Dopamine agonist serum concentrations and impulse control disorders in Parkinson's disease.

BACKGROUND AND PURPOSE: Impulse control disorders (ICDs) are common among Parkinson's disease patients using dopamine agonists. We wanted to determine whether ICD patients have higher dopamine agonist serum concentrations than those without any sign of ICD. METHODS: Patients who used either pramipexole or ropinirole depot once daily were screened for ICDs using the validated Questionnaire for Impulsive-Compulsive Disorders in Parkinson's Disease-Rating Scale. Those who scored above the cut-off for one or more of the four defined ICDs (gambling, compulsive sexual behavior, compulsive shopping, and binge-eating) were compared in a case-control study to patients who scored zero points (no evidence of ICD) on the same items. They were examined clinically and evaluated using relevant scales. Three blood samples were taken on the same day: before daily dose, and then 6 and 12 h later. RESULTS: Forty-six patients were included: 19 ICD-positive and 27 controls. Ropinirole serum concentrations 6 h after daily intake (Cmax ) were higher in the case group compared to the control group, as was the daily ropinirole dosage. No differences were observed in serum concentrations, dosage or total drug exposure for pramipexole. Disease duration and length of dopamine agonist treatment was significantly longer among ICD patients for ropinirole, but not for pramipexole. CONCLUSIONS: The use of pramipexole may in itself confer high ICD risk, whereas ICDs among ropinirole users depend more on serum concentration and drug exposure. The pharmacokinetic properties of ropinirole make it challenging to predict its effects on patients, which supports the need for therapeutic drug monitoring to reduce risk of ICD.

Moderate but not severe hypothermia increases intracellular cyclic AMP through preserved production and reduced elimination.

Rewarming from accidental hypothermia could be complicated by acute cardiac dysfunction but providing supportive pharmacotherapy at low core temperatures is challenging. Several pharmacological strategies aim to improve cardiovascular function by increasing cAMP in cardiomyocytes as well as cAMP and cGMP levels in vascular smooth muscle, but it is not clear what effects temperature has on cellular elimination of cAMP and cGMP. We therefore studied the effects of differential temperatures from normothermia to deep hypothermia (37 °C-20 °C) on cAMP levels in embryonic H9c2 cardiac cells and elimination of cAMP and cGMP by PDE-enzymes and ABC-transporter proteins. Our experiments showed significant elevation of intracellular cAMP in H9c2-cells at 30 °C but not 20 °C. Elimination of both cAMP and cGMP through ABC transport-proteins and PDE-enzymes showed a temperature dependent reduction. Accordingly, the increased cardiomyocyte cAMP-levels during moderate hypothermia appears an effect of preserved production and reduced elimination at 30 °C. This correlates with earlier in vivo findings of a positive inotropic effect of moderate hypothermia.

Sex Differences in Age-Related Loss of Kidney Function.

BACKGROUND: CKD is more prevalent in women, but more men receive kidney replacement therapy for kidney failure. This apparent contradiction is not well understood. METHODS: We investigated sex differences in the loss of kidney function and whether any sex disparities could be explained by comorbidity or CKD risk factors. In the Renal Iohexol Clearance Survey (RENIS) in northern Europe, we recruited 1837 persons (53% women, aged 50-62 years) representative of the general population and without self-reported diabetes, CKD, or cardiovascular disease. Participants' GFR was measured by plasma iohexol clearance in 2007-2009 (n=1627), 2013-2015 (n=1324), and 2018-2020 (n=1384). At each study visit, healthy persons were defined as having no major chronic diseases or risk factors for CKD. We used generalized additive mixed models to assess age- and sex-specific GFR decline rates. RESULTS: Women had a lower GFR than men at baseline (mean [SD], 90.0 [14.0] versus 98.0 [13.7] ml/min per 1.73 m2; P<0.001). The mean GFR change rate was -0.96 (95% confidence interval [CI], -0.88 to -1.04) ml/min per 1.73 m2 per year in women and -1.20 (95% confidence interval [CI], -1.12 to -1.28) in men. Although the relationship between age and GFR was very close to linear in women, it was curvilinear in men, with steeper GFR slopes at older ages (nonlinear effect; P<0.001). Healthy persons had a slower GFR decline, but health status did not explain the sex difference in the GFR decline. CONCLUSION: Among middle-aged and elderly individuals in the general population, decline in the mean GFR in women was slower than in men, independent of health status.

