Glutathione species and metabolomic prints in subjects with liver disease as biological markers for the detection of hepatocellular carcinoma.

BACKGROUND: The incidence of liver disease is increasing in USA. Animal models had shown glutathione species in plasma reflects liver glutathione state and it could be a surrogate for the detection of hepatocellular carcinoma (HCC). METHODS: The present study aimed to translate methods to the human and to explore the role of glutathione/metabolic prints in the progression of liver dysfunction and in the detection of HCC. Treated plasma from healthy subjects (n = 20), patients with liver disease (ESLD, n = 99) and patients after transplantation (LTx, n = 7) were analyzed by GC- or LC/MS. Glutathione labeling profile was measured by isotopomer analyzes of 2H2O enriched plasma. Principal Component Analyzes (PCA) were used to determined metabolic prints. RESULTS: There was a significant difference in glutathione/metabolic profiles from patients with ESLD vs healthy subjects and patients after LTx. Similar significant differences were noted on patients with ESLD when stratified by the MELD score. PCA analyses showed myristic acid, citric acid, succinic acid, l-methionine, d-threitol, fumaric acid, pipecolic acid, isoleucine, hydroxy-butyrate and glycolic, steraric and hexanoic acids were discriminative metabolites for ESLD-HCC+ vs ESLD-HCC- subject status. CONCLUSIONS: Glutathione species and metabolic prints defined liver disease severity and may serve as surrogate for the detection of HCC in patients with established cirrhosis.

Multiple mass isotopomer tracing of acetyl-CoA metabolism in Langendorff-perfused rat hearts: channeling of acetyl-CoA from pyruvate dehydrogenase to carnitine acetyltransferase.

We developed an isotopic technique to assess mitochondrial acetyl-CoA turnover (≈citric acid flux) in perfused rat hearts. Hearts are perfused with buffer containing tracer [(13)C2,(2)H3]acetate, which forms M5 + M4 + M3 acetyl-CoA. The buffer may also contain one or two labeled substrates, which generate M2 acetyl-CoA (e.g. [(13)C6]glucose or [1,2-(13)C2]palmitate) or/and M1 acetyl-CoA (e.g. [1-(13)C]octanoate). The total acetyl-CoA turnover and the contributions of fuels to acetyl-CoA are calculated from the uptake of the acetate tracer and the mass isotopomer distribution of acetyl-CoA. The method was applied to measurements of acetyl-CoA turnover under different conditions (glucose ± palmitate ± insulin ± dichloroacetate). The data revealed (i) substrate cycling between glycogen and glucose-6-P and between glucose-6-P and triose phosphates, (ii) the release of small excess acetyl groups as acetylcarnitine and ketone bodies, and (iii) the channeling of mitochondrial acetyl-CoA from pyruvate dehydrogenase to carnitine acetyltransferase. Because of this channeling, the labeling of acetylcarnitine and ketone bodies released by the heart are not proxies of the labeling of mitochondrial acetyl-CoA.

Multicentre results of stereotactic body radiotherapy for secondary liver tumours.

BACKGROUND: Surgical resection is the standard treatment for liver metastases, although for the majority of patients this is not possible. Stereotactic body radiotherapy (SBRT) is an alternative local-regional therapy. The purpose of this study was to evaluate the results of SBRT for secondary liver tumours from a combined multicentre database. METHODS: Variables from patients treated with SBRT from four Academic Medical Centres were entered into a common database. Local tumour control and 1-year survival rates were calculated. RESULTS: In total, 153 patients (91 women) 59 ± 8.4 years old with 363 metastatic liver lesions were treated with SBRT. The underlying primary tumour arose from gastrointestinal (GI), retroperitoneal and from extra-abdominal primaries in 56%, 8% and 36% of patients, respectively. Metastases, with a gross tumour volume (GTV) of 138.5 ± 126.8 cm(3) , were treated with a total radiation dose of 37.5 ± 8.2 Gy in 5 ± 3 fractions. The 1-year overall survival was 51% with an overall local control rate of 62% at a mean follow-up of 25.2 ± 5.9 months. A complete tumour response was observed in 32% of patients. Grade 3-5 adverse events were noted in 3% of patients. CONCLUSION: Secondary liver tumours treated with SBRT had a high rate of local control with a low incidence of adverse events.

4-Hydroxy-2(E)-nonenal (HNE) catabolism and formation of HNE adducts are modulated by ß oxidation of fatty acids in the isolated rat heart.

We previously reported that a novel metabolic pathway functionally catabolizes 4-hydroxy-2(E)-nonenal (HNE) via two parallel pathways, which rely heavily on ß-oxidation pathways. The hypothesis driving this report is that perturbations of ß oxidation will alter the catabolic disposal of HNE, favoring an increase in the concentrations of HNE and HNE-modified proteins that may further exacerbate pathology. This study employed Langendorff perfused hearts to investigate the impact of cardiac injury modeled by ischemia/reperfusion and, in a separate set of perfusions, the effects of elevated lipid (typically observed in obesity and type II diabetes) by perfusing with increased fatty acid concentrations (1mM octanoate). During ischemia, HNE concentrations doubled and the glutathione-HNE adduct and 4-hydroxynonanoyl-CoA were increased by 7- and 10-fold, respectively. Under conditions of increased fatty acid, oxidation to 4-hydroxynonenoic acid was sustained; however, further catabolism through ß oxidation was nearly abolished. The inhibition of HNE catabolism was not compensated for by other disposal pathways of HNE, rather an increase in HNE-modified proteins was observed. Taken together, this study presents a mechanistic rationale for the accumulation of HNE and HNE-modified proteins in pathological conditions that involve alterations to ß oxidation, such as myocardial ischemia, obesity, and high-fat diet-induced diseases.

Metabolism of γ-hydroxybutyrate in perfused rat livers.

GHB (γ-hydroxybutyrate) is both a neurotransmitter and a drug of abuse (date-rape drug). We investigated the catabolism of this compound in perfused rat livers. Using a combination of metabolomics and mass isotopomer analysis, we showed that GHB is metabolized by multiple processes, in addition to its previously reported metabolism in the citric acid cycle via oxidation to succinate. A substrate cycle operates between GHB and γ-aminobutyrate via succinic semialdehyde. Also, GHB undergoes (i) ß-oxidation to glycolyl-CoA+acetyl-CoA, (ii) two parallel processes which remove C-1 or C-4 of GHB and form 3-hydroxypropionate from C-2+C-3+C-4 or from C-1+C-2+C-3 of GHB, and (iii) degradation to acetyl-CoA via 4-phosphobutyryl-CoA. The present study illustrates the potential of the combination of metabolomics and mass isotopomer analysis for pathway discovery.

Multicenter results of stereotactic body radiotherapy (SBRT) for non-resectable primary liver tumors.

BACKGROUND: An excess of 100 000 individuals are diagnosed with primary liver tumors every year in USA but less than 20% of those patients are amenable to definitive surgical management due to advanced local disease or comorbidities. Local therapies to arrest tumor growth have limited response and have shown no improvement on patient survival. Stereotactic body radiotherapy (SBRT) has emerged as an alternative local ablative therapy. The purpose of this study was to evaluate the tumor response to SBRT in a combined multicenter database. STUDY DESIGN: Patients with advanced hepatocellular carcinoma (HCC, n = 21) or intrahepatic cholangiocarcinoma (ICC, n = 11) treated with SBRT from four Academic Medical Centers were entered into a common database. Statistical analyses were performed for freedom from local progression (FFLP) and patient survival. RESULTS: The overall FFLP for advanced HCC was 63% at a median follow-up of 12.9 months. Median tumor volume decreased from 334.2 to 135 cm(3) (p < 0.004). The median time to local progression was 6.3 months. The 1- and 2-years overall survival rates were 87% and 55%, respectively. Patients with ICC had an overall FFLP of 55.5% at a median follow-up of 7.8 months. The median time to local progression was 4.2 months and the six-month and one-year overall survival rates were 75% and 45%, respectively. The incidence of grade 1-2 toxicities, mostly nausea and fatigue, was 39.5%. Grade 3 and 4 toxicities were present in two and one patients, respectively. CONCLUSION: Higher rates of FFLP were achieved by SBRT in the treatment of primary liver malignancies with low toxicity.

Stereotactic body radiation therapy for nonresectable tumors of the pancreas.

BACKGROUND: Stereotactic body radiation therapy (SBRT) has emerged as a potential treatment option for local tumor control of primary malignancies of the pancreas. We report on our experience with SBRT in patients with pancreatic adenocarcinoma who were found not to be candidates for surgical resection. METHODS: The prospective database of the first 20 consecutive patients receiving SBRT for unresectable pancreatic adenocarcinomas and a neuroendocrine tumor under an IRB approved protocol was reviewed. Prior to SBRT, cylindrical solid gold fiducial markers were placed within or around the tumor endoscopically (n = 13), surgically (n = 4), or percutaneously under computerized tomography (CT)-guidance (n = 3) to allow for tracking of tumor during therapy. Mean radiation dose was 25 Gray (Gy) (range 22-30 Gy) delivered over 1-3 fractions. Chemotherapy was given to 68% of patients in various schedules/timing. RESULTS: Patients had a mean gross tumor volume of 57.2 cm(3) (range 10.1-118 cm(3)) before SBRT. The mean total gross tumor volume reduction at 3 and 6 mo after SBRT were 21% and 38%, respectively (P < 0.05). Median follow-up was 14.57 mo (range 5-23 mo). The overall rate of freedom from local progression at 6 and 12 mo were 88% and 65%. The probability of overall survival at 6 and 12 mo were 89% and 56%. No patient had a complication related to fiducial markers placement regardless of modality. The rate of radiation-induced adverse events was: grade 1-2 (11%) and grade 3 (16%). There were no grade 4/5 adverse events seen. CONCLUSION: Our preliminary results showed SBRT as a safe and likely effective local treatment modality for pancreatic primary malignancy with acceptable rate of adverse events.

Disturbances in the glutathione/ophthalmate redox buffer system in the woodchuck model of hepatitis virus-induced hepatocellular carcinoma.

Purpose. The incidence of liver tumors is rising in USA. The purpose of this study was to evaluate liver oxido-reductive status in the presence of chronic liver disease and hepatocellular carcinoma (HCC). Methods. Glutathione species and ophthalmate (OA) concentrations were measured by LC-MS in processed plasma and red blood cells (RBC) from infected Woodchuck with hepatitis virus (WHV). Blood samples were obtained from: (i) infected animals with tumors (WHV+/HCC+), (ii) infected animals without tumors (WHV+/HCC-) and (iii) healthy animals (WHC-/HCC-). Results. The concentration of reduced glutathione (GSH) and the ratio GSH/GSG were lower in plasma from WHV+/HCC+ animals when compared to WHV+/HCC- and WHV-/HCC- (P < 0.01). In contrast, the concentration of oxidized glutathione (GSSG) was found to be higher in plasma from WHV+/HCC+ animals when compared to WHV+/HCC- and WHV-/HCC- (P < 0.01). The Glutathione species and its ratio from the RBC compartment were similar among all groups. OA concentration in both plasma and RBC was significantly higher from WHV+/HCC+ when compared to WHV+/HCC- and WHV-/HCC- (P < 0.01). Conclusions. Disturbances of the glutathione redox buffer system and higher concentrations of OA were found in the WCV+/HCC+ animal model. The role of these compounds as biomarkers of early tumor development in patients with end stage liver disease remains to be determined.

Experiencia cubana con el uso terapéutico de células madre adultas / Cuban experience with the therapeutic use of adult stem cells

Las investigaciones básicas y clínicas realizadas en los últimos años sobre las células madre y sus posibilidades terapéuticas, son en la actualidad uno de los temas más excitantes de la medicina contemporánea. Ya se han obtenido importantes avances en el estudio y aplicación de las células madre adultas que muestran notables ventajas sobre las embrionarias, pues su manipulación resulta más simple, económica y se pueden obtener del propio individuo que va a ser tratado. Para la introducción en Cuba de la terapia celular regenerativa, en el Instituto de Hematología e Inmunología se seleccionaron como fuentes celulares las células madre adultas derivadas de la médula ósea y las movilizadas a la sangre periférica. Para facilitar la extensión del tratamiento a otros centros hospitalarios, se estandarizó una técnica para la movilización de las células madre hematopoyéticas a la sangre periférica, mediante un factor estimulador de colonias de granulocitos (Filgrastim, de producción nacional) y se desarrolló un método simple, económico y también más tolerable para los enfermos. De esta forma, se ha extendido la terapia celular a 6 provincias cubanas y hasta abril del año 2009 se habían tratado 563 casos con trasplante de células madre adultas autólogas, de los cuales el 81,7 por ciento corresponde a pacientes con enfermedades angiológicas, en los que se ha logrado disminuir significativamente la indicación de amputaciones mayores. También los resultados han sido muy prometedores en las lesiones óseas y procesos periodontales, entre otras enfermedades tratadas. Los resultados obtenidos hasta el momento se pueden considerar como un nuevo logro de la ciencia revolucionaria y de nuestros sistemas nacionales de salud y de ciencia y técnica. El método empleado es un proceder económico y factible para instituciones con recursos limitados

The basic and clinical researches carried out during past years on the stem cells and its therapeutic possibilities are at present times, one of the most interesting subjects of the contemporaneous medicine. There are advances in the study and application of adult stem cells showing remarkable advantages on the embryonic ones, since its handling is more simple, economic and they are obtained from the own subject to be treated. For the introduction in Cuba of the regenerative cellular therapy in the Institute of Hematology and Immunology the cellular sources selected were the adult stem cells derived from bone marrow and the mobilized ones to the peripheral blood. To make easy the expansion of treatment to other hospital centers, authors standardized a technique for the mobilization of the hematopoietic stem cells to peripheral blood using a granulocyte colony-stimulating factor (Filgrastim, of national production) developing a simple, economic and more tolerable method for patients. In this way, the cellular therapy has been expanded to 6 Cuban provinces and until April, 2009 562 cases with autologous adult stem cells transplant have been treated, from which the 81.7 percent to correspond to patients presenting with Angiology diseases with a significant reduction of major amputations. Also, the results have been very promising in the bone lesions and periodontal processes among other diseases treated. The results obtained until now may be considered as a new achievement of revolutionary science and of our national health systems and of science and technique. The method used is an economic and feasible procedure for the institutions with scarce resources

Experiencia cubana con el uso terapéutico de células madre adultas / Cuban experience with the therapeutic use of adult stem cells

Las investigaciones básicas y clínicas realizadas en los últimos años sobre las células madre y sus posibilidades terapéuticas, son en la actualidad uno de los temas más excitantes de la medicina contemporánea. Ya se han obtenido importantes avances en el estudio y aplicación de las células madre adultas que muestran notables ventajas sobre las embrionarias, pues su manipulación resulta más simple, económica y se pueden obtener del propio individuo que va a ser tratado. Para la introducción en Cuba de la terapia celular regenerativa, en el Instituto de Hematología e Inmunología se seleccionaron como fuentes celulares las células madre adultas derivadas de la médula ósea y las movilizadas a la sangre periférica. Para facilitar la extensión del tratamiento a otros centros hospitalarios, se estandarizó una técnica para la movilización de las células madre hematopoyéticas a la sangre periférica, mediante un factor estimulador de colonias de granulocitos (Filgrastim, de producción nacional) y se desarrolló un método simple, económico y también más tolerable para los enfermos. De esta forma, se ha extendido la terapia celular a 6 provincias cubanas y hasta abril del año 2009 se habían tratado 563 casos con trasplante de células madre adultas autólogas, de los cuales el 81,7 por ciento corresponde a pacientes con enfermedades angiológicas, en los que se ha logrado disminuir significativamente la indicación de amputaciones mayores. También los resultados han sido muy prometedores en las lesiones óseas y procesos periodontales, entre otras enfermedades tratadas. Los resultados obtenidos hasta el momento se pueden considerar como un nuevo logro de la ciencia revolucionaria y de nuestros sistemas nacionales de salud y de ciencia y técnica. El método empleado es un proceder económico y factible para instituciones con recursos limitados(AU)

The basic and clinical researches carried out during past years on the stem cells and its therapeutic possibilities are at present times, one of the most interesting subjects of the contemporaneous medicine. There are advances in the study and application of adult stem cells showing remarkable advantages on the embryonic ones, since its handling is more simple, economic and they are obtained from the own subject to be treated. For the introduction in Cuba of the regenerative cellular therapy in the Institute of Hematology and Immunology the cellular sources selected were the adult stem cells derived from bone marrow and the mobilized ones to the peripheral blood. To make easy the expansion of treatment to other hospital centers, authors standardized a technique for the mobilization of the hematopoietic stem cells to peripheral blood using a granulocyte colony-stimulating factor (Filgrastim, of national production) developing a simple, economic and more tolerable method for patients. In this way, the cellular therapy has been expanded to 6 Cuban provinces and until April, 2009 562 cases with autologous adult stem cells transplant have been treated, from which the 81.7 percent to correspond to patients presenting with Angiology diseases with a significant reduction of major amputations. Also, the results have been very promising in the bone lesions and periodontal processes among other diseases treated. The results obtained until now may be considered as a new achievement of revolutionary science and of our national health systems and of science and technique. The method used is an economic and feasible procedure for the institutions with scarce resources(AU)

Isotopomer enrichment assay for very short chain fatty acids and its metabolic applications.

The present work illustrated an accurate GC/MS measurement for the low isotopomer enrichment assay of formic acid, acetic acid, propionic aicd, butyric acid, and pentanoic acid. The pentafluorobenzyl bromide derivatives of these very short chain fatty acids have high sensitivity of isotopoic enrichment due to their low natural isotopomer distribution in negative chemical ionization mass spectrometric mode. Pentafluorobenzyl bromide derivatization reaction was optimized in terms of pH, temperature, reaction time, and the amount of pentafluorobenzyl bromide versus sample. The precision, stability, and accuracy of this method for the isotopomer analysis were validated. This method was applied to measure the enrichments of formic acid, acetic acid, and propionic acid in the perfusate from rat liver exposed to Krebs-Ringer bicarbonate buffer only, 0-1mM [3,4-(13)C(2)]-4-hydroxynonanoate, and 0-2mM [5,6,7-(13)C(3)]heptanoate. The enrichments of acetic acid and propionic acid in the perfusate are comparable to the labeling pattern of acetyl-CoA and propionyl-CoA in the rat liver tissues. The enrichment of the acetic acid assay is much more sensitive and precise than the enrichment of acetyl-CoA by LC-MS/MS. The reversibility of propionyl-CoA from succinyl-CoA was confirmed by the low labeling of M1 and M2 of propionic acid from [5,6,7-(13)C(3)]heptanoate perfusates.

Comparison of some reproductive characteristics of farmed and wild white shrimp males Litopenaeus vannamei (Decapoda: Penaeidae)

We rated some reproductive characteristics of white shrimp Litopenaeus vannamei (Boone, 1931) males using 46 farmed individuals (weighing 21.42 +/- 0.56 g) and 40 wild individuals (weighing 36.10 +/- 0.72 g). In farmed shrimps, spermatophore mean weight was 8.94 +/- 0.51 mg; total mean sperm count was 3.90 +/- 0.27 x 10(6) in each spermatophore; and mean percentage of normal sperm was 86.9 +/- 0.37%. In wild individuals, the respective values were 30.68 +/- 2.32 mg; 6.22 +/- 1.09 x 10(6); and 62.1 +/- 3.56%. In both groups, the differences between right and left spermatophore were not significant (p < 0.01). There were significant differences in spermatophore weight and percentage of normal sperm between farmed and wild shrimps; sperm counts differences, however, were not significant (p < 0.01). The relationship between spermatophore weight (Ws) and individual weight (Wo) was Ws (mg)=1.23 (Wo)-17.34 (r2=0.89), in farmed shrimps; and Ws (mg) = 2.57 (Wo)-60.04 (r2 = 0.64), in wild ones. In cultivated organisms, the relationship between sperm counts (Cs) and individual weight (Wo) was Cs (x 10(6)) = 1.13 * 10(-4*) (Wo) 3.361 (r2 = 0.85); and versus spermatophores weight was Cs (x 10(6)) = 0.439* (Ws) 0.984 (r2 = 0.90). In wild organisms, there was no correlation. The proportion of normal sperm ranged from 79.8 to 95.2 % (86.9 +/- 0.37%) and from 14.0 to 91.5% (62.1 +/- 2.52%), in farmed and wild shrimps, respectively. The most frequent abnormalities in both farm and wild animals were sperm without spike (49.3% and 76.6%, respectively) and irregular shape (35.8 % and 17.7 %). The less frequent occurrences were those of bent (10.2 % and 4.29%) and double spike (4.7% and 1.41%).

Comparison of some reproductive characteristics of farmed and wild white shrimp males Litopenaeus vannamei (Decapoda: Penaeidae).

We rated some reproductive characteristics of white shrimp Litopenaeus vannamei (Boone, 1931) males using 46 farmed individuals (weighing 21.42 +/- 0.56 g) and 40 wild individuals (weighing 36.10 +/- 0.72 g). In farmed shrimps, spermatophore mean weight was 8.94 +/- 0.51 mg; total mean sperm count was 3.90 +/- 0.27 x 10(6) in each spermatophore; and mean percentage of normal sperm was 86.9 +/- 0.37%. In wild individuals, the respective values were 30.68 +/- 2.32 mg; 6.22 +/- 1.09 x 10(6); and 62.1 +/- 3.56%. In both groups, the differences between right and left spermatophore were not significant (p < 0.01). There were significant differences in spermatophore weight and percentage of normal sperm between farmed and wild shrimps; sperm counts differences, however, were not significant (p < 0.01). The relationship between spermatophore weight (Ws) and individual weight (Wo) was Ws (mg)=1.23 (Wo)-17.34 (r2=0.89), in farmed shrimps; and Ws (mg) = 2.57 (Wo)-60.04 (r2 = 0.64), in wild ones. In cultivated organisms, the relationship between sperm counts (Cs) and individual weight (Wo) was Cs (x 10(6)) = 1.13 * 10(-4*) (Wo) 3.361 (r2 = 0.85); and versus spermatophores weight was Cs (x 10(6)) = 0.439* (Ws) 0.984 (r2 = 0.90). In wild organisms, there was no correlation. The proportion of normal sperm ranged from 79.8 to 95.2 % (86.9 +/- 0.37%) and from 14.0 to 91.5% (62.1 +/- 2.52%), in farmed and wild shrimps, respectively. The most frequent abnormalities in both farm and wild animals were sperm without spike (49.3% and 76.6%, respectively) and irregular shape (35.8 % and 17.7 %). The less frequent occurrences were those of bent (10.2 % and 4.29%) and double spike (4.7% and 1.41%).

Escherichia coli K-12 undergoes adaptive evolution to achieve in silico predicted optimal growth.

Annotated genome sequences can be used to reconstruct whole-cell metabolic networks. These metabolic networks can be modelled and analysed (computed) to study complex biological functions. In particular, constraints-based in silico models have been used to calculate optimal growth rates on common carbon substrates, and the results were found to be consistent with experimental data under many but not all conditions. Optimal biological functions are acquired through an evolutionary process. Thus, incorrect predictions of in silico models based on optimal performance criteria may be due to incomplete adaptive evolution under the conditions examined. Escherichia coli K-12 MG1655 grows sub-optimally on glycerol as the sole carbon source. Here we show that when placed under growth selection pressure, the growth rate of E. coli on glycerol reproducibly evolved over 40 days, or about 700 generations, from a sub-optimal value to the optimal growth rate predicted from a whole-cell in silico model. These results open the possibility of using adaptive evolution of entire metabolic networks to realize metabolic states that have been determined a priori based on in silico analysis.

Miastenia gravis: presentación de tres casos / Myasthenia gravis: report of 3 cases

Se informan de tres casos de pacientes con miastenia gravis y embarazo. Todas las pacientes recibieron un régimen de medicación anticolineasterásica con buena respuesta terapéutica. En cada paciente se efectuaron estudios ecosonográficos y cardiotocográficos seriados. La prueba sin estrés (PSS) fue normal en todos los casos. El estudio ultrasonográfico mostró un feto con retraso en el crecimiento intrauterino (RCIU) que desarrolló miastenia gravis neonatal (MGN). Dos pacientes requirieron operación cesárea. La paciente restante inició trabajo de parto espontáneo que finalizó en parto eutócico. Dos fetos nacieron con buenas puntuaciones de Apgar y no desarrollaron MGN. En todas las pacientes se efectuó bloqueo epidural (BED) como método de analgesia-anestesia