinogen) using the immunoperoxidase technique. Retrieved grafts of Biomer and B-PEO-4K showed mural thrombi along the graft length, while thrombus formation on B-PEO-4K-HEP and H-S grafts was limited to the anastomotic sites. SEM pictures of B-PEO-4-HEP and H-S surfaces demonstrated clear morphology, with minimal platelet adhesion and activation, and microthrombi. Biomer and B-PEO-4K demonstrated a thick proteinaceous layer (1000-2000 A), whereas B-PEO-4K-HEP and H-S showed what can be described as a monolayer protein thickness (200-300 A). B-PEO-4K-HEP and H-S showed a monolayer-like adsorbed protein pattern, with high concentrations of albumin and IgG, and less fibrinogen, while Biomer and B-PEO-4K showed multilayered patterns with relatively high concentrations of fibrinogen, and less albumin. These results suggest that the surface properties of polymer may control protein adsorption pattern, and the composition of adsorbed protein is essential to in vivo long-term blood compatibility.

Albumin absorption by canine bronchial epithelium.

Albumin concentrations in airway surface liquid are low compared with plasma. To investigate the mechanisms that generate albumin gradients across airway epithelia, we have investigated whether active albumin absorption is a feature of bronchial epithelia. Freshly excised canine bronchi were mounted in Ussing chambers and short-circuited. Permeability coefficients of 14C-labeled canine albumin (Palb) were measured in the mucosal-to-submucosal (M----S) and submucosal-to-mucosal (S----M) directions in conductance-matched tissues. Mean steady-state values for Palb in the absorptive (M----S) direction (5.97 +/- 1.89 x 10(-7) cm/s) were significantly greater than rates in the S----M direction (1.09 +/- 0.41 x 10(-7) cm/s). Simultaneous measurements detected no asymmetry of transport of the fluid phase marker [3H]inulin. Gel filtration chromatography demonstrated that the majority of the radiolabel released into the submucosal bathing solution represented albumin fragments. Albumin fragments per se were not transported because no asymmetries in permeabilities of albumin fragments isolated from spontaneous degradation of tracer were detected. Decreasing the temperature of the bathing solution from 37 to 4 degrees C completely inhibited net albumin absorption. [14C]albumin transport was saturated by addition of high concentrations of unlabeled albumin (estimated Michaelis constant = 1.6 x 10(-3) M). These results demonstrate that albumin is absorbed by a low-affinity process that may contribute to the maintenance of low albumin concentrations in secretions.

Albumin, a biologically active protein acting on Leydig cells

The objective of the present study was to characterize further the albumin fraction of rat testicular fluid (rTF), which can enhance luteinizing hormone (LH)-stimulated pregnenolone production by immature Leydig cells in vitro. Testicular fluid, obtained from 300 rat testes was fractionated by sequential ammonium sulfate precipitation, dye-affinity, anion-exchange chromatography, chromatofocussing and an additional heat treatment. The final fraction showed a single band when analyzed on a silver-stained sodium dodecyl sulfate-polyacrylamide gel and isoelectric focussing gel. The protein had a molecular weight of 67 kDa, an isoelectric point of 5.0 and was identified as albumin after Western blotting using an antibody against rat serum albumin. Albumin in this fraction gave a dose-dependent (0.1–2% protein, w/v) increase in LH-induced pregnenolone production, up to 4-fold, and the increase in specific bioactivity when compared to rTF was 1.4-fold. Selective depletion of albumin from testicular fluid was used as another approach to confirm that albumin itself is the main biologically active component in rTF. rTF from mutant analbuminemic rats (albumin content 0.02 mg/ml) did not stimulate LH-induced steroid production in our assay, in contrast to rTF from normal rats (albumin content 40 mg/ml). Albumin fractions obtained from rat, bovine and human sera were also effective in stimulation of steroid production in the presence of LH, in contrast to chicken serum albumin which gave no stimulation. The stimulatory effect of albumin is not caused by bound fatty acids, nor by the presence of modified forms of albumin such as testibumin or the albumin-bilirubin complex. Our results indicate that Leydig cells are more active in steroid production when surrounded by high but physiological concentrations of albumin.

Mechanism for the inhibitory and stimulatory actions of proteins on the activity of phospholipase A 2

The influence of proteins on phospholipase A 2 was found to depend strongly on the enzyme assay system. We have used three different systems to measure phospholipase A 2 which represent the different assay conditions used by a number of previous investigators. Two distinct stimulatory and two distinct inhibitory effects of proteins were observed. (1) A previous investigators. Two distinct stimulatory and two distinct inhibitory effects of proteins were observed. (1) A number of proteins - such as albumin, γ-globulin and lysozyme - were found to inhibit phospholipase A 2 activity only at very low substrate concentrations. This ‘substrate depletion’ was recently proposed as the mode of action for lipocortin. We therefore suggest that substrate depletion is not sufficiently specific to serve as a physiological regulatory mechanism and that the observed inhibition by lipocortin and other proteins more recently reported to mimic it are unlikely to be of physiological significance. (2) Use of liposomes at higher concentrations led to a nonlinear time-course. In this assay system, albumin (and other protein) stimulation can be accounted for as relief of product inhibition. (3) With high concentrations of phospholipids in the presence of cholate (mixed micelles), the behavior of proteins in the assay was complex. The assay time-course appeared linear in the absence of added protein, but at concentrations of added albumin up to 1 mg/ml, stimulation of phospholipase A 2 activity was observed. Concentrations greater than this led to diminution of enzyme activity to the original activity. No effect whatever was observed when lysozyme was substituted for albumin. Since this biphasic result was not observed with liposomes, we suggest that the product whose inhibition is being relieved is the lysophosphatidylcholine, and not the free fatty acid. The inhibitory effect at high albumin concentrations is probably the result of removal of free fatty acids from the micelle: fatty acids are known to cause stimulation of phospholipase A 2 by providing a negative charge to the lipid/water interface. (4) A different type of phospholipase A 2 stimulation was apparent with melittin. This was found to be more specific than generally believed: we found no melittin stimulation of pancreatic phospholipase A 2, yet confirmed a several-fold stimulation of bee venom phospholipase A 2. We also found that high (millimolar) concentrations of calcium suppressed the melittin stimulation of bee venom phospholipase A 2, and that a cationic detergent mimicked the stimulation by melittin. (5) We conclude that the effects of proteins on phospholipase A 2 studied here can all be explained by proteins binding to substrate or product rather than enzyme-protein interactions.

