Sex differences in renal angiotensin converting enzyme 2 (ACE2) activity are 17β-oestradiol-dependent and sex chromosome-independent

Background

Angotensin converting enzyme 2 (ACE2) is a newly discovered monocarboxypeptidase that counteracts the vasoconstrictor effects of angiotensin II (Ang II) by converting Ang II to Ang-(1-7) in the kidney and other tissues.

Methods

ACE2 activity from renal homogenates was investigated by using the fluorogenic peptide substrate Mca-YVADAPK(Dnp)-OH, where Mca is (7-methoxycoumarin-4-yl)-acetyl and Dnp is 2,4-dinitrophenyl.

Results

We found that ACE2 activity expressed in relative fluorescence units (RFU) in the MF1 mouse is higher in the male (M) compared to the female (F) kidney [ACE2 (RFU/min/μg protein): M 18.1 ± 1.0 versus F 11.1 ± 0.39; P < 0.0001; n = 6]. Substrate concentration curves revealed that the higher ACE2 activity in the male was due to increased ACE2 enzyme velocity (V_max) rather than increased substrate affinity (K_m). We used the four core genotypes mouse model in which gonadal sex (ovaries versus testes) is separated from the sex chromosome complement enabling comparisons among XX and XY gonadal females and XX and XY gonadal males. Renal ACE2 activity was greater in the male than the female kidney, regardless of the sex chromosome complement [ACE2 (RFU/min/μg protein): intact-XX-F, 7.59 ± 0.37; intact-XY-F, 7.43 ± 0.53; intact-XX-M, 12.1 ± 0.62; intact-XY-M, 12.7 ± 1.5; n = 4-6/group; P < 0.0001, F versus M, by two-way ANOVA]. Enzyme activity was increased in gonadectomized (GDX) female mice regardless of the sex chromosome complement whereas no effect of gonadectomy was observed in the males [ACE2 (RFU/min/μg protein): GDX-XX-F, 12.4 ± 1.2; GDX-XY-F, 11.1 ± 0.76; GDX-XX-M, 13.2 ± 0.97; GDX-XY-M, 11.6 ± 0.81; n = 6/group]. 17β-oestradiol (E₂) treatment of GDX mice resulted in ACE2 activity that was only 40% of the activity found in the GDX mice, regardless of their being male or female, and was independent of the sex chromosome complement [ACE2 (RFU/min/μg protein): GDX+E₂-XX-F, 5.56 ± 1.0; GDX+E₂-XY-F, 4.60 ± 0.52; GDX+E₂-XX-M, 5.35 ± 0.70; GDX+E₂-XY-M, 5.12 ± 0.47; n = 6/group].

Conclusions

Our findings suggest sex differences in renal ACE2 activity in intact mice are due, at least in part, to the presence of E₂ in the ovarian hormone milieu and not to the testicular milieu or to differences in sex chromosome dosage (2X versus 1X; 0Y versus 1Y). E₂ regulation of renal ACE2 has particular implications for women across their life span since this hormone changes radically during puberty, pregnancy and menopause.     

Treatment with 50000 IU Vitamin D2 Every Other Week and Effect on Serum 25-Hydroxyvitamin D2, 25-Hydroxyvitamin D3, and Total 25-Hydroxyvitamin D in Aclinical Setting

ObjectiveTo examine the effect of 50 000 IU-vitamin D₂ supplementation in a clinical setting on serum total 25-hydroxyvitamin D (25[OH]D), 25-hydroxyvitamin D₂ (25[OH]D₂), and 25-hydroxyvitamin D₃ (25[OH]D₃).MethodsThis retrospective cohort study was performed in an urban tertiary referral hospital in Boston, Massachusetts. Patients who had been prescribed 50 000 IU vitamin D₂ repletion and maintenance programs were identified through a search of our electronic medical record. Baseline and follow-up total serum 25(OH)D, 25(OH)D₂, and 25(OH)D₃ levels were compared.ResultsWe examined the medical records of 48 patients who had been prescribed 50 000 IU vitamin D₂ in our clinic. Mean ± standard deviation baseline total 25(OH) D was 31.0 ± 10.6 ng/mL and rose to 48.3 ± 13.4 ng/mL after treatment (P <.001). 25(OH)D₂ increased from 4.2 ± 4.3 ng/mL to 34.6 ± 12.3 ng/mL after treatment (P <.001), for an average of 158 days (range, 35-735 days). Serum 25(OH)D3 decreased from 26.8 ± 10.8 ng/mL to 13.7 ± 7.9 ng/mL (P <.001).ConclusionsFifty thousand IU vitamin D₂ repletion and maintenance therapy substantially increases total 25(OH)D and 25(OH)D2 despite a decrease in serum 25(OH)D3. This treatment program is an appropriate and effective strategy to treat and prevent vitamin D deficiency.(Endocr Pract. 2012;18:399-402)     

Simultaneous measurement of unbound testosterone and estradiol fractions in undiluted plasma at 37 degrees C by steady-state gel filtration

A method is described for the simultaneous measurement of percent unbound testosterone (T) and estradiol (E₂) using steady-state gel filtration (SSGF), a technique which maintains physiological conditions of pH, temperature and plasma constituents, thus avoiding alterations in the original equilibria. Combined with specific assays of their total plasma levels it probably yields a closer estimate of the true circulating unbound (biologically-active) concentration of T and E₂ than previous methods. Its main application lies in the study of sex-hormone disorders which may result in an imbalance between unbound T and E₂ concentrations. Results of percent unbound are: normal men - T, 1.71 ± 0.29 (SD); E₂, 1.79 ± 0.32: normal women - T, 1.02 ± 0.17; E₂, 1.49 ± 0.35: hirsute women - T, 1.68 ± 0.52; E₂, 1.55 ± 0.29; women on estrogens - T, 0.67 ± 0.06; E₂, 1.02 ± 0.13. As predicted from earlier studies in hirsute females the fall in SHBG level is associated with a significant rise in percent unbound T, but no rise in percent unbound E₂.     

Reduced Parathyroid Hormone-Stimulated 1,25-Dihydroxyvitamin D Production in Vitamin D Sufficient Postmenoposual Women with Low Bone Mass and Idiopathic Secondary Hyperparathyroidism

ObjectiveDistinguishing secondary hyperparathyroidism (sHPT) from eucalcemic primary hyperparathyroidism (EC-pHPT) is important. The objective of this study was to measure parathyroid hormone (PTH)-stimulated production of 1α,25-dihydroxyvitamin D (1,25[OH]₂D) in early postmenopausal patients with idiopathic sHPT, who also fit the criteria for EC-pHPT, compared to age-matched controls.MethodsIn this pilot case-control study, postmenopausal women aged 44 to 55 years with normal serum calcium (Ca), glomerular filtration rate (GFR) ≥65 mL/min, and 25-hydroxyvitamin D (25[OH]D) ≥75 nmol/L (30 ng/mL) were given an 8 hour infusion of PTH(1-34), 12 pmol/kg/h. Patients (n = 5) had elevated PTH, normal 1,25(OH)₂D, and no hypercalciuria. Controls (n = 5) had normal PTH. At baseline, 4, and 8 hours, serum Ca, creatinine (Cr), phosphorus (P), 1,25(OH)₂D, fibroblast growth factor (FGF23), and 24,25(OH)₂D as well as urine Ca, P, Cr, and cAMP/GFR were measured. The fractional excretion of calcium (FeCa) and tubular reabsorption of phosphorus (TMP)/GFR were calculated.ResultsPatients had lower 1,25(OH)₂D levels (± SD) than controls at 4 (39.8 ± 6.9 versus 58.8 ± 6.7; P = .002) and 8 hours (56.4 ± 9.2 versus 105 ± 2.3; P = .003) of PTH infusion, attenuated after adjusting for higher body mass index (BMI) in patients (P = .05, .04), respectively. The 24,25(OH)2D levels were lower in patients than controls (1.9 ± 0.6 versus 3.4 ± 0.6, respectively; P = .007). No differences were seen in serum Ca or P, urine cAMP/GFR, TRP/GFR, FeCa, or PTH suppression at 8 hours (patients 50%, controls 64%).ConclusionVitamin D sufficient patients who fit the criteria for EC-pHPT had reduced PTH-stimulated 1,25(OH)₂D compared to controls, partially attributable to their higher BMI. Other causes of reduced 1,25(OH)₂D production ruled out were excessive catabolism of vitamin D metabolites, elevated FGF23, and CYP27B1 mutation. Elevated BMI and idiopathic reduced PTH-stimulated 1,25(OH)₂D production should be considered in the differential of sHPT. (Endocr Pract. 2013;19:91-99)     

An improved radioreceptor assay for 1,25-dihydroxyvitamin D in human plasma

We describe a modified assay technique for quantitating 1,25-dihydroxyvitamin D in plasma. The method involves a rapid extraction of the hormone using minicolumn (made of granular diatomaceous earth) chromatography followed by single-step purification on high-performance liquid chromatography. Quantitation of plasma 1,25-dihydroxyvitamin D is achieved by a radioligand receptor assay employing lyophilized cytosolic receptor protein from chick intestine and high-specific-activity 1,25-dihydroxy[³H]vitamin D₃ (166 Ci/mmol). A new incubation medium including an ethanol extract of vitamin D-deficient chick serum yields high specific binding and improves the precision of the radioassay. Bound and free hormone are separated with dextran-coated charcoal of equivalent particle size. The method is sensitive to 0.5 pg/tube with a practical detection range of 1–20 pg/tube, permitting duplicate assay of endogenous 1,25-dihydroxyvitamin D in plasma volumes as small as 0.5 ml. The intra- and interassay coefficient of variation are 5 and 9%, respectively, and the method is valid over a wide-range sample dilution. This assay technique was applied to the measurement of plasma 1,25-dihydroxyvitamin D hormone concentration in normal young adults (55.2 ± 13.6 pg/ml; n = 20) and in patients with chronic renal failure (13.5 ± 5.2 pg/ml; n = 9) and primary hyperparathyroidism (83.3 ± 18 pg/ml; n = 10).     

Production,clearance rates and metabolic fate of estradiol-17β in the plasma of the laying hen

A single dose of tritiated estradiol-17β (³H-E₂β) was injected i.v. into 5 high egg producing White Leghorn hens, 31 weeks of age, at 19.2 ± 2.1 (mean ± S.D.) hr before oviposition. Blood (2 ml) was sampled at approximately 5 min intervals over 40 min. Whenever possible, metabolites were monitored and identified by the double isotope technique with the addition of the corresponding ¹⁴C-labelled standards to plasma prior to analysis. The metabolic half-life and clearance rate of ³H-E₂β in plasma were 10.9 ± 1.9 min and 118 ± 18 ml/min/kg body weight, respectively. The calculated production rate of E₂β at 19.2 hr before oviposition was 19.5 ± 5.7 ng/min based on the plasma level (93±22 pg/ml) measured at that time. The relative concentrations (% of plasma radioactivity) of the major metabolites isolated at 5.7 ± 0.6 min post injection were, in descending order: estradiol-17β-3-sulfate (E₂β-3S : 14.9 ± 2.7), estradiol-17α-3-sulfate (E₂α-3S; 5.7 ± 0.3), estrone (E₁; 4.6 ± 0.5), estrone sulfate (E₁S; 2.2 ± 0.5), and estradiol-17 α (E₂α; 1.2 ± 0.4). As time proceeded, the relative concentration of E₂α-3S gradually increased so that by 43.2 ± 1.0 min it became the most abundant identifiable metabolite (12.3 ± 1.1) followed by E₂β-3S (9.1 ± 1.7), E₂S (1.2 ± 0.6), E₁ (0.7 ± 0.4) and E₂α (0.3 ± 0.2). These findings are consistent with the view that one of the major pathways of E₂β metabolism in the circulation of the hen is via E₂β

E₂β?3S ?E₁S

E₂α-3S.     

