RESUMEN
Normal human peripheral blood mononuclear cells (MNCs), particularly T lymphocytes (T cells), are a rich source of granulocyte-macrophage colony-stimulating factor (GM-CSF). Glucocorticoids are known to inhibit GM-CSF production in in vitro cultures of a human fibroblast cell line and in normal human blood monocytes and alveolar macrophages. To determine whether glucocorticoids also inhibit GM-CSF production from normal human MNCs and T cells, we set up cultures of normal human MNCs and T cells in a liquid system in the presence and absence of 5, 50, and 250 microg/dL of hydrocortisone, and an hour later, a constant dose of 50-ng/mL Escherichia coli lipopolysaccharide (LPS) or 10-microg/mL phytohemagglutinin (PHA) was added. After three days, cell counts and GM-CSF levels were determined. Administering 50- and 250-microg/dL hydrocortisone decreased lymphocyte recovery from MNC cultures with LPS (p < or = 0.01), and 250 microg/dL of hydrocortisone decreased lymphocyte recovery from MNC and T-cell cultures with PHA (p < or = 0.03). The amount of GM-CSF produced from PHA-stimulated MNCs was about 100-fold higher than that produced from LPS-stimulated MNCs. The magnitude of GM-CSFs produced in MNC and T-cell cultures stimulated by PHA was comparable (p=0.88). Administering hydrocortisone at 5, 50, and 250 pg/dL decreased GM-CSF production (p < 0.003) in LPS- or PHA-stimulated MNC cultures and in PHA-stimulated T-cell cultures. PHA (not tested with LPS)-stimulated GM-CSF messenger RNA (mRNA) expression was blocked by hydrocortisone. These results indicate that lower concentrations of hydrocortisone inhibit GM-CSF production from normal human blood MNCs and T cells entirely by inhibiting the expression of GM-CSF mRNA, and higher concentrations of hydrocortisone inhibit by a combined effect of inhibiting the expression of GM-CSF mRNA and decreasing the lymphocyte count.
Asunto(s)
Complejo CD3/análisis , Factor Estimulante de Colonias de Granulocitos y Macrófagos/biosíntesis , Hidrocortisona/farmacología , Leucocitos Mononucleares/metabolismo , Linfocitos T/metabolismo , Apoptosis , Células Cultivadas , Escherichia coli , Expresión Génica/efectos de los fármacos , Factor Estimulante de Colonias de Granulocitos y Macrófagos/genética , Humanos , Hidrocortisona/administración & dosificación , Leucocitos Mononucleares/efectos de los fármacos , Lipopolisacáridos/farmacología , Fitohemaglutininas/farmacología , ARN Mensajero/metabolismo , Linfocitos T/efectos de los fármacos , Linfocitos T/inmunologíaRESUMEN
This study was undertaken to determine whether measurements of serum total homocysteine (Hcys) and bound B12 absorption are useful in determining which patients with low- or low-normal levels of serum B12 are B12 deficient. In 40 patients with low or borderline serum levels of B12, food-bound B12 absorptions were determined using a body counter in an iron room, and were related to serum total Hcys levels. Food-bound B12 absorption was decreased in 16 patients and in an additional four, absorption of the free vitamin was also decreased. Homocysteine levels were elevated in four of the 16; in three of the four who had both decreased bound and free B12 absorptions, Hcys was elevated. If elevation of the Hcys level indicates tissue deficiency of B12, the 75% incidence of normal levels of Hcys in these patients with low food-bound B12 absorptions suggests the existence of a cohort of patients who may be at risk to develop, but have not yet developed, B12 deficiency. Only long term follow-up will reveal how many ultimately will become B12 deficient.
