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This article explores the lived experience of informal caregivers in cancer care, focusing on the perceived burden and needs of individuals seeking support from an informal group for next of kin. A total of 28 individuals who were closely related to a patient with cancer participated in focus group interviews. Three themes were identified: setting aside one’s own needs, assuming the role of project manager, and losing one’s sense of identity. Together they form the framing theme: being co-afflicted. The characteristics of informal caregivers are shown to be similar to those of people with codependency, motivating development of targeted interventions from this perspective.

The physician's communication skill influences the patient's mental and physical wellbeing, as well as the physician's own experience of stress. Most patients wish to be informed about their disease, by physicians who are honest, gives time, sustains hope, listens and shows compassion and empathy. Even though there are established guidelines on how to break bad news, the physician must find out and respond to the unique reactions and needs of each individual, in order to communicate successfully. There is no consensus on how to construct and evaluate communication skills training programs for physicians, and more RCT-studies are requested.

Omission of extracellular Ca2+ for 15 min from the incubation medium of cultured hippocampal slices stimulated the efflux of glutathione, phosphoethanolamine, hypotaurine, and taurine. The efflux was reduced by several blockers of gap junctions, i.e. carbenoxolone, flufenamic acid, and endothelin-1, and by the connexin43 hemichannel blocking peptide Gap26 but was unchanged by the P2X7 receptor inhibitor oxidized ATP, a pannexin1 hemichannel blocking peptide and an inactive analogue of carbenoxolone. Pretreatment of the slices with the neurotoxin N-methyl-d -aspartate left the efflux by Ca2+ omission unchanged, indicating that the stimulated efflux primarily originated from glia. Elevated glutamate efflux was detected when Ca2+ omission was combined with the glutamate uptake blocker l-trans-pyrrolidine-2,4-dicarboxylate and when both Ca2+ and Mg2+ were omitted from the medium. Omission of Ca2+ for 15 min alone did not induce delayed toxicity, but in combination with blocked glutamate uptake, significant cell death was observed 24 h later. Our results indicate that omission of extracellular Ca2+ stimulates efflux of glutathione and specific amino acids including glutamate via opening of glial hemichannels. This type of efflux may have protective functions via glutathione efflux but can aggravate toxicity in situations when glutamate reuptake is impaired, such as following a stroke.

N -acetylaspartate (NAA) was discovered in mammalian brain 50 years ago but its functions remain debated. One reason for the relatively slow progress of NAA research is the paucity of tools to specifically modify NAA concentrations. In this work we evaluated the use of the monomethyl ester of NAA (NAA MME) to increase the relatively low level of NAA in cultured hippocampal slices. When slices were treated with 30 mM NAA MME for 3 days the NAA concentration increased from 31.6 to 185.3 nmol/mg protein. Incubation with NAA alone increased the NAA concentration non-significantly to 65.6 nmol/mg protein. NAA MME treatment increased NAA in neurons and the increase was non-toxic as determined by the low uptake of propidium iodide, a dye that only enters damaged cells. NMDA-mediated excitotoxicity which is initiated by influx of Ca 2+ was unaltered by increased NAA levels indicating poor intracellular Ca 2+ -chelation by NAA.

Intrauterine growth restriction (IUGR) represents an important risk factor for perinatal complications and for adult disease. IUGR is associated with a down-regulation of placental amino acid transporters; however, whether these changes are primary events directly contributing to IUGR or a secondary consequence is unknown. We investigated the time course of changes in placental and fetal growth, placental nutrient transport in vivo and the expression of placental nutrient transporters in pregnant rats subjected to protein malnutrition, a model for IUGR. Pregnant rats were given either a low protein (LP) diet (n = 64) or an isocaloric control diet (n = 66) throughout pregnancy. Maternal insulin, leptin and IGF-I levels decreased, whereas maternal amino acid concentrations increased moderately in response to the LP diet. Fetal and placental weights in the LP group were unaltered compared to control diet at gestational day (GD) 15, 18 and 19 but significantly reduced at GD 21. Placental system A transport activity was reduced at GD 19 and 21 in response to a low protein diet. Placental protein expression of SNAT2 was decreased at GD 21. In conclusion, placental amino acid transport is down-regulated prior to the development of IUGR, suggesting that these placental transport changes are a cause, rather than a consequence, of IUGR. Reduced maternal levels of insulin, leptin and IGF-1 may link maternal protein malnutrition to reduced fetal growth by down-regulation of key placental amino acid transporters.

Proton-translocating mitochondrial nicotinamide nucleotide transhydrogenase (NNT) was investigated regarding its physiological role in Caenorhabditis elegans. NNT catalyzes the reduction of NADP(+) by NADH driven by the electrochemical proton gradient, Deltap, and is thus a potentially important source of mitochondrial NADPH. Mitochondrial detoxification of reactive oxygen species (ROS) by glutathione-dependent peroxidases depends on NADPH for regeneration of reduced glutathione. Transhydrogenase may therefore be directly involved in the defense against oxidative stress. nnt-1 deletion mutants of C. elegans, nnt-1(sv34), were isolated and shown to grow essentially as wild type under normal laboratory conditions, but with a strongly lowered GSH/GSSG ratio. Under conditions of oxidative stress, caused by the superoxide-generating agent methyl viologen, growth of worms lacking nnt-1 activity was severely impaired. A similar result was obtained by using RNAi. Reintroducing nnt-1 in the nnt-1(sv34) knockout mutant led to a partial rescue of growth under oxidative stress conditions. These results provide evidence for the first time that nnt-1 is important in the defense against mitochondrial oxidative stress.

N-Methyl-D-aspartate (NMDA)-receptor stimulation evoked a selective and partly delayed elevated efflux of glutathione, phosphoethanolamine, and taurine from organotypic rat hippocampus slice cultures. The protein kinase inhibitors H9 and staurosporine had no effect on the efflux. The phospholipase A2 inhibitors quinacrine and 4-bromophenacyl bromide, as well as arachidonic acid, a product of phospholipase A2 activity, did not affect the stimulated efflux. Polymyxin B, an antimicrobal agent that inhibits protein kinase C, and quinacrine in high concentration (500 microM), blocked efflux completely. The stimulated efflux after but not during NMDA incubation was attenuated by a calmodulin antagonist (W7) and an anion transport inhibitor (DNDS). Omission of calcium increased the spontaneous efflux with no or small additional effects by NMDA. In conclusion, NMDA receptor stimulation cause an increased selective efflux of glutathione, phosphoethanolamine and taurine in organotypic cultures of rat hippocampus. The efflux may partly be regulated by calmodulin and DNDS sensitive channels.

A method for the determination of AZD0837 and its two metabolites AR-H069927 and AR-H067637 in human bile was developed and validated. All three analytes and their stable isotope-labeled internal standards were isolated from bile using solid phase extraction on a mixed mode reversed phase/anion exchange column. Elution was done at high ionic strength with 0.125 M ammoniumacetate in 50% methanol. The extraction recoveries were >75%. Due to the high concentration of AR-H067637 a portion of the extract was diluted before injection on to the LC column, while undiluted extract was directly injected for the analysis of AZD0837 and AR-H069927. Chromatographic separation of all three analytes was achieved in a single system utilizing a C18 column based on fused core particle technology at high flow rate. The two metabolites were eluted when a gradient from 30 to 57% methanol was applied while the more hydrophobic pro-drug, AZD0837, eluted during a steeper second gradient from 57 to 80% methanol with the ammonium acetate concentration and acetic acid concentration kept constant at 3.8 mmol/L and 0.1%, respectively. The total cycle time was 3.2 min. Detection was performed using positive electrospray ionization tandem mass spectrometry. The linearity range was 0.02–20 μmol/L for AZD0837 and AR-H069927, and 1–1000 μmol/L for AR-H067637. The repeatability and the overall precision were less than 15% (RSD) and the accuracy was within the interval 93–100%.

Omission of extracellular Ca2+ for 15 min from the incubation medium of cultured hippocampal slices stimulated the efflux of glutathione, phosphoethanolamine, hypotaurine, and taurine. The efflux was reduced by several blockers of gap junctions, i.e. carbenoxolone, flufenamic acid, and endothelin-1, and by the connexin43 hemichannel blocking peptide Gap26 but was unchanged by the P2X7 receptor inhibitor oxidized ATP, a pannexin1 hemichannel blocking peptide and an inactive analogue of carbenoxolone. Pretreatment of the slices with the neurotoxin N-methyl-d -aspartate left the efflux by Ca2+ omission unchanged, indicating that the stimulated efflux primarily originated from glia. Elevated glutamate efflux was detected when Ca2+ omission was combined with the glutamate uptake blocker l-trans-pyrrolidine-2,4-dicarboxylate and when both Ca2+ and Mg2+ were omitted from the medium. Omission of Ca2+ for 15 min alone did not induce delayed toxicity, but in combination with blocked glutamate uptake, significant cell death was observed 24 h later. Our results indicate that omission of extracellular Ca2+ stimulates efflux of glutathione and specific amino acids including glutamate via opening of glial hemichannels. This type of efflux may have protective functions via glutathione efflux but can aggravate toxicity in situations when glutamate reuptake is impaired, such as following a stroke.

S-sulfo-cysteine (SSC) is an agonist of glutamate receptors which could be involved in cysteine-induced neurotoxicity. Here we analyzed SSC by HPLC and demonstrated that the concentration of SSC in cortex of cysteine-injected rats increased to 1.4 microM, about four times the value of control rats. The neurotoxic effect of SSC was evaluated in slice cultures of rat hippocampus and compared to NMDA and cysteine. The neurotoxicity threshold of SSC was well above the tissue concentration. Our results show that SSC increases in neonatal rat brain after cysteine injection but reaches a tissue concentration far below concentrations that induce neurotoxicity in vitro. Thus, even if all the tissue SSC after cysteine injection was extracellular it would be below the threshold for toxicity, indicating that SSC is not a main excitotoxin involved in cysteine toxicity.

In an earlier study, using hippocampal slice cultures from rat, we showed that N-methyl-d-aspartate (NMDA)1 receptor activation caused release of N-acetylaspartate (NAA), an organic anion of predominantly neuronal origin that is frequently used as a marker of neuronal death and/or dysfunction [1,2]. A specific number of other organic anions, including the antioxidant GSH, were also released after NMDA receptor activation. The efflux of these anions was Ca2+ dependent and correlated with delayed nerve cell death measured 24 h later, indicating a role of the efflux in the nerve cell death. Interestingly, the efflux of NAA, as determined by reversed-phase HPLC coupled to UV detection, was accompanied by the release of a few unknown UV-absorbing components. The tight correlation between the efflux and delayed nerve cell death prompted us to try to identify the unknown components. In this work, we identified one of the earlier unknown and released substances as creatine (Cr) and further developed the previously used reversed-phase HPLC method [1]. The HPLC system consisted of a Varian 5000 pump, a Waters 717 autosampler, and an Applied Biosystems 757 UV detector. For multiple wavelength measurements, a Waters 2996 photodiode array detector was used. The sampling rate was 1 spectrum/s, and the sampling range was 190–400 nm with a resolution of 1.2 nm. Data were processed with Millennium32 software (Waters, Milford, MA, USA). If not indicated, chemicals were from Sigma (St. Louis, MO, USA). NAA (Aldrich, Milwaukee, WI, USA) and Cr were separated at room temperature on a TSK gel ODS-80TM column (250 × 4.6 mm, 5 μm particle size, Tosoh, Tokyo, Japan) fitted with a guard column packed with C18 Nucleosil material. The mobile phase consisted of 50 mM NaH2PO4 (pH 2.15) and was degassed with N2 before use. A flow rate of 1 ml/min was used, and peaks were detected by absorbance at 210 nm. After each sample set (typically 20–40 injections of 90 μl each), the column was washed with 20 and 100% methanol (Rathburn, Walkersburn, UK) for 40 min each. For NAA analysis, the current method was compared with that of Koller and coworkers. This method was carried out essentially as described previously [3]. The detection limits at a signal-to-noise ratio of 2 were 9 pmol (0.1 μM) injected for NAA and 1.8 pmol (0.02 μM) injected for Cr. To determine the linearity and intraassay precision of the method, six sets of eight combined standards, ranging from 0.5 to 200 μM for NAA and from 0.1 to 40 μM for Cr, were injected. Interassay precision was determined by injecting six sets of the combined standards (stored at −20 °C) over a period of 32 days. Calibration curves were plotted as peak height (μV) versus analyte concentration (μM). At a signal-to-noise ratio of 10, the method was linear from 45 pmol up to at least 18 nmol injected (0.5–200 μM) for NAA and from 9 pmol up to at least 3.6 nmol injected (0.1–40 μM) for Cr. The inter- and intraassay coefficients of variance (CVs) for NAA and Cr were generally less than or slightly greater than 10%, and calibration curves showed R2 values greater than 0.999, demonstrating good precision and linearity. The accuracy was determined by adding a known amount of NAA and Cr to samples prior to injection. The recoveries for NAA and Cr were 101.6% (7.6% CV, n = 6) and 99.5% (6.2% CV, n = 6), respectively. A chromatogram of a mixture of NAA, Cr, and possible interfering compounds is shown in Fig. 1A. As can be seen, a large number of organic ions were separated. NAA and Cr eluted with retention factors (k) of 3.4 and 1.3, respectively (k = (tR − t0)/t0, where tR is the retention time of the analyte and t0 is the elution time for unretained solutes). The lack of an absorbance maximum at 217 nm confirmed that the Cr peak was free of creatinine (Fluka, Buchs, Switzerland) (Fig. 1B) [4]. Furthermore, for NAA analysis, the current method was compared with that of Koller and coworkers [3]. No significant difference was found between the concentrations obtained with the two methods when brain tissue extracts were analyzed (P = 0.67, n = 29, paired t test). Fig. 1 (A) Chromatogram of a standard of NAA, Cr, and possible interfering compounds (nmol injected in parentheses): 1, hypotaurine (0.5); 2, aspartate (45); 3, NMDA (90); 4, glutamate (45); 5, l-carnosine (0.9); 6, γ-aminobutyric acid (90); 7, Cr (11); ... By lowering the pH in the mobile phase to 2.15, organic acids such as NAA and Cr are protonated and retained on a reversed-phase column. Although the use of pure aqueous buffer in combination with silica-based columns is generally not recommended, we found that our method was robust in terms of peak shape and column lifetime. We chose NAA and Cr detection at 210 nm, although both compounds show higher absorbance at lower wavelengths. However, at lower wavelengths, the increasing absorbance of the void interfered with Cr analysis. The separation of Cr and creatinine could be confirmed by the characteristic absorbance of creatinine at 217 nm in low-pH solution (Fig. 1B) [4]. In addition, no difference between concentrations obtained with our method and that of Koller and coworkers was found when NAA was analyzed. This further confirms the validation of both methods. The method is comparable in sensitivity to earlier methods using UV detection for NAA analysis and is better when compared with some recently published reversed-phase methods for Cr determination [3,5–7]. Previous methods for NAA analysis recommended purification before injection or use of ion-pairing reagents and extensive washing between injections [3,7]. Also, it was reported that a high K+ concentration interfered with NAA determination using the method of Koller and coworkers [8]. With our method, a high K+ concentration (100 mM) did not interfere with NAA or Cr analysis (data not shown), and we showed previously that high salt-containing samples, such as artificial cerebrospinal fluid (ACSF), can be analyzed with the current method [1]. Thus, in some applications, our method may be a suitable complement, particularly to the method based on anion exchange, which is the most commonly used HPLC method for NAA determination today [3]. We have also determined NAA and Cr in extracts from rat hippocampus developed in vivo and in vitro (Fig. 2 and Table 1). The increasing concentrations of NAA and Cr during in vivo development are in agreement with previous studies; however, as can be noted, the NAA concentration, but not the Cr concentration, is lower in hippocampus developed in vitro than in vivo [3,9,10]. Reversed-phase HPLC methods for the separate determination of NAA and Cr have been reported previously, but to our knowledge the current method is the first one that evaluates potential interferences with NAA analysis and allows the simultaneous determination of NAA and Cr [5,6,11,12]. In addition, a large number of other metabolites, including N-acetylaspartylglutamate, were separated, making the method a possible choice in a variety of different types of neuro-chemical studies. Fig. 2 (A) Chromatogram of UV-absorbing species at 210 nm in an extract of a rat hippocampus. (B) and (C) show the absorbance profiles of the NAA and Cr peaks, respectively, in a standard injection and in an extract of a cultured hippocampal slice from rat. Table 1 Content (nmol/mg protein ± SEM) of NAA and Cr in hippocampus (in vivo, n = 6) and in cultured hippocampal slices from rat (in vitro, n = 12–14) In conclusion, the method described is a relatively simple reversed-phase HPLC method for the simultaneous determination of NAA and Cr. The method shows good precision and linearity and can be used for NAA and Cr determination in ACSF as well as in brain extracts and, thus, is a good complement to existing methods for NAA and Cr determination.

N-Acetylaspartate (NAA) is a largely neuron specific dianionic amino acid present in high concentration in vertebrate brain. Many fundamental questions concerning N-acetylaspartate in brain remain unanswered. One such issue is the predominantly neuronal synthesis and largely glial catabolism which implies the existence of a regulated efflux from neurons. Here we show that transient (5 min) NMDA-receptor activation (60 microM) induces a long lasting Ca2+ -dependent efflux of N-acetylaspartate from organotypic slices of rat hippocampus. The NMDA-receptor stimulated efflux was unaffected by hyper-osmotic conditions (120 mM sucrose) and no efflux of N-acetylaspartate was evoked by high K+ -depolarization (50 mM) or kainate (300 microM). These results indicate that the efflux induced by NMDA is not related directly to either cell swelling or depolarization but is coupled to Ca2+ -influx via the NMDA-receptor. The efflux of N-acetylaspartate persisted at least 20 min after the omission of NMDA, similar to the efflux of the organic anions glutathione and phosphoethanolamine. The efflux of taurine and hypotaurine was also stimulated by NMDA but returned more quickly to basal levels. The NMDA-receptor stimulated efflux of N-acetylaspartate, glutathione, phosphoethanolamine, taurine and hypotaurine correlated with delayed nerve cell death measured 24 h after the transient NMDA-receptor stimulation. However, exogenous administration of high concentrations of N-acetylaspartate to the culture medium was non-toxic. The results suggest that Ca2+ -influx via the NMDA-receptor regulates the efflux of N-acetylaspartate from neurons which may have both physiological and pathological importance.