The concentration of 16x-hydroxy androgens in serum and cyst fluid of women with gross cystic disease of the breast

The concentrations of 16 alpha-hydroxydehydroepi-androsterone sulfate (16 alpha-OHDHAS) and androst-5-ene-3 beta, 16 alpha-, 17 beta-triol-3-sulfate (A-TriolS) were measured in the plasma and breast cyst fluid (BCF) of women with gross cystic disease of the breast. In the 19 BCF samples analyzed, the 16 alpha-OHDHAS and A-TriolS concentrations ranged from 15 to 1130 ng/mL, and 12 to 871 ng/mL, respectively. However the concentrations of these steroids in the sera of these women were lower (15-179 ng/mL, 8-80 ng/mL, respectively). Estriol-3-sulfate (E3-3S) concentrations in the BCF samples ranged from barely detectable (0.2 ng/mL) to 3 micrograms/mL. In BCF or serum a positive linear correlation was observed in the concentration of 16 alpha-OHDHAS and A-TriolS (p less than 0.001 and 0.05, respectively). However, in the same patients no statistical significance was observed in the BCF vs serum concentrations of these two steroids. When the specimens from this and previous studies were combined, positive correlation was found between potassium ion concentration and E3-3S or 16 alpha-OHDHAS. The origin of the high concentration of E3-3S is still obscure. Although no linear correlation between 16 alpha-OHDHAS and E3-3S was observed, the possibility of a precursor-product relationship between the two is not elimnated.     

Correlation of concentrations of estriol-3-sulfate with those of potassium and sodium in human breast cyst fluid

Estriol-3-sulfate (E3-3S) was assayed in 92 specimens of human breast cyst fluid (BCF) obtained by needle aspiration from women with fibrocystic disease. The concentrations of K+ and Na+ were determined in the same samples. The median concentration of E3-3S in the fluids from premenopausal women under 51 years of age (69 cases) was 4.4 ng/mL. Based on the K+ levels the samples were divided into two groups, above 50 mM (Type I) and below 50 mM (Type II). Correlations were made between the concentrations of the estrogen conjugate and the univalent ions. In the premenopausal women, Type I cysts were associated with above median E3-3S and Type II cysts with below median E3-3S (P less than 0.01). A K+/Na+ ratio of more than one was also related to elevated E3-3S (P less than 0.025). The BCF obtained from postmenopausal women and women older than 50 years tended to be low in E3-3S (median 1.64 ng/mL) and high in K+ but there were too few cases to permit statistical comparisons to be made. Since fibrocystic disease constitutes a risk factor for the development of breast cancer, it will be of interest to determine retrospectively whether any of the above subsets of BCF may be useful in identifying a patient at such risk.     

Radioimmunoassay of 16alpha-hydroxy-dehydroepiandrosterone and its sulfate.

A simple and reliable radioimmunoassay for plasma 3beta, 16alpha-dihydroxy-5-androsten-17-one(16alpha-OH-DHEA) and its sulfate has been developed. The antiserum against 16alpha-OH-DHEA and its sulfate (16alpha-OH-DHEA-3-sulfate) was produced in rabbits immunized with 16alpha-OH-DHEA-3-succinate-bovine serum albumin. This antiserum reacted well with both 16alpha-OH-DHEA and its sulfate and only slightly cross reacted with DHEA and its sulfate. The coefficient of variation (C.V.) of the intra assay was 10.26% for 16alpha-OH-DHEA and 12.32% for 16alpha-OH-DHEA-S. The C.V. of the interassay were 14.34% for 16alpha-OH-DHEA and 15.64% for 16alpha-OH-DHEA-S. The umbilical artery concentrations for 16alpha-OH-DHEA and 16alpha-OH-DHEA-S were 7.20 +/- 6.71 ng/ml and 4490 +/- 2140 ng/ml, and the umbilical vein concentrations were 14.20 +/- 11.27 ng/ml and 2970 +/- 1450 ng/ml respectively.     

Evidence for 16alpha-hydroxylation of estrone 3-sulphate by guinea pig liver slices.

[6,7-3H,35S]Estrone 3-sulfate (E13S) of 3H/35S = 3.57 was incubated with female guinea pig liver slices. Small amounts of free steroid and estrone-3-glucuronide, each containing 3H, were found. In addition, E13S, 17beta-estradiol 3-sulfate, and a 'disulfate' fraction, with 3H/35S = 4.4, 4.3, and 4.7, respectively, were also isolated from the incubated tissue. The latter fraction was a major metabolite and about 45% of it consisted of 'disulfates' of 16alpha-hydroxyestrone and estriol, thus providing strong evidence for 16alpha-hydroxylation in guinea pig liver slices.     

Determination of estradiol-17-sulfate in human urine by a direct radioimmunoassay: urinary levels throughout the menstrual cycle

The measurement of urinary estradiol-17-sulfate concentration by direct radioimmunoassay was established. The urinary estradiol-17-sulfate levels measured by the radioimmunoassay correlated well with those determined by high-performance liquid chromatography equipped with electrochemical detector. Estradiol-17-sulfate concentrations in early morning urine of six healthy adult men was 8.2 +/- 2.0 ng/mL, or 5.7 +/- 1.8 ng/mg creatinine. The urinary levels in women throughout the menstrual cycle showed a characteristic three-peak excretion pattern: the first and the second peaks appeared just after and three days before the urinary LH peak, and the third peak appeared a few days before menstruation.     

Development and validation of a high-performance liquid chromatography assay for the quantitative determination of 7-oxo-dehydroepiandrosterone-3β-sulfate in human plasma

A new, simple, reproducible and reliable high-performance liquid chromatography method with ultraviolet absorbance detection at 240 nm was developed and validated for the determination of 7-oxo-dehydroepiandrosterone-3β-sulfate in human plasma. The method was based upon solid-phase (C₁₈) extraction of plasma after addition of 17β-hydroxy-3β-methoxyandrost-5-en-7-one as internal standard. Using 1 ml of plasma for extraction, the detection limit of the assay was 3 ng/ml. The standard curve was linear over the concentration range 10–1000 ng/ml. Stored at −20°C for about 4 months at various concentrations in plasma, 7-oxo-dehydroepiandrosterone-3β-sulfate did not reveal any appreciable degradation. Also included herein is a method for the simultaneous detection and determination of 7-oxo-dehydroepiandrosterone and 7-oxo-dehydroepiandrosterone-3β-acetate in plasma.     

Quantification of fat-soluble vitamins in human breast milk by liquid chromatography-tandem mass spectrometry

Sensitive quantification method for fat-soluble vitamins in human breast milk by liquid chromatography-tandem mass spectrometry was developed. Vitamins A, D and E were extracted from 10.0 mL of breast milk after saponifying by basic condition. Vitamin K derivatives were extracted from 3.0 mL of breast milk after lipase treatment. The corresponding stable isotope-labeled compounds were used as internal standards. For the determination of vitamin D compounds, derivatization with a Cookson-type reagent was performed. All fat-soluble vitamins were determined by liquid chromatography-tandem mass spectrometry in the positive ion mode. The detection limits of all analytes were 1-250 pg per 50 microL. The recoveries of fat-soluble vitamins were 91-105%. Inter-assay CV values of each vitamin were 1.9-11.9%. The mean concentrations of retinol, vitamin D3, 25-hydroxyvitamin D3, alpha-tocopherol, phylloquinone and menaquinone-4 were 0.455 microg/mL, 0.088 ng/mL, 0.081 ng/mL, 5.087 microg/mL, 3.771 ng/mL, and 1.795 ng/mL, respectively (n=82). This method makes possible to determine fat-soluble vitamins with a wide range of polarities in human breast milk. The assay may be useful for large-scale studies.     

Are plasma insulin-like growth factor I (IGF-I) and IGF-binding protein 3 (IGFBP-3) useful markers of prostate cancer?

Increased concentrations of insulin-like growth factor I (IGF-I) and decreased insulin-like growth factor binding protein 3 (IGFBP-3) in serum have been proposed as markers of prostate cancer (CaP). The evidence for this, however, is contradictory. We assayed serum for IGF-I, IGFBP-3 and prostate-specific antigen (PSA) in patients with CaP and benign prostatic hyperplasia (BPH) and in healthy controls (HC). The mean +/- SD concentration of IGF-I in CaP (98.3 +/- 39.3 ng/mL; n = 15) was lower than in BPH (119 +/- 31.1 ng/mL; n=24) and HC (119 +/- 36.1 ng/mL; n=46), but the differences between the three groups were not statistically significant (p > 0.05). The mean IGFBP-3 concentrations in CaP (2691 +/- 1105 ng/mL; n = 16; p = 0.029) and BPH (2618 +/- 816 ng/mL; n = 26; p = 0.006) patients were significantly lower than that of the HC (3119 +/- 618 ng/mL; n=59), but the difference between the two groups of patients was not significant (p > 0.05). PSA concentrations in CaP (median = 80.8 ng/mL; n = 25) were significantly higher than those in BPH (median = 8.6 ng/mL; n = 39) (p < 0.001). Ninety-six percent of CaP and 72% of BPH patients had PSA concentrations >4.0 ng/mL; the proportions of patients with concentrations exceeding 20 ng/mL were 76% and 10%, respectively. We conclude that IGF-I and IGFBP-3 are inferior to PSA for CaP detection.     

Pretreatment serum concentrations of 25-hydroxyvitamin D and breast cancer prognostic characteristics: a case-control and a case-series study

Background

Results from epidemiologic studies on the relationship between vitamin D and breast cancer risk are inconclusive. It is possible that vitamin D may be effective in reducing risk only of specific subtypes due to disease heterogeneity.

Methods and Findings

In case-control and case-series analyses, we examined serum concentrations of 25-hydroxyvitamin D (25OHD) in relation to breast cancer prognostic characteristics, including histologic grade, estrogen receptor (ER), and molecular subtypes defined by ER, progesterone receptor (PR) and HER2, among 579 women with incident breast cancer and 574 controls matched on age and time of blood draw enrolled in the Roswell Park Cancer Institute from 2003 to 2008. We found that breast cancer cases had significantly lower 25OHD concentrations than controls (adjusted mean, 22.8 versus 26.2 ng/mL, p<0.001). Among premenopausal women, 25OHD concentrations were lower in those with high- versus low-grade tumors, and ER negative versus ER positive tumors (p≤0.03). Levels were lowest among women with triple-negative cancer (17.5 ng/mL), significantly different from those with luminal A cancer (24.5 ng/mL, p = 0.002). In case-control analyses, premenopausal women with 25OHD concentrations above the median had significantly lower odds of having triple-negative cancer (OR = 0.21, 95% CI = 0.08–0.53) than those with levels below the median; and every 10 ng/mL increase in serum 25OHD concentrations was associated with a 64% lower odds of having triple-negative cancer (OR = 0.36, 95% CI = 0.22–0.56). The differential associations by tumor subtypes among premenopausal women were confirmed in case-series analyses.

Conclusion

In our analyses, higher serum levels of 25OHD were associated with reduced risk of breast cancer, with associations strongest for high grade, ER negative or triple negative cancers in premenopausal women. With further confirmation in large prospective studies, these findings could warrant vitamin D supplementation for reducing breast cancer risk, particularly those with poor prognostic characteristics among premenopausal women.     

Cytokines in human breast cyst fluid

Gross cystic breast disease is a common benign disorder in which palpable cysts occur in the breast and are normally treated by aspiration of the contents. The cysts are classified as either Type 1, containing a high level of potassium ions and a low level of sodium ions, or as Type 2, with low potassium and high sodium ion concentrations. Steroid sulphatase activity in MDA-MB-231 and MCF-7 cell lines is regulated by exogenous breast cyst fluid (BCF), possibly because of cytokines in the BCF. A screening method was used to determine the range of cytokines in eight BCFs, four of each type. This was an array system, which uses antibodies immobilised on a membrane to qualitatively detect 79 different cytokines or growth factors. Nine cytokines were detected well above background levels: all were found in both types of BCF, but only epidermal growth factor (EGF) was higher in Type 1. All the other factors were higher in Type 2 BCF. Two of these cytokines, IL-6 and EGF, have previously been suggested to affect steroid sulphatase expression and several (MIP-1β, IL-8, NAP-2) are known to affect MCF-7 cell chemotaxis. In addition two cytokines were measured by ELISA in 57 BCFs, and both IL-1β and IL-13 were found in BCF, with significantly higher amounts of IL-1β in Type 1 than Type 2 BCF (35.5 ± 4.4 pg/ml versus 9.9 ± 2.9 pg/ml).     

Splanchnic and intestinal uptake and formation of estriol and estriol conjugates in the dog in vivo.

A loading dose of 3H-estriol was given to male dogs followed by a constant infusion. The concentrations of total radioactivity, conjugated estriol metabolites, estriol, estriol-o-glucosiduronate, estriol-16alpha-glucosiduronate, estriol-3-sulfate and estriol-3-sulfate, 16alpha-glucosiduronate were determined in plasma from the femoral artery(A), hepatic vein(HV) and superior mesenteric vein (SMV). From these values the splanchnic (100[1-HV/A]) and intestinal (100[1-SMV/A]) extractions were calculated. The mean splanchnic extraction of total radioactivity was positive (23, SE 3, P less than .01), indicating net uptake by the splanchnic area, possibly due to biliary excretion. The mean splanchnic extraction of estriol was 77, SE 1, P less than .01, also indicating net uptake. The splachnic extractions of estriol-3-glucosiduronate, estriol-16alpha-glucosiduronate and estriol-3-sulfate were negative (-15, SE 3, P less than .01; -23, SE 6, P less than .01; -31, SE 8, P less than .01 respectively) indicating net formation of these conjugates for release into the systemic circulation. The mean intestinal extraction of estriol was 12, SE 4, P less than .01, indicating net uptake by the intestine. This net uptake was associated with mean negative intestinal extractions of estriol-3-glucosiduronate (-15, SE 7, P approximately .05), estriol-3-sulfate (-33, SE 10, P less than .01) and estriol-3-sulfate, 16alpha-glucosiduronate (-53, SE 13, P less than .01), indicating net formation of these conjugates by the intestine.     

Human Pentraxin 3 (PTX3) as a Novel Biomarker for the Diagnosis of Pulmonary Arterial Hypertension

Background

Although inflammation is an important feature of pulmonary arterial hypertension (PAH), the usefulness of local inflammatory markers as biomarkers for PAH is unknown. In this study, we tested whether plasma concentrations of human pentraxin 3 (PTX3), a local inflammatory marker, would be a useful biomarker for detecting PAH.

Methods

Plasma PTX3 concentrations were evaluated in 50 PAH patients (27 with idiopathic PAH, 17 with PAH associated with connective tissue disease (CTD-PAH), and six with congenital heart disease), 100 age and sex-matched healthy controls, and 34 disease-matched CTD patients without PAH. Plasma concentrations of B-type natriuretic peptide (BNP) and C-reactive protein (CRP) were also determined.

Results

Mean PTX3 levels were significantly higher in all PAH patients than in the healthy controls (4.40±0.37 vs. 1.94±0.09 ng/mL, respectively; P<0.001). Using a threshold level of 2.84 ng/mL, PTX3 yielded a sensitivity of 74.0% and a specificity of 84.0% for the detection of PAH. In CTD-PAH patients, mean PTX3 concentrations were significantly higher than in CTD patients without PAH (5.02±0.69 vs. 2.40±0.14 ng/mL, respectively; P<0.001). There was no significant correlation between plasma levels of PTX3 and BNP or CRP. Receiver operating characteristic (ROC) curves for screening PAH in patients with CTD revealed that PTX3 (area under the ROC curve 0.866) is superior to BNP. Using a PTX3 threshold of 2.85 ng/mL maximized true-positive and false-negative results (sensitivity 94.1%, specificity 73.5%).

Conclusion

Plasma concentrations of PTX3 may be a better biomarker of PAH than BNP, especially in patients with CTD.     

FSH and LH plasma levels in bitches with differences in risk for urinary incontinence

To determine whether the height of the plasma gonadotropin levels after spaying is associated with urinary incontinence, the concentrations of plasma follicle stimulating hormone (FSH) and luteinizing hormone (LH) were determined once in 191 intact and 308 spayed bitches. The bitches were grouped according to their risk for urinary incontinence and the medians of their respective gonadotropin levels were compared. For intact anestrous bitches, the FSH- and LH-plasma concentrations were 5.2 (4, 8) ng/mL (median (Q1, Q3)) and 0.5 (0.5-0.5) ng/mL, respectively. In the first year after spaying, the gonadotropin concentrations rose significantly, then stabilised at a level around 10 times those of intact bitches (FSH 62.5 (44, 91) ng/mL; LH 6.1(4, 11) ng/mL). The plasma gonadotropin concentrations of long-term spayed (>12 months) continent bitches (n=209) were higher (FSH 66.8 (46, 104) ng/mL; LH 6.5 (4, 11) ng/mL) than in spayed incontinent bitches (n=60) (FSH 51.5 (38, 74) ng/mL; LH 5.5 (3, 8) ng/mL), the latter also had a higher body weight. Multiple regression analysis showed that the FSH-plasma concentration and not the body weight was decisive for the occurrence of urinary incontinence. The results of this study suggest that levels of gonadotropins are associated, directly or indirectly in the pathophysiology of urinary incontinence after spaying.     

Human pharmacokinetics of ethynyl estradiol 3-sulfate and 17-sulfate

Pharmacokinetic parameters of ethynyl estradiol 3-sulfate (EE-3) and 17-sulfate (EE-17) were estimated. Each sulfate was administered orally and intravenously to five ovariectomized volunteer women. Blood samples were taken over a period of 24 h. Radioimmunoassay for free and sulfoconjugated ethynyl estradiol (EE) was performed. The analysis of the plasma concentrations obtained after administration of EE-3 and EE-17 indicates significant differences in their pharmacokinetic profiles. EE-3 is cleared more rapidly from the central compartment (systemic circulation), which may indicate that differences in protein binding, tissue binding, metabolism, and distribution exist between EE-3 and EE-17. It has been suggested that these conjugates are a slow-release reservoir for maintenance of blood levels of free EE itself. However, previous studies in baboons have shown that the half-lives of the free and sulfoconjugated EE are similar (ranging from 8.8 to 11.2 h), which is not consistent with this hypothesis. The t1/2 beta (mean 9.28 h) of the 17-sulfate after IV administration was almost identical in women and baboons, and similar to the t1/2 beta of free EE, confirming the previous observation. Only 3.4% of IV and 11.4% of the orally administered 17-sulfate appeared in the blood as free EE; with the 3-sulfate, the conversions were 13.7 and 20.7%, respectively, suggesting that these sulfates are not important slow-release reservoirs. The similarity of pharmacokinetic parameters between women and baboons suggests that this species of nonhuman primate is, in important respects, a suitable animal model for clinical pharmacology.     

Validation of a serum immunoassay to measure progesterone and diagnose pregnancy in the West Indian manatee (Trichechus manatus)

The objective was to validate a high-sensitivity chemiluminescent assay of serum progesterone concentrations for pregnancy diagnosis in manatees. Assay analytical sensitivity was 0.1 ng/mL, with mean intra- and inter-assay coefficients of variation of 9.7 and 9.2%, respectively, and accuracy had a mean adjusted R(2) of 0.98. Methods comparison (relative to Siemen's Coat-A-Count RIA) demonstrated r=0.98, Deming regression slope of 0.95, and an intercept of 0.01. Based on ROC analysis, a progesterone concentration >or=0.4 ng/mL was indicative of pregnancy. Assay results were not significantly altered by two freeze-thaw cycles of samples. Characteristic progesterone concentrations during pregnancy were Months 1-4 (1.7-4.7 ng/mL), 5-8 ( approximately 1.0 ng/mL), and 10 and 11 (0.3-0.5 ng/mL), whereas two late-pregnant females with impending abortion had progesterone concentrations of 0.1 ng/mL. Among pregnant females, maximum progesterone concentrations occurred in autumn (3.9+/-1.8 ng/mL), and were greater during all seasons than concentrations in non-pregnant females (0.1-0.2 ng/mL). Progesterone concentrations were also significantly higher in pregnant females than in non-pregnant females and males. This highly sensitive, specific, and diagnostic assay will be valuable for monitoring pregnancy and abortion in manatees.     

Electrolytes and Trace Elements in Human Breast Cyst Fluid

Gross cystic breast disease (GCBD) is one of the most common breast diseases, and women with apocrine (type I) cysts are at higher risk of developing breast cancer than women with flattened (type II) cysts. Type I cysts contain fluid with an electrolyte composition similar to that of intracellular fluid (Na/K ratio <3), whereas type II cysts fluid’s content resembles that of plasma (Na/K ratio >3). The electrolyte composition of breast cyst fluid (BCF) has been investigated intensively; however, there have been only a few studies in literature reporting the content of trace elements in BCF. The aim of this study was to compare the concentrations of Na, K, Ca, P, Zn, Cu, Fe, and Na/K and trace element ratios in breast cyst fluid in two subgroups of breast cysts. Sixty-three BCF were obtained by needle aspiration from premenopausal women with GCBD diagnosed by clinical, xheromammographic, and cytological studies. After separation of cells for cytological evaluation, the cyst fluid was centrifuged and supernatant stored at −80°C until the analysis. Sodium, potassium, calcium, phosphorus, and iron were measured using Roche Diagnostics commercial kits on Hitachi 747-200 autoanalyzer. Measurements of copper and zinc were performed by flame atomic absorption spectrophotometer on Shimadzu AAS 680. We found statistically significant higher K, lower Na, higher phosphorus concentrations, and lower Na/K ratios in type I cysts when compared with type II cysts’ values. Median values of Na/K ratio in type I and in type II were 0.32 and 6.2, respectively. Higher Zn, Cu, and Fe concentrations with respect to median values were noted in type I cysts; higher [Na.Cu/K.Zn], [Na.Cu/K.Fe], and [Na.Zn/K.Fe] ratios were found in type II cysts. A significant negative correlation existed between Na/K and Cu, and a significant positive correlation between Na/K and Fe in type II cysts (r = −0.660, p = 0.007; r = 0.615, p = 0.014, respectively). We can conclude that the trace elements content of BCF, in addition to electrolytes, could be useful in classifying the breast cyst. Future studies in larger series are needed to confirm these data.     

Quantitative aspects of lipopolysaccharide and cytokine requirements to generate nitric oxide in macrophages from LPS-hyporesponsive (<Emphasis Type="Italic">Lps</Emphasis><Superscript>d</Superscript>) C3H/HeJ mice

Due to a gene defect (Lps(d)), C3H/HeJ mice are known to be hyporesponsive to the immunobiological potential of lipopolysaccharide (LPS). We studied dose requirements for LPS, IFN-gamma, and cytokines TNF-alpha and IL-10 to produce nitric oxide (NO) in peritoneal macrophages (Mphi) from these animals. In contrast to the Lps(n) C3H/HeN mice, high concentrations of LPS (up to 5 microg/mL) or IFN-gamma (up to 5 ng/mL) by themselves were unable to activate NO production in C3H/HeJ Mphi. The failure to produce NO could not be overcome by addition of L-arginine or tetrahydropterin. The high-output NO biosynthesis was dose-dependently stimulated by combined administration of varying concentrations of IFN-gamma (50-5000 pg/mL) and LPS (approximately 1 ng/mL) or to a lesser extent by IFN-gamma plus TNF-alpha or TNF-alpha/IL-10. Formation of NO in C3H/HeJ MCO triggered by high concentration of LPS (approximately 1 microg/mL) given together with IFN-gamma (0.2-5 ng/mL) reached the values typical for Lps(n) C3H/HeN mice. While Mphi from C3H/HeN mice secreted TNF-alpha, IL-10, and IL-10 upon contact with a low dose of LPS (1 ng/mL), C3H/HeJ Mphi required high concentration of LPS (5 microg/mL) to enhance the secretion of the cytokines. Yet, this dose remained ineffective to stimulate IFN-gamma in Mphi from C3H/HeJ mice. It can be presumed that one of the important factors influencing their deficient ability to form NO is a failure of Mphi to produce IFN-gamma upon LPS contact.     

Plasma progesterone and prolactin concentrations in overtly pseudopregnant bitches: a clinical study

Plasma concentrations of progesterone (P(4)) and prolactin (PRL) were measured in 35 bitches presented at veterinary clinics for symptoms of overt pseudopregnancy (PSP) between 50 and 95 days after the onset of proestrus. Results were compared to those from samples collected from 35 control bitches at comparable stages of the ovarian cycle (expressed as days after the onset of observed signs of proestrus). In the PSP bitches at 71.4+/-1.6 (mean+/-S.E.M.) days of the cycle, P(4) (1.5+/-0.2ng/mL) was lower (P<0.01) and PRL (16.0+/-1.9ng/mL) was higher (P<0.01), compared to P(4) (2.7+/-0.4ng/mL) and PRL (2.9+/-0.6ng/mL) in control bitches at 70.6+/-1.5 days of the cycle. Low P(4) was not a prerequisite for elevated PRL. Although elevated (> or =10ng/mL) PRL (20.9+/-2.0ng/mL) occurred more often with low (<2ng/mL) P(4) (20 of 24 cases) it also occurred with P(4) above 3ng/mL in two affected bitches and in two control bitches. Whether the occurrence of relatively low PRL concentrations (<4ng/mL) in samples obtained from 4 of the 35 pseudopregnant bitches reflected variable and often elevated PRL secretion or increased sensitivity to PRL in the absence of elevated prolactin in those animals was not determined. We inferred that elevated plasma PRL was often involved in the etiology of overt PSP; furthermore, a premature decline in circulating P(4) concentrations may be a factor in some instances.     

Development and validation of a simple reversed-phase HPLC method for the determination of camptothecin in animal organs following administration in solid lipid nanoparticles

A simple, sensitive and specific high-performance liquid chromatography (HPLC) assay for the quantification of camptothecin (CPT), a potent anticancer candidate, incorporated into solid lipid nanoparticles (SLN) in several rat organs (brain, heart, kidneys liver, lung, spleen) and serum was developed and validated. The sample pre-treatment involved organs homogenisation followed by CPT extraction. The samples were injected onto an analytical reversed-phase (RP) Mediterranea? Sea18 column maintained at 30°C. The chromatographic separation was achieved by gradient elution consisting of triethyamine buffer pH 5.5 and acetonitrile at a flow rate of 1.2 mL/min in 16 min of run time and retention time of 9.8 min (lactone). Fluorescence detection was used at the excitation and emission of 360 and 440 nm, respectively. The calibration curves in the different organs, serum and in PB3 were linear (r(2)>0.9999) over CPT concentrations ranging from 1 to 200 ng/mL or 0.5 to 200 ng/mL (n=6), respectively. The method was shown to be specific, accurate (between 94.4±4.5% and 108.9±0.6%) and precise at the intra-day and inter-day levels as reflected by the coefficient of variation (CV<6.3%) at three different concentrations (10, 50 and 100 ng/mL) in all matrices. Stability studies showed that CPT was stable in all matrices after 24h of incubation at room temperature (RT), after 24 h in the autosampler or after three freeze/thaw cycles. The mean recoveries of CPT in suspension, loaded into SLN and in a physical mixture with SLN at three concentrations of 10, 50 and 200 ng/mL were higher than 86.4%. The detection limit (DL) was ≤0.2 ng/mL and the quantification limit (QL) was ≤0.5 ng/mL. The method developed is reliable, precise and accurate and can be used in the determination of CPT amount in rat organ samples after i.v. administration of CPT in suspension, in physical mixture with SLN and incorporated in SLN.     

Flow-injection determination of carbaryl and carbofuran based on KMnO(4)-Na(2)SO(3) chemiluminescence detection.

A flow-injection method is described for the determination of carbaryl and carbofuran. It was found that a strong chemiluminescence (CL) signal was generated when these pesticides were mixed with Na(2)SO(3) and KMnO(4) in acidic medium. Under the optimum experimental conditions, the enhanced CL intensity was linear, with the concentrations in the range 0.1-2.0 microg/mL (r(2) = 0.9996 and 0.9993, n = 6) with relative standard deviation (n = 4) in the range 1.0-2.3%. The limits of detection (3sigma blank) were 10 and 50 ng/mL, respectively, with a sample throughput of 180/h. The proposed method was applied to determine carbaryl and carbofuran in freshwaters with satisfactory results. Most metal and non-metal ions and some pesticides, such as carbophenothion and aldicarb, do not interfere with the determination. Dinoseb, diazinon and malathion calibration graphs (in the range 0.2-2.0 microg/mL, r(2) = 0.9966-0.9988, n = 6) were also established with relative standard deviations (n = 4) in the range 1.2-2.0% with limits of detection (3sigma blank) in the range 100-300 ng/mL.