Correction to: The MINDMAP project: mental well-being in urban environments : Design and first results of a survey on healthcare planning policies, strategies and programmes that address mental health promotion and mental disorder prevention for older people in Europe.

Correction to: Z Gerontol Geriat 2017 https://doi.org/10.1007/s00391-017-1290-7 The article "The MINDMAP project: mental well-being in urban environments. Design and first results of a survey on healthcare planning policies, strategies and programmes that address mental health promotion and mental disorder prevention for older people in …The original article was corrected.

The MINDMAP project: mental well-being in urban environments : Design and first results of a survey on healthcare planning policies, strategies and programmes that address mental health promotion and mental disorder prevention for older people in Europe.

BACKGROUND: The MINDMAP consortium (2016-2019) aims to identify opportunities provided by the urban environment for the promotion of mental well-being and functioning of older people in Europe by bringing together European cities with urban longitudinal ageing studies: GLOBE, HAPIEE, HUNT, LASA, LUCAS, RECORD, Rotterdam Study, Turin Study. A survey on mental healthcare planning policies and programmes dedicated to older persons covering the range from health promotion to need of nursing care was performed for profound data interpretation in Amsterdam, Eindhoven, Hamburg, Helsinki, Kaunas, Krakow, London, Nord-Trøndelag, Paris, Prague, Rotterdam and Turin. OBJECTIVES: To collect detailed information on healthcare planning policies and programmes across these European cities to evaluate variations and to delineate recommendations for sciences, policies and planners using experience from evidence-based practice feedback from the MINDMAP cities. MATERIALS AND METHODS: The MINDMAP partners identified experts in the 12 cities with the best background knowledge of the mental health sector. After pretesting, semi-structured telephone interviews (1-2 h) were performed always by the same person. A structured evaluation matrix based on the geriatric functioning continuum and the World Health Organization (WHO) Public Health Framework for Healthy Ageing was applied. RESULTS: A complete survey (12 out of 12) was performed reporting on 41 policies and 280 programmes on the city level. It appeared from extensive analyses that the focus on older citizens, specific target groups, and multidimensional programmes could be intensified. CONCLUSION: There is a broad variety to cope with the challenges of ageing in health, and to address both physical and mental capacities in older individuals and their dynamic interactions in urban environments.

Different glycosylation pattern of human IgG1 and IgG3 antibodies isolated from transiently as well as permanently transfected cell lines.

The effector functions of IgG depend on the presence of carbohydrates attached to asparagine 297 in the Fc-portion. In this report, glycosylation profiles of recombinant wild-type and mutant IgG1 and IgG3 antibodies produced from three cell lines were analysed using LC-ESI-Orbitrap. Clear differences were detected between IgG1 and IgG3 glycoforms, where IgG1 generally contained fucosylated glycoforms, whilst IgG3 mainly were non-fucosylated. When using NS-0 and J558L cells for permanent transfection, IgG1 wt glycoforms differed between the two cell lines, whilst IgG3 wt glycoforms did not. Transiently transfected HEK 293E cells were used to produce IgG1 and IgG3 wt and mutants, affecting complement activation. Cell supernatants were harvested at early and late time points and analysed separately. IgGs harvested late showed simpler and less developed glycosylation structure compared to those harvested early. The IgG harvested early was slightly more effective in complement activation than those harvested late, whilst the antibody-dependent cell-mediated cytotoxicity was unaltered. Generally, the glycosylation pattern of the mutants tested, including a hinge truncate mutant of IgG3, did not differ significantly from the wild-type IgGs. The striking difference in glycosylation pattern of IgG1 compared to IgG3 therefore appears not to be due to the long hinge region of IgG3 (62 amino acids) relative to the IgG1 hinge region (15 amino acids). Furthermore, mutation variants at or near the C1q binding site showed similar glycosylation structure and difference in their complement activation activity observed earlier is thus most likely due to differences in protein structure only.

Lysosomes as a possible target of enniatin B-induced toxicity in Caco-2 cells.

