Plasma Binding Research Articles

Microdialysis of Triamcinolone Acetonide in Rat Muscle

The objective of this study was to compare plasma and muscle concentrations of triamcinolone acetonide (TA) in the rat by microdialysis. Microdialysis experiments were carried out at steady state in rats after an initial I.V. bolus 50 mg/kg of the phosphate ester of TA (TAP) followed by 23 mg/kg/h infusion. In vivo recovery was calculated by retrodialysis. The concentration determined at steady state in microdialysate, corrected for recovery, was 2.73 +/- 0.42 microg/mL compared to 21.9 +/- 2.3 microg/mL in plasma. The pharmacokinetics of TA in plasma was described by an open two-compartment model with a terminal half-life of 2.7 h. The clearance of TA in rats determined by compartmental analysis was 0.94 L/h/kg. The measured microdialysate levels of TA in muscle, corrected for recovery, were comparable to the predicted free drug levels in the peripheral compartment. Protein binding in rat plasma, measured by ultrafiltration, was 90.1%. The microdialysis in vivo recovery in muscle was similar to the in vitro recovery under stirred conditions. The results show the applicability of microdialysis to measure free tissue concentrations of TA in rats.

Hydromorphone transfer into breast milk after intranasal administration.

To determine the distribution of hydromorphone into breast milk and the potential exposure of the suckling infant, and whether the distribution of hydromorphone into milk can be predicted accurately by a passive diffusion model. Single-dose, pharmacokinetic study. University clinical research unit. Eight lactating, nonsmoking, healthy women aged 24-32 years. Hydromorphone HCl 2 mg was given intranasally to the women to characterize its pharmacokinetics and extent of its transfer into breast milk. Plasma and milk samples were analyzed using liquid chromatography with tandem mass spectrometry detection. The milk:plasma ratio (M:P) was calculated as the total area under the concentration-time curve (AUC) of the milk divided by the total AUC of the plasma. Predicted in vitro M:P ratios were calculated using a diffusion model. Protein binding in milk and plasma, partitioning into milk fat (whole milk:skim milk ratios), as well as pH partitioning between plasma and milk were incorporated in the model. Protein binding was determined by equilibrium dialysis. Protein binding was minimal in both milk and plasma, with unbound fractions of 1 and 0.84, respectively There was little partitioning into milk fat, as demonstrated by the whole milk:skim milk ratio of 0.98. The observed and predicted M:P ratios +/- SD for hydromorphone were 2.57 +/- 0.47 and 1.11 +/- 0.28, respectively. The 95% confidence interval for the observed M:P ratio overlapped the confidence interval of the predicted M:P ratio, a finding that supports a role for both passive diffusion and active transport as mechanisms of hydromorphone transfer into milk. Hydromorphone distributes rapidly from plasma into breast milk; however, the drug does not partition into fat. The suckling infant would receive approximately 0.67% of the maternal dose of hydromorphone (adjusted for body weight). As this is a limited exposure, further studies are needed to determine any potential impact to an infant who is fed breast milk from a mother treated with hydromorphone.

Specificity of plasma membrane targeting by the rous sarcoma virus gag protein.

Budding of C-type retroviruses begins when the viral Gag polyprotein is directed to the plasma membrane by an N-terminal membrane-binding (M) domain. While dispersed basic amino acids within the M domain are critical for stable membrane association and consequent particle assembly, additional residues or motifs may be required for specific plasma membrane targeting and binding. We have identified an assembly-defective Rous sarcoma virus (RSV) Gag mutant that retains significant membrane affinity despite having a deletion of the fourth alpha-helix of the M domain. Examination of the mutant protein's subcellular distribution revealed that it was not localized to the plasma membrane but instead was mistargeted to intracytoplasmic membranes. Specific plasma membrane targeting was restored by the addition of myristate plus a single basic residue, by multiple basic residues, or by the heterologous hydrophobic membrane-binding domain from the cellular Fyn protein. These results suggest that the fourth alpha-helix of the RSV M domain promotes specific targeting of Gag to the plasma membrane, either through a direct interaction with plasma membrane phospholipids or a membrane-associated cellular factor or by maintaining the conformation of Gag to expose specific plasma membrane targeting sequences.

Enzymology and Molecular Biology of Glucocorticoid Metabolism in Humans

Glucocorticoids (GCs) are a vital class of steroid hormones that are secreted by the adrenal cortex and that are regulated by ACTH largely under the control of the hypothalamic-pituitary-adrenal axis. GCs mediate profound and diverse physiological effects in vertebrates, ranging from development, metabolism, neurobiology, anti-inflammation and programmed cell death to many other fuctions. Multiple factors "downstream" of GC secretion, such as glucocorticoid receptor (GR) number and the abundance of plasma binding proteins have originally been considered as modulators of GC action. However, in the last decade the role of tissue-specific GC activating and inactivating enzymes have been identified as additional determinants in GC signalling pathways. On the cellular level, they function as important pre-receptor regulators by acting as "molecular switches" for receptor-active and receptor-inactive GC hormones. According to their biologic activity to catalyze the interconversion of C11-hydroxyl and C11-oxo GCs these enzymes have been named 11beta-hydroxysteroid dehydrogenase (11beta-HSD; EC 1.1.1.146). Two isoforms of 11beta-HSD have been cloned and characterized so far. 11beta-HSD type 1 is found in a wide range of tissues, acts predominantly as a reductase in intact cells and tissues by regenerating active cortisol from cortisone, and has been described to regulate GC access to the GR. 11beta-HSD type 2 is found mainly in mineralocorticoid target tissues such as kidney and colon, acts only as a dehydrogenase by producing inactive cortisone, and has been found to protect the mineralocorticoid receptor from high levels of receptor-active cortisol. Recently, 11beta-HSD 1 has become highly topical due to the finding that 11beta-HSD 1 plays a pivotal role in the pathogenesis of central obesity and the appearance of the metabolic syndrome. This review provides an overview on the components involved in GC signalling of 11beta-HSD type 1 as an important pre-receptor control enzyme that modulates activation of the GR.

Protein binding of isofluorophate in vivo after coexposure to multiple chemicals.

Full toxicologic profiles of chemical mixtures, including dose-response extrapolations to realistic exposures, is a prohibitive analytical problem, even for a restricted class of chemicals. We present an approach to probing in vivo interactions of pesticide mixtures at relevant low doses using a monitor compound to report the response of biochemical pathways shared by mixture components. We use accelerator mass spectrometry (AMS) to quantify [14C]-diisopropylfluorophosphate as a tracer at attomole levels with 1-5% precision after coexposures to parathion (PTN), permethrin (PER), and pyridostigmine bromide separately and in conjunction. Pyridostigmine shows an overall protective effect against tracer binding in plasma, red blood cells, muscle, and brain that is not explained as competitive protein binding. PTN and PER induce a significant 25-30% increase in the amount of tracer reaching the brain with or without pyridostigmine. The sensitivity of AMS for isotope-labeled tracer compounds can be used to probe the physiologic responses of specific biochemical pathways to multiple compound exposures.

Endocrine disrupting chemicals: interference of thyroid hormone binding to transthyretins and to thyroid hormone receptors

We examined the effects of industrial, medical and agricultural chemicals on 3,5,3′- l-[ 125I]triiodothyronine ([ 125I]T 3) binding to transthyretins (TTRs) and thyroid hormone receptors (TRs). Among the chemicals investigated diethylstilbestrol (DES) was the most powerful inhibitor of [ 125I]T 3 binding to chicken and bullfrog TTR (cTTR and bTTR). Inhibition of [ 125I]T 3 binding was more apparent in cTTR than bTTR. Scatchard analysis revealed DES, pentachlorophenol and ioxynil as competitive inhibitors of [ 125I]T 3 binding to cTTR and bTTR. However, cTTR's affinity for the three chemicals was higher than its affinity for T 3. A miticide dicofol (10 −10–10 −7 M) activated [ 125I]T 3 binding to bTTR up to 170%. However, at 4×10 −5 M it inhibited [ 125I]T 3 binding by 83%. Dicofol's biphasic effect upon [ 125I]T 3 binding was not detected in TTRs from other species. DES and pentachlorophenol, in the presence of plasma, increased cellular uptake of [ 125I]T 3 in vitro, by displacing [ 125I]T 3 from its plasma binding sites. These chemicals did not, however, affect the association of cTTR with chicken retinol-binding protein. All chemicals investigated had little or no influence on [ 125I]T 3 binding to chicken TR (cTR) and bullfrog TR (bTR). Several endocrine disrupting chemicals that were tested interfered with T 3 binding to TTR rather than to TR. Binding of the endocrine disrupting chemicals to TTR may weaken their intrinsic effects on target cells by depressing their free concentrations in plasma. However, this may affect TH homeostasis in vivo by altering the free concentrations of plasma THs.

Plasma binding proteins as mediators of corticosteroid action in vertebrates.

Stressors elicit a complex but variable suite of endocrine events. Comparative studies of the stress response have focused primarily on the adrenocortical response to stress, in particular the measurement of plasma levels of glucocorticoids. However, a number of other factors contribute to and modify cellular and organismal responses to glucocorticoids. Notably, plasma corticosteroid binding globulins (CBGs) can regulate the general availability of steroid to tissues, and/or direct the delivery of hormones to specific sites. In this paper, we discuss possible functions of CBG and mechanisms of CBG action, review CBG characteristics among vertebrates, and discuss our recent studies indicating that CBG may indeed modulate responses to stressors. For example, in house sparrows, we found that basal and stress-induced concentrations of total corticosteroid (cortisol or corticosterone) (CORT) vary seasonally, but CBG concentrations change proportionally, so that free CORT concentrations appear static year-round. In contrast, in white-crowned sparrows and tree lizards, CBG concentrations change under conditions when total CORT levels do not, resulting in significant changes in circulating free CORT. These differences in free CORT are masked if CBG is not accounted for. We have also found that the binding properties of CBG vary considerably between species and need to be determined empirically. Such studies led to the observation that CBG in several species may also serve as a functional androgen binding protein; this is especially important for birds, because previous studies had concluded that birds lack androgen binding globulins. We propose that consideration of CBG is paramount to understanding the role of glucocorticoids in mediating behavioral and physiological responses to stress.

Comparative phenotypic analyses of human plasma and urinary retinol binding protein using mass spectrometric immunoassay

Mass spectrometric immunoassay (MSIA) is a proteomics technology that combines the selectivity of affinity capture with the sensitivity and resolution of mass spectrometric detection. This unique approach allows for intact protein identification therefore is readily capable of discriminating between protein variants, i.e., mutations, posttranslational modifications, and truncations. In this work, MSIA is used in the comparative analyses of retinol binding protein (RBP) from the plasma and urine of a small study population. Detailed RBP profiles were obtained from both biological fluids, resulting in the identification of several catabolic RBP products (present in urine) that have not been previously reported. In addition, comparative analysis of urine samples taken from healthy and renally impaired individuals revealed different breakdown profiles. These results illustrate the use of MSIA for the rapid, sensitive, and accurate profiling of RBP both within and between individuals.

Endocrine Disruptors: Can Biological Effects and Environmental Risks Be Predicted?

A large number of diverse nonsteroidal chemicals, referred to as xenoestrogens, bind to the estrogen receptor (ER) and evoke biological responses. The activity of most xenoestrogens is weak (from about 1/1000th to 1/1,000,000th that of estradiol). These substances interact with the binding pocket of the ER because they have chemical similarities to estradiol (usually a phenolic A-ring). Reduced activity of xenoestrogens probably results from lack of fit of the remainder of the molecule within the binding pocket. ER binding per se has only limited influence on endocrine disruption. The nature (estrogenic or antiestrogenic) or magnitude of the response is a function of the substance itself, complexities within the various stages of the ER signaling pathway, as well as other factors (such as, plasma binding of xenoestrogens, cross-talk between ER and other signaling pathways, androgen antagonism, and alternate modes of estrogen action). Whereas there is general agreement that high doses of nonsteroidal chemicals can evoke endocrine disruptive effects, there is no consensus that such substances produce low-dose effects or that humans are at risk of endocrine disruption due to exposure to environmentally relevant levels of such chemicals. Furthermore, screening programs to identify hormonally active chemicals (such as the Endocrine Disruptor Screening Program) may be premature in view of the complexity of the mechanisms involved.

Pharmacokinetic modelling of total and unbound plasma carboplatin--a population study in 75 children.

A compartmental open model was developed to describe the relationship between plasma unbound (C.) and bound (CT) carboplatin concentrations. A population pharmacokinetic study was then undertaken to investigate the effect of demographic covariates on unbound and bound carboplatin clearance and volume parameters. Carboplatin and demographic data were collected from 75 children (1-17 years old, 10 children with unilateral nephrectomy) treated using 1-hour daily infusions for various malignancies. Concentration-time data, C(U) and C(T), from children with rich data were used to develop the model. The data from all children were then simultaneously analyzed using a population approach. The average population values for total unbound carboplatin clearance, CL(U), and distribution volume of unbound carboplatin, VI, were 3.87 l/h and 6.26 l/h, respectively. The clearance of plasma-bound carboplatin was comparatively low, 0.11 l/h. CL(U) was dependent on weight, nephrectomy status and serum creatinine. A constant fraction of CL(U), 0.17 l/h, included the disappearance of unbound compound due to irreversible plasma binding. V1 was dependent on body weight. The unbound plasma carboplatin fraction (fu) was simulated and rapidly decreased with post-infusion time. The body weight was a better predictor for unbound carboplatin clearance than body surface area, and UNP and SCr caused a reduction in clearance of unbound carboplatin, as previously reported. The rate ofcarboplatin plasma binding was low and not dependent on demographic patient characteristics. The f(U) of plasma carboplatin could be predicted as a function of time, infusion rate and covariates affecting CL(U), weight, UNP and SCr.

Assay methodology for the quantitation of unbound ertapenem, a new carbapenem antibiotic, in human plasma

Ertapenem is a new once-a-day antibiotic with excellent coverage of common community gram negative and gram positive aerobes and anaerobes. It demonstrates nonlinear protein binding in human plasma (about 94% bound). An assay for unbound drug was developed to study the pharmacokinetics of unbound ertapenem in plasma. Unbound drug is separated from plasma samples (1.0 ml) by ultrafiltration using a Centrifree ® centrifugal filter device. Ertapenem (vulnerable to hydrolysis of the beta-lactam moiety) is stabilized in the filtrate by adding an equal volume of 0.1 M MES buffer, pH 6.5 and then is analyzed by reversed-phase high-performance liquid chromatography (HPLC) with ultraviolet (UV) absorbance detection (300 nm). Non-specific binding to the Centrifree ® device is <3%. A suitable internal standard is not available. The assay is specific and linear over the concentration range of 0.25 to 100 μg/ml in plasma filtrate. The lower limit of quantitation (LLOQ) is 0.25 μg/ml. Intra-day precision is C.V.<10% and accuracy ranges from 97 to 101% of nominal concentration. Inter-day precision and accuracy were determined using quality control samples (QCs) prepared in plasma ultrafiltrate at 0.5, 12 and 80 μg/ml and stored at −70 °C with stabilizer. Inter-day assay accuracy and precision ranged from 100 to 111% of nominal concentration and 1.8 to 5.3% C.V. ( n=40), respectively. The assay has been used to analyze plasma samples from subjects receiving 500 and 2000 mg i.v. doses of ertapenem (30 min infusion).

Plasma insulin-like growth factor 1, insulin-like growth factor binding protein 3, and risk of colorectal cancer: a prospective study in northern Sweden.

Insulin-like growth factor 1 (IGF-1) has antiapoptotic and mitogenic effects on various cell types, and raised IGF-1 levels are increasingly being implicated as potential risk factors for cancer. To examine the relationship between IGF-1 and its major plasma binding protein, IGF binding protein 3 (IGFBP-3), and the risk of colorectal cancer. We conducted a case-control study nested within the Northern Sweden Health and Disease Cohort. IGF-1 and IGFBP-3 were measured in prediagnostic plasma samples from 168 men and women who developed cancers of the colon (n=110) or rectum (n=58), and from 336 matched controls. Conditional logistic regression analyses showed an increase in colon cancer risk with increasing levels of IGF-1 (odds ratios (ORs) 1.00, 1.89, 2.30, 2.66; p(trend)=0.03) and IGFBP-3 (ORs 1.00, 0.91, 1.80, 1.93; p(trend)=0.02). Rectal cancer risk was inversely related to levels of IGF-1 (ORs 1.00, 0.45, 0.33, 0.33; p(trend)=0.09) and IGFBP-3 (ORs 1.00, 0.75, 0.66, 0.49; p(trend)=0.21). Mutual adjustments between IGF-1 and IGFBP-3 did not materially alter these relationships. These results support earlier findings of increased risk of colon cancer in subjects with elevated plasma IGF-1. Our results however do not support the hypothesis that the risk of rectal cancer could also be directly related to IGF-1 levels.

High-performance liquid chromatographic method for the quantification of unbound evernimicin in human plasma ultrafiltrate

A rapid HPLC method was developed for quantification of unbound evernimicin in human plasma. Protein-free samples prepared by ultrafiltration were injected directly onto a polymeric reversed-phase column and the eluent monitored at 302 nm. Evernimicin that eluted within 3.5 min was well resolved from endogenous components. Linearity was established between peak height and evernimicin concentration from 25 to 2500 ng/ml. Assay precision (C.V.) was within 5% while bias was no greater than 3%. This method has been used for the ex vivo assessment of evernimicin protein binding in human plasma from safety and tolerance as well as liver dysfunction and renal insufficiency studies.

Protein binding predictions in infants.

Plasma binding protein levels are lower in the newborn than in the adult and gradually increase with age. At birth, human serum albumin (HSA) concentrations are close to adult levels (75%-80%), while alpha 1-acid glycoprotein (AAG) is initially half the adult concentration. As a result, the extent of drug binding to HSA is closer to that of the adult than are those drugs bound largely to AAG. A model that incorporates the fraction unbound in adults and the ratio of the binding protein concentration between infants and adults successfully predicted the fraction unbound in infants and children.

Pharmacokinetic and pharmacodynamic considerations when treating patients with sepsis and septic shock.

Sepsis and septic shock are accompanied by profound changes in the organism that may alter both the pharmacokinetics and the pharmacodynamics of drugs. This review elaborates on the mechanisms by which sepsis-induced pathophysiological changes may influence pharmacological processes. Drug absorption following intramuscular, subcutaneous, transdermal and oral administration may be reduced due to a decreased perfusion of muscles, skin and splanchnic organs. Compromised tissue perfusion may also affect drug distribution, resulting in a decrease of distribution volume. On the other hand, the increase in capillary permeability and interstitial oedema during sepsis and septic shock may enhance drug distribution. Changes in plasma protein binding, body water, tissue mass and pH may also affect drug distribution. For basic drugs that are bound to the acute phase reactant alpha(1)-acid glycoprotein, the increase in plasma concentration of this protein will result in a decreased distribution volume. The opposite may be observed for drugs that are extensively bound to albumin, as the latter protein decreases during septic conditions. For many drugs, the liver is the main organ for metabolism. The determinants of hepatic clearance of drugs are liver blood flow, drug binding in plasma and the activity of the metabolic enzymes; each of these may be influenced by sepsis and septic shock. For high extraction drugs, clearance is mainly flow-dependent, and sepsis-induced liver hypoperfusion may result in a decreased clearance. For low extraction drugs, clearance is determined by the degree of plasma binding and the activity of the metabolic enzymes. Oxidative metabolism via the cytochrome P450 enzyme system is an important clearance mechanism for many drugs, and has been shown to be markedly affected in septic conditions, resulting in decreased drug clearance. The kidneys are an important excretion pathway for many drugs. Renal failure, which often accompanies sepsis and septic shock, will result in accumulation of both parent drug and its metabolites. Changes in drug effect during septic conditions may theoretically result from changes in pharmacodynamics due to changes in the affinity of the receptor for the drug or alterations in the intrinsic activity at the receptor. The lack of valid pharmacological studies in patients with sepsis and septic shock makes drug administration in these patients a difficult challenge. The patient's underlying pathophysiological condition may guide individual dosage selection, which may be guided by measuring plasma concentration or drug effect.

Expression and Function of C1q Receptors and C1q Binding Proteins at the Cell Surface

C1q is the recognition unit of the first component of complement that binds not only IgG and IgM containing immune complexes, but also recognizes foreign structures such as the lipid A of endotoxin, and molecules expressed at the surface of apoptotic cells. In this review, the plasma membrane receptors and binding proteins for C1q are discussed and new data are presented on calreticulin expression on human peripheral blood cells. Although much is known about C1q receptors and binding molecules there are still many questions regarding their role in vivo.

Influence of the ORM1 phenotypes on serum unbound concentration and protein binding of quinidine

Background: Only unbound or free drug in plasma can be transported to its site of action. The fraction of unbound drug in plasma varies widely for highly bound drugs among individuals. The genetic polymorphism of orosomucoid (ORM) could be related to the interindividual variability in plasma binding of basic drugs, as ORM is the transport protein for these drugs in plasma. The ORM is a major binding protein in plasma for various basic drugs and is coded by two loci, ORM1 and ORM2, which are closely linked on chromosome 9q31→34.1. ORM1 locus is highly polymorphic and the ORM2 locus is monomorphic in most population. Methods: Twenty-eight healthy volunteers were selected with three ORM1 phenotypes, containing homozygotes ORM1 F1 ( n=10) and ORM1 S ( n=8), and heterozygote ORM1 F1S ( n=10), identified by isoelectric focusing on polyacrylamide gels followed immunoblotting after desialylation of sera. After a single oral dose of quinidine 200 mg, serum total (HPLC) and unbound concentrations in ultrafiltrate (ultrafiltration/HPLC) were determined, and the pharmacokinetic parameters and protein binding rate were calculated. Results: Serum concentrations of ORM (553.8–573.2 mg/l) and albumin proteins (57.5–58.4 mg/l) were similar in the three groups ( P>0.05). Unbound quinidine concentration in ORM1 F1 phenotype subjects was higher than that in ORM1 S and ORM1 F1S phenotype; the free drug percentage for the subjects with ORM1 F1 phenotype (19.79%) was twice as high as that with ORM1 S phenotype (10.96%) ( P<0.01) at 24 h after administration of oral quinidine when the state of disposition equilibrium occurred. The elimination t 1/2 values and the other pharmacokinetic parameters of quinidine were not affected by the different ORM1 phenotypes. Conclusions: Different ORM1 phenotypes may affect the disposition of quinidine, a basic drug, rather than its hepatic metabolism and elimination. The functional heterogeneity of ORM1 could be responsible for the differences in plasma binding of quinidine. Therefore, monitoring of the unbound quinidine concentration would be important for the patients with different ORM1 phenotypes who are treated with quinidine.

Finite element model of antibody penetration in a prevascular tumor nodule embedded in normal tissue

We have developed a pharmacokinetic model for monoclonal antibodies (mAb) to aid in investigating protocols for targeting small primary tumors or sites of metastatic disease. The model describes the uptake of systemically-administered antibody by a prevascular spherical tumor nodule embedded in normal tissue. The model incorporates plasma kinetics, transcapillary transport, interstitial diffusion, binding reactions, and lymphatic clearance. Antigen internalization can easily be incorporated. Simulations obtained from a three-dimensional finite element analysis are used to assess errors in predictions from earlier models in which the influence of the normal tissue was collapsed into a boundary condition at the tumor surface. The model employing a Dirichlet boundary condition substantially overpredicted the mean total tumor mAb concentration at all times. Although the model with a concentration-dependent flux (composite) boundary condition underpredicted mAb concentration, the discrepancy with finite element results is only notable at early times. Sensitivity analyses were performed on mAb dose and on the coefficients for mAb diffusion in the tissue regions, since reported antibody diffusivity values have varied over 30-fold. The results of the study suggest that mAb diffusivity and mAb binding site density in tumors should have major influences on optimizing doses and scheduling of mAb administration in tumor targeting protocols.

Binding of 13-HODE and 15-HETE to Phospholipid Bilayers, Albumin, and Intracellular Fatty Acid Binding Proteins: IMPLICATIONS FOR TRANSMEMBRANE AND INTRACELLULAR TRANSPORT AND FOR PROTECTION FROM LIPID PEROXIDATION

Transport and utilization of fatty acids (FA) in cells is a multistep process that includes adsorption to and movement across the plasma membrane and binding to intracellular fatty acid binding proteins (FABP) in the cytosol. We monitored the transbilayer movement of several polyunsaturated FA and oxidation products (13-hydroxy octadecadienoic acid (HODE) and 15-hydroxytetraenoic acid (HETE)) in unilamellar protein-free phospholipid vesicles containing a fluorescent pH probe. All FA diffused rapidly by the flip-flop mechanism across the model membrane, as revealed by pH changes inside the vesicle. This result suggests that FA oxidation products generated in the cell could cross the plasma or nuclear membrane spontaneously without a membrane transporter. To illuminate features of extra- and intracellular transport, the partitioning of unsaturated FA and oxidized FA between phospholipid vesicles and albumin or FABP was studied by the pyranin assay. These experiments showed that all polyunsaturated FA and oxidized FA (13-HODE and 15-HETE) desorbed rapidly from the phospholipid bilayer to bind to bovine serum albumin, which showed a slight preference for the unsaturated FA over the oxidized FA. FABP rapidly bound FA in the presence of phospholipid bilayers, with a preference of 13-HODE over the unsaturated FA and with a specificity depending on the type of FABP. Liver FABP was significantly more effective than intestinal FABP in binding 13-HODE in the presence of vesicles. The more effective binding of the FA metabolite, 13-HODE, than its precursor 18:2 by FABP may help protect cellular membranes from potential damage by monohydroxy fatty acids and may contribute a pathway for entry of 13-HODE into the nucleus.

Increase of plasma eCG binding rate after administration of repeated high dose of eCG to cows.

Equine chorionic gonadotrophin (eCG) is still used to promote follicular growth in cattle and, more recently with an increased frequency of administration, in ovum pick-up protocols. The aim of this experiment was to verify the possible effect of high frequency of administration on the immune response to eCG. The profiles of eCG binding rate, in the blood of two groups (A, B) of 4 primiparous cross breed beef cows (3-3.5 years old) submitted weekly for 5 to 10 weeks to repeated high doses (1000-2000 IU) of equine chorionic gonadotrophin, are presented in this paper. A sensitive radiometric method was used to detect antibodies in plasma. The profiles clearly indicated a marked increase of eCG binding rate after 3 to 5 injections of the exogenous hormone to the females. The statistical analysis of the results established that treatments induced a significant increase (P < 0.01) in binding rates after 6 and 3 injections in group A and B respectively. These binding rates remained elevated for at least 1 week following the last injection and decreased afterwards. The values of plasma binding rates following repeated eCG administration differed significantly between groups (0.90+/-1.04 and 1.04+/-0.11 for groups A and B before treatment versus 11.77+/-0.92, 6.70+/-0.85 for groups A and B after treatment, P < 0.01) and from one cow to another (P < 0.01) with some cows presenting no significant immune response while others were more reactive against the hormone (at least 3 injections).