Stability of Direct Oral Anticoagulants and Antiarrhythmic Drugs in Serum Collected in Standard (Nongel) Serum Tubes Versus Tubes Containing Gel Separators.

BACKGROUND: Separation gels are often used in collection tubes, but adsorption of drugs onto the gel may cause falsely low concentrations in therapeutic drug monitoring. In this study, the stability of apixaban, edoxaban, rivaroxaban, flecainide, amiodarone, and desethylamiodarone was assessed in tubes, with and without gel separators. METHODS: Drug-free blood was spiked and stored for up to 7 days in nongel tubes and gel tubes from 2 manufacturers (Vacuette and Vacutainer). The samples were analyzed in triplicates using ultra-high-pressure liquid chromatography-tandem mass spectrometry. RESULTS: At ambient temperature conditions, the serum concentrations of apixaban, edoxaban, and rivaroxaban in a tube with acrylic-based gel had already decreased at baseline, whereas it took 6 hours to observe the same result in a tube with olefin-based gel. At 4°C, the reduction in serum concentration was considerably slower. For flecainide, the gel tube concentrations were stable at ambient temperature for 3 days, but decreased after 7 days in acrylic-based gel tubes. Amiodarone and desethylamiodarone stored in gel tubes at 4°C showed decrease in concentrations after 24 hours and 6 hours, respectively. CONCLUSIONS: Acrylic-based gel tubes should not be used for any of the tested drugs. Although olefin-based gel tubes may be used for anticoagulants and flecainide, it is advisable to prefer nongel tubes as a general precaution.

Pre- and post-diagnostic blood profiles of chlorinated persistent organic pollutants and metabolic markers in type 2 diabetes mellitus cases and controls; a pilot study.

OBJECTIVE: Several risk factors for type 2 diabetes mellitus (T2DM) are also associated with blood concentrations of persistent organic pollutants (POPs), and factors related to the disease may affect POP concentrations, and subsequent associations between POPs and T2DM. The purpose of this pilot study was to investigate the change in concentrations of lipids, hormones and POPs pre- and post-diagnosis in T2DM cases compared to healthy controls and their associations with T2DM. METHODS: We measured POPs, lipids, and thyroid and steroid hormones in plasma from 44 female cases collected prior to (pre-diagnostic) and following (post-diagnostic) T2DM diagnosis, and in 44 healthy female age-matched controls. We compared cross-sectional differences and longitudinal changes within and between matched cases and controls with t-tests and multivariable linear regression models. Associations between POP concentrations and T2DM were investigated using conditional logistic regression. RESULTS: Between the pre- and post-diagnostic measurement, cases developed more favorable lipid profiles and the longitudinal changes in lipid-normalized concentrations of non-dioxin-like polychlorinated biphenyls (PCBs), dioxin-like PCBs, beta-hexachlorocyclohexane (HCH), HCB, and 1,1,1-trichloro-2,2-bis(4-chlorophenyl) ethane (p,p'-DDE) differed significantly between cases and controls. The longitudinal changes in POPs were mainly driven by changes in bodyweight, total lipids and T2DM status. Cases had significantly higher pre-diagnostic concentrations of POPs and triglycerides, and lower concentrations of high-density lipoprotein cholesterol and free thyroxin than controls. Pre-diagnostic POP concentrations were not significantly associated with incident T2DM, whereas several post-diagnostic POP concentrations were significantly positively associated with prevalent T2DM. CONCLUSIONS: This pilot study suggests that factors related to T2DM affect blood concentrations of POPs and may partly explain the positive associations between POPs and T2DM.

Skeletal muscle mitochondria exhibit decreased pyruvate oxidation capacity and increased ROS emission during surgery-induced acute insulin resistance.