成分献血時に用いる抗凝固剤ＳＣＳ‐４（４％クエン酸ナトリウム液）の有用性の検討

ACD-A solution, anticoagulant for preserved blood, has been used for donor plasmapheresis (DP), howerver it has some undesirable problems for the puropose.This time, we examined usefulness of SCS-4 (4% sodium citrate solution, USP XXI) as an anticoagulant for DP in 85 healthy volunteers, 75 cases by membrane method and 10 cases by centrifugal method.SCS-4 was safely used as an anticoagulant for DP in both methods, in less volume than the conventional ACD-A solution because of the higher citrate concentration. The blood picture, biochemistry, coagulation factors and immunoglobulin of the donors returned normal promptly in both methods. We could obtain plasma with high concentration of total protein, albumin, immunoglobulin and coagulation factors.These results indicate that SCS-4 is useful as an anticoagulant for DP.

Effect of Altered Albumin Concentrations on Elimination of Unbound Prazosin In Vivo in the Rat and in the Isolated Perfused Rat Liver

Albumin is known to affect the intrinsic clearance of many compounds in the isolated perfused rat liver, but little is known of its effect in vivo. The influence of decreased albumin concentrations on clearance of unbound prazosin and intrinsic clearance of prazosin was therefore studied in vivo in rats that had undergone plasmapheresis. An approximate 30% reduction in the plasma albumin concentration was achieved in animals not given plasma expanders and an approximate 50% reduction was achieved in animals given Ficol 70 as a plasma expander. No differences were seen in the intrinsic clearances or in the clearances of unbound prazosin between control animals and plasmapheretic animals. The reason for this lack of effect is apparent from parallel studies in isolated perfused rat livers. An increase in the clearance of unbound prazosin of 27% was seen when the albumin concentration was increased from 0 to 30 microM, and of 49% when the concentration of albumin was increased to 90 microM, with no further increase at higher albumin concentration. These results, therefore, suggest that changes in the albumin plasma concentrations normally encountered clinically may have little effect on the intrinsic elimination of drugs.

Study of platelet activating factor and its antagonists on rat colon strip with a new method avoiding tachyphylaxis

Platelet activating factor induced slow, but sustained contraction of isolated rat colon in a dose-dependent manner. The contraction persisted even when the strips were washed several times with Tyrode solution. However, the addition of high concentrations of bovine serum albumin to the washing solution led to the rapid relaxation of the strips to the basal level, possibly by removing platelet activating factor tightly bound to rat colon. These strips then responded normally to a second addition of platelet activating factor. Neither the cholinergic nervous system nor release of histamine, serotonin, prostaglandins or leukotriene D 4 were significantly involved in the contraction induced by platelet activating factor. FR-900452 and CV-3988, recently found to be antagonists of platelet activating factor, selectively blocked the contraction of rat colon induced by the active phospholipid, indicating that it induced ontraction by a direct stimulatory effect on the smooth muscle of rat in a receptor-mediated manner.

Fluorescence labeling and sorting of hepatocyte subpopulations to determine the intralobular heterogeneity of paraoxon-metabolizing enzymes in DDE-treated and control rats

To obtain hepatocyte subpopulations of defined intralobular regional origin, rat livers were perfusion-labeled with fluorescein diacetate. A high concentration of fluorescein diacetate solution was prepared with the aid of porcine serum albumin and pulse-infused into the rat liver in situ in an antero- or a retrograde mode. Fluorescein diacetate was rapidly and reversibly taken up by hepatocytes of afferent regions and migrated “chromatographically” along the sinusoid while undergoing very rapid hydrolysis to fluorescein. The migration rate was accelerated by higher albumin concentrations in the infusate and perfusate and by higher flow rates. Flrorescence photomicrographs showed a distinct labeling of afferent regions which appeared as the brightest labeled population in a flow cytometry plot of frequency vs log (fluorescence). The cytometry plot showed unlabeled hepatocytes as a major broad peak above autofluorescence and fluorescence-labeled cells as a plateau above the peak. The centrilobular hepatocytes sorted in a fluorescence-activated cell sorter contained about twofold as much activity for paraoxonase, paraoxon deethylase, and fluorescein diacetate hydrolase as the periportal hepatocytes of the liver from both control and DDE-treated rats. DDE-treated rat livers had about 15-fold higher paraoxon deethylase activity than controls while paraoxonase and fluorescein diacetate hydrolase were elevated about 1.5 times.