Effect of vitamin D treatments on plasma metabolism and immune parameters of healthy dairy cows

The objective of this study was to investigate the possible beneficial effect of vitamin D repletion on certain immune parameters of vitamin D insufficient dairy cows. Twenty dairy cows in late lactation were treated daily with vitamin D in five different ways: sunlight exposure (SUN), D₂ supplementation combined with sunlight exposure (D2SUN), D₂ supplementation (D2), D₃ supplementation (D3), and D₂ and D₃ supplementation combined (D2D3). The cows had very low vitamin D levels at d 0 because of the vitamin D deprivation before the study. After 1 month of vitamin D repletion, all cows had plasma 25(OH)D levels within the normal range. Total 25(OH)D concentration was significantly higher in SUN, D2SUN and D2D3 than D2 or D3 at the end of the study. However, milk yield, as well as protein and fat content of the milk, was not influenced by vitamin D treatments. There was no difference obtained in the measured immune parameters: Leucocyte populations, somatic cell count, immunoglobulin concentrations in plasma and milk, and antigen-stimulated cytokine productions did not change in response to vitamin D repletion or difference in vitamin D sources, and no relations to plasma 25(OH)D levels were identified. Despite the fact that plasma 25(OH)D increased from a very low level to normal range, the present study did not show any effect of vitamin D repletion on the tested immune parameters of healthy dairy cows. Therefore, in this study, it was concluded that repletion to physiologically normal plasma 25-hydroxyvitamin D levels of vitamin D-depleted healthy dairy cows had no influence on immune parameters.     

The metabolism of synthetic estrogens in non-users and users of oral contraceptives

Using both pulse injections and constant infusions of ³H-mestranol (³H-ME) (1) and ³H-ethinyl estradiol (³H-EE) we have studied the metabolism of these compounds in non-users and users of oral contraceptives. Following pulse injection of ³H-ME the disappearance of radioactivity could be described as a function which was the sum of two exponentials. Studied by both types of administration there was no difference in the metabolism of ³H-ME in the two groups; the overall mean ± SE metabolic clearance rate (MCR) was 690 ± 45 1/day/m², the mean ratio of the concentrations of radioactivity as EE following administration of ME (CR_BB^{M, E}) was 0. 23 ± 0. 02 and the mean [ρ]_BB^{M, E} (fraction of administered ME measured in blood as EE) was 0. 19 (95% confidence limits = 0.15 – 0. 23).Following pulse injection of ³H-EE the disappearance of radioactivity was best described as a function which is the sum of three exponentials. Results from both types of administration revealed no difference in the metabolism of ³H-EE between non-users. The overall mean ± SE MCR^EE was 630 ± 30 I/day/m². The MCR^EE is significantly (0. 02 > P > 0. 01) less than the mean MCR for estradiol reported previously, in both non-users and users of oral contraceptives. The use of oral contraceptives containing estrogens and progestins does not appear to influence the metabolism of the estrogen used. Approximately 20% of mestranol is converted to and appears in the blood as ethinyl estradiol.     

Biochemical properties of A-homovitamin D derivatives: 1,4-dihydroxy-3-deoxy-A-homo-19-nor-9, 10-secocholesta-5,7-dienes

A study has been made in the chick of the stereostructural requirements of A-ring-functionalized vitamin D analogs which elicit vitamin D₃ and 1,25-(OH)₂D₃-dependent biological responses of intestinal calcium absorption (ICA) and bone calcium mobilization (BCM). Ring expansion of vitamin D₃ to produce (1S,4S), (1S,4R), or (1R,4S)-(7E)-1,4-dihydroxy-3-deoxy-A-homo-19-nor-9,10-secocholesta-5,7-dienes resulted in the loss of both ICA and BCM biological activity at dose levels of steroid of up to 650 nmol/0.1 kg birds. Accordingly the three A-homo analogs of vitamin D₃ were assessed for their ability to inhibit or increase the ICA or BCM responses of D₃ and 1,25-(OH)₂D₃. Only (1R,4S)-(7E)-diol-C, maintaining a cis-β,β-hydroxyl orientation showed antagonistic biological activity. Intraperitoneal doses (65–325 nmol) of diol-C administered in conjunction with D₃ (0.8–3.25 nmol) inhibited the BCM responses selectively and had no effect on the ICA response. Doses of analog-C (16.3-3.25 nmol) injected before and after the active hormone 1,25-(OH)₂D₃ (0.13–01.30 nmol) stimulated the ICA response of the latter above its normal levels (a synergistic response) when administered alone.     

hCG-induced prostaglandin E2 and F2 alpha release in adult rat testis: role in Leydig cell desensitization to hCG.

Testicular levels of prostaglandin E₂ and F_2α were measured in decapsulated adult rat testis following hCG stimulation. Basal levels were, respectively, 342 ± 74 and 502 ± 89 pg/testis. Following hCG administration these basal values are not significantly modified up to 2 hours. From 2 to 24 hours the concentrations are clearly increased above the basal level: at 12 hrs they are 1925 ± 165 for E₂ and 3200 ± 190 for F_2α. Levels are back to normal at 48 hrs and remain so until 144 hrs. An identical pattern of prostaglandin release is observed in vitro in Leydig cell preparations isolated at different times following in vivo hCG injection. This suggests that prostaglandins are secreted by Leydig cells. In hypophysectomized animals the release of both prostaglandins E₂ and F_2α is similar to controls indicating that prostaglandin secretion is not directly linked to testosterone production. alternatively testosterone injections (10 mg) does not modify prostaglandin levels. Binding sites for prostaglandins E₁, E₂ and F_2α are present on the Leydig cells and consequently Leydig cell function may be modulated by endogenous or exogenous prostaglandins. Their level is slightly increased at 24 hrs following hCG stimulation. Since the acute changes in prostaglandin E₂ and F_2α secretion occur during the period of “desensitization” and of acute “down regulation” of the LH-hCG receptor in the Leydig cells it is suggested that prostaglandins are involved in both phenomena.     

ARPE-19-derived VEGF-containing exosomes promote neovascularization in HUVEC: the role of the melanocortin receptor 5

ARPE-19 retinal pigment epithelial cells cultured in a medium containing 35 mM D-glucose led to an augmented ROS formation and release of vascular endothelial factor (VEGF)-containing exosomes compared to ARPE-19 cells cultured in a medium containing 5 mM D-glucose (standard medium). Exposing these cells to the melanocortin 5 receptor agonist (MCR₅) PG-901 (10^?10M), for 9 d reduced ROS generation, the number of exosomes released and their VEGF content. In contrast, incubating the cells with the melanocortin receptor MCR₁ agonist BMS-470539 (10^?5 M) or with the mixed MCR_3/4 agonist MTII (0.30 nmol) did not produce any significant decrease in ROS levels. ARPE-19-derived VEGF-containing exosomes promoted neovascularization in human umbilical vein endothelial cells (HUVEC), an effect that was markedly reduced by PG-901 (10^?10M) but not by the MCR_3/4 agonist MTII (0.30 nmol) or the MCR₁ agonist BMS-470539 (10^?5 M). The MCR₅-related action in the ARPE-19 cells was accompanied by the increased expression of two coupled factors, cytochrome p4502E1 (CYP2E1) and nuclear factor kappa b (Nf-κB). These are both involved in high glucose signalling, in ROS generation and, interestingly, were reduced by the MCR₅ agonist in the ARPE-19 cells. Altogether, these data suggest that MCR₅ is a modulator of the responses stimulated by glucose in ARPE-19 cells, which might possibly be translated into a modulation of the retinal pigment epithelium response to diabetes in vivo.     

The determination of the vitamin D metabolites on a single plasma sample: changes during parturition in dairy cows.

Methods have been developed for the precise measurement of the major known vitamin D metabolites in a single sample of cow plasma (~5 ml). The procedure involves initial extraction with methylene chloride-methanol followed by chromatography on Sephadex LH-20. 25-Hydroxyvitamin D₂ and 25-hydroxyvitamin D₃ were determined using high-pressure liquid chromatography and comparing ultraviolet absorption peak height with absorption peak heights of standards. The dihydroxylated metabolites were further purified and resolved by high-pressure liquid chromatography and determined by radioligand binding assays. The assays were employed to measure the total vitamin D metabolite levels in the plasma of paretic and normal dairy cows at parturition. Parturition had no effect on 25-hydroxyvitamin D levels in either group of cows (paretic, 37–44 ng/ml; normal, 35–38 ng/ml). However, normal cows did show lower mean 25-hydroxyvitamin D levels at every sampling period with the lowest levels in both groups occurring at 7 days postpartum. Plasma 25, 26-dihydroxyvitamin D was higher in paretic animals prepartum and at parturition (0.7–1.0 ng/ml) when compared to nonparetic animals (0.4–0.45 ng/ml). Similar levels (0.6 ng/ml) were observed in both groups postpartum. Cows developing parturient paresis showed a significant (P < 0.05) elevation of 1,25-dihydroxyvitamin D at parturition with a maximum level of 350 pg/ml attained at 1 day postpartum compared to prepartum levels of 60 pg/ml. Normal animals also showed a rise in plasma levels of 1,25-dihydroxyvitamin D with a maximum level of 185 pg/ml observed at 1 day postpartum. Plasma 24,25-dihydroxyvitamin D was initially higher in paretic cows (1.9 ng/ml) with a significant (P < 0.05) drop to 1.05 ng/ml occurring at parturition. This level was maintained for 7 days postpartum. The levels of this steroid were maintained at 1.3–1.4 ng/ml in the normal cows throughout the entire sampling period.     

Hemodialysis Decreases Serum Brain-Derived Neurotrophic Factor Concentration in Humans

In the present study we have evaluated the effect of a single hemodialysis session on the brain-derived neurotrophic factor levels in plasma [BDNF]_pl and in serum [BDNF]_s as well as on the plasma isoprostanes concentration [F₂ isoprostanes]_pl, plasma total antioxidant capacity (TAC) and plasma cortisol levels in chronic kidney disease patients. Twenty male patients (age 69.8?±?2.9?years (mean?±?SE)) with end-stage renal disease undergoing maintenance hemodialysis on regular dialysis treatment for 15?C71?months participated in this study. A single hemodialysis session, lasting 4.2?±?0.1?h, resulted in a decrease (P?=?0.014) in [BDNF]_s by ~42?% (2,574?±?322 vs. 1,492?±?327?pg?ml^?1). This was accompanied by an increase (P?<?10^?4) of [F₂-Isoprostanes]_pl (38?±?3 vs. 116?±?16?pg?ml^?1), decrease (P?<?10^?4) in TAC (1,483?±?41 vs. 983?±?35 trolox equivalents, ??mol?l^?1) and a decrease (P?=?0.004) in plasma cortisol level (449.5?±?101.2 vs. 315.3?±?196.3?nmol?l^?1). No changes (P?>?0.05) in [BDNF]_pl and the platelets count were observed after a single dialysis session. Furthermore, basal [BDNF]_s in the chronic kidney disease patients was significantly lower (P?=?0.03) when compared to the age-matched control group (n?=?23). We have concluded that the observed decrease in serum BDNF level after hemodialysis accompanied by elevated [F₂-Isoprostanes]_pl and decreased plasma TAC might be caused by enhanced oxidative stress induced by hemodialysis.     

Reevaluation of circulating prostacyclin and thromboxane in diabetes

Although several investigators have attempted to measure the plasma levels of prostacyclin (PGI₂) and thromboxane A₂ (TXA₂) in diabetes and normal subjects, their results have been controversial. In this study, we measured plasma PGI₂ and TXA₂ levels in diabetic patients and normal subjects. The plasma PGI₂ and TXA₂ were determined by RIA as 6-keto-PGF_1a and TXB₂, respectively. The plasma levels of 6-keto-PGF_1a were significantly reduced in diabetics with microangiopathy (52.5 ± 18.9 pg/ml, mean ± SE, p<0.05) compared with those of normal subjects. Diabetics as a whole also showed lower levels of 6-keto-PGF_1a than normal subjects (57.8 ± 26.1 vs. 70.2 ± 20.7 pg/ml), though this was not significant statistically. The plasma 6-keto-PGF_1a levels did not significantly correlate with either age of the patients or duration of diabetes in diabetics. Interestingly, however, hemoglobin Alc significantly correlated inversely with 6-keto-PGF_1a levels in diabetics without microangiopathy (r=−0.60, p<0.05). The plasma levels of TXB₂ in diabetics were significantly higher than those of normal subjects (155.2 ± 69.5 vs. 108.0 ± 30.0 pg/ml, p<0.05). These data suggest that an imbalance of circulating PGI₂ and TXA₂ may contribute to the development of diabetic microangiopathy.     

Glycosaminoglycan-binding properties and kinetic characterization of human heparin cofactor II expressed in Escherichia coli

Irreversible inactivation of α-thrombin (T) by the serpin, heparin cofactor II (HCII), is accelerated by ternary complex formation with the glycosaminoglycans (GAGs) heparin and dermatan sulfate (DS). Low expression of human HCII in Escherichia coli was optimized by silent mutation of 27 rare codons and five secondary Shine-Dalgarno sequences in the cDNA. The inhibitory activities of recombinant HCII, and native and deglycosylated plasma HCII, and their affinities for heparin and DS were compared. Recombinant and deglycosylated HCII bound heparin with dissociation constants (K_D) of 6 ± 1 and 7 ± 1 μM, respectively, ∼6-fold tighter than plasma HCII, with K_D 40 ± 4 μM. Binding of recombinant and deglycosylated HCII to DS, both with K_D 4 ± 1 μM, was ∼4-fold tighter than for plasma HCII, with K_D 15 ± 4 μM. Recombinant HCII, lacking N-glycosylation and tyrosine sulfation, inactivated α-thrombin with a 1:1 stoichiometry, similar to plasma HCII. Second-order rate constants for thrombin inactivation by recombinant and deglycosylated HCII were comparable, at optimal GAG concentrations that were lower than those for plasma HCII, consistent with its weaker GAG binding. This weaker binding may be attributed to interference of the Asn¹⁶⁹N-glycan with the HCII heparin-binding site.     

Treatment with 17β-oestradiol does not influence age and weight at puberty in Bos indicus heifers

The working hypothesis was that treatment of heifers with 17β-oestradiol (E₂) during specific periods of prepuberty would reduce the response of the hypothalamic–pituitary axis to E₂ negative feedback and induce an earlier onset of puberty. The effects of chronic treatment with exogenous E₂ administered at specific maturational phases on the age and weight at puberty were studied in 96 prepubertal Brahman (3/4–7/8 Bos indicus) heifers (187.0±3.3 days of age, mean±SEM), weighing 149.9±2.5 kg. Heifers were randomly assigned to one of six groups (n=16 per group). Groups 2–6 received E₂ implants (Compudose 200®) for 90-day periods starting at 10, 13, 16, 19 and 22 months of age, while animals in group 1 remained untreated. Implants were placed subcutaneously at the base of the ear. Blood was collected for progesterone (P₄) determination by radioimmunoassay (RIA) and the animals were weighed at monthly intervals from 6 to 15 months then weekly from 15 to 28 months of age. Puberty was defined by concentrations of P₄>1 ng/ml in plasma and identification of a corpus luteum (CL) by transrectal ultrasonography (Aloka 210DX:7.5 MHZ probe). Treatment with exogenous E₂ at any of the ages/treatment intervals evaluated in this study did not reduce age or weight at puberty (P>0.7). The mean age and weight at puberty of control heifers was 735.3±19.7 days (range: 597–861) and 299.2±10.2 kg (range: 233–382), respectively, which is greater than the age and weight at puberty of 481 days and 246 kg, that was previously reported for B. indicus heifers [Post, T.B., Reich, M.M., 1980. Puberty in tropical breeds of heifers as monitored by plasma progesterone. Proceedings of the Australian Society of Animal Production 13, 61–62.]. The large variation in age and weight at puberty that was observed in the present study among heifers might indicate an individual animal effect to E₂ treatment among some of the treated animals. The lengthy interval from birth to puberty observed in this study, as compared to other studies, reflects the effects of other factors such as genotype, environmental or nutritional influences on puberty.     

Clinical results of an EPID-based in-vivo dosimetry method for pelvic cancers treated by intensity-modulated radiation therapy

The purpose of our work was to investigate the feasibility of using an EPID-based in-vivo dosimetry method initially designed for conformal fields on pelvic dynamic IMRT fields. The method enables a point dose delivered to the patient to be calculated from the transit signal acquired with an electronic portal imaging device (EPID). After defining a set of correction factors allowing EPID pixel values to be converted into absolute doses, several tests on homogeneous water-equivalent phantoms were performed to estimate the validity of the method in reference conditions. The effects of different treatment parameters, such as delivered dose, field size dependence and patient thickness were also studied. The model was first evaluated on a group of 53 patients treated by 3D conformal radiotherapy (3DCRT) and then on 92 patients treated by IMRT, both for pelvic cancers. For each measurement, the dose was reconstructed at the isocenter (D_REC) and compared with the dose calculated by our treatment planning system (D_TPS). Excellent agreement was found between D_REC and D_TPS for both techniques. For 3DCRT treatments, the mean deviation between D_REC and D_TPS for the 211 in-vivo dose verifications was equal to −1.0  ±  2.2% (1SD). Concerning IMRT treatments, the averaged deviation for the 418 fields verified was equal to −0.3 ± 2.6% (1SD) proving that the method is able to reconstruct a dose for dynamic IMRT pelvic fields. Based on these results, tolerance criteria and action levels were established before its implementation in clinical routine.     

Effect of thyroid state on estradiol-17β metabolism in the rabbit

It is well known that the metabolic clearance rate (MCR) of a hormone is Influenced highly by the level of its specific binding protein. It was interesting, therefore, to study the metabolism of estradiol-17β (E₂) in an animal model such as the rabbit where there is a lack for a highly specific binding protein for the steroid. The kinetics of the hormone was studied in relation to the thyroid state, namely in rabbits receiving thyroxin or propylthiouracil.In the absence of any significant decrease of the level of the rabbit androgen binding protein (R-ABP), the accelerated MCR^E2 and the elevated conversion ratio of estradiol to estrone (CR ^E₂→E₁) observed in hyperthyroid rabbits were attributed to the important role of metabolizing enzymes in the liver and/or extrahepatic tissues. In hypothyroid rabbits, while the CR ^E₂→E₁ decreased significantly the MCR^E2 was not altered.