Development of acute insulin resistance represents a negative factor after surgery, but the underlying mechanisms are not fully understood. We investigated the postoperative changes in insulin sensitivity, mitochondrial function, enzyme activities, and release of reactive oxygen species (ROS) in skeletal muscle and liver in pigs on the 2nd postoperative day after major abdominal surgery. Peripheral and hepatic insulin sensitivity were assessed by D-[6,6-²H2]glucose infusion and hyperinsulinemic euglycemic step clamping. Surgical trauma elicited a decline in peripheral insulin sensitivity (∼34%, P<0.01), whereas hepatic insulin sensitivity remained unchanged. Intramyofibrillar (IFM) and subsarcolemma mitochondria (SSM) isolated from skeletal muscle showed a postoperative decline in ADP-stimulated respiration (V(ADP)) for pyruvate (∼61%, P<0.05, and ∼40%, P<0.001, respectively), whereas V(ADP) for glutamate and palmitoyl-L-carnitine (PC) was unchanged. Mitochondrial leak respiration with PC was increased in SSM (1.9-fold, P<0.05) and IFM (2.5-fold, P<0.05), indicating FFA-induced uncoupling. The activity of the pyruvate dehydrogenase complex (PDC) was reduced (∼32%, P<0.01) and positively correlated to the decline in peripheral insulin sensitivity (r=0.748, P<0.05). All other mitochondrial enzyme activities were unchanged. No changes in mitochondrial function in liver were observed. Mitochondrial H2O2 and O2·â» emission was measured spectrofluorometrically, and H2O2 was increased in SSM, IFM, and liver mitochondria (∼2.3-, ∼2.5-, and ∼2.3-fold, respectively, all P<0.05). We conclude that an impairment in skeletal muscle mitochondrial PDC activity and pyruvate oxidation capacity arises in the postoperative phase along with increased ROS emission, suggesting a link between mitochondrial function and development of acute postoperative insulin resistance.

Estimated GFR is biased by non-traditional cardiovascular risk factors.

BACKGROUND: Estimated glomerular filtration rate (eGFR) based on either cystatin C or creatinine performs similarly in estimating measured GFR, but associate differently with cardiovascular disease (CVD) and mortality. This could be due to confounding by non-GFR-related traits associated with cystatin C and creatinine levels. We investigated non-GFR-related associations between eGFR and two types of nontraditional risk factors for CVD and death: L-arginine/dimethylarginine metabolism and insulin resistance. METHODS: GFR was measured via iohexol clearance in a cross-sectional study of 1,624 middle-aged persons from the general population without CVD, diabetes or chronic kidney disease. The dimethylarginines were measured using liquid chromatography tandem mass spectrometry (LC-MSMS). Insulin resistance was determined by the homeostasis model assessment (HOMA-IR). RESULTS: Asymmetric dimethylarginine (ADMA), symmetric dimethylarginine (SDMA), the L-arginine/ADMA ratio and insulin resistance were associated with creatinine-based eGFR after accounting for measured GFR in multivariable adjusted analyses. The cystatin C-based eGFR showed a similar residual association with SDMA; an oppositely directed, borderline significant association with ADMA; and a stronger residual association with insulin resistance compared with eGFR based on creatinine. CONCLUSION: Both creatinine- and cystatin C-based eGFR are influenced by nontraditional risk factors, which may bias risk prediction by eGFR in longitudinal studies.

L-Ornithine phenylacetate reduces ammonia in pigs with acute liver failure through phenylacetylglycine formation: a novel ammonia-lowering pathway.

Glycine is an important ammoniagenic amino acid, which is increased in acute liver failure (ALF). We have previously shown that L-ornithine phenylacetate (OP) attenuates ammonia rise and intracranial pressure in pigs suffering from ALF but failed to demonstrate a stoichiometric relationship between change in plasma ammonia levels and excretion of phenylacetylglutamine in urine. The aim was to investigate the impact of OP treatment on the phenylacetylglycine pathway as an alternative and additional ammonia-lowering pathway. A well-validated and -characterized large porcine model of ALF (portacaval anastomosis, followed by hepatic artery ligation), which recapitulates the cardinal features of human ALF, was used. Twenty-four female pigs were randomized into three groups: (1) sham operated + vehicle, (2) ALF + vehicle, and (3) ALF + OP. There was a significant increase in arterial glycine concentration in ALF (P < 0.001 compared with sham), with a three-fold increase in glycine release into the systemic circulation from the kidney compared with the sham group. This increase was attenuated in both the blood and brain of the OP-treated animals (P < 0.001 and P < 0.05, respectively), and the attenuation was associated with renal removal of glycine through excretion of the conjugation product phenylacetylglycine in urine (ALF + vehicle: 1,060 ± 106 µmol/l; ALF + OP: 27,625 ± 2,670 µmol/l; P < 0.003). Data from this study provide solid evidence for the existence of a novel, additional pathway for ammonia removal in ALF, involving glycine production and removal, which is targeted by OP.

A sensitive method for the analysis of glutathione in porcine hepatocytes.

BACKGROUND: Reduced glutathione (γ-glutamylcysteinylglycine), GSH, is essential when protecting cells from oxidative stress and also an indicator of disease risk. Reported concentrations of GSH and its oxidized form, glutathione disulfide (GSSG), varies considerably, primarily due to the instability of GSH and various analytical methods. METHODS: We designed a sensitive method to analyze GSH and GSSG in porcine hepatocytes using liquid chromatography-tandem mass spectrometry (LC-MS/MS) after stabilization with N-ethylmaleimide (NEM). This method includes stable isotope labeled internal standards and simple synthesis of labeled GSSG which commercial sources rarely offer. GSH and GSSG were analyzed in porcine liver biopsies giving a reference interval based on a large number of samples (26 pigs; 3 parallels). RESULTS: The LC-MS/MS results revealed excellent linearity for both GSH and GSSG, with interday coefficient of variation (%CV) for GSH-NEM and GSSG <10 %. Accuracy for recovery tests was between 95.6% and 106.7% (n = 3) for GSH and between 92.3% and 107.7% (n = 3) for GSSG. The limits of quantification were 0.1 µM for GSH-NEM and 0.08 µM for GSSG. The mean concentration of GSH was 3.5 (95% CI = 1.5-8.1) mmol/liter and of GSSG 0.0023 (95% CI = 0.0003-0.019) mmol/liter. CONCLUSION: For the first time GSH and GSSG are analyzed in porcine hepatocytes by LC-MS/MS yielding a reference level of GSH and GSSG. The method is reproducible in any laboratory with LC-MS/MS service and will probably be applicable in all soft tissues and cell suspensions, essentially with no modification.

Longitudinal assessment of classic and 11-oxygenated androgen concentrations and their association with type 2 diabetes mellitus development: the Tromsø study.

AIM: We aimed to investigate changes in pre-diagnostic concentrations of classic and 11-oxygenated androgens in type 2 diabetes (T2DM) cases and healthy controls, associations between androgen concentrations and T2DM, and the potential for androgens to improve the prediction of T2DM when considered in combination with established risk factors. METHODS: Androgen concentrations were analysed in serum samples from 116 T2DM cases and 138 controls at 3, pre-diagnostic time-points: 1986/87 (T1), 1994/95 (T2), and 2001 (T3). Generalised estimating equations were used to longitudinally examine androgen concentrations, and logistic regression models were used to estimate the odds ratios (OR) of T2DM at each time-point. Logistic regression models were also used to calculate area under the receiver operating characteristics curve (AROC) from models including established risk factors alone (ERF model) and established risk factors plus each androgen, respectively, which were compared to identify improvements in predictive ability. RESULTS: For women, no significant associations were observed between any of the investigated androgens and T2DM after adjusting for confounders. For men, after adjusting for confounders, concentrations of all investigated 11-oxygenated androgens were higher in cases than controls at one or several time-points. We observed associations between T2DM and concentrations of 11-ketoandrostenedione (OR: 1.59) and 11-ketotestosterone (OR: 1.62) at T1; and 11-hydroxyandrostenedione (OR: 2.00), 11-hydroxytestosterone (OR: 1.76), 11-ketoandrostenedione (OR: 1.84), 11-ketotestosterone (OR: 1.78) and testosterone (OR: 0.45) at T3 in men. The addition of these androgens (including 11-hydroxytestosterone at T2) to the ERF model resulted in an improved ability to predict T2DM in men (AROC: 0.79-0.82). We did not observe significant differences in changes in androgen concentrations over time between cases and controls in either sex. CONCLUSION: Our results demonstrate that testosterone and 11-oxygenated androgens are associated with T2DM in men before diagnosis and may be potential biomarkers in T2DM risk assessment.

HOW SHOULD TRANEXAMIC ACID BE ADMINISTERED IN HEMORRHAGIC SHOCK? CONTINUOUS SERUM CONCENTRATION MEASUREMENTS IN A SWINE MODEL.

ABSTRACT: Background : Tranexamic acid (TXA) reduces mortality in trauma patients. Intramuscular (IM) administration could be advantageous in low-resource and military settings. Achieving the same serum concentration as intravenous (IV) administration is important to achieve equal mortality reduction. Therefore, we aimed to investigate whether dividing an IM dose of TXA between two injection sites and whether an increase in dose would lead to serum concentrations comparable to those achieved by IV administration. Methods : Norwegian landrace pigs (n = 29) from a course in hemostatic emergency surgery were given TXA 1 h after start of surgery. Blood samples were drawn at 0, 5, 10, 15, 20, 25, 35, 45, 60, and 85 min. The samples were centrifuged and serum TXA concentrations quantified with liquid chromatography-tandem mass spectrometry. The use of two injection sites was compared with distributing the dose on one injection site, and a dose of 15 mg/kg was compared with a dose of 30 mg/kg. All IM groups were compared with IV administration. Results : The groups were in a similar degree of shock. Increasing the IM dose from the standard of 15 mg/kg to 30 mg/kg resulted in significantly higher serum concentrations of TXA, comparable to those achieved by IV administration. Distributing the IM dose on two injection sites did not affect drug uptake, as shown by equal serum concentrations. Conclusions : For IM administration of TXA, 30 mg/kg should be the standard dose. With a short delay, IM administration will provide equal serum concentrations as IV administration, above what is considered necessary to inhibit fibrinolysis.

Longitudinal changes in vitamin D concentrations and the association with type 2 diabetes mellitus: the Tromsø Study.

AIM: We aimed to investigate the relationship between pre- and post-diagnostic 25-hydroxyvitamin D (25(OH)D) concentrations and type 2 diabetes (T2DM) over a period of 30 years in individuals who developed T2DM compared to healthy controls. METHODS: This case-control study included 254 participants with blood samples collected at five different time-points (T1-T5) between 1986 and 2016. Of the 254 participants, 116 were diagnosed with T2DM between T3 and T4, and were considered cases; the remaining 138 were controls. Linear mixed regression models were used to examine pre- and post-diagnostic changes in 25(OH)D concentrations, and logistic regression was used to examine associations between these concentrations and T2DM at each time-point. RESULTS: 25(OH)D concentrations at different time-points and the longitudinal change in concentrations differed between cases and controls, and by sex. For women, each 5-nmol/l increase in 25(OH)D concentrations was inversely associated with T2DM at T3 (odds-ratio, OR, 0.79), whereas for men, this same increase was positively associated with T2DM at T1 (OR 1.12). Cases experienced a significant decrease in pre-diagnostic 25(OH)D concentrations (p value < 0.01 for women, p value = 0.02 for men) and a significant increase in post-diagnostic 25(OH)D concentrations (p value < 0.01 for women, p value = 0.01 for men). As such, each 1-unit increase in month-specific z-score change between T1 and T3 was significantly inversely associated with T2DM (OR 0.51 for women, OR 0.52 for men), and each such increase between T3 and T5 was significantly positively associated with T2DM in women (OR 2.48). CONCLUSIONS: 25(OH)D concentrations seem to be affected by disease progression and type 2 diabetes diagnosis.

Nitric Oxide Precursors and Dimethylarginines as Risk Markers for Accelerated Measured GFR Decline in the General Population.

Introduction: Nitric oxide (NO) deficiency is associated with endothelial dysfunction, hypertension, atherosclerosis, and chronic kidney disease (CKD). Reduced NO bioavailability is hypothesized to play a vital role in kidney function impairment and CKD. We investigated the association of serum levels of endogenous inhibitors of NO, asymmetric dimethylarginine (ADMA) and symmetric dimethylarginine (SDMA), and precursors of NO, arginine, citrulline, and ornithine, with a decline in glomerular filtration rate (GFR) and new-onset CKD. Methods: In a prospective cohort study of 1407 healthy, middle-aged participants of Northern European origin in the Renal Iohexol Clearance Survey (RENIS), GFR was measured repeatedly with iohexol clearance during a median follow-up time of 11 years. GFR decline rates were analyzed using a linear mixed model, new-onset CKD (GFR < 60 ml/min per 1.73 m2) was analyzed with interval-censored Cox regression, and accelerated GFR decline (the 10% with the steepest GFR decline) was analyzed with logistic regression. Results: Higher SDMA was associated with slower annual GFR decline. Higher levels of citrulline and ornithine were associated with accelerated GFR decline (odds ratio [OR], 1.43; 95% confidence interval [CI] 1.16-1.76 per SD higher citrulline and OR 1.23; 95% CI 1.01 to 1.49 per SD higher ornithine). Higher citrulline was associated with new-onset CKD, with a hazard ratio of 1.33 (95% CI 1.07-1.66) per SD higher citrulline. Conclusions: Associations between NO precursors and the outcomes suggest that NO metabolism plays a significant role in the pathogenesis of age-related GFR decline and the development of CKD in middle-aged people.

Longitudinal changes in blood biomarkers and their ability to predict type 2 diabetes mellitus-The Tromsø study.

INTRODUCTION: Identification of individuals at high risk of developing type 2 diabetes mellitus (T2DM) is important for early prevention of the disease. Once T2DM is established, it is difficult to treat and is associated with cardiovascular complications and increased mortality. We aimed to describe pre- and post-diagnostic changes in blood biomarker concentrations over 30 years in individuals with and without T2DM, and to determine the predictive potential of pre-diagnostic blood biomarkers. METHODS: This nested case-control study included 234 participants in the Tromsø Study who gave blood samples at five time points between 1986 and 2016: 130 did not develop T2DM and were used as controls; 104 developed T2DM after the third time point and were included as cases. After stratifying by sex, we investigated changes in pre- and post-diagnostic concentrations of lipids, thyroid hormones, HbA1c , glucose and gamma-glutamyltransferase (GGT) using linear mixed models. We used logistic regression models and area under the receiver operating characteristic curve (AROC) to assess associations between blood biomarker concentrations and T2DM, as well as the predictive ability of blood biomarkers. RESULTS: Cases and controls experienced different longitudinal changes in lipids, free T3 , HbA1c , glucose, and GGT. The combination of selected blood biomarker concentrations and basic clinical information displayed excellent (AROC 0.78-0.95) predictive ability at all pre-diagnostic time points. A prediction model that included HDL (for women), HbA1c , GGT, and basic clinical information demonstrated the strongest discrimination 7 years before diagnosis (AROC 0.95 for women, 0.85 for men). CONCLUSION: There were clear differences in blood biomarker concentrations between cases and controls throughout the study, and several blood biomarkers were associated with T2DM. Selected blood biomarkers (lipids, HbA1c , GGT) in combination with BMI, physical activity, elevated blood pressure, and family history of T2DM had excellent predictive ability 1-7 years before T2DM diagnosis and acceptable predictive ability up to 15 years before diagnosis.

Pharmacodynamic properties for inhibition of cAMP- and cGMP elimination by pentoxifylline remain unaltered in vitro during hypothermia.

BACKGROUND: Rewarming from hypothermia is associated with severe complications, one of which is hypothermia-induced cardiac dysfunction. This condition is characterized by decreased cardiac output accompanied by increased total peripheral resistance. This contributes to mortality rate approaching 40%. Despite this, no pharmacological interventions are recommended for these patients below 30 °C. Raising the intracellular levels of cAMP and/or cGMP, through PDE3- and PDE5-inhibitors respectively, have showed the ability to alleviate hypothermia-induced cardiac dysfunction in vivo. Drugs that raise levels of both cAMP and cGMP could therefore prove beneficial in patients suffering from hypothermia-induced cardiac dysfunction. METHODS: The unselective PDE-inhibitor pentoxifylline was investigated to determine its ability to reach the intracellular space, inhibit PDE3 and PDE5 and inhibit cellular efflux of cAMP and cGMP at temperatures 37, 34, 30, 28, 24 and 20 °C. Recombinant human PDE-enzymes and human erythrocytes were used in the experiments. IC50-values were calculated at all temperatures to determine temperature-dependent changes. RESULTS: At 20 °C, the IC50-value for PDE5-mediated enzymatic breakdown of cGMP was significantly increased compared to normothermia (IC50: 39.4 µM ± 10.9 µM vs. 7.70 µM ± 0.265 µM, p-value = 0.011). No other significant changes in IC50-values were observed during hypothermia. CONCLUSIONS: This study shows that pentoxifylline has minimal temperature-dependent pharmacodynamic changes, and that it can inhibit elimination of both cAMP and cGMP at low temperatures. This can potentially be effective treatment of hypothermia-induced cardiac dysfunction. TRIAL REGISTRATION: Not applicable.

Treatment of Cardiovascular Dysfunction with PDE3-Inhibitors in Moderate and Severe Hypothermia-Effects on Cellular Elimination of Cyclic Adenosine Monophosphate and Cyclic Guanosine Monophosphate.

Introduction: Rewarming from accidental hypothermia is often complicated by hypothermia-induced cardiovascular dysfunction, which could lead to shock. Current guidelines do not recommend any pharmacological treatment at core temperatures below 30°C, due to lack of knowledge. However, previous in vivo studies have shown promising results when using phosphodiesterase 3 (PDE3) inhibitors, which possess the combined effects of supporting cardiac function and alleviating the peripheral vascular resistance through changes in cyclic nucleotide levels. This study therefore aims to investigate whether PDE3 inhibitors milrinone, amrinone, and levosimendan are able to modulate cyclic nucleotide regulation in hypothermic settings. Materials and methods: The effect of PDE3 inhibitors were studied by using recombinant phosphodiesterase enzymes and inverted erythrocyte membranes at six different temperatures-37°C, 34°C, 32°C, 28°C, 24°C, and 20°C- in order to evaluate the degree of enzymatic degradation, as well as measuring cellular efflux of both cAMP and cGMP. The resulting dose-response curves at every temperature were used to calculate IC50 and Ki values. Results: Milrinone IC50 and Ki values for cGMP efflux were significantly lower at 24°C (IC50: 8.62 ± 2.69 µM) and 20°C (IC50: 7.35 ± 3.51 µM), compared to 37°C (IC50: 22.84 ± 1.52 µM). There were no significant changes in IC50 and Ki values for enzymatic breakdown of cAMP and cGMP. Conclusion: Milrinone, amrinone and levosimendan, were all able to suppress enzymatic degradation and inhibit extrusion of cGMP and cAMP below 30°C. Our results show that these drugs have preserved effect on their target molecules during hypothermia, indicating that they could provide an important treatment option for hypothermia-induced cardiac dysfunction.

Comprehensive UHPLC-HR-MSE screening workflow optimized for use in routine laboratory medicine: Four workflows in one analytical method.

A comprehensive HR-MS screening can be used to identify thousands of drugs from a single analysis, which makes it a valuable tool for broad-scope component-resolved toxicological analysis. However, it is common practice in clinical toxicology to perform restricted data analysis to avoid examining and/or reporting data not requested for examination. In this study, a HR-MS screening workflow was developed to allow a comprehensive toxicological evaluation, but also restricted and levelled data analysis to fit in a clinical setting. Following precipitation and reconstitution, samples were injected on an UHPLC-HR-MS and data were analyzed with the data processing software UNIFI. Analytical validation of 38 selected drugs of abuse (DoA), included determination of matrix effect, recovery, process efficiency, and limit of identification (LOI). The method was tested on 49 authentic samples and matrix-matched ranges of calibrators for 95 drugs. The LOI ranged from 0.3 to 1426.7 ng mL-1 for most analytes which was within expected concentration range for authentic samples with THC-COOH (>1722.0 ng mL-1) and morphine (1426.7 ng mL-1) as notable exceptions. Four individual screening workflows were developed: 1) a targeted workflow to serve as orthogonal identification of the 38 selected DOAs from another in-house method, 2) a general toxicology workflow, 3) an extended toxicology workflow including new psychoactive substances (NPS), and 4) a workflow for NPS based on the online HighResNPS library. Our study presents a comprehensive LC-HR-MS toxicology screening method optimized for laboratory medicine. The workflow allows for levelled data reviewing when requested without compromising the ability to perform full toxicological analyses.

Transfer of triptans into human breast milk and estimation of infant drug exposure through breastfeeding.

Clinical data on the transfer of triptans into human breast milk remain scarce. In a lactation study including 19 breastfeeding women with migraine, we examined the excretion of six different triptans into milk. Following intake of a single dose, each participant collected seven breast milk samples at predefined intervals up to 24 hours after dose. Triptan concentrations in milk were measured using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Infant drug exposure was estimated by calculating the relative infant dose (RID). Twenty-two breast milk sample sets were obtained for sumatriptan (n = 8), rizatriptan (n = 5), zolmitriptan (n = 4), eletriptan (n = 3), almotriptan (n = 1) and naratriptan (n = 1). Based on the average concentration in milk throughout the day, estimated mean RIDs (with range in parenthesis) were as follows: eletriptan 0.6% (0.3%-0.8%), sumatriptan 0.7% (0.2%-1.8%), rizatriptan 0.9% (0.3%-1.4%), almotriptan 1.8% (-), zolmitriptan 2.1% (0.7%-5.3%) and naratriptan 5.0% (-). Infant drug exposure through breastfeeding appears to be low and indicates that use of the triptans in this study is compatible with breastfeeding. Naratriptan may not be first choice in breastfeeding mothers initiating triptans during the neonatal period.

Intramuscular uptake of tranexamic acid during haemorrhagic shock in a swine model.

BACKGROUND: Tranexamic acid (TXA) reduce mortality in bleeding trauma patients, with greater effect if administered early. Serum concentrations above 10 µg/mL are considered sufficient to inhibit fibrinolysis. Normally administered intravenously (i.v.), TXA can also be administered intramuscularly (i.m.). This could be advantageous in low resource and military settings, if sufficient serum concentrations can be reached in shocked patients with reduced muscular blood perfusion. Accordingly, we aimed to: (1) Determine the impact of shock on the pharmacokinetics of i.m. TXA, and (2) Compare the pharmacokinetics of i.v. versus i.m. TXA in ongoing shock. MATERIALS AND METHODS: In a prospective experimental study, N = 18 Norwegian landrace pigs (40-50 kg), utilised in a surgical course in haemostatic emergency surgery, were subjected to various abdominal and thoracic trauma. After 1 h of surgery the animals were given 15 mg/kg TXA either i.v. or i.m. A control group without injury, or surgery, received intramuscular TXA. Blood samples were drawn at 0, 5, 15, 25, 35, 45, 60 and 85 min. The samples were centrifuged and analysed with liquid chromatography-tandem mass spectrometry (LC-MS/MS) for TXA serum-concentrations. RESULTS: In shocked pigs, i.m. administration resulted in a mean maximum serum concentration (Cmax) of 20.9 µg/mL, and i.v. administration a Cmax of 48.1 µg/mL. Cmax occurred 15 min after i.m. administration and 5 min after i.v. administration. In non-shocked swine, i.m. administration resulted in a Cmax of 36.9 µg/mL after 15 min. In all groups, mean TXA serum concentrations stayed above 10 µg/mL from administration to end of experiments. CONCLUSIONS: I.m. administration of TXA in shocked pigs provides serum concentrations associated with inhibition of fibrinolysis. It may be an alternative to i.v. and intraosseous administration during stabilisation and transport of trauma patients to advanced medical care.

Treatment of Cardiovascular Dysfunction With PDE5-Inhibitors - Temperature Dependent Effects on Transport and Metabolism of cAMP and cGMP.

INTRODUCTION: Cardiovascular dysfunction is a potentially lethal complication of hypothermia. Due to a knowledge gap, pharmacological interventions are not recommended at core temperatures below 30°C. Yet, further cooling is induced in surgical procedures and survival of accidental hypothermia is reported after rewarming from below 15°C, advocating a need for evidence-based treatment guidelines. In vivo studies have proposed vasodilation and afterload reduction through arteriole smooth muscle cGMP-elevation as a favorable strategy to prevent cardiovascular dysfunction in hypothermia. Further development of treatment guidelines demand information about temperature-dependent changes in pharmacological effects of clinically relevant vasodilators. MATERIALS AND METHODS: Human phosphodiesterase-enzymes and inverted erythrocytes were utilized to evaluate how vasodilators sildenafil and vardenafil affected cellular efflux and enzymatic breakdown of cAMP and cGMP, at 37°C, 34°C, 32°C, 28°C, 24°C, and 20°C. The ability of both drugs to reach their cytosolic site of action was assessed at the same temperatures. IC50- and K i -values were calculated from dose-response curves at all temperatures, to evaluate temperature-dependent effects of both drugs. RESULTS: Both drugs were able to reach the intracellular space at all hypothermic temperatures, with no reduction compared to normothermia. Sildenafil IC50 and K i -values increased during hypothermia for enzymatic breakdown of both cAMP (IC50: 122 ± 18.9 µM at 37°C vs. 269 ± 14.7 µM at 20°C, p < 0.05) and cGMP (IC50: 0.009 ± 0.000 µM at 37°C vs. 0.024 ± 0.004 µM at 32°C, p < 0.05), while no significant changes were detected for vardenafil. Neither of the drugs showed significant hypothermia-induced changes in IC50 and K i- values for inhibition of cellular cAMP and cGMP efflux. CONCLUSION: Sildenafil and particularly vardenafil were ableto inhibit elimination of cGMP down to 20°C. As the cellular effects of these drugs can cause afterload reduction, they show potential in treating cardiovascular dysfunction during hypothermia. As in normothermia, both drugs showed higher selectivity for inhibition of cGMP-elimination than cAMP-elimination at low core temperatures, indicating that risk for cardiotoxic side effects is not increased by hypothermia.