Asunto(s)
Homocisteína/sangre , Deficiencia de Vitamina B 12/sangre , Vitamina B 12/farmacocinética , Adulto , Anciano , Anciano de 80 o más Años , Recuento de Células Sanguíneas , Femenino , Ácido Fólico/sangre , Alimentos , Gastrinas/sangre , Humanos , Absorción Intestinal , Masculino , Persona de Mediana Edad , Vitamina B 12/sangreRESUMEN
OBJECTIVES: To address the need for clinical preventive services for 11- to 21-year-old males and females and provide cost estimates for those services under a fee-for-service system. Preventive services include screening, health promotion, and immunizations. DESIGN: The prevalence of adolescent morbidities was derived from national surveys. Estimated costs of these morbidities were obtained from published data and adjusted for 1992 dollars using the Consumer Price Index. The estimated costs of preventive services for adolescents under a fee-for-service system were derived from a 1993 survey of nine Blue Cross and Blue Shield plans and four insurance companies. MAIN OUTCOME MEASURES: The cost of adolescent morbidities includes only direct medical costs for a single year and excludes long-term and indirect costs. The cost of clinical preventive services is calculated at 100% participation levels. RESULTS: Each year, an estimated $33.5 billion is spent on medical treatment for select adolescent morbidities, approximately $859 per adolescent per year; this is a conservative estimate. The average cost of clinical preventive services per adolescent per year would be approximately $130 in a fee-for-service system, although these are not entirely "new" costs because payers already incur screening costs for some conditions. CONCLUSION: The cost-effectiveness of clinical interventions for various health risk behaviors among adolescents is unknown. It appears that preventive interventions would have to eliminate 15% of adolescent morbidities overall to break even in economic terms.
Asunto(s)
Medicina del Adolescente/economía , Atención Integral de Salud/economía , Servicios Preventivos de Salud/economía , Accidentes de Tránsito/prevención & control , Síndrome de Inmunodeficiencia Adquirida/prevención & control , Adolescente , Alcoholismo/prevención & control , Niño , Atención Integral de Salud/estadística & datos numéricos , Femenino , Seropositividad para VIH/transmisión , Promoción de la Salud , Encuestas Epidemiológicas , Humanos , Inmunización , Masculino , Servicios de Salud Mental/normas , Servicios de Salud Mental/estadística & datos numéricos , Embarazo , Embarazo en Adolescencia , Servicios Preventivos de Salud/estadística & datos numéricos , Asunción de Riesgos , Enfermedades de Transmisión Sexual/prevención & controlRESUMEN
Astrocytes derived from human brain tissue secreted a single cobalamin (vitamin B12, Cbl) binding protein over a 4 day period in culture. Cycloheximide reversibly inhibited the release, and the binding protein was identified as transcobalamin II (TCII) based on molecular size, reaction with anti-human TCII antiserum, precipitation with 2.0 M ammonium sulfate and its ability to bind radioactive cyanocobalamin. It also enhanced the cellular incorporation of the vitamin. Our data show that cultured cells from human brain synthesize and secrete TCII and suggests that at least some of the TCII known to be present in cerebrospinal fluid may originate from within the central nervous system.
Asunto(s)
Astrocitos/metabolismo , Química Encefálica/fisiología , Encéfalo/citología , Transcobalaminas/biosíntesis , Astrocitos/efectos de los fármacos , Astrocitoma/metabolismo , Química Encefálica/efectos de los fármacos , Neoplasias Encefálicas/metabolismo , Medios de Cultivo , Cicloheximida/farmacología , Humanos , Unión Proteica/efectos de los fármacos , Transcobalaminas/inmunología , Transcobalaminas/farmacología , Células Tumorales Cultivadas , Vitamina B 12/metabolismoRESUMEN
We investigated the nitrous oxide-induced inactivation of methionine synthase and the concurrent homocysteine (Hcy) export in mutant fibroblasts with defects in the homocysteine catabolizing enzyme, cystathionine beta-synthase, or in methionine synthase, which carries out homocysteine remethylation. The fibroblasts were incubated in various concentrations of methionine to create conditions favoring methionine conservation or catabolism. In cystathionine beta-synthase-deficient cells, high medium methionine partly protected the enzyme against inactivation, as previously found in normal fibroblasts. The Hcy export rate at low methionine levels was low (0.2-0.6 nmol/h/10(6) cells), and increased 2-3-fold at high methionine levels. Nitrous oxide enhanced Hcy export rate at low methionine, so that in the presence of nitrous oxide, the Hcy export became less dependent of methionine. In cb1G cells, the enzyme inactivation was moderate and independent of medium methionine. The Hcy export rate was intermediate (0.5-0.8 nmol/h/10(6) cells) at low methionine levels, and increased moderately (< 2-fold) at high methionine levels or following nitrous oxide exposure. In cb1E mutants, the enzyme activity was not affected by nitrous oxide, and the Hcy export was high (0.8-1.6 nmol/h/10(6) cells) and independent of methionine and nitrous oxide. These data suggest that Hcy remethylation and cystathionine beta-synthase activity are major determinants of Hcy export at low and high methionine, respectively. The low susceptibility of methionine synthase to nitrous oxide in the presence of high methionine or in cb1G or cb1E mutants is probably related to low catalytic turnover.
