Characterization of the vitamin E-binding properties of human plasma afamin

Human plasma afamin, the fourth member of the albumin gene family, is shown to be a specific binding protein for vitamin E. A radio ligand-binding assay followed by Scatchard and Hill analysis are used to show that afamin has a binding affinity for both alpha-tocopherol and gamma-tocopherol, two of the most important forms of vitamin E, in vitro. The binding-dissociation constant was determined to be 18 microM, indicating that afamin plays a role as vitamin E carrier in body fluids such as human plasma and follicular fluid under physiological conditions. Additionally, we demonstrate that afamin has multiple binding sites for both alpha- and gamma-tocopherol. Due to the large binding capacity of afamin for vitamin E, it might take over the role of vitamin E transport in body fluids under conditions where the lipoprotein system is not sufficient for vitamin E transport. To confirm the experimental results, we performed homology modeling and docking calculations on the predicted tertiary structure, which showed coincidence between calculated and in vitro results.     

Parameters of dietary selenium and vitamin E deficiency in growing rabbits.

4 x 5 growing female rabbits (New Zealand White) with an initial live weight of 610 +/- 62 g were fed a torula yeast based semisynthetic diet low in selenium (<0.03 mg/kg diet) and containing <2 mg alpha-tocopherol per kg (group I). Group II received a vitamin E supplementation of 150 mg alpha-tocopherylacetate per kg diet, whereas for group III 0.40 mg Se as Na-selenite and for group IV both supplements were added. Selenium status and parameters of tissue damage were analyzed after 10 weeks on experiment (live weight 2,355 +/- 145 g). Selenium depletion of the Se deficient rabbits (groups I and II) was indicated by a significantly lower plasma Se content (group I: 38.3 +/- 6.23 microg Se/mL plasma, group II: 42.6 +/- 9.77, group III: 149 +/- 33.4, group IV: 126 +/- 6.45) and a significantly lower liver Se content (group I: 89.4 +/- 18.2 microg/kg fresh matter, group II: 111 +/- 26.2) as compared to the Se supplemented groups III (983 +/- 204) and IV (926 +/- 73.9). After 5 weeks on the experimental diets differences in the development of plasma glutathione peroxidase were observed. As compared to the initial status group (45.2 +/- 4.50) pGPx activity in mU/mg protein was decreased in group I (19.1 +/- 7.08), remained almost stable in the vitamin E supplemented group II (46.3 +/- 11.2) whereas an elevated enzyme activity was measured in the Se supplemented groups III (62.4 +/- 23.9) and IV (106 +/- 19.9). In the rabbit organs investigated 10 weeks of Se deficiency caused a significant loss of Se dependent cellular glutathione peroxidase activity (GPx1) of 94% (liver), 80% (kidney), 50% (heart muscle) and 60% (musculus longissimus dorsi) in comparison to Se supplemented control animals. Damage of cellular lipids and proteins in the liver was due to either Se or vitamin E deficiency. However damage was most severe under conditions of a combined Se and vitamin E deficiency. It can be concluded that the activity of plasma glutathione peroxidase is a sensitive indicator of Se deficiency in rabbits. The loss of GPx1 activity indicates the selenium depletion in various rabbit organs. Both selenium and vitamin E are essential and highly efficient antioxidants which protect rabbits against lipid and protein oxidation.     

Plasma protein carbonyl concentration is not enhanced by chronic intake of high-protein diets in adult rats

We tested the hypothesis of whether high dietary protein intake is linked to oxidative stress as measured by the concentration of reactive carbonyl residues in plasma proteins. Three groups of male Wistar rats ( approximately 230 g, n = 10) were fed either 15% (15C), 30% (30C), or 60% (60C) casein diets over a period of 18 weeks. For comparison, a vitamin E deficient diet (60C-E) based on diet 60C was given to an additional group to provoke oxidative stress. Concentrations of alpha-tocopherol in plasma and of reactive carbonyl residues in total plasma proteins were measured by high performance liquid chromatography using fluorescence and by diode array detection after 2,4-dinitrophenylhydrazine reaction, respectively. After 1 week the concentration of reactive carbonyl residues in plasma proteins was found to be significantly (P < 0.05) higher in the 60C and 60C-E groups ( approximately 2.7 nmol/mg protein) compared with the 15C and 30C groups ( approximately 1.7 nmol/mg protein). After 14 weeks the 15C (3.4 +/- 1.2 nmol/mg protein) and 60C-E groups (3.9 +/- 1.7 nmol/mg protein) showed a significantly increased concentration of reactive carbonyl residues in plasma protein compared with the 30C and 60C groups (2.5 +/- 1.0 nmol/mg protein; 2.6 +/- 0.8 nmol/mg protein). As expected, chronic vitamin E deficiency (60C-E) resulted in significantly decreased alpha-tocopherol concentrations (3.91 +/- 2.42 micromol/mL vs. 31.3 +/- 4.8 micromol/mL) and a higher concentration of reactive carbonyl residues in plasma proteins. These results do not support the hypothesis that a chronic intake of high-protein diets leads to oxidative stress in adult rats. However, in the non-adapted state (1 week) a high protein intake contributes to oxidative modifications of protein-bound amino acid residues.     