Enniatins are cyclic hexadepsipeptidic mycotoxins with ionophoric, antibiotic, and insecticidal activity. Enniatin B (EnnB), the most important analogue, is produced by many Fusarium species and is a common contaminant in grain-based foods. The compound's cytotoxic potential has been shown in different experiments; however, the mode of action has not been detailed so far. In the present study, several mutually confirmative experiments have been performed indicating that EnnB-initiated cytotoxicity could be connected with lysosomal membrane permeabilization (LMP). Lysosomal functionality, as assessed by the Neutral Red assay, was already affected after 3 h of toxin exposure. After 24 h, cell proliferation was decreased, and there was indication for a cell cycle arrest in the G(2)/M phase leading to the initiation of apoptosis or necrosis. Intracellular ROS-production was observed. However, antioxidants did not alter the observed EnnB-induced loss of lysosomal functionality leading to the conclusion that ROS was not an initial factor but one produced later in the event cascade. The collected data suggested that lysosomal destabilization is an upstream event in EnnB-initiated cytotoxicity followed by a certain extent of translocation of cathepsins into the cytosol, which was observed using immunological and proteomic methods. It appeared that cell death induced by EnnB was delayed and occurred not as a massive lysosomal breakdown but was probably progressing and leading to partial and selective LMP, starting a nonapoptotic cell death pathway with morphological features that had been previously considered as necrotic. The molecular mechanism of EnnB-triggered lysosomal destabilization, and the cellular processes leading to mitochondrial permeabilization and cell death are still unknown. They may, however, be connected to the compound's ionophoric properties.

Brain tissue oxygen monitoring to assess reperfusion after intra-arterial treatment of aneurysmal subarachnoid hemorrhage-induced cerebral vasospasm: a retrospective study.

BACKGROUND AND PURPOSE: Cerebral vasospasm resistant to medical management frequently requires intra-arterial spasmolysis. Angiographic resolution of vasospasm does not provide physiologic data on the adequacy of reperfusion. We recorded pre- and postspasmolysis PbO(2) data in the endovascular suite to determine whether this physiologic parameter could be used to determine when successful reperfusion was established. MATERIALS AND METHODS: Eight patients with 10 Licox monitors and cerebral vasospasm underwent intra-arterial spasmolysis. Pre- and postspasmolytic PbO(2) was recorded for comparison. Other physiologic parameters, such as CPP, ICP, SaO(2), and Fio(2), were also recorded. RESULTS: Mean prespasmolysis PbO(2) recordings were 35.2 and 27.3 for the mild-to-moderate and moderate-to-severe vasospasm group, respectively. Mean postspasmolysis PbO(2) increased to 40.3 and 38.4, respectively, which was statistically significant (P < .05) for both groups. In 100% of instances in the moderate-to-severe group and 83% of instances in mild-to-moderate group, the mean PbO(2) increased after spasmolysis and correlated with improvement in angiographic vasospasm. Other physiologic parameters, such as CPP, ICP, SaO(2), and Fio(2), did not show any statistically significant difference before and after spasmolysis. CONCLUSIONS: PbO(2) monitoring provides the interventionalist with an objective physiologic parameter to determine adequate spasmolysis. Further investigation is needed to establish target PbO(2) rates indicative of adequate reperfusion, which can be used in the endovascular suite.

ORF 2 from the Bacillus cereus linear plasmid pBClin15 encodes a DNA binding protein.

AIMS: To isolate and identify DNA-binding protein(s) with affinity for the mobile chromosomal repeat element bcr1 in Bacillus cereus group bacteria. METHODS AND RESULTS: A biotinylated bcr1 element was immobilized to streptavidin-coated magnetic beads and used to pull out a 20 kDa DNA-binding protein from a whole cell protein extract of B. cereus ATCC 14579. The protein was identified as the product of ORF 2 encoded by the bacteriophage-related autonomously replicating linear genetic element pBClin15 carried by the strain. DNA binding was not bcr1-specific. By Northern blotting ORF 2 was co-transcribed with ORF 1, and also in certain instances with ORF 3 by transcriptional readthrough of the terminator located between ORF 2 and ORF 3. CONCLUSIONS: ORF 2 from pBClin15 encodes a DNA-binding protein. ORF 2 is co-transcribed with its upstream gene ORF 1, and in a subset of the transcripts also with the downstream gene ORF 3 through alternative transcription termination. SIGNIFICANCE AND IMPACT OF THE STUDY: The B. cereus group contains bacterial species of medical and economic importance. Bacteriophages or phage-encoded proteins from these bacteria have been suggested as potential therapeutic agents. Understanding the biology of bacteriophage-related genetic elements through functional characterization of their genes is of high relevance.

Thiopentone and methohexital, but not pentobarbitone, reduce early focal cerebral ischemic injury in rats.

PURPOSE: Although barbiturates are considered to be cerebral protectants, little is known regarding the relative efficacy of different barbiturates to reduce ischemic brain injury. In a model of middle cerebral artery occlusion (MCAo), we compared the relative effects of 1.0 and 0.4 burst-suppression doses of thiopentone, methohexital, and pentobarbitone on cerebral infarct. METHODS: During isoflurane anesthesia, MCAo was achieved via a temporal craniotomy. Thirty minutes before MCAo the rats were randomized to receive one of the following which was maintained throughout the study. Halothane (n=20)-1.2 MAC halothane, thiopentone (n=20), methohexital (n=20), or pentobarbitone (n=20). The first ten animals in each barbiturate group received the respective barbiturate in a dose sufficient to maintain burst-suppression of the electroencephalogram (3-5 bursts x min(-1)). The subsequent ten animals in each barbiturate group received 40% of the burst-suppression dose. After 180 min of MCAo and 120 min of reperfusion, cerebral injury was assessed. RESULTS: For the burst-suppression animals, injury volume (mm3, mean +/- SD) was less in the thiopentone group (88 +/- 14) than the halothane (133 +/- 17), methohexital (126 +/- 19), or pentobarbitone (130 +/- 17) groups (P <0.05). For 0.4 burst-suppression animals, injury volume was less for the methohexital group (70 +/- 22) than the halothane (124 +/- 24), thiopentone (118 +/- 15), or pentobarbitone (121 +/- 20) groups (P <0.05). CONCLUSIONS: These data are inconsistent with the longstanding assumption that electrophysiologically comparable doses of the various classes of barbiturates have equivalent protective efficacy. They in turn suggest that mechanisms other than, or at least in addition to, metabolic suppression may contribute to the protective effect of barbiturates.

Sirolimus, but not the structurally related RAD (everolimus), enhances the negative effects of cyclosporine on mitochondrial metabolism in the rat brain.

Clinical studies have shown enhancement of cyclosporine toxicity when co-administered with the immunosuppressant sirolimus. We evaluated the biochemical mechanisms underlying the sirolimus/cyclosporine interaction on rat brain metabolism using magnetic resonance spectroscopy (MRS) and compared the effects of sirolimus with those of the structurally related RAD. Two-week-old rats (25 g) were allocated to the following treatment groups (all n=6): I. control, II. cyclosporine (10 mg kg(-1) d(-1)), III. sirolimus (3 mg kg(-1) d(-1)), IV. RAD (3 mg kg(-1) d(-1)), V. cyclosporine+sirolimus and VI. cyclosporine+RAD. Drugs were administered by oral gavage for 6 days. Twelve hours after the last dose, metabolic changes were assessed in brain tissue extracts using multinuclear MRS. Cyclosporine significantly inhibited mitochondrial glucose metabolism (glutamate: 78+/-6% of control; GABA: 67+/-12%; NAD(+): 76+/-3%; P<0.05), but increased lactate production. Sirolimus and RAD inhibited cytosolic glucose metabolism via lactate production (sirolimus: 81+/-3% of control, RAD: 69+/-2%; P<0.02). Sirolimus enhanced cyclosporine-induced inhibition of mitochondrial glucose metabolism (glutamate: 60+/-4%; GABA: 59+/-8%; NAD(+): 45+/-5%; P<0.02 versus cyclosporine alone). Lactate production was significantly reduced. In contrast, RAD antagonized the effects of cyclosporine (glutamate, GABA, and NAD(+), not significantly different from controls). The results can partially be explained by pharmacokinetic interactions: co-administration increased the distribution of cyclosporine and sirolimus into brain tissue, while co-administration with RAD decreased cyclosporine brain tissue concentrations. In addition RAD, but not sirolimus, distributed into brain mitochondria. The combination of cyclosporine/RAD compares favourably to cyclosporine/sirolimus in regards to their effects on brain high-energy metabolism and tissue distribution in the rat.

Metabolism of sirolimus and its derivative everolimus by cytochrome P450 3A4: insights from docking, molecular dynamics, and quantum chemical calculations.

A combination of quantum chemical calculations and molecular simulations (DOCKing and molecular dynamics) is used to investigate the metabolism of sirolimus (rapamycin) and its derivative everolimus (SDZ-RAD) by cytochrome P450 3A4. Both molecules are drugs with high immunosuppressive activity. Our calculations yield qualitative predictions of the regiospecificities of the hydroxylations and O-dealkylations occurring in these two substrates which are in good agreement with recent experimental results. An analysis of the modeled enzyme-substrate interactions allows us to rationalize the reduced metabolic activity of the larger substrate everolimus compared to sirolimus. Moreover, our simulations suggest that hydrogen donor functionalities close to the metabolic site are important for anchoring the substrate at the active center of the enzyme. In particular, we predict that replacing one hydroxyl group by a fluorine atom should considerably suppress the major metabolic reaction in sirolimus, 39-O-demethylation.

Pharmacokinetics of SDZ RAD and cyclosporin including their metabolites in seven kidney graft patients after the first dose of SDZ RAD.

AIMS: The aim of the study was to investigate the pharmacokinetics and metabolism of the new immunosuppressant SDZ RAD during concomitant therapy with cyclosporin in stable renal transplant patients. Furthermore, we studied the influence of SDZ RAD on the pharmacokinetics of cyclosporin at steady state levels. METHODS: SDZ RAD was administered orally in different doses (0.25-15 mg day-1) to seven patients, who were on standard cyclosporin-based immunosuppression. The blood concentrations of both drugs including their main groups of metabolites were measured simultaneously by LC/electrospray-mass spectrometry. RESULTS: The mean area under the blood concentration-time curve to 12 h (AUC(0,12 h)) was 4244 +/- 1311 microg l-1 h for cyclosporin before SDZ RAD treatment and 4683 +/- 1174 microg l-1 h (P = 0.106) on the day of SDZ RAD treatment (95% CI for difference -126, 1003). On both study days Cmax, and tmax of cyclosporin were not significantly different. The metabolite pattern of cyclosporin did not change. The pharmacokinetic data of SDZ RAD dose-normalized to 1 mg SDZ RAD were as follows: AUC(0,24 h): 35.4 +/- 13.1 microg l-1 h, Cmax: 7.9 +/- 2.7 microg l-1 and tmax: 1.5 +/- 0.9 h. The metabolites of SDZ RAD found in blood were hydroxy-SDZ RAD, dihydroxy-SDZ RAD, demethyl-SDZ RAD, and a ring-opened form of SDZ RAD. CONCLUSIONS: A single dose of SDZ RAD did not influence significantly the pharmacokinetics of cyclosporin. The most important metabolite of SDZ RAD was the hydroxy-SDZ RAD, its AUC(0,24 h) being nearly half that of the parent compound SDZ RAD.

Automated, fast and sensitive quantification of drugs in blood by liquid chromatography-mass spectrometry with on-line extraction: immunosuppressants.

We developed a universal LC-mass spectrometry assay with automated online extraction (LC/LC-MS) to quantify the immunosuppressants cyclosporine, tacrolimus, sirolimus and SDZ-RAD alone or in combination in whole blood. After protein precipitation, samples were loaded on a C18 extraction column, were washed and, after activation of the column-switching valve, were backflushed onto the C8 analytical column. [M+Na]+ ions were detected in the selected ion mode. For tacrolimus, sirolimus and SDZ-RAD, the assay was linear from 0.25 to 100 microg/l and for cyclosporine from 7.5 to 1250 microg/l (all r2>0.99). Analytical recovery was >85% and, in general, inter-day, intra-day variability for precision and accuracy were <10%.

In vitro evaluation of the disposition of A novel cysteine protease inhibitor.

K11777 (N-methyl-piperazine-Phe-homoPhe-vinylsulfone-phenyl) is a potent, irreversible cysteine protease inhibitor. Its therapeutic targets are cruzain, a cysteine protease of the protozoan parasite Trypanosoma cruzi, and cathepsins B and L, which are associated with cancer progression. We evaluated the metabolism of K11777 by human liver microsomes, isolated cytochrome P450 (CYP) enzymes, and flavin-containing monooxygenase 3 (FMO3) in vitro. K11777 was metabolized by human liver microsomes to three major metabolites: N-oxide K11777 (apparent K(m) = 14.0 +/- 4.5 microM and apparent V(max) = 3460 +/- 3190 pmol. mg(-1). min(-1), n = 4), beta-hydroxy-homoPhe K11777 (K(m) = 16.8 +/- 3.5 microM and V(max) = 1260 +/- 1090 pmol. mg(-1). min(-1), n = 4), and N-desmethyl K11777 (K(m) = 18.3 +/- 7.0 microM and V(max) = 2070 +/- 1830 pmol. mg(-1). min(-1), n = 4). All three K11777 metabolites were formed by isolated CYP3A and their formation by human liver microsomes was inhibited by the CYP3A inhibitor cyclosporine (50 microM, 54-62% inhibition) and antibodies against human CYP3A4/5 (100 microg of antibodies/100 microg microsomal protein, 55-68% inhibition). CYP2D6 metabolized K11777 to its N-desmethyl metabolite with an apparent K(m) (9.2 +/- 1.4 microM) lower than for CYP3A4 (25.0 +/- 4.0 microM) and human liver microsomes. The apparent K(m) for N-oxide K11777 formation by cDNA-expressed FMO3 was 109 +/- 11 microM. Based on the intrinsic formation clearances and the results of inhibition experiments (CYP2D6, 50 microM bufuralol; FMO3 mediated, 100 mM methionine) using human liver microsomes, it was estimated that CYP3A contributes to >80% of K11777 metabolite formation. K11777 was a potent (IC(50) = 0.06 microM) and efficacious (maximum inhibition 85%) NADPH-dependent inhibitor of human CYP3A4 mediated 6'beta-hydroxy lovastatin formation, suggesting that K11777 is not only a substrate but also a mechanism-based inhibitor of CYP3A4.

Lactonization is the critical first step in the disposition of the 3-hydroxy-3-methylglutaryl-CoA reductase inhibitor atorvastatin.

In an in vitro study, we compared the cytochrome P450 (CYP)-dependent metabolism and drug interactions of the acid and lactone forms of the 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase inhibitor atorvastatin. Metabolism of atorvastatin acid and lactone by human liver microsomes resulted in para-hydroxy and ortho-hydroxy metabolites. Both substrates were metabolized mainly by CYP3A4 and CYP3A5. Atorvastatin lactone had a significantly higher affinity to CYP3A4 than the acid (K(m): para-hydroxy atorvastatin, 25.6 +/- 5.0 microM; para-hydroxy atorvastatin lactone, 1.4 +/- 0.2 microM; ortho-hydroxy atorvastatin, 29.7 +/- 9.4 microM; and ortho-hydroxy atorvastatin lactone, 3.9 +/- 0.2 microM). Compared with atorvastatin acid, CYP-dependent metabolism of atorvastatin lactone to its para-hydroxy metabolite was 83-fold higher [formation CL(int) (V(max)/K(m)): lactone 2949 +/- 3511 versus acid 35.5 +/- 48.1 microl. min(-1). mg(-1)] and to its ortho-hydroxy metabolite was 20-fold higher (CL(int): lactone 923 +/- 965 versus acid 45.8 +/- 59. 1 microl. min(-1). mg(-1)). Atorvastatin lactone inhibited the metabolism of atorvastatin acid by human liver microsomes with an inhibition constant (K(i)) of 0.9 microM while the K(i) for inhibition of atorvastatin by atorvastatin lactone was 90 microM. Binding free energy calculations of atorvastatin acid and atorvastatin lactone complexed with CYP3A4 revealed that the smaller desolvation energy of the neutral lactone compared with the anionic acid is the dominant contribution to the higher binding affinity of the lactone rather than an entropy advantage. Because atorvastatin lactone has a significantly higher metabolic clearance and the lactone is a strong inhibitor of atorvastatin acid metabolism, it can be expected that metabolism of the lactone is the relevant pathway for atorvastatin elimination and drug interactions. We hypothesize that most of the open acid metabolites present in human plasma are generated by interconversion of lactone metabolites.

Tissue distribution and clinical monitoring of the novel macrolide immunosuppressant SDZ-RAD and its metabolites in monkey lung transplant recipients: interaction with cyclosporine.

We report the tissue distribution and clinical monitoring of the novel macrolide immunosuppressant SDZ-RAD ¿40-O-(2-hydroxyethyl)-rapamycin and its metabolites in monkey lung transplant recipients as well as its interaction with cyclosporine as the Neoral formulation. After left unilateral lung transplantation, cynomolgus monkeys received by oral administration either 1) 1.5 mg/kg/day SDZ-RAD (n = 4); 2) 100 mg/kg/day cyclosporine (n = 4); 3) 0.3 mg/kg/day SDZ-RAD + 100 mg/kg/day cyclosporine (n = 6); 4) 1.5 mg/kg/day SDZ-RAD + 50 mg/kg/day cyclosporine (n = 5); or 5) SDZ-RAD and cyclosporine doses adjusted according to trough blood concentration measurements (n = 6). At the end of the observation period (usually 29 days after transplantation), and 24 h after the last doses, tissue samples were collected and analyzed with HPLC/mass spectrometry. Gall bladder, pancreas, the transplant lung, cerebellum, kidneys, and spleen had the highest SDZ-RAD concentrations. Coadministration of cyclosporine increased SDZ-RAD concentrations in most tissues as well as tissue-to-blood distribution coefficients. In contrast, SDZ-RAD had only a small effect on cyclosporine blood and tissue concentrations. Rejection in lung grafts in monkeys treated with either of the cyclosporine/SDZ-RAD combinations was significantly less than in the monotherapy groups (P <.002). Histological rejection scores were inversely correlated with SDZ-RAD concentrations in blood (r = -0. 68; P <.001; n = 24), lymph nodes (P = -0.58; P <.003; n = 24), thymus (r = -0.63; P <.001; n = 23) and transplant lung tissue (r = -0.58; P <.003; n = 24). We conclude that, in addition to the synergistic pharmacodynamic interaction, a pharmacokinetic interaction resulting in higher SDZ-RAD tissue concentrations contributed to the significantly better immunosuppressive efficacy when both drugs were combined compared with monotherapy.

Small intestinal metabolism of the 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitor lovastatin and comparison with pravastatin.

We compared the intestinal metabolism of the structurally related 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors lovastatin and pravastatin in vitro. Human small intestinal microsomes metabolized lovastatin to its major metabolites 6'beta-hydroxy (apparent K(m) = 11.2 +/- 3.3 microM) and 6'-exomethylene (apparent K(m) = 22.7 +/- 9.0 microM) lovastatin. The apparent K(m) values were similar for lovastatin metabolism by human liver microsomes. 6'beta-Hydroxylovastatin formation by pig small intestinal microsomes was inhibited with the following inhibition K(i) values: cyclosporine, 3.3 +/- 1.2 microM; ketoconazole, 0.4 +/- 0.1 microM; and troleandomycin, 0.8 +/- 0.9 microM. K(i) values for 6'-exomethylene lovastatin were similar. Incubation of pravastatin with human small intestinal microsomes resulted in the generation of 3'alpha,5'beta, 6'beta-trihydroxypravastatin (apparent K(m) = 4560 +/- 1410 microM) and hydroxypravastatin (apparent K(m) = 5290 +/- 1740 microM). In addition, as in the liver, pravastatin was metabolized in the small intestine by sulfation and subsequent degradation to its main metabolite 3'alpha-iso-pravastatin. It was concluded that lovastatin is metabolized by cytochrome P-450 3A enzymes in the small intestine. Compared with lovastatin, the cytochrome P-450-dependent intestinal intrinsic clearance of pravastatin was >5000-fold lower and cannot be expected to significantly affect its oral bioavailability or to be a significant site of drug interactions.

Subarachnoid hemorrhage in rats: effect of singular or sustained hemodilution with alpha-alpha diaspirin crosslinked hemoglobin on cerebral hypoperfusion.

OBJECTIVE: To evaluate the effect of singular or sustained hemodilution, with alpha-alpha diaspirin crosslinked hemoglobin (DCLHb), on the area of hypoperfusion after subarachnoid hemorrhage. DESIGN: Prospective animal study. SETTING: Animal research laboratory. SUBJECTS: Isoflurane anesthetized, mechanically ventilated rats. INTERVENTIONS: Subarachnoid hemorrhage was induced by injecting 0.3 mL of blood into the cisterna magna. The animals were randomly assigned to one of the following groups (n = 16 in each hemodilution group; eight animals received a single treatment of hemodilution after subarachnoid hemorrhage; and, for eight animals, treatment was sustained for 48 hrs): control group (n = 8), no hematocrit (45%) manipulation; DCLHb group (n = 16), hematocrit decreased to 30% with DCLHb; or Alb group (n = 16), hematocrit decreased to 30% with human serum albumin. After 48 hrs, the area of hypoperfusion (cerebral blood flow < 40 ml/100g/min) was determined with 14C-iodoantipyrine in five coronal brain sections. MEASUREMENTS AND MAIN RESULTS: For both singular and sustained treatment, the area of hypoperfusion was less in both hemodilution groups than in the control group (p<.05). For four of the five coronal brain sections, no differences were found between the DCLHb and Alb groups within a given hemodilution protocol. In addition, in four of the five coronal brain sections for the DCLHb hemodilution groups and in all five sections for the albumin hemodilution groups, the area of hypoperfusion was less for rats that received sustained hemodilution compared with their respective groups in the singular treatment protocol (p<.05). CONCLUSIONS: These data support the hypothesis that hemodilution with molecular hemoglobin decreases hypoperfusion after subarachnoid hemorrhage and that sustained hemodilution is more effective than singular treatment. The data do not support the notion that intravascular DCLHb has an adverse effect on cerebral ischemia after subarachnoid hemorrhage.

Simultaneous on-line extraction and analysis of sirolimus (rapamycin) and ciclosporin in blood by liquid chromatography-electrospray mass spectrometry.

We developed a sensitive and specific semi-automated liquid chromatography-electrospray mass spectrometric (HPLC-ESI-MS) assay for the simultaneous quantification of sirolimus and ciclosporin in blood. Following a simple protein precipitation step, the supernatants were injected into the HPLC system and extracted on-line. After column switching, the analytes were backflushed from the extraction column onto the analytical narrow-bore column and eluted into the ESI-MS system. The assay was linear from 0.4 to 100 microg/l sirolimus and from 2 to 1500 microg/l ciclosporin. The mean recoveries of sirolimus and ciclosporin were 98 and 96%, respectively. The mean interday precision/accuracy was 8.6%/-4.8% for sirolimus and 9.3%/-2.9% for ciclosporin.