Asunto(s)
Cistationina betasintasa/deficiencia , Homocisteína/metabolismo , 5-Metiltetrahidrofolato-Homocisteína S-Metiltransferasa/antagonistas & inhibidores , 5-Metiltetrahidrofolato-Homocisteína S-Metiltransferasa/metabolismo , Transporte Biológico Activo/efectos de los fármacos , Línea Celular , Medios de Cultivo , Cistationina betasintasa/genética , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Homocistinuria/genética , Homocistinuria/metabolismo , Humanos , Cinética , Metionina/biosíntesis , Metionina/farmacología , Metilación , Mutación , Óxido Nitroso/farmacologíaRESUMEN
In advanced cases of superficial siderosis of the human central nervous system, the clinical triad of hearing loss, cerebellar ataxia, and myelopathy permits the diagnosis at the bedside, and magnetic resonance imaging readily confirms the hemosiderin deposits in brainstem, cerebellum, and spinal cord. To study the pathogenesis of this condition and explain the selective vulnerability of the cerebellum, experimental siderosis was induced in rabbits by the repeated intracisternal injection of autologous red blood cells. The earliest cellular response in the cerebellar molecular layer was hyperplasia and hypertrophy of microglia as displayed by immunocytochemistry for ferritin. Microglia also contained iron, but ferritin biosynthesis appeared to proceed without commensurate iron accumulation. This early apoferritin response probably occurred due to the presence of heme, rather than iron, in the cerebrospinal fluid and subpial tissue. Ferritin biosynthesis is accelerated when the ferritin repressor protein is dissociated from ferritin messenger ribonucleic acid. A specific antiserum localized ferritin repressor protein predominantly to astrocytes including Bergmann glia. It is proposed that abundance and proximity of ferritin repressor protein--immunoreactive Bergmann glia and ferritin-containing microglia in the cerebellar molecular layer permit prompt cellular interaction in the conversion of heme to ferritin and ultimately hemosiderin.
Asunto(s)
Cerebelo/patología , Corteza Cerebral/patología , Siderosis/etiología , Animales , Enfermedades del Sistema Nervioso Central/líquido cefalorraquídeo , Enfermedades del Sistema Nervioso Central/etiología , Enfermedades del Sistema Nervioso Central/patología , Cerebelo/química , Corteza Cerebral/química , Ferritinas/análisis , Proteína Ácida Fibrilar de la Glía/análisis , Hiperplasia , Hipertrofia , Inmunohistoquímica , Microglía/química , Microglía/patología , Conejos , Valores de Referencia , Siderosis/líquido cefalorraquídeo , Siderosis/patología , Transferrina/análisisRESUMEN
We show that hydroxocobalamin bound to human serum albumin can dissociate and bind to transcobalamin II present in serum. Human liver cells in culture exposed to hydroxocobalamin bound to albumin incorporated less of the vitamin than when similar amounts of unbound hydroxocobalamin or cyanocobalamin were present. In the presence of transcobalamin II, a 4.5-fold increase in cellular uptake occurred, but this amount was less than when hydroxocobalamin or cyanocobalamin were added to transcobalamin II. These results indicate that albumin, by binding hydroxocobalamin, can alter the dynamics of binding to transcobalamin II and the subsequent cellular incorporation of this form of the vitamin.
