RESUMEN
OBJECTIVE: To conduct patient and public involvement (PPI) to gain insight into the experience of healthy eating and weight management advice during pregnancy. DESIGN: PPI in the planning and development of health interventions, aiming to ensure patient-centred care. Optimum nutrition and weight management are vital for successful pregnancy outcomes, yet many services report poor attendance and engagement. SETTING: Community venues in Liverpool and Ulster (UK). PARTICIPANTS: Two PPI representatives were involved in all aspects of the study: design, interview questions, recruitment and collection/analysis of feedback. INTERVENTION: Feedback was collected via note taking during group discussions, two in Liverpool (n = 10 & 5); two in Ulster (n = 7 & 9) and an interview (n = 1, in Ulster). MAIN OUTCOME MEASURES: Transcript data were collated and thematic analysis was applied in analysis. RESULTS: Thematic analysis identified three themes: (i) weight gain is inevitable in pregnancy; (ii) healthy eating advice is important but currently lacks consistency and depth and (iii) expectations regarding the type of knowledge/support. CONCLUSIONS: PPI provides opportunity to enhance research design and offers valuable insight towards the needs of healthcare users. Pregnant women want positive health messages, with a focus on what they can/should do, rather than what they should not do. Midwives need to consider their communication with pregnant women, to ensure that their unique relationship is maintained, especially when the topics of diet and weight management are addressed. A well-designed digital intervention could improve access to pregnancy-specific nutrition information; empowering midwives to communicate patient-centred, healthy eating messages with confidence. This has the potential to change dietary and weight management behaviour in pregnant women.
Asunto(s)
Dieta Saludable , Ganancia de Peso Gestacional , Partería/métodos , Embarazo , Adulto , Participación de la Comunidad , Femenino , Promoción de la Salud/métodos , Humanos , Participación del Paciente , Atención Dirigida al Paciente/métodos , Atención Prenatal/métodos , Reino UnidoRESUMEN
STUDY DESIGN: A prospective double blind cross over trial of intravenous 4-Aminopyridine (4-AP). OBJECTIVE: To determine the efficacy of this drug in the treatment of spinal cord injured (SCI) patients for neurologic impairment, pain and spasticity. SETTING: The post anesthesia care unit (PACU) of a tertiary care acute hospital. METHODS: Twelve paraplegic patients were enrolled in a double blind cross over intravenous trial of 4-Aminopyridine (4-AP). Thirty milligrams of 4-AP or placebo were administered over a 2 h period. Patients were serially examined during and after the infusion clinically for pain, sensorimotor function, hypertonicity and motor control using electromyography (EMG). Samples of blood and cerebrospinal fluid (CSF) were also analyzed at similar intervals. RESULTS: Despite penetration of 4-AP into the CSF, no significant differences were noted in the clinical and EMG parameters at the times measured. Individual changes in sensory function were reported by some patients in both the placebo and 4-AP trials, however mean values were not robust. Frequently, patients complained of unpleasant symptoms during the 4-AP infusion. CONCLUSION: The intravenous route may not be the best way to administer this drug as no short term benefits were observed.
Asunto(s)
4-Aminopiridina/administración & dosificación , Traumatismos de la Médula Espinal/tratamiento farmacológico , 4-Aminopiridina/efectos adversos , 4-Aminopiridina/líquido cefalorraquídeo , 4-Aminopiridina/uso terapéutico , Adulto , Anciano , Enfermedad Crónica , Estudios Cruzados , Método Doble Ciego , Electromiografía , Femenino , Humanos , Inyecciones Intravenosas , Masculino , Persona de Mediana Edad , Movimiento , Músculos/fisiopatología , Sistema Nervioso/fisiopatología , Dolor/fisiopatología , Estudios Prospectivos , Sensación , Traumatismos de la Médula Espinal/fisiopatologíaRESUMEN
BACKGROUND: The cause of severe acquired hyperammonemia, an uncommon but often fatal complication of organ transplantation and chemotherapy for cancer, is obscure. OBJECTIVE: To test the hypothesis that liver glutamine synthetase deficiency may explain hyperammonemia in patients who have had organ transplantation or are receiving chemotherapy. DESIGN: Case report. PATIENTS: Two patients who had fatal hyperammonemia after orthotopic lung transplantation. MEASUREMENTS: Liver tissue was analyzed to determine the activities of two urea cycle enzymes and glutamine synthetase. Western blot assays for hepatic glutamine synthetase were performed to determine whether glutamine synthetase deficiency resulted from reduced enzyme levels. RESULTS: Activities of carbamoyl phosphate synthetase I and ornithine carbamoyltransferase in the liver were normal. The activity of hepatic glutamine synthetase was markedly reduced (in patient 1, 12% of the mean value in controls; in patient 2, 28% of the mean value in controls), and a concomitant reduction in the amount of glutamine synthetase protein was observed. CONCLUSION: Hyperammonemia after transplantation was associated with hepatic glutamine synthetase deficiency in two patients, but the causal relation between these two conditions must be further studied.
Asunto(s)
Amoníaco/sangre , Glutamato-Amoníaco Ligasa/deficiencia , Hígado/enzimología , Trasplante de Pulmón/efectos adversos , Carbamoil-Fosfato Sintasa (Amoniaco)/metabolismo , Resultado Fatal , Femenino , Humanos , Persona de Mediana Edad , Ornitina Carbamoiltransferasa/metabolismoRESUMEN
The crystal structure of Escherichia coli ornithine transcarbamoylase (OTCase, EC 2.1.3.3) complexed with the bisubstrate analog N-(phosphonacetyl)-L-ornithine (PALO) has been determined at 2.8-A resolution. This research on the structure of a transcarbamoylase catalytic trimer with a substrate analog bound provides new insights into the linkages between substrate binding, protein-protein interactions, and conformational change. The structure was solved by molecular replacement with the Pseudomonas aeruginosa catabolic OTCase catalytic trimer (Villeret, V., Tricot, C., Stalon, V. & Dideberg, O. (1995) Proc. Natl. Acad. Sci. USA 92, 10762-10766; Protein Data Bank reference pdb 1otc) as the model and refined to a crystallographic R value of 21.3%. Each polypeptide chain folds into two domains, a carbamoyl phosphate binding domain and an L-ornithine binding domain. The bound inhibitor interacts with the side chains and/or backbone atoms of Lys-53, Ser-55, Thr-56, Arg-57, Thr-58, Arg-106, His-133, Asn-167, Asp-231, Met-236, Leu-274, Arg-319 as well as Gln-82 and Lys-86 from an adjacent chain. Comparison with the unligated P. aeruginosa catabolic OTCase structure indicates that binding of the substrate analog results in closure of the two domains of each chain. As in E. coli aspartate transcarbamoylase, the 240s loop undergoes the largest conformational change upon substrate binding. The clinical implications for human OTCase deficiency are discussed.
Asunto(s)
Ornitina Carbamoiltransferasa/química , Conformación Proteica , Secuencia de Aminoácidos , Cristalografía por Rayos X , Escherichia coli/enzimología , Humanos , Datos de Secuencia Molecular , Relación Estructura-Actividad , Especificidad por SustratoRESUMEN
Although many mutations in the ornithine transcarbamylase gene have been correlated with 'late onset' of hyperammonemia in patients, the effects of these mutations on enzyme function are largely unknown. Three recurrent mutations (R40H, R277W and R277Q) found in patients with 'late onset' disease were incorporated into 'mature' human ornithine transcarbamylase cDNA and overexpressed in Escherichia coli. The three recombinant mutant enzymes were purified to homogeneity on an affinity column and their biochemical characteristics were compared to the wild type enzyme. The R277W and R277Q mutants display markedly reduced affinity for L-ornithine, loss of substrate inhibition, alkaline shift of pH optimum, and reduced thermal stability compared to the wild type enzyme. These differences, particularly the reduced affinity for L-ornithine, are sufficient to account for their biochemical effects. In contrast, the 'mature' R40H mutant was biochemically indistinguishable from the wild type enzyme in vitro.
Asunto(s)
Enfermedad por Deficiencia de Ornitina Carbamoiltransferasa , Edad de Inicio , Humanos , Mutación , Ornitina Carbamoiltransferasa/química , Ornitina Carbamoiltransferasa/genética , Ornitina Carbamoiltransferasa/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismoRESUMEN
Ornithine Transcarbamylase Deficiency, an X-linked disorder, is the most common cause of inherited urea cycle disorders. Approx. 90 mutations that produce reduced levels of ornithine transcarbamylase (OTCase) activity have been identified in patients [Tuchman (1993) Hum. Mutat. 2, 174-178; Tuchman and Plante (1995) Hum. Mutat. 5, 293-295]. A model of the three-dimensional structure of OTCase, developed on the basis of its homology to the catalytic subunit of Escherichia coli aspartate transcarbamylase (ATCase) [Tuchman, Morizono, Reish, Yuan and Allewell (1995) J. Med. Genet. 32, 680-688], and in good agreement with the crystal structure of Pseudomonas aeruginosa OTCase [Villeret, Tricot, Stalon and Dideberg (1995) Proc. Natl. Acad. Sci. U.S.A. 92, 10762-10766], indicates that many mutations that produce severe clinical symptoms are at the active site or buried in the interior of the protein. However, one of the few recurrent mutations, R277W, an alteration that produces a milder phenotype of ornithine transcarbamylase deficiency, is located in the model in a loop remote from the active site that is analogous to a similar loop (the 240's loop, a flexible loop of the catalytic chain of Escherichia coli aspartate transcarbamylase, comprised of residues 230-250) of ATCase. Human wild-type OTCase and the R277W mutant have been cloned and overexpressed in E. coli and a rapid and efficient purification method utilizing the bisubstrate analogue, Ndelta-(phosphonacetyl)-L-ornithine, has been developed and used to purify both proteins. Gel chromatography indicates both are trimeric. The pH dependence of the kinetic parameters of the wild-type enzyme is similar to that of E. coli OTCase [Kuo, Herzberg and Lipscomb (1985) Biochemistry 24, 4754-4761], suggesting that its catalytic mechanism is similar, although its maximal activity is approx. 10-fold less. Compared with the wild-type, the R277W mutant has nearly 70-fold lower affinity for L-ornithine, shows no substrate inhibition, and its thermal stability is reduced by 5 degrees C. Its reduced affinity for L-ornithine, which in turn results in lower activity at physiological concentrations of ornithine, as well as its reduced stability, may contribute to the clinical effects that it produces.
Asunto(s)
Amoníaco/sangre , Enfermedades Genéticas Congénitas/enzimología , Mutación , Ornitina Carbamoiltransferasa/genética , Cromatografía de Afinidad , Clonación Molecular , Estabilidad de Enzimas , Enfermedades Genéticas Congénitas/genética , Humanos , Concentración de Iones de Hidrógeno , Modelos Moleculares , Ornitina Carbamoiltransferasa/metabolismo , Proteínas RecombinantesRESUMEN
Escherichia coli strains capable of enhanced synthesis of arginine and urea were produced by derepression of the arginine regulon and simultaneous overexpression of the E. coli carAB and argI genes and the Bacillus subtilis rocF gene. Plasmids expressing carAB driven by their natural promoters were unstable. Therefore, E. coli carAB and argI genes with and without the B. subtilis rocF gene were constructed as a single operon under the regulation of the inducible promoter ptrc. Arginine operator sequences (Arg boxes) from argI were also cloned into the same plasmids for titration of the arginine repressor. Upon overexpression of these genes in E. coli strains, very high carbamyl phosphate synthetase, ornithine transcarbamylase, and arginase catalytic activities were achieved. The biosynthetic capacity of these engineered bacteria when overexpressing the arginine biosynthetic enzymes was 6- to 16-fold higher than that of controls but only if exogenous ornithine was present (ornithine was rate limiting). Overexpression of arginase in bacteria with a derepressed arginine biosynthetic pathway resulted in a 13- to 20-fold increase in urea production over that of controls with the parent vector alone; in this situation, the availability of carbamyl phosphate was rate limiting.
Asunto(s)
Arginina/biosíntesis , Escherichia coli/genética , Escherichia coli/metabolismo , Urea/metabolismo , Arginasa/genética , Arginasa/metabolismo , Arginina/genética , Bacillus subtilis/genética , Secuencia de Bases , Carbamoil-Fosfato Sintasa (Glutamina-Hidrolizante)/genética , Carbamoil-Fosfato Sintasa (Glutamina-Hidrolizante)/metabolismo , Clonación Molecular , Cartilla de ADN/genética , ADN Bacteriano/genética , Escherichia coli/enzimología , Expresión Génica , Genes Bacterianos , Ingeniería Genética , Operón , Ornitina Carbamoiltransferasa/genética , Ornitina Carbamoiltransferasa/metabolismo , Plásmidos/genética , Regiones Promotoras Genéticas , RegulónRESUMEN
A bacterial expression system has been engineered for human glutamine synthetase (EC 6.3.1.2) that produces approximately 60 mg of enzyme (20% of the bacterial soluble protein) and yields approx. 8 mg of purified enzyme per litre of culture. The recombinant enzyme was purified 5-fold to apparent homogeneity and characterized. It has a subunit molecular mass of approx. 45000 Da. The Vmax value obtained using a radioactive assay with ammonia and l-[G-3H]glutamic acid as substrates was 15.9 micromol/min per mg, 40% higher than that obtained in the colorimetric assay (9.9 micromol/min per mg) with hydroxylamine replacing ammonia as a substrate. Km values for glutamate were 3.0 mM and 3.5 mM, and for ATP they were 2.0 mM and 2. 9 mM for the radioactive and spectrophotometric assays respectively. The Km for ammonia in the radioactive assay was 0.15 mM. The midpoint of thermal inactivation was 49.7 degrees C. Hydroxylamine, Mg(II) and Mg(II)-ATP stabilized the enzyme against thermal inactivation, whereas ATP promoted inactivation. The pure enzyme is stable for several months in storage and provides a source for additional studies, including X-ray crystallography.
Asunto(s)
Glutamato-Amoníaco Ligasa/biosíntesis , Glutamato-Amoníaco Ligasa/genética , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/aislamiento & purificación , Clonación Molecular , Activación Enzimática , Escherichia coli/genética , Vectores Genéticos/genética , Glutamato-Amoníaco Ligasa/química , Glutamato-Amoníaco Ligasa/aislamiento & purificación , Calor , Humanos , Cinética , Proteínas Recombinantes/química , Especificidad por Sustrato/genéticaRESUMEN
A specific method for quantification of methylmalonic acid (MMA) from urine samples dried onto filter paper is described. The method involves stable isotope dilution gas chromatography-mass spectrometry with [methyl-2H3]-MMA as the internal standard. MMA is stable in dry paper samples stored at room temperature for at least 2 weeks. The extraction efficiency of MMA from paper was 56-58%. The concentration of urinary MMA in dried filter paper specimens from 190 normal controls was 1.21 +/- 1.34 (mean +/- SD) mmol/mol of urinary creatinine. Age-related reference values are also reported. The concentrations, normalized to the urinary creatinine concentration, decrease with age. The applicability of this method to rapid screening for cobalamin (vitamin B12)-related disorders and methylmalonic aciduria is demonstrated.
Asunto(s)
Cromatografía de Gases y Espectrometría de Masas/métodos , Ácido Metilmalónico/orina , Papel , Adolescente , Adulto , Errores Innatos del Metabolismo de los Aminoácidos/orina , Niño , Preescolar , Deuterio , Cromatografía de Gases y Espectrometría de Masas/estadística & datos numéricos , Humanos , Lactante , Valores de Referencia , Deficiencia de Vitamina B 12/orinaRESUMEN
A rapid, sensitive, and specific method for quantification of orotic acid from dried filter-paper urine samples is described. The method involves stable isotope dilution with 1,3-[15N2]orotic acid analysis by gas chromatography-mass spectrometry. The assay is sufficiently sensitive to be used with solvent extraction techniques commonly used for urinary organic acid analysis. Extraction efficiencies of both native and isotopic orotic acid from dried filter paper and from water were 31% and 28%, respectively. The concentration of orotic acid in dried filter-paper urine specimens from 50 healthy controls was 1.1 +/- 0.67 (mean +/- SD) mmol/mol of urinary creatinine. The same 50 urine samples, analyzed directly from a 5-mL aliquot of liquid urine, gave values of 0.93 +/- 0.51. The correlation coefficient between the results obtained by the two different collection methods was 0.87. Age-related reference values in filter-paper samples are also reported. The concentrations, which are normalized to urinary creatinine, decrease with age. This method is applicable to rapid screening for urea cycle disorders and may also be used for carrier testing of ornithine transcarbamylase deficiency.
Asunto(s)
Técnicas de Dilución del Indicador , Ácido Orótico/orina , Papel , Adolescente , Adulto , Envejecimiento/orina , Niño , Preescolar , Creatinina/orina , Cromatografía de Gases y Espectrometría de Masas , Humanos , Lactante , Recién Nacido , Espectrometría de Masas , Persona de Mediana Edad , Isótopos de Nitrógeno , Enfermedad por Deficiencia de Ornitina Carbamoiltransferasa , Valores de ReferenciaAsunto(s)
Mutación , Ornitina Carbamoiltransferasa/genética , Adolescente , Adulto , Errores Innatos del Metabolismo de los Aminoácidos/enzimología , Errores Innatos del Metabolismo de los Aminoácidos/genética , Amoníaco/sangre , Secuencia de Bases , Niño , Preescolar , Codón/genética , ADN/genética , Análisis Mutacional de ADN , ADN de Cadena Simple/genética , Femenino , Humanos , Lactante , Recién Nacido , Masculino , Datos de Secuencia Molecular , Enfermedad por Deficiencia de Ornitina Carbamoiltransferasa , Fenotipo , Polimorfismo GenéticoRESUMEN
Urinary methylmalonic acid (MMA) and 4-hydroxyphenyllactic acid (HPL) have been determined in 3345 and 2498 3-week-old newborns, respectively. Urine was collected onto filter paper and assayed by a rapid gas chromatography-mass spectrometry method. Forty-six infants (1.7%) had elevated MMA levels (greater than 58.5 micrograms/mg creatinine, means + 5 SD) and 31 infants (1.2%) had elevated levels of HPL (greater than 87.7 micrograms/mg creatinine, means + 5 SD). Fifteen infants with elevated values of MMA were retested from one to several months after the first test. In 12 infants the MMA levels normalized, while in the remaining three, elevated methylmalonic acid persisted. Nine infants with elevated values of HPL were retested, and in all except one, HPL levels normalized. No access to clinical evaluation of the infants was available. Transient methylmalonic aciduria and transient tyrosyluria affect a substantial number of infants and the clinical significance of this phenomenon has yet to be determined.
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Ácido Metilmalónico/orina , Fenilacetatos/orina , Humanos , Recién Nacido , Tamizaje NeonatalRESUMEN
Screening urine for inherited and acquired organic acidurias in newborns has the potential of preventing severe disease, mental retardation, and death. A method for screening dried urine filter paper samples for acidic markers of at least 20 different metabolic conditions has been developed. These conditions include, among others, maple syrup urine disease; methylmalonic, propionic, isovaleric, glutaric, and hydroxymethylglutaric acidurias; methylcrotonylglycinuria; medium-chain acyl-CoA dehydrogenase deficiency; inherited vitamin responsive disorders B12, biotin, B2), and acquired deficiencies of these vitamins. The preparation of the urine extract is identical to the method we use to screen infants for neuroblastoma. Screening is based on a highly sensitive and specific determination of eight organic acid markers by an automated computerized gas chromatography mass spectrometry system using selected ion monitoring. The markers used for screening are methylmalonic acid, 2-hydroxyisocaproic acid, glutaric acid, propionylglycine, isovalerylglycine, 3-methylcrotonylglycine, hexanoylglycine, and 3-phenylpropionylglycine. The extraction efficiencies of these acids from dried filter paper were similar to extraction from water, ranging from about 40% to 80%, except for propionylglycine which showed a low extraction efficiency of 11-13%. The stability of these acids on filter paper exposed to room air and temperature over a period of 15 d was adequate for the use of this collection method for organic aciduria screening. Normal levels, adjusted to urinary creatinine, were established for these acids in 519 urine filter paper samples obtained from 3-wk-old newborns. This screening method was tested on samples obtained from 12 patients with known organic acidurias including stored urine filter paper collected at 3-wk of age from two infants later found to have organic acidurias.
Asunto(s)
Aminoácidos/orina , Glutaratos/orina , Tamizaje Masivo/métodos , Ácido Metilmalónico/orina , Cromatografía de Gases y Espectrometría de Masas , Humanos , Recién Nacido , Discapacidad Intelectual/orina , Tiras ReactivasRESUMEN
A large neuroblastoma screening study was recently started in the province of Quebec, Canada. This project, a collaboration between the Quebec Network for Genetic Medicine and the University of Minnesota, is studying the impact of screening infants for the preclinical detection of neuroblastoma on the population-based mortality caused by this tumor. All infants born in Quebec during a 5-year period will be screened twice, at 3 weeks and at 6 months. Urinary homovanillic acid and vanillylmandelic acid determination from dried filter paper samples is used for screening. Initial qualitative screening is done by means of thin-layer chromatography with confirmatory quantitative screening by gas chromatography-mass spectrometry (GC-MS). During the initial 6 months of 3-week screening, 41,673 neonates (92% compliance rate) were screened and 10.6% of them were tested also by GC-MS. Nine of these neonates had positive results on two GC-MS tests and were referred for evaluation to rule out the presence of neuroblastoma. Four had the tumor, 1 had a calcified adrenal gland, and 4 had no tumor detected. Three additional neonates had clinical diagnosis of neuroblastoma before they reached the screening age of 3 weeks. A neuroblastoma that did not secrete homovanillic acid or vanillylmandelic acid was diagnosed clinically in 1 additional patient who tested negative by screening.