Successful treatment of a cohort of infants with neonatal diabetes using insulin pumps including data on genetics and estimated incidence.

AIM: Neonatal diabetes is rare, and treatment is challenging. We present aspects on treatment, genetics and incidence. METHOD: This was a prospective cohort study including all cases in our study area in Sweden. We compared with data from the National Diabetes Registry, the Neonatal Quality Register and the National Patient Register. RESULTS: In the 19-year study period January 1, 1998 to December 31, 2016, we treated seven infants, five of them boys. Six patients used a subcutaneous insulin pump, and the smallest patient started at a weight of 938 g. Most important was for the pump to deliver minute doses of insulin and the design of cannulas and tubing. All patients could stop insulin treatment at 17-145 days of age. One patient relapsed at age 4.5 years. Four patients used the insulin pump after discharge. A mutation was identified in five patients, and this included all patients born after 30 weeks of gestation. The incidence of neonatal diabetes was 2/1 00 000, higher than previously estimated for Europe. Similar but lower incidences were reported in the registries. CONCLUSION: Insulin pumps were safe in neonatal diabetes. All seven cases were transient. Neonatal diabetes was more common in our area than reported from Europe.

Tetramethylpyrazine prevents contrast-induced nephropathy by inhibiting p38 MAPK and FoxO1 signaling pathways.

BACKGROUND: Apoptosis is recognized as an important mechanism in contrast-induced nephropathy (CIN). As tetramethylpyrazine (TMP) has been recently found to be renoprotective and anti-apoptotic in multiple kidney injuries, we hypothesized that TMP would prevent CIN. METHODS: An experimental model of CIN was established in rats. Serum creatinine, blood urea nitrogen, plasma cystatin C, urinary N-acetyl-ß-glucosaminidase, and urinary γ-glutamyl transpeptidase were measured to evaluate kidney function. Apoptosis was assessed by transmission electron microscopy, transferase-mediated deoxyuridine triphosphate nick end-labeling staining, and poly-ADP-ribose polymerase cleavage. Fork-head box O1 transcriptional factor (FoxO1) mRNA expression was evaluated by quantitative real-time PCR. Phospho-p38 mitogen-activated protein kinase (MAPK) protein expression was assessed by immunohistochemistry and Western blotting. RESULTS: TMP significantly attenuated the resulting renal dysfunction and renal tubular cell apo-ptosis. Mechanistically, TMP decreased the expression of phospho-p38 MAPK protein and attenuated the increased FoxO1 mRNA and nuclear protein expression. In addition, TMP inhibited inducible nitric oxide synthase and Bax protein expression while it upregulated Bcl-2. CONCLUSION: In summary, this study demonstrated the protective role of TMP against CIN and indicated the effects of TMP may be mediated by the inhibition of p38 MAPK and FoxO1 pathways. Thus, TMP may be a new potential therapeutic agent to prevent CIN.

Protective effects of N-acetylcysteine amide (NACA) on gentamicin-induced apoptosis in LLC-PK1 cells.

AIM: Apoptosis plays a critical role in the pathogenesis of gentamicin (Gen)-induced nephrotoxicity. However, the underlying molecular mechanisms still remain unclear. In this study, we addressed the role of p38 mitogen-activated protein kinase (MAPK)/inducible nitric oxide synthase (iNOS) signaling pathway in Gen-induced nephrotoxicity and evaluated the protective effect of the free-radical scavenger N-acetylcysteine amide (NACA). METHODS: Pig kidney epithelial cells (LLC-PK1) cells were exposed to Gen for variable times and doses. Cytotoxicity was assessed by morphology and by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Protein expression was assessed by Western blotting. RESULTS: Exposure to Gen-induced apoptosis in a dose-dependent and time-dependent manner was assessed by DNA content analysis and poly ADP ribose polymerase (PARP) cleavage. Gen caused increased phosphorylation of p38 MAPK and induction of iNOS. This was accompanied by a significant upregulation of Bax and nuclear factor κB (NF-κB) and a downregulation of Bcl-2 expression. Pretreatment with SB203580, aminoguanidine (AG), and NACA inhibited apoptosis. Furthermore, pretreatment with SB203580 and NACA not only attenuated the pro-apoptotic effect of Gen, but also significantly reversed its effects on p38 MAPK phosphorylation and iNOS induction. The Gen-induced effects on Bcl-2, Bax, and NF-κB expression were also reversed by SB203580, AG, and NACA. CONCLUSION: In conclusion, NACA can attenuate Gen-induced apoptotic injury in LLC-PK1 cells through inhibiting p38 MAPK/iNOS signaling pathway.