Asunto(s)
Hidroxocobalamina/metabolismo , Hígado/metabolismo , Transcobalaminas/metabolismo , Humanos , Técnicas In Vitro , Unión Proteica , Albúmina Sérica/metabolismo , Células Tumorales CultivadasRESUMEN
Serial dilution susceptibility testing of imipenem against 59 clinical isolates of Pseudomonas aeruginosa, conducted simultaneously on single lots of Difco and BBL Mueller-Hinton agar (MHA), resulted in MICs for 90% of strains tested of 8 and 16 micrograms/ml, respectively. MICs for Escherichia coli, Klebsiella pneumoniae, and Pseudomonas spp. were also higher on BBL MHA. Quantification of the cation content of the two MHAs by atomic absorption spectroscopy demonstrated that the zinc concentration in BBL MHA was 15 times greater than that measured in Difco MHA (2.61 and 0.17 micrograms/ml, respectively). Concentrations of calcium, magnesium, iron, manganese, and copper in the two agars were similar. Addition of zinc to Difco MHA resulted in increases in MICs of imipenem for P. aeruginosa but not in the MICs of ceftazidime or cefpirome for P. aeruginosa (P < 0.01). A lesser zinc effect was seen on the activity of imipenem against E. coli, K. pneumoniae, and Pseudomonas spp. The activities of ceftazidime and cefpirome were similar on both MHAs when tested against all gram-negative organisms in this study. Thus, the effect of zinc in MHA was clearly demonstrated by a significant increase in the MICs of imipenem for P. aeruginosa, and, to a lesser extent, for other gram-negative bacilli.
Asunto(s)
Imipenem/farmacología , Pseudomonas aeruginosa/efectos de los fármacos , Zinc/farmacología , Agar , Pruebas de Sensibilidad MicrobianaRESUMEN
Cobalamin (Cbl, vitamin B12) metabolism was analyzed in cultures of human chorionic villus (CV) cells obtained at 9-10 weeks of gestation. CV cells were shown to synthesize transcobalamin II (TCII) and to possess a high affinity receptor for that molecule. The cells bound and internalized radioactive cyanocobalamin (CN[57Co]Cbl) complexed to TCII. This internalized CN[57Co]Cbl was found to be converted to both methylCbl and adenosylCbl, the two intracellular coenzyme forms of Cbl, and bound to the two known intracellular Cbl requiring enzymes, methionine synthase (MS) and methylmalonyl-CoA mutase. Both enzyme systems were found to be functional in the intact cell by demonstrating the incorporation of the radioactive label from both [14C]CH3-tetrahydrofolate and [14C]propionate into acid insoluble products. MS activity was also detected in lysed cell material. CV cells were shown not to be auxotrophic for methionine since they were able to utilize homocysteine in place of methionine for cell division. Since CV cells are capable of performing many of the complex events associated with Cbl metabolism, it may be possible to use these cells to diagnose genetic defects of Cbl metabolism.
Asunto(s)
Vellosidades Coriónicas/metabolismo , Transcobalaminas/metabolismo , Vitamina B 12/metabolismo , 5-Metiltetrahidrofolato-Homocisteína S-Metiltransferasa/metabolismo , Apoenzimas/metabolismo , Biopsia , División Celular , Células Cultivadas , Homocisteína/metabolismo , Humanos , Técnicas In Vitro , Metionina/metabolismo , Metilmalonil-CoA Mutasa/metabolismo , Receptores de Superficie Celular/metabolismoRESUMEN
The effect of supplying exogenous methylcobalamin (MeCbl), a methyl donor to methