Exogenous gonadotropins do not increase the blood-follicular transportation capacity of extra-ovarian hormones such as prolactin and cortisol.

BACKGROUNDS In vitro fertilization involves high dosage gonadotropin stimulation, which apparently has some negative impact on follicular endocrine function. As chorionic gonadotropin stimulation has been shown to increase the blood-follicular permeability in animal models, this raises the question if such an effect also applies to gonadotropins in humans, possibly affecting the endocrine follicular milieu. FINDINGS Follicular fluid and serum were collected at the time of follicular aspiration in in vitro fertilisation without (Natural cycle IVF, n = 24) and with (conventional gonadotropin stimulated IVF, n = 31) gonadotropin stimulation. The concentration of the extra-ovarian hormones prolactin and cortisol were analysed by immunoassays. RESULTS Median serum prolactin and cortisol concentrations were 12.3 ng/mL and 399 nmol/L without versus 32.2 ng/mL and 623 nmol/L with gonadotropin stimulation. The corresponding concentrations in follicular fluid were 20.6 ng/mL and 445 nmol/L versus 28.8 ng/ml and 456 nmol/L for prolactin and cortisol. As a consequence, mean follicular fluid:serum ratios were significantly reduced under gonadotropin stimulation (prolactin p = 0.0138, cortisol p = 0.0001). As an enhanced blood-follicular permeability and transportation, induced by gonadotropin stimulation, would result in increased instead of decreased follicular fluid:serum ratios as found in this study, it can be assumed that this does not affect extra-ovarian protein and steroid hormones as illustrated by prolactin and cortisol. CONCLUSIONS The model of serum follicular fluid:serum ratio of hormones, produced outside the ovaries, did not reveal a gonadotropin induced increased blood-follicular transportation capacity. Therefore it can be assumed that the effect of gonadotropins on follicular endocrine function is not due to an increased ovarian permeability of extra-ovarian hormones.

The basel cocktail for simultaneous phenotyping of human cytochrome P450 isoforms in plasma, saliva and dried blood spots.

BACKGROUND AND OBJECTIVE Phenotyping cocktails use a combination of cytochrome P450 (CYP)-specific probe drugs to simultaneously assess the activity of different CYP isoforms. To improve the clinical applicability of CYP phenotyping, the main objectives of this study were to develop a new cocktail based on probe drugs that are widely used in clinical practice and to test whether alternative sampling methods such as collection of dried blood spots (DBS) or saliva could be used to simplify the sampling process. METHODS In a randomized crossover study, a new combination of commercially available probe drugs (the Basel cocktail) was tested for simultaneous phenotyping of CYP1A2, CYP2B6, CYP2C9, CYP2C19, CYP2D6 and CYP3A4. Sixteen subjects received low doses of caffeine, efavirenz, losartan, omeprazole, metoprolol and midazolam in different combinations. All subjects were genotyped, and full pharmacokinetic profiles of the probe drugs and their main metabolites were determined in plasma, dried blood spots and saliva samples. RESULTS The Basel cocktail was well tolerated, and bioequivalence tests showed no evidence of mutual interactions between the probe drugs. In plasma, single timepoint metabolic ratios at 2 h (for CYP2C19 and CYP3A4) or at 8 h (for the other isoforms) after dosing showed high correlations with corresponding area under the concentration-time curve (AUC) ratios (AUC0-24h parent/AUC0-24h metabolite) and are proposed as simple phenotyping metrics. Metabolic ratios in dried blood spots (for CYP1A2 and CYP2C19) or in saliva samples (for CYP1A2) were comparable to plasma ratios and offer the option of minimally invasive or non-invasive phenotyping of these isoforms. CONCLUSIONS This new combination of phenotyping probe drugs can be used without mutual interactions. The proposed sampling timepoints have the potential to facilitate clinical application of phenotyping but require further validation in conditions of altered CYP activity. The use of DBS or saliva samples seems feasible for phenotyping of the selected CYP isoforms.

Therapeutic drug monitoring of once daily aminoglycoside dosing: comparison of two methods and investigation of the optimal blood sampling strategy.

PURPOSE Therapeutic drug monitoring of patients receiving once daily aminoglycoside therapy can be performed using pharmacokinetic (PK) formulas or Bayesian calculations. While these methods produced comparable results, their performance has never been checked against full PK profiles. We performed a PK study in order to compare both methods and to determine the best time-points to estimate AUC0-24 and peak concentrations (C max). METHODS We obtained full PK profiles in 14 patients receiving a once daily aminoglycoside therapy. PK parameters were calculated with PKSolver using non-compartmental methods. The calculated PK parameters were then compared with parameters estimated using an algorithm based on two serum concentrations (two-point method) or the software TCIWorks (Bayesian method). RESULTS For tobramycin and gentamicin, AUC0-24 and C max could be reliably estimated using a first serum concentration obtained at 1 h and a second one between 8 and 10 h after start of the infusion. The two-point and the Bayesian method produced similar results. For amikacin, AUC0-24 could reliably be estimated by both methods. C max was underestimated by 10-20% by the two-point method and by up to 30% with a large variation by the Bayesian method. CONCLUSIONS The ideal time-points for therapeutic drug monitoring of once daily administered aminoglycosides are 1 h after start of a 30-min infusion for the first time-point and 8-10 h after start of the infusion for the second time-point. Duration of the infusion and accurate registration of the time-points of blood drawing are essential for obtaining precise predictions.

Determination of Δ9-tetrahydrocannabinolic acid A (Δ9-THCA-A) in whole blood and plasma by LC–MS/MS and application in authentic samples from drivers suspected of driving under the influence of cannabis

Delta-9-tetrahydrocannabinolic acid A (THCA-A) is the biosynthetic precursor of delta-9-tetrahydrocannabinol (THC) in cannabis plants, and has no psychotropic effects. THCA-A can be detected in blood and urine, and several metabolites have been identified. THCA-A was also shown to be incorporated in hair by side stream smoke to a minor extent, but incorporation via blood stream or sweat seems unlikely. The detection of THCA-A in biological fluids may serve as a marker for differentiating between the intake of prescribed THC medication – containing only pure THC – and cannabis products containing THC besides THC-acid A and other cannabinoids. However, the knowledge about its usefulness in forensic cases is very limited. The aim of the present work was the development of a reliable method for THCA-A determination in human blood or plasma using LC–MS/MS and application to cases of driving under the influence of drugs. Fifty eight (58) authentic whole blood and the respective plasma samples were collected from drivers suspected of driving under the influence of cannabis from the region of Bern (Switzerland). Samples were first tested for THC, 11-OH-THC and THC-COOH, and then additionally for THCA-A. For this purpose, the existing LC–MS/MS method was modified and validated, and found to be selective and linear over a range of 1.0 to 200 ng/mL (the correlation coefficients were above 0.9980 in all validation runs). Limit of detection (LOD) and limit of quantification (LOQ) were 0.3 ng/mL and 1.0 ng/mL respectively. Intra- and inter-assay accuracy were equal or better than 90% and intra- and inter-assay precision were equal or better than 11.1%. The mean extraction efficiencies were satisfactory being equal or higher than 85.4%. THCA-A was stable in whole blood samples after 3 freeze/thaw cycles and storage at 4 °C for 7 days. Re-injection (autosampler) stability was also satisfactory. THC was present in all blood samples with levels ranging from 0.7 to 51 ng/mL. THCA-A concentrations ranged from 1.0 to 496 ng/mL in blood samples and from 1.4 to 824 ng/mL in plasma samples. The plasma:blood partition coefficient had a mean value of 1.7 (±0.21, SD). No correlation was found between the degree of intoxication or impairment stated in the police protocols or reports of medical examinations and the detected THCA-A-concentration in blood.

Concentration, Working Speed and Memory: Cognitive Problems in Young Childhood Cancer Survivors and Their Siblings.

BACKGROUND Cognitive problems can have a negative effect on a person's education, but little is known about cognitive problems in young childhood cancer survivors (survivors). This study compared cognitive problems between survivors and their siblings, determined if cognitive problems decreased during recent treatment periods and identified characteristics associated with the presence of a cognitive problem in survivors. METHODS As part of the Swiss Childhood Cancer Survivor Study, a questionnaire was sent to all survivors, aged 8-20 years, registered in the Swiss Childhood Cancer Registry, diagnosed at age <16 years, who had survived ≥5 years. Parent-reported (aged 8-15 years) and self-reported (aged 16-20 years) cognitive problems (concentration, working speed, memory) were compared between survivors and siblings. Multivariable logistic regression was used to identify characteristics associated with cognitive problems in survivors. RESULTS Data from 840 survivors and 247 siblings were analyzed. More often than their siblings, survivors reported problems with concentration (12% vs. 6%; P = 0.020), slow working speed (20% vs. 8%; P = 0.001) or memory (33% vs. 15%; P < 0.001). Survivors from all treatment periods were more likely to report a cognitive problem than were siblings. Survivors of CNS tumors (OR = 2.82 compared to leukemia survivors, P < 0.001) and those who had received cranial irradiation (OR = 2.10, P = 0.010) were most severely affected. CONCLUSION Childhood cancer survivors, even those treated recently (2001-2005), remain at risk to develop cognitive problems, suggesting a need to improve therapies. Survivors with cognitive problems should be given the opportunity to enter special education programs. Pediatr Blood Cancer © 2014 Wiley Periodicals, Inc.

Hypoglycemia despite hyperglycemia. Is a cerebral glucose deficiency possible even with raised blood sugar levels?

Hypoglycemia represents the most frequent endocrinologic emergency situation in prehospital patient care. As the patients are usually unconscious on arrival of emergency medical personnel, often the only way to establish a diagnosis is by determination of the blood glucose concentration. However, even normoglycemic or hyperglycemic levels cannot definitively exclude the diagnosis of a previous hypoglycemia as the cause of the acute cerebral deficiency. Therefore, and especially in the case of insulin-dependent diabetes mellitus, a differential diagnosis should be considered. We report a case of emergency treatment of a hypoglycemic episode in a female patient with prolonged neuroglycopenia together with cerebrovascular dementia and Alzheimer's disease.

The sub-lethal effects and tissue concentration of the human pharmaceutical atenolol in rainbow trout (Oncorhynchus mykiss).

Atenolol is a highly prescribed anti-hypertensive pharmaceutical and a member of the group of β-blockers. It has been detected at concentrations ranging from ng L(-1) to low μg L(-1) in waste and surface waters. The present study aimed to assess the sub-lethal effects of atenolol on rainbow trout (Oncorhynchus mykiss) and to determine its tissue-specific bioconcentration. Juvenile rainbow trout were exposed for 21 and 42 days to three concentration levels of atenolol (1 μg L(-1) - environmentally relevant concentration, 10 μg L(-1), and 1000 μg L(-1)). The fish exposed to 1 μg L(-1) atenolol exhibited a higher lactate content in the blood plasma and a reduced haemoglobin content compared with the control. The results show that exposure to atenolol at concentrations greater than or equal to 10 μg L(-1) significantly reduces both the haematocrit value and the glucose concentration in the blood plasma. The activities of the studied antioxidant enzymes (catalase and superoxide dismutase) were not significantly affected by atenolol exposure, and only the highest tested concentration of atenolol significantly reduced the activity of glutathione reductase. The activities of selected CYP450 enzymes were not affected by atenolol exposure. The histological changes indicate that atenolol has an effect on the vascular system, as evidenced by the observed liver congestion and changes in the pericardium and myocardium. Atenolol was found to have a very low bioconcentration factor (the highest value found was 0.27). The bioconcentration levels followed the order liver>kidney>muscle. The concentration of atenolol in the blood plasma was below the limit of quantification (2.0 ng g(-1)). The bioconcentration factors and the activities of selected CYP450 enzymes suggest that atenolol is not metabolised in the liver and may be excreted unchanged.

Milk fatty acids as possible biomarkers to early diagnose elevated concentrations of blood plasma nonesterified fatty acids in dairy cows

Most cows encounter a state of negative energy balance during the periparturient period, which may lead to metabolic disorders and impaired fertility. The aim of this study was to assess the potential of milk fatty acids as diagnostic tools of detrimental levels of blood plasma nonesterified fatty acids (NEFA), defined as NEFA concentrations beyond 0.6 mmol/L, in a data set of 92 early lactating cows fed a glucogenic or lipogenic diet and subjected to 0-, 30-, or 60-d dry period before parturition. Milk was collected in wk 2, 3, 4, and 8 (n = 368) and blood was sampled weekly from wk 2 to 8 after parturition. Milk was analyzed for milk fatty acids and blood plasma for NEFA. Data were classified as "at risk of detrimental blood plasma NEFA" (NEFA ≥ 0.6 mmol/L) and "not at risk of detrimental blood plasma NEFA" (NEFA <0.6 mmol/L). Concentrations of 45 milk fatty acids and milk fat C18:1 cis-9-to-C15:0 ratio were subjected to a discriminant analysis. Milk fat C18:1 cis-9 revealed the most discriminating variable to identify detrimental blood plasma NEFA. A false positive rate of 10% allowed us to diagnose 46% of the detrimental blood plasma NEFA cases based on a milk fat C18:1 cis-9 concentration of at least 230 g/kg of milk fatty acids. Additionally, it was assessed whether the milk fat C18:1 cis-9 concentrations of wk 2 could be used as an early warning for detrimental blood plasma NEFA risk during the first 8 wk in lactation. Cows with at least 240 g/kg of C18:1 cis-9 in milk fat had about 50% chance to encounter blood plasma NEFA values of 0.6 mmol/L or more during the first 8 wk of lactation, with a false positive rate of 11.4%. Profit simulations were based on costs for cows suffering from detrimental blood plasma NEFA, and costs for preventive treatment based on daily dosing of propylene glycol for 3 wk. Given the relatively low incidence rate (8% of all observations), continuous monitoring of milk fatty acids during the first 8 wk of lactation to diagnose detrimental blood plasma NEFA does not seem cost effective. On the contrary, milk fat C18:1 cis-9 of the second lactation week could be an early warning of cows at risk of detrimental blood NEFA. In this case, selective treatment may be cost effective.

Continuous milking of dairy cows disrupts timing of peak IgG concentration appearance in mammary secretions

The length of the dry period in commercial dairy production is under close scrutiny. While the main concern is the composition and volume of milk produced, the evaluation of colostrum quality under these new paradigms has suggested a decline in IgG concentrations, while some reports indicate no change. Colostrum quality has been defined as an adequate concentration (>50 mg/ml) of immunoglobulin in the secretions to provide the newborn with maximal disease resistance. We investigated the appearance of IgG in mammary pre- and post partum secretions in cows without a dry period (continuously milked, Dry0) and compared the secretions with cows that experienced a dry period of 60 d (Dry60). Blood was collected during the experimental period and plasma analysed for progesterone (P4) and prolactin (Prl). Approximately -6 d relative to parturition, the Dry0 animals exhibited increased concentration of IgG in their secretions to an average of ∼35 mg/ml that remained rather constant through subsequent pregnancy and following parturition. Dry0 cows were producing an average IgG concentration in parturition colostrum of 44·2±17·6 mg/ml that was not different than that of controls (66·86±16·8 mg/ml). However, Dry0 cows exhibited high variation, different peak times (day) of IgG concentration including times that occurred both pre and post parturition. IgG mass of the Dry0 cows remained rather constant pre- and post partum and did not show the same declining mass following parturition that was shown for the Dry60 cows. The change in plasma P4 and Prl were shown to have no timing effect on colostrum IgG concentration.

Effect of intramammary administration of prednisolone on the blood-milk barrier during the immune response of the mammary gland to lipopolysaccharide

OBJECTIVE: To investigate effects of intramammary administration of prednisolone on the immune response of mammary glands in cows. ANIMALS: 5 lactating Red Holsteins. PROCEDURES: Cows received a different intramammary infusion in each mammary gland (10 mg of prednisolone, 100 μg of lipopolysaccharide [LPS], 100 μg of LPS and 10 mg of prednisolone, or saline [0.9% NaCl] solution). Milk samples were collected before (time 0) and 3, 6, 9, 12, 24, and 36 hours after treatment. Somatic cell count (SCC), lactate dehydrogenase (LDH) activity, and concentrations of serum albumin (SA) and tumor necrosis factor (TNF)-α in milk and mRNA expression of TNF-α, interleukin (IL)-8, and IL-1β in milk somatic cells were analyzed. RESULTS: Saline solution or prednisolone did not change SCC, LDH activity, and SA and TNF-α concentrations in milk and mRNA expression of TNF-α, IL-1β, and IL-8 in milk somatic cells. The SCC and TNF-α concentration in milk increased similarly in glands infused with LPS, independent of prednisolone administration. However, the increase of LDH activity and SA concentration in milk after LPS infusion was diminished by prednisolone administration. The mRNA expression of TNF-α, IL-8, and IL-1β in milk somatic cells increased after LPS infusion and was unaffected by prednisolone. CONCLUSIONS AND CLINICAL RELEVANCE: Intramammary administration of prednisolone did not induce an immune response and did not change mRNA expression of TNF-α, IL-8, and L-1β during the response to intramammary administration of LPS. However, prednisolone reduced disruption of the blood-milk barrier. This could influence the severity and cure rate of mastitis.

Development of a rapid column-switching LC-MS/MS method for the quantification of THCCOOH and THCCOOH-glucuronide in whole blood for assessing cannabis consumption frequency

The concentration of 11-nor-9-carboxy-Δ(9)-tetrahydrocannabinol (THCCOOH) in whole blood is used as a parameter for assessing the consumption behavior of cannabis consumers. The blood level of THCCOOH-glucuronide might provide additional information about the frequency of cannabis use. To verify this assumption, a column-switching liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the rapid and direct quantification of free and glucuronidated THCCOOH in human whole blood was newly developed. The method comprised protein precipitation, followed by injection of the processed sample onto a trapping column and subsequent gradient elution to an analytical column for separation and detection. The total LC run time was 4.5 min. Detection of the analytes was accomplished by electrospray ionization in positive ion mode and selected reaction monitoring using a triple-stage quadrupole mass spectrometer. The method was fully validated by evaluating the following parameters: linearity, lower limit of quantification, accuracy and imprecision, selectivity, extraction efficiency, matrix effect, carry-over, dilution integrity, analyte stability, and re-injection reproducibility. All acceptance criteria were analyzed and the predefined criteria met. Linearity ranged from 5.0 to 500 μg/L for both analytes. The method was successfully applied to whole blood samples from a large collective of cannabis consumers, demonstrating its applicability in the forensic field.

Sources of familial aggregation and co-aggregation of fasting blood glucose and nutritional factors among Mexican -Americans in Starr County, Texas

The purpose of this study was to determine the effects of nutrient intake, genetic factors and common household environmental factors on the aggregation of fasting blood glucose among Mexican-Americans in Starr County, Texas. This study was designed to determine: (a) the proportion of variation of fasting blood glucose concentration explained by unmeasured genetic and common household environmental effects; (b) the degree of familial aggregation of measures of nutrient intake; and (c) the extent to which the familial aggregation of fasting blood glucose is explained by nutrient intake and its aggregation. The method of path analysis was employed to determine these various effects.^ Genes play an important role in fasting blood glucose: Genetic variation was found to explain about 40% of the total variation in fasting blood glucose. Common household environmental effects, on the other hand, explained less than 3% of the variation in fasting blood glucose levels among individuals. Common household effects, however, did have significant effects on measures of nutrient intake, though it explained only about 10% of the total variance in nutrient intake. Finally, there was significant familial aggregation of nutrient intake measures, but their aggregation did not contribute significantly to the familial aggregation of fasting blood glucose. These results imply that similarities among relatives for fasting blood glucose are not due to similarities in nutrient intake among relatives. ^

(Table 1) Cortisol concentration in hair of East Greenland polar bears (Ursus maritimus)

To demonstrate the ability to assess long-term hypothalamic-pituitary-adrenocortical (HPA) axis activity in polar bears (Ursus maritimus), a pilot study was conducted in which cortisol concentration was analyzed in hair from 7 female (3-19 years) and 10 male (6-19 years) East Greenland polar bears sampled in 1994-2006. The hair was chosen as matrix as it is non-invasive, seasonally harmonized, and has been validated as an index of long-term changes in cortisol levels. The samples were categorized according to contamination: eight were clean (2 females, 6 males), 5 had been contaminated with bear blood (2 F, 3 M), and 4 with bear fat (3 F, 1 M). There was no significant difference in cortisol concentration between the three categories after external contamination was removed. However, contaminated hair samples should be cleaned before cortisol determination. Average hair cortisol concentration was 8.90 pg/mg (range: 5.5 to 16.4 pg/mg). There was no significant correlation between cortisol concentration and age (p = 0.81) or sampling year (p = 0.11). However, females had higher mean cortisol concentration than males (females mean: 11.0 pg/mg, males: 7.3 pg/mg; p = 0.01). The study showed that polar bear hair contains measurable amounts of cortisol and that cortisol in hair may be used in studies of long-term stress in polar bears.

(Table 1) Lipid content and organohalogen contaminant concentration in captive sledge dogs (Canis familiaris) and wild female polar bears (Ursus maritimus) from Greenland

The limited knowledge and/or the inability to control physiological condition parameters that influence the fate of organohalogen contaminants (OHCs) has been the foremost confounding aspect in monitoring programs and health risk assessments of wild top predators in the Arctic such as the polar bear (Ursus maritimus). In the present comparative study, we used a potential surrogate Canoidea species for the East Greenland polar bear, the captive sledge dog (Canis familiaris), to investigate some factors that may influence the bioaccumulation and biotransformation of major chlorinated and brominated OHCs in adipose tissue and blood (plasma) of control (fed commercial pork fat) and exposed (fed West Greenland minke whale (Balaenoptera acutorostrata) blubber) adult female sledge dogs. Furthermore, we compared the patterns and concentrations of OHCs and their known or suggested hydroxylated (OH) metabolites (e.g., OH-PCBs) in sledge dogs with those in adipose tissue and blood (plasma) of East Greenland adult female polar bears, and blubber of their main prey species, the ringed seal (Pusa hispida). The two-year feeding regime conducted with sledge dogs led to marked differences in overall adipose tissue (and plasma) OHC residue accumulation between the control and exposed groups. Characteristic prey-to-predator OHC bioaccumulation dynamics for major PCB and PBDE congeners (patterns and concentrations) and biotransformation capacity with respect to PCB metabolite formation and OH-PCB retention distinguished, to some extent, captive sledge dogs and wild polar bears. Based on the present findings, we conclude that the use of surrogate species in toxicological investigations for species in the Canoidea family should be done with great caution, although they remain essential in the context of contaminants research with sensitive arctic top carnivore species such as the polar bear.

(Table 1) Concentration of organohalogenated compounds in clean and contaminated hair of polar bears (Ursus maritimus) from East Greenland

In this pilot study, we report on levels of persistent organohalogenated contaminants (OHCs) in hair of polar bears (Ursus maritimus) from East Greenland sampled between 1999 and 2001. To our knowledge, this is the first study on the validation of polar bear hair as a non-invasive matrix representative of concentrations and profiles in internal organs and blood plasma. Because of low sample weights (13-140 mg), only major bioaccumulative OHCs were detected above the limit of quantification: five polychlorinated biphenyl (PCB) congeners (CB 99, 138, 153, 170 and 180), one polybrominated diphenyl ether (PBDE) congener (BDE 47), oxychlordane, trans-nonachlor and ß-hexachlorocyclohexane. The PCB profile in hair was similar to that of internal tissues (i.e. adipose, liver, brain and blood), with CB 153 and 180 as the major congeners in all matrices. A gender difference was found for concentrations in hair relative to concentrations in internal tissues. Females (n = 6) were found to display negative correlations, while males (n = 5) showed positive correlations, although p-values were not found significant. These negative correlations in females may reflect seasonal OHC mobilisation from periphery adipose tissue due to, for example, lactation and fasting. The lack of significance in most correlations may be due to small sample sizes and seasonal variability of concentrations in soft tissues. Further research with larger sample weights and sizes is therefore necessary to draw more definitive conclusions on the usefulness of hair for biomonitoring OHCs in polar bears and other fur mammals.