The role of electronic assessment of adherence in the education and counseling of children taking growth hormone: progress and challenges.

There are numerous issues surrounding adherence in children taking recombinant human growth hormone (rh-GH). New technologies capable of accurately recording/monitoring may highlight some of these issues, and have value in optimizing adherence levels through education and counseling. The intention of this review is to guide healthcare professionals (HCPs). PubMed, Google Scholar and citations in published papers were used to substantiate the views expressed by the authors. Both perceptional and practical factors influence the adherence levels of children taking rh-GH. Understanding such factors may help to identify the characteristics of ideal rh-GH devices and their potential impact on adherence. New technologies, such as electronic monitors, may facilitate patient-provider discussions on adherence, and help identify barriers that are amenable to change. Monitoring adherence may also help differentiate nonadherence from biological low response to rh-GH therapy. However, the medical, psychological, social and ethical aspects of electronic assessment require further investigation.

Glutathione is essential for early embryogenesis--analysis of a glutathione synthetase knockout mouse.

Glutathione (GSH) is present in all mammalian tissues and plays a crucial role in many cellular processes. The second and final step in the synthesis involves the formation of GSH from gamma-glutamylcysteine (γ-GC) and glycine and is catalyzed by glutathione synthetase (GS). GS deficiency is a rare autosomal recessive disorder, and is present in patients with a range of phenotypes, from mild hemolytic anemia and metabolic acidosis to severe neurologic disorders or even death in infancy. The substrate for GS, γ-GC, has been suggested as playing a protective role, by substituting for GSH as an antioxidant in GS deficient patients. To examine the role of GS and GSH metabolites in development, we generated mice deficient in GSH by targeted disruption of the GS gene (Gss). Homozygous mice died before embryonic day (E) 7.5, but heterozygous mice survived with no distinct phenotype. GS protein levels and enzyme activity, as well as GSH metabolites, were investigated in multiple tissues. Protein levels and enzyme activity of GS in heterozygous mice were diminished by 50%, while GSH levels remained intact. γ-GC could not be detected in any investigated tissue. These data demonstrate that GSH is essential for mammalian development, and GSH synthesis via GS is an indispensable pathway for survival.

Growth hormone and treatment outcomes: expert review of current clinical practice.

Although there are guidelines for treatment of short stature, open questions regarding optimal management of growth hormone therapy still exist. Experts attending six international meetings agree that successful therapy results in the patient attaining mid-parental height, and relies on correct diagnosis and early intervention. Experts advocate patient followup every 3-6 months, and that growth and adherence should be monitored at each visit. Growth response is variable, and an accepted definition of good/poor response is lacking. Combined with patient education and regular patient follow-up, a definition of treatment response could lead to improved treatment outcomes. Few experts use prediction models in clinical practice, but all agree that pharmacogenetics might improve prediction, enable early therapy modulation, and promote growth. Poor growth is often due to low adherence. Guidance on optimal management of growth hormone therapy is required, with focus on early diagnosis, dosing, treatment monitoring, adherence, and motivation.

Tissue-specific knockout of TSHr in white adipose tissue increases adipocyte size and decreases TSH-induced lipolysis.

BACKGROUND: The primary function of TSH is to activate TSH receptors (TSHr) in the thyroid gland and thereby stimulate thyroid hormone synthesis and secretion. TSHr are also expressed in other organs, but their physiological importance is still unclear. We have previously shown that TSHr, expressed in adipocytes, are of potential importance for lipolysis and extrauterine adaptation of the neonate. METHODOLOGY: To further study the role of TSHr in adipocytes we selectively removed the TSHr gene in mice adipocytes by using the Cre-loxP recombination system (B6.Cg-Tg (Fabp4-Cre) 1Rev/J. TSHr knockout (KO) newborn mice were phenotypically characterized. Isolated adipocytes from 8-week-old male mice were studied in term of adipocyte size and metabolism. RESULTS: Mice lacking TSHr in adipocytes were apparently normal at birth and no differences in thyroid gland function or histology were observed. Sensitivity to TSH-induced lipolysis was ten times lower in adipocytes from targeted animals compared to wild-type. This indicates that adipocytes from targeted animals are refractory to stimulation of physiological concentrations of TSH. Catecholamine-induced lipolysis and insulin-induced inhibition of lipolysis were unaltered. Adipocyte size was increased in the targeted animals. Basal lipolysis was increased as an effect of the increased adipocyte size. CONCLUSION: Our results indicate that adipocyte TSHr under normal conditions affects adipocyte growth and development.

N-acetylcysteine amide protects renal proximal tubular epithelial cells against iohexol-induced apoptosis by blocking p38 MAPK and iNOS signaling.

BACKGROUND: The pathogenesis of contrast-induced nephropathy (CIN) is still poorly understood and apoptosis via oxidative stress has been proposed as one possible mechanism. We therefore studied the apoptotic signaling mechanism in CIN and also tested whether the new antioxidant N-acetylcysteine amide (NACA) could prevent CIN. METHODS: LLC-PK1 cells were exposed to a widely used contrast agent, iohexol (IH). Cytotoxicity was assessed with morphology and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Cell death was analyzed by the DNA content analysis and PARP cleavage. Protein expression was assessed with Western blotting. RESULTS: We observed cell death with apoptotic features in a dose- and time-dependent manner. Initiation of IH-induced apoptosis was mediated by upregulation of Bax and downregulation of Bcl-2 and Mcl-1, which was preceded by p38 MAPK activation and iNOS induction. Inhibitors of p38 MAPK and iNOS partially abolished IH-induced apoptosis. Furthermore, we found pretreatment with NACA partially protected cells from IH-induced death by reverting the expression of Bcl-2, Mc1-1 and Bax expression through inhibition of p38 MAPK and iNOS pathway. CONCLUSIONS: This study demonstrates that apoptosis occurs during CIN. Apoptosis is associated with activations of p38 MAPK and iNOS. Pretreatment with the antioxidant NACA could prevent IH-induced cell death by blocking the p38 MAPK/iNOS signaling pathway.

HOMA-IR and QUICKI: decide on a general standard instead of making further comparisons.

AIM: To limit further comparisons between the two fasting indices Homeostasis Model Assessment for Insulin Resistance (HOMA-IR) and Quantitative Insulin Sensitivity Check Index (QUICKI), and to examine their robustness in assessing insulin sensitivity. METHODS: A total of 191 obese children and adolescents (age 13.9 ± 2.9 years, BMI SDS 6.1 ± 1.6), who had undergone a Frequently Sampled Intravenous Glucose Tolerance Test (FSIVGTT), were included. Receiver operating characteristic curve (ROC) analysis was used to compare indices in detecting insulin resistance and Bland-Altman plots to investigate agreement between three consecutive fasting samples when compared to using single samples. RESULTS: ROC analysis showed that the diagnostic accuracy was identical for QUICKI and HOMA-IR [area under the curve (AUC) boys 0.80, 95%CI 0.70-0.89; girls 0.80, 0.71-0.88], while insulin had a nonsignificantly lower AUC (boys 0.76, 0.66-0.87; girls 0.75, 0.66-0.84). Glucose did not perform better than chance as a diagnostic test (boys 0.47, 0.34-0.60; girls 0.57, 0.46-0.68). Indices varied with consecutive sampling, mainly attributable to fasting insulin variations (mean maximum difference in HOMA-IR -0.8; -0.9 to -0.7). CONCLUSIONS: Using both HOMA-IR and QUICKI in further studies is superfluous as these indices function equally well as predictors of the FSIVGTT sensitivity index. Focus should be on establishing a general standard for research and clinical purposes.

Pharmacokinetic profile of N-acetylcysteine amide and its main metabolite in mice using new analytical method.

N-acetylcysteine amide (NACA) is the amide derivative of N-acetylcysteine (NAC) that is rapidly converted to NAC after systemic administration. It has emerged as a promising thiol antioxidant for multiple indications; however, the pharmacokinetic property is yet unclear due to lack of an accurate quantification method. The present investigation aimed to develop an analytical method for simultaneous quantification of NACA and NAC in plasma. A new reagent (2-(methylsulfonyl)-5-phenyl-1,3,4-oxadiazole, MPOZ) was introduced for thiol stabilization during sample processing and storage. Further, we utilized tris (2-carboxyethyl) phosphine (TCEP) to reduce the oxidized forms of NACA and NAC. After derivatization, NACA-MPOZ and NAC-MPOZ were quantified using liquid chromatography-mass spectrometry (LC-MS). The new method was validated and found to have high specificity, linearity, accuracy, precision, and recovery for the quantification of NACA and NAC in plasma. Furthermore, the formed derivatives of NACA and NAC were stable for 48 h under different conditions. The method was utilized in pharmacokinetic study which showed that the bioavailability of NACA is significantly higher than NAC (67% and 15%, respectively). The pharmacokinetic of NACA obeyed a two-compartment open model. The glutathione (GSH)-replenishing capacity was found to be three to four-fold higher after the administration of NACA compared to that observed after the administration of NAC. In conclusion, the present method is simple, robust and reproducible, and can be utilized in both experimental and clinical studies. NACA might be considered as a prodrug for NAC. Furthermore, this is the first report describing the pharmacokinetics and bioavailability of NACA in mouse.

Insulin sensitivity, VO2max and body composition in severely obese Swedish children and adolescents.

AIM: The aim of this study was to identify relationships between insulin sensitivity (SI), cardiorespiratory fitness and body composition in severely obese Swedish children and adolescents. METHODS: Two hundred and twenty-eight obese children (119 girls, 8-16 years, body mass index (BMI) 23.2-57.0 kg/m(2)) performed a frequently sampled intravenous glucose tolerance test (FSIVGTT), a submaximal bicycle ergometry test and a dual-energy X-ray absorptiometry (DEXA). RESULTS: Mean SI (SD) was 0.38 (0.32) (x10(-5)/min/pM). SI correlated positively with relative body mass (BM) VO(2)max (r = 0.42) (p < 0.001), relative fat-free mass (FFM) VO(2)max (r = 0.36) (p < 0.001) and negatively with body mass index standard deviation score (BMI SDS) (r =-0.22) (p = 0.001). SI did not correlate with percent body fat (r =-0.01) and absolute VO(2)max (r = 0.01). In multiple regression analyses with SI as dependent variable, VO(2)max and body composition, together with gender, age and Tanner stage, explained 20-26% of the variance. CONCLUSION: Relative (BM) VO(2)max and relative (FFM) VO(2)max were stronger predictors of SI than percent body fat in severely obese children and adolescents. The study confirms that cardiorespiratory fitness is of importance for the metabolic syndrome in the studied population. Efforts to improve SI should include physical activity targeting cardiorespiratory fitness also in severely obese children and adolescents.

Adherence remains a challenge for patients receiving growth hormone therapy.

Patients receiving growth hormone therapy have the best growth results when they have good adherence, but adherence rates are often low. Adherence has been difficult to measure and is often measured indirectly as direct methods are inconvenient, expensive or both. However, use of electronic monitors that log the dose and the time it was delivered provides a simple and reliable means of gathering data and may facilitate the discussion of adherence in the clinic. Although many factors impact on adherence, there are some 'red flags' to which a physician should be particularly alert, such as history of poor attendance at consultations. Based on the author's own clinical experience and opinion, as well as published studies, this article describes factors associated with non-adherence and how most, if not all, barriers to good adherence can be overcome by maintaining a good non-judgemental relationship with the patient, and delivering useful and clear education and training soon after diagnosis.

Impact sibutramine therapy in children with hypothalamic obesity or obesity with aggravating syndromes.

OBJECTIVE: Behavioral treatment of children suffering from hypothalamic obesity or uncomplicated obesity in combination with syndromes that aggravate this condition has proven to be ineffective. The combination of comorbidities and severe obesity lower the quality of these children's lives drastically. The present goal was to determine whether treatment with sibutramine has a beneficial effect on such children. DESIGN AND SUBJECTS: A double-blind, placebo-controlled, cross-over study (20 + 20 wk), followed by a 6-month open phase, was performed. The primary indicator of efficacy was the body mass index (BMI) sd score (SDS) value, which was analyzed using an ANOVA repeated-measures design [intention to treat (ITT)]. The 50 children (7-20 yr of age) involved included 22 with hypothalamic obesity and 28 with uncomplicated obesity plus aggravating syndromes. Forty-five patients completed the first phase, and 42 participated in the entire study. RESULTS: The group that initially received the placebo demonstrated an insignificant decrease (-0.06) in BMI SDS during this treatment but a significant decrease (-0.68; P < 0.001) when treated with sibutramine. The other group demonstrated a reduction in their BMI SDS of -0.72 during administration of sibutramine and a rebound of +0.43 when placed on the placebo (P < 0.001 in both cases). The response of children with hypothalamic obesity was also significant but was less pronounced than that of children with nonhypothalamic obesity. During the open phase, a continuous reduction in weight was observed. The treatment was tolerated well. CONCLUSION: The clinically and statistically significant weight reduction caused by sibutramine in this short-term study indicates that treatment of hypothalamic and syndromal obesity with this drug may be beneficial.

N-Acetylcysteine Amide Exerts Possible Neuroprotective Effects in Newborn Pigs after Perinatal Asphyxia.

BACKGROUND: Perinatal asphyxia and ensuing reoxygenation change the antioxidant capacity of cells and organs. OBJECTIVES: To analyze the neuroprotective effect of the antioxidant N-acetylcysteine amide (NACA) after perinatal hypoxia-reoxygenation with an emphasis on proinflammatory cytokines and the transcription factor NF-κB in the prefrontal cortex of neonatal pigs. METHODS: Twenty-nine newborn pigs, aged 12-36 h, were subjected to global hypoxia and hypercapnia. One sham-operated group (n = 5) and 2 experimental groups (n = 12) were exposed to 8% oxygen, until the base excess was -20 mmol/l or the mean arterial blood pressure fell to <20 mm Hg (asphyxia with NACA or saline). The pigs were observed for 9.5 h after hypoxia. Samples of prefrontal cortex and plasma were analyzed. RESULTS: Cortex: there was no significant difference in mRNA expression between the intervention groups regarding IL-1ß, IL6, TNFα, MMP2, MMP9 or IL18. Pigs exposed to hypoxia-reoxygenation and treatment with NACA (NACA-pigs) had a significantly lower protein concentration of IL-1ß than pigs treated with saline (placebo controls), i.e. 8.8 ± 3.9 versus 16.8 ± 10.5 pg/mg protein (p = 0.02). The activation of the transcription factor NF-κB (measured as the fold-change of phosphorylated p65Ser 536), was reduced in the NACA-pigs when compared to the placebo controls (5.2 ± 4.3 vs. 16.0 ± 13.5; p = 0.02). No difference between the intervention groups regarding brain histopathology or in the levels of 8-oxoguanine measured in the prefrontal cortex were observed. Plasma: the NACA-pigs had a stronger reduction of TNFα in the first 30 min following asphyxia compared with the placebo controls, i.e. 36 (30-44) versus 24 (14-32)% (p = 0.01). CONCLUSION: The reduced levels of the pivotal inflammatory markers IL-1ß and TNFα and the transcription factor NF-κB may indicate that NACA has possible neuroprotective effects after perinatal asphyxia.

Nephroprotective Effects of N-Acetylcysteine Amide against Contrast-Induced Nephropathy through Upregulating Thioredoxin-1, Inhibiting ASK1/p38MAPK Pathway, and Suppressing Oxidative Stress and Apoptosis in Rats.

Contrast-induced nephropathy (CIN) is a leading cause of hospital-acquired acute kidney injury (AKI) due to apoptosis induced in renal tubular cells. Our previous study demonstrated the novel N-acetylcysteine amide (NACA); the amide form of N-acetyl cysteine (NAC) prevented renal tubular cells from contrast-induced apoptosis through inhibiting p38 MAPK pathway in vitro. In the present study, we aimed to compare the efficacies of NACA and NAC in preventing CIN in a well-established rat model and investigate whether thioredoxin-1 (Trx1) and apoptosis signal-regulating kinase 1 (ASK1) act as the potential activator for p38 MAPK. NACA significantly attenuated elevations of serum creatinine, blood urea nitrogen, and biomarkers of AKI. At equimolar concentration, NACA was more effective than NAC in reducing histological changes of renal tubular injuries. NACA attenuated activation of p38 MAPK signal, reduced oxidative stress, and diminished apoptosis. Furthermore, we demonstrated that contrast exposure resulted in Trx1 downregulation and increased ASK1/p38 MAPK phosphorylation, which could be reversed by NACA and NAC. To our knowledge, this is the first report that Trx1 and ASK1 are involved in CIN. Our study highlights a renal protective role of NACA against CIN through modulating Trx1 and ASK1/p38 MAPK pathway to result in the inhibition of apoptosis among renal cells.

Mortality Is Not Increased in Recombinant Human Growth Hormone-treated Patients When Adjusting for Birth Characteristics.

OBJECTIVE: This study aimed to investigate whether reported high mortality in childhood recombinant human GH (rhGH)-treated patients was related to birth-characteristics and/or rhGH treatment. DESIGN AND SETTING: We sought to develop a mortality model of the Swedish general population born between 1973 and 2010, using continuous-hazard functions adjusting for birth characteristics, sex, age intervals, and calendar year to estimate standardized mortality ratio (SMR) and to apply this model to assess expected deaths in Swedish rhGH-treated patients with idiopathic isolated GH deficiency (IGHD), idiopathic short stature (ISS) or born small for gestational age (SGA). PARTICIPANTS: The general population: Swedish Medical Birth Register (1973-2010: 1 880 668 males; 1 781 131 females) and Cause of Death Register (1985-2010). Intervention Population: Three thousand eight hundred forty-seven patients starting rhGH treatment between 1985 and 2010 and followed in the National GH Register and/or in rhGH trials diagnosed with IGHD (n = 1890), ISS (n = 975), or SGA (n=982). MAIN OUTCOME MEASURES: Death. RESULTS: Using conventional models adjusting for age, sex, and calendar-year, the SMR was 1.43 (95% confidence interval, 0.89-2.19), P = .14, observed/expected deaths 21/14.68. The rhGH population differed (P < .001) from the general population regarding birth weight, birth length, and congenital malformations. Application of an Advanced Model: When applying the developed mortality model of the general population, the ratio of observed/expected deaths in rhGH-treated patients was 21/21.99; SMR = 0.955 (0.591-1.456)P = .95. Model Comparison: Expected number of deaths were 14.68 (14.35-14.96) using the conventional model, and 21.99 (21.24-22.81) using the advanced model, P < .001, which had at all ages a higher gradient of risk per SD of the model, 24% (range, 18-42%; P < .001). CONCLUSIONS: Compared with the general Swedish population, the ratio of observed/expected deaths (21/21.99) was not increased in childhood rhGH-treated IGHD, ISS, and SGA patients when applying an advanced sex-specific mortality model adjusting for birth characteristics.

Human hereditary glutathione synthetase deficiency: kinetic properties of mutant enzymes.

Patients with hereditary glutathione synthetase deficiency suffer from haemolytic anaemia, 5-oxoprolinuria, metabolic acidosis, recurrent bacterial infections and various degrees of central nervous system dysfunction. To investigate the molecular basis of the mutations associated with this disease, seven naturally occurring missense mutations [L188P (Leu188-->Pro), D219A, D219G, Y270C, Y270H, R283C and P314L] were expressed using a His-tagged, Escherichia coli-based expression system. Effects of the mutations on kinetic properties, including negative co-operative binding of gamma-glutamyl substrate, were evaluated. The mutation P314L did not have any major effect on these parameters and was classified as a neutral mutation. The remaining mutations decreased V(max) to 2-27% of wild-type activity. Negative co-operativity for gamma-gluABA (L-gamma-glutamyl-L-alpha-aminobutyric acid) was abolished in five mutant recombinant enzymes, whereas for one mutant enzyme, this co-operativity changed from negative to positive. The structural consequences of the mutations were interpreted on the basis of the known structure of the wild-type enzyme.

Neuroprotective effects of N-acetylcysteine amide on experimental focal penetrating brain injury in rats.

We examined the effects of N-acetylcysteine amide (NACA) in the secondary inflammatory response following a novel method of focal penetrating traumatic brain injury (TBI) in rats. N-acetylcysteine (NAC) has limited but well-documented neuroprotective effects after experimental central nervous system ischemia and TBI, but its bioavailability is very low. We tested NACA, a modified form of NAC with higher membrane and blood-brain barrier permeability. Focal penetrating TBI was produced in male Sprague-Dawley rats randomly selected for NACA treatment (n=5) and no treatment (n=5). In addition, four animals were submitted to sham surgery. After 2 hours or 24 hours the brains were removed, fresh frozen, cut in 14 µm coronal sections and subjected to immunohistochemistry, immunofluorescence, Fluoro-Jade and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) analyses. All treated animals were given 300 mg/kg NACA intraperitoneally (IP) 2 minutes post trauma. The 24 hour survival group was given an additional bolus of 300 mg/kg IP after 4 hours. NACA treatment decreased neuronal degeneration by Fluoro-Jade at 24 hours with a mean change of 35.0% (p<0.05) and decreased TUNEL staining indicative of apoptosis at 2 hours with a mean change of 38.7% (p<0.05). Manganese superoxide dismutase (MnSOD) increased in the NACA treatment group at 24 hours with a mean change of 35.9% (p<0.05). Levels of migrating macrophages and activated microglia (Ox-42/CD11b), nitric oxide-producing inflammatory enzyme iNOS, peroxynitrite marker 3-nitrotyrosine, NFκB translocated to the nuclei, cytochrome C and Bcl-2 were not affected. NACA treatment decreased neuronal degeneration and apoptosis and increased levels of antioxidative enzyme MnSOD. The antiapoptotic effect was likely regulated by pathways other than cytochrome C. Therefore, NACA prevents brain tissue damage after focal penetrating TBI, warranting further studies towards a clinical application.

Diagnostics in patients with glutathione synthetase deficiency but without mutations in the exons of the GSS gene.

The synthesis of the ubiquitous tripeptide glutathione is impaired in patients with glutathione synthetase deficiency. The defect is inherited in an autosomal recessive manner, and the diagnosis is based on clinical, biochemical, and genetic criteria. In seven of our 30 index cases, however, no disease causing mutations could be identified in the coding exons or exon-intron boundaries of the glutathione synthetase gene GSS. These patients had severely decreased glutathione synthetase activities in lysates of cultured fibroblasts, and the levels of the enzyme were undetectable using a polyclonal antibody raised against human glutathione synthetase. RT-PCR mediated sequence analysis revealed previously not reported splice mutations in all patients. Thus, we conclude that in the investigation of patients with glutathione synthetase deficiency, and probably other genetic diseases as well, it might be time saving to initiate mutation analysis with sequencing of mRNA.

Age-dependent regulation of lipogenesis in human and rat adipocytes.

The regulation of adipocyte metabolism is of importance for adipose tissue growth and therefore also for the development of obesity. This study was designed to investigate the regulation of basal and insulin-induced lipogenesis, glucose transport, and glucose transporter protein expression in human and rat adipocytes from different age groups. The study included 21 infants, 21 children, nine adults, and 80 male weaned and 20 male adult Fischer rats. The lipogenesis experiments were performed under conditions at which glucose transport is rate limiting. Basal lipogenesis was approximately three times higher in infants and children than in adults, whereas insulin-induced lipogenesis was two times higher in infants than in children and adults. In rats, basal lipogenesis, insulin-induced lipogenesis, and insulin sensitivity were two times higher in weaned than in adult animals. Moreover, basal and insulin-induced glucose transport were two times higher in weaned than in adult rats. No differences were detected in GLUT1 or GLUT4 content between any of the age groups in human or in rat adipocytes. In conclusion, basal and insulin-stimulated lipogenesis are increased in adipocytes early in life. This may promote adipose tissue growth in early age. The data indicate that age-dependent variation in basal and insulin-stimulated lipogenesis is differently regulated.