Inhibition of human P450 enzymes by multiple constituents of the Ginkgo biloba extract

The Ginkgo biloba extract EGb761 was tested for its ability to inhibit the major human cytochrome P450 enzymes (CYPs). The full extract was found to strongly inhibit CYP2C9 (Ki = 14+/- 4 microg/mL), and to a lesser extent, CYP1A2 (Ki = 106 +/- 24 microg/mL), CYP2E1 (Ki = 127 +/- 42 microg/mL), and CYP3A4 (Ki = 155 +/- 43 microg/mL). The terpenoidic and flavonoidic fractions of the extract were tested separately against the same P450s to identify the source of inhibition by EGb761. The terpenoidic fraction inhibited only CYP2C9 (Ki = 15 +/-6 microg/mL) whereas the flavonoidic fraction of EGb761 showed high inhibition of CYP2C9, CYP1A2, CYP2E1, and CYP3A4 (Ki's between 4.9 and 55 microg/mL). The flavonoidic fraction was further fractionated using extraction and chromatography. Inhibition studies indicated that the majority of these fractions inhibited P450s at a significant level (IC50 < 40 microg/mL).     

Cystatin C secretion by rat glomerular mesangial cells: autocrine loop for in vitro growth-promoting activity.

Cystatin C, the major inhibitor of the cysteine proteinases found in human and rat body fluids, is particularly abundant in seminal plasma and cerebrospinal fluid. In a precedent report, we have evidenced noteworthy levels of cystatin C in rat kidney cortex. In the present study, we show that rat mesangial glomerular cells produce cystatin C. Immunoprecipitation of extracts of metabolically labeled cells and culture media showed that the synthesized cystatin C is a 15.5 +/- 0.5 kDa protein. The protein was released into the culture supernatant (1.6 +/- 0.26 micrograms/10(6) cells/24 h). Urinary rat cystatin C and PPPR synthetic peptide (5-8 N-terminal sequence of rat cystatin C) increased mesangial cell proliferation. Affinity chromatography on Ultrogel-avidin-biotin-PPPR of extracts of metabolically labeled cells indicate the existence of a PPPR binding protein of 46 kDa. The results described in this work suggest, for glomerular rat mesangial cells in vitro, an autocrine regulation of proliferation by cystatin C.     

The effect of vitamins C and E on biomarkers of oxidative stress depends on baseline level

Oxidative stress is elevated in obesity, and may be a major mechanism for obesity-related diseases. Nonsmokers (n=396) were randomized to 1000 mg/day vitamin C, 800 IU/day vitamin E, or placebo, for 2 months. Treatment effect was examined in multiple regression analyses using an intention-to-treat approach. Vitamin C (P=0.001) and vitamin E (P=0.043) reduced plasma F2-isoprostanes. In the overall sample, changes from baseline were +6.8, -10.6, and -3.9% for placebo, vitamin C, and vitamin E groups, respectively. However, a significant interaction with baseline F2-isoprostane was found. When baseline F2-isoprostane was >50 microg/mL, vitamin C reduced F2-isoprostane by 22% (P=0.01). Vitamin E reduced it by 9.8% (P=0.46). Below that cut point, neither treatment produced further reductions. F2-isoprostane>50 microg/mL was strongly associated with obesity, and was present in 42% of the sample. Change in malondialdehyde concentration was minimal. These findings suggest a role for vitamin C in reducing lipid peroxidation. Future research on effects of vitamins C or E on plasma F2-isoprostane should limit participants to those with baseline levels >50 mug/mL. Further studies are needed to establish whether treatment with vitamins C or E in persons with concentrations above that cut point could slow the development of cardiovascular disease.     

Comparative plasma pharmacokinetics of meloxicam in sheep and goats following intravenous administration

Meloxicam, a novel cyclooxygenase-2 selective nonsteroidal anti-inflammatory drug (NSAID), has been used extensively in humans and recently in some domestic animal species. Although it is an attractive NSAID for use in small ruminants, meloxicam pharmacokinetics have not been investigated in sheep and goats and this information is essential for rational therapeutic use of the drug in these species. In this investigation, comparative pharmacokinetic properties of meloxicam were studied in sheep and goats after a single intravenous dose of 0.5 mg kg(-1) body mass. Blood samples were collected via jugular venepuncture into heparinised tubes at predetermined times after drug administration. Plasma concentrations of meloxicam were determined by reversed-phase high performance liquid chromatography. The plasma concentrations of meloxicam were detectable in sheep and goats up to 72 and 48 h, respectively. The plasma concentration versus time data of meloxicam in both sheep and goats were adequately described by a two-compartment open model. The values obtained for sheep and goats for distribution half-life, volume of distribution at steady state and volume of the central compartment were almost similar in sheep and goats. The elimination half-life (t(1/2beta)), area under the plasma concentration-time curve (AUC), mean residence time (MRT) and total systemic clearance (Cl(B)) in sheep were significantly different from those of goats. The mean+/-S.E. values of t(1/2beta), MRT, AUC and Cl(B) in sheep were 10.85+/-1.21 h, 15.13+/-1.67 h, 31.88+/-2.97 microg h mL(-1) and 0.016+/-0.002 L h(-1) kg(-1), respectively whereas the respective values in goats were 6.73+/-0.58 h, 9.37+/-0.83 h, 19.23+/-2.23 microg h mL(-1) and 0.03+/-0.01 L h(-1) kg(-1). The results indicate that elimination kinetics of meloxicam differ significantly between sheep and goats and the elimination of the drug tends to be faster in goats compared to sheep.     

A capillary gas chromatography-selected ion monitoring mass spectrometry method for the analysis of atractylenolide I in rat plasma and tissues, and application in a pharmacokinetic study

The aim of this paper is to investigate the characteristics of atractylenolide I (AO-I) in the body by a GC-MS method. All bio-samples were cleared up with a liquid-liquid extraction procedure. The calibration curves were linear within a range of 5-1000 ng/mL for plasma samples, 0.06-16.00 microg/g for cerebellum samples, and 0.03-8.00 microg/g for other tissue samples. The limit of quantification (LOQ) for AO-I was 1.0 ng/mL or 1.0 ng/g (S/N>micro=10) in the bio-samples. In the applications, the main pharmacokinetic parameters were firstly obtained as follows: Tmax=0.37+/-0.19 h, Cmax=0.26+/-0.05 microg/mL, AUC=1.95+/-0.30 microgh/mL and ka=10.08+/-5.60 h(-1). The tissue distribution of AO-I in rats after the oral administration of 50.0mg/kg was from 0.225 to 0.031microg/g with a decreasing tendency in different tissues like liver>kidney>spleen>cerebellum>heart>cerebrum>lung. The protein binding in rat plasma, human plasma and bovine serum albumin was 80.8+/-3.9, 90.6+/-3.1 and 60.9+/-5.1%, respectively.     

Quantification of meropenem in plasma and cerebrospinal fluid by micellar electrokinetic capillary chromatography and application in bacterial meningitis patients

A high-performance micellar electrokinetic capillary chromatography (MEKC) has been demonstrated for the determination of meropenem in human plasma and in cerebrospinal fluid (CSF) and application in meningitis patients after intravenous (IV) administration. Plasma sample was pretreated by means of solid-phase extraction (SPE) on C(18) cartridge and CSF sample was by direct injection without sample pretreatment, with subsequent quantitation by MEKC. The separation of meropenem was carried out in an untreated fused-silica capillary (40.2 cm x 50 microm I.D., effective length 30 cm) and was performed at 25 degrees C using a background electrolyte consisting of Tris buffer (40 mM, pH 8.0) solution with sodium dodecyl sulfate (SDS) as the running buffer and on-column detection at 300 nm. Several parameters affecting the separation and sensitivity of the drug were studied, including pH, the concentrations of Tris buffer and surfactant. Using cefotaxime as an internal standard (IS), the linear ranges of the method for the determination of meropenem in plasma and in CSF were all over 0.5-50 microg/mL; the detection limits (signal-to-noise ratio=3) of meropenem in plasma and in CSF were 0.2 microg/mL and 0.3 microg/mL, respectively.     

A high-performance liquid chromatographic method for the determination of tocopherol in plasma and cellular elements of the blood.

A rapid sensitive, and reproducible procedure is described for the analysis of alpha-tocopherol in blood cells and plasma using high-performance liquid chromatography and fluorometric detection. The cardinal feature for the increased sensitivity of this high-performance liquid chromatographic procedure is that the fluorometric analysis was carried out at a short excitation wavelength (205 nm) which increased the sensitivity of 20-fold over the usual excitation wavelength of 295 nm. Tocopherol levels can be measured in as little as 50 microliters of plasma and 200 microliters of erythrocytes. The tocopherol contentof plasma, red blood cells, platelets, polymorphonuclear leukocytes, and lymphocytes of normal subjects and subjects ingesting additional quantitites of vitamin E are reported. The values for the white cells are approximately 30 times higher than those of the red blood cells (polymorphonuclear leukocytes 4.47 +/- 0.62 micrograms/10(9), lymphocytes 3.89 +/- 0.85 micrograms/10(9), and erythrocytes 1.40 +/- 0.14 micrograms/10(10) cells). The tocopherol contents of the plasma and all the cellular elements of the blood were increased by oral feeding with vitamin E.     

Steroid concentrations in follicular fluid aspirated repeatedly from transitional and cyclic mares

The objectives of the present study were to determine follicular progesterone (P4) and estradiol-17beta (E2) in transitional mares and to compare follicular steroid concentrations between transitional and cyclic mares. Follicles > 8 mm were aspirated under transvaginal ultrasound-guidance 4 times at 3 to 4 day intervals (T1-T4) in Norwegian pony mares during vernal transition. During the breeding season, follicular aspirations were conducted in each mare on Day 6, Day 14 and Day 18 after ovulation of 3 separate estrous cycles (Day of ovulation = Day 0). Plasma and follicular fluids were analyzed for P4 and E2 with ELISA and RIA, respectively. Plasma P4 concentrations remained below 1 ng/mL throughout T1-T4, while the follicular P4 concentrations increased significantly to cyclic levels after the first transitional aspiration. Plasma E2 concentrations similarly remained at low levels during the course of the transitional aspirations, while the follicular E2 concentrations increased gradually over the 4 aspirations to cyclic concentrations. The mares ovulated on average 9.8 +/- 1.6 (mean +/- SEM) days after the last transitional aspiration, and 16.6 +/- 0.2, 11.3 +/- 1.5 and 23.2 +/- 4.4 days after aspirations conducted on Day 6, 14 and 18, respectively. The present study demonstrates that in the transitional mare newly developing follicles exhibit biosynthesis of P4 and E2. Furthermore, an increase in follicular steroid concentrations is not necessarily reflected in the peripheral steroid concentrations.     

Quantitative determination of trans-polydatin, a natural strong anti-oxidative compound, in rat plasma and cellular environment of a human colon adenocarcinoma cell line for pharmacokinetic studies

A simple, accurate, precise, specific and reproducible high-performance liquid chromatography (HPLC) method was developed for determination of trans-polydatin, a natural strong anti-oxidative compound, in rat plasma and cell suspension. The assay procedure involved simple liquid-liquid extraction, the supernatant liquid was added an equal volume of water to avoid solvent effect. The detection of the analyte peak was achieved by monitoring the eluate using a UV detector set at 303 nm. The analysis used a Hypersil ODS2 C18 column (5 microm, 4.6 mm x 250 mm) and methanol/distilled water as the mobile phase (flow rate=1 mL/min). A total analytical run was achieved within 6.0 min and calibration curve was linear over a wide concentration range of 0.25-40 microg/mL for plasma sample and 1.0-500 microM for cell suspension, the coefficients of correlation were 0.9997 and 0.9999 or better, respectively. There was 80.7+/-7.86%, 96.8+/-3.20% and 102.7+/-9.72% recovery from 0.5, 10, and 40 microg/mL plasma samples, respectively. Intra- and inter-batch accuracy and precision were acceptable for the both matrices. The RSD of intra- and inter-day assay variations were all less than 10%. Both analyte and IS were stable in the battery of stability studies, freeze-thaw cycles. The described assay method was applied to pharmacokinetic studies in rats and a human colon adenocarcinoma cell line (Caco-2) successfully. The application of the assay to determine the pharmacokinetic is described.     

Physiological changes in platelet aggregation and nitric oxide levels during menstrual cycle in healthy women.

Hormonal levels, mainly those of estrogens, protect women from the appearance of cardiovascular diseases by an increasing nitric oxide (NO) activity. NO is an endogenous vasodilator and antiaggregating substance. We decided to investigate platelet function and plasma levels of nitric oxide during preovulatory and midluteal phases in young and healthy women with normal menstrual cycles (MCs). Nine young, healthy female subjects had recorded three consecutive MCs before entering this program. Platelet-rich plasma (PRP) was used for the determination of platelet aggregation and NO measurements. Moreover, platelet sensitivity to the inhibitory effect of exogenous NO was tested. The EC(50) of collagen showed no differences between the preovulatory (1.36+/-0.16 microg/mL) and the midluteal (1.31+/-0.08 microg/mL; P, NS) phases. However, the EC(90) during the preovulatory phase was higher (2.05+/-0.2 microg/mL) than during the midluteal phase (1.8+/-0.6 microg/mL). Plasma levels of NO were lower during the preovulatory phase (19.1+/-2 microM) in comparison to the midluteal phase (20.9+/-2.3 microM). Interestingly, the exogenous amount of NO to produce at least half of the inhibition of an EC(90) collagen-induced aggregation was higher at the preovulatory phase (323.3+/-60.9 nM) than during the midluteal phase (240.0+/-37.5 nM; P, NS). We propose that during the follicular phase platelets rather use NO produced by the endothelium; therefore, it is necessary to add more agonist to activate those, but it results in higher consumption of circulating NO, whereas during luteal-phase platelets are not able to use NO, requiring lower amounts of agonist and thus resulting in higher plasma levels of NO. This is an interesting fact in research on cardiovascular diseases of women.     

Cigarette smokers have decreased lymphocyte and platelet alpha-tocopherol levels and increased excretion of the gamma-tocopherol metabolite gamma-carboxyethyl-hydroxychroman (gamma-CEHC)

Cigarette smoking is associated with increased oxidative stress and increased risk of degenerative disease. As the major lipophilic antioxidant, requirements for vitamin E may be higher in smokers due to increased utilisation. In this observational study we have compared vitamin E status in smokers and non-smokers using a holistic approach by measuring plasma, erythrocyte, lymphocyte and platelet alpha- and gamma-tocopherol, as well as the specific urinary vitamin E metabolites alpha- and gamma-carboxyethyl-hydroxychroman (CEHC). Fifteen smokers (average age 27 years, smoking time 7.5 years) and non-smokers of comparable age, gender and body mass index (BMI) were recruited. Subjects completed a 7-day food diary and on the final day they provided a 24 h urine collection and a 20 ml blood sample for measurement of urinary vitamin E metabolites and total vitamin E in blood components, respectively. No significant differences were found between plasma and erythrocyte alpha- and gamma-tocopherol in smokers and non-smokers. However, smokers had significantly lower alpha-tocopherol (mean+/-SD, 1.34+/-0.31 micromol/g protein compared with 1.94+/-0.54, P = 0.001) and gamma-tocopherol (0.19+/-0.04 micromol/g protein compared with 0.26+/-0.08, P = 0.026) levels in their lymphocytes, as well as significantly lower alpha-tocopherol levels in platelets (1.09+/-0.49 micromol/g protein compared with 1.60+/-0.55, P = 0.014; gamma-tocopherol levels were similar). Interestingly smokers also had significantly higher excretion of the urinary gamma-tocopherol metabolite, gamma-CEHC (0.49+/-0.25mg/g creatinine compared with 0.32+/-0.16, P = 0.036) compared to non-smokers, while their alpha-CEHC (metabolite of alpha-tocopherol) levels were similar. There was no significant difference between plasma ascorbate, urate and F2-isoprostane levels. Therefore in this population of cigarette smokers (mean age 27 years, mean smoking duration 7.5 years), alterations to vitamin E status can be observed even without the more characteristic changes to ascorbate and F2-isoprostanes. We suggest that the measurement of lymphocyte and platelet vitamin E may represent a valuable biomarker of vitamin E status in relation to oxidative stress conditions.     

Afamin is synthesized by cerebrovascular endothelial cells and mediates α-tocopherol transport across an in vitro model of the blood–brain barrier

α-Tocopherol (αTocH), a member of the vitamin E family, is essential for normal neurological function. Despite the importance of αTocH transport into the CNS, transfer mechanisms across the blood–brain barrier (BBB) are not entirely clear. We here investigate whether afamin, a known αTocH-binding protein, contributes to αTocH transport across an in vitro model of the BBB consisting of primary porcine brain capillary endothelial cells (BCEC) and basolaterally cultured astrocytoma cells. Exogenously added afamin had no adverse effects on BCEC viability or barrier function and was transported across BCEC Transwell cultures. Furthermore, αTocH transport across polarized BCEC cultures to astrocytoma cells is facilitated by afamin, though to a lesser extent than by high-density lipoprotein-mediated transport, an essential and in vivo operating αTocH import pathway at the cerebrovasculature. We also demonstrate that porcine BCEC endogenously synthesize afamin. In line with these in vitro findings, afamin was detected by immunohistochemistry in porcine, human postmortem, and mouse brain, where prominent staining was observed almost exclusively in the cerebrovasculature. The demonstration of afamin mRNA expression in isolated brain capillaries suggests that afamin might be a new family member of binding/transport proteins contributing to αTocH homeostasis at the BBB in vivo .     

Plasma transport and tissue distribution of beta-carotene, vitamin A and retinol-binding protein in domestic cats.

The objective of this study was to determine retinol, retinyl esters and retinol-binding protein (RBP) as well as carotenoids in plasma, urine, liver and kidneys of randomly selected domestic cats. Retinol (240+/-64 ng/ml, mean+/-S.D.) represented one-third of total retinyl esters (736+/-460 ng/ml) in plasma. Retinyl esters were stearate, palmitate and oleate representing 61+/-6, 36+/-13 and 5+/-3% of total retinyl esters, respectively. In half of the cats, retinyl esters (22+/-21 ng/ml) were found in the urine. Vitamin A in the livers (4317+/-1956 microg/g) was significantly higher than in the kidney cortex and medulla (14.16+/-8.92 and 7.59+/-4.52 microg/g, respectively, both P<0.001). RBP was detected in the plasma but not in the urine. Immunoreactive RBP was observed in hepatocytes and in the cells of the proximal tubules. beta-Carotene was present in plasma but never in tissues. The results show that similar to canines differences in vitamin A metabolism in cats are related to the occurrence of retinyl esters in plasma. They differ, however, with regard to the tissue distribution of beta-carotene and the excretion of vitamin A in the urine.     

The role of sub-optimal preovulatory oestradiol secretion in the aetiology of premature luteolysis during the short oestrous cycle in the cow

Premature regression of the corpus luteum, following the first post partum ovulation, is often preceded by sub-optimal preovulatory oestradiol secretion and accompanied by elevated levels of oxytocin receptors early in the luteal phase. We have investigated the role of preovulatory oestradiol in the control of subsequent oxytocin receptor concentration and activity by treating ovariectomised cows, over a simulated 48 h follicular phase, with high (600 microg per day) medium (300 microg per day) or low (150 microg per day) levels of oestradiol. These doses of oestradiol generated mean+/-S.E.M. plasma oestradiol concentrations of 12.1+/-1.0, 4.9+/-0.5 and 2.9+/-0.4 pg ml(-1), respectively. In Study 1 (n=4 per group), we found that by day 4 following oestrus there was a significant (P< 0.05) effect of the level of oestradiol on the inhibition of oxytocin binding activity measured in endometrial biopsy samples. This had fallen to mean+/-S.E.M. concentrations of 25+/-2 fmol per mg protein in the high group, 47+/-8 fmol per mg protein in the medium group and 65+/-12 fmol per mg protein in the low group. In Study 2, cows (n=3 per group) were treated with the same three levels of oestradiol followed by treatment with increasing levels of progesterone from days 3 to 6 following oestrus, generating mean+/-S.E.M. plasma concentrations of 2.17+/-0.18 ng ml(-1) by day 6. On day 6, there was a significant (P< 0.01) effect of the level of oestradiol on PGF(2alpha) release in response to oxytocin challenge. High, medium and low oestradiol groups exhibiting mean+/-S.E.M., increase plasma PGF(2alpha) metabolite concentrations of 10.0+/-2.2, 21.3+/-4.3 and 41.3+/-1.2 pg ml(-1), respectively, during the hour after oxytocin administration. From these results, we postulate that at the first post partum ovulation a low level of preovulatory oestradiol can result in the early generation of a luteolytic mechanism during the subsequent luteal phase due to impaired inhibition of oxytocin receptors allowing increased PGF(2alpha) release.     

Quantitative determination of individual teicoplanin components in human plasma and cerebrospinal fluid by high-performance liquid chromatography with electrochemical detection

We have developed a simple, rapid and highly sensitive method for determining a plasma or cerebrospinal fluid (CSF) concentrations of individual teicoplanin components using reversed-phase high-performance liquid chromatography followed by electrochemical detection. A linear relationship was observed between concentrations and peak heights for the teicoplanin concentration range of 0.025-10microg/mL. The correlation coefficients of all standard curves were greater than or equal to 0.999. The limit of detection for the major component of teicoplanin was 1.0ng/mL (signal/noise ratio >3). Daily fluctuations of standard curves (n=5) were small, with coefficients of variation of 3.3%. The intra-assay precision was 5.9% (n=5). Inter-assay precision ranged from 2.6 to 6.8%. The method described here is suitable for clinical monitoring of teicoplanin levels in plasma or CSF level and for use in studies involving pharmacokinetics of individual teicoplanin component.     

The evaluation of the role of beta-hydroxy fatty acids on chronic inflammation and insulin resistance

Beta-hydroxy fatty acids are a major component of lipid A moiety of lipopolysaccharide. We aimed to investigate the role of free beta-hydroxy fatty acids on inflammation, as well as to evaluate their effects on cytokine release from human blood cells, and whether they exist in plasma of patients with chronic inflammatory diseases with/without insulin resistance. Peripheral venous blood was incubated with beta-hydroxy lauric and beta-hydroxy myristic acids (each 100 ng, 1 microg, 10 microg/mL) up to 24 hours. Cytokines were measured from culture media and plasma. Free fatty acids and biochemical parameters were also measured from patients' plasma. Only beta-hydroxy lauric acid significantly stimulated interleukin-6 production at 10 microg/mL compared to control (533.9 +/- 218.1 versus 438.3 +/- 219.6 pg/mL, P < .05). However, free beta-hydroxy lauric and myristic acids were not found in patients' plasma. Therefore, free beta-hydroxy lauric and myristic acids do not seem to have a role on sterile inflammation in chronic inflammatory diseases associated with insulin resistance.     

Modulation of the response to estradiol-17beta of rat vascular tissues by a non calcemic vitamin D analog

Estradiol-17beta (E(2)) increases creatine kinase (CK) specific activity in aorta (Ao) and left ventricle of the heart (Lv) from rat females. In the present study, we analyzed the effects of pretreatment with the non calcemic analog of vitamin D, JK 1624 F2-2 (JKF) on the response to E(2) (either 0.5 or 5 microg/rat) of Ao and Lv from prepubertal female rats. JKF did not affect CK in either organ. However, pretreatment with JKF (0.1 ng/g body weight for 1 or 2 weeks) increased the CK response to E(2) (0.5 microg/rat) by 50 +/- 10% in Ao and by 150 +/- 12% in Lv. The CK response to 5 microg/rat of E(2) in intact female rats, was increased by 118 +/- 15% and 99 +/- 11% in the Ao and by 89 +/- 6% and 112 +/- 13% in the Lv, in animals treated daily with JKF for 1 or 2 weeks, respectively, before administration of E(2). JKF also increased the response to 500 microg/rat raloxifene (Ral) by 47 +/- 8% in Ao and by 56 +/- 12% in Lv. Preliminary experiments showed that JKF treatment induced a approximately 50% increase in estradiol receptor ERalpha in both organs. The results indicate that the vitamin D analog JKF upregulates the response and sensitivity of vascular tissues to E(2), in association with increased expression of their ERalpha. These results should prompt examination of the possibility that the effects estrogen therapy in postmenopausal women can be augmented by vitamin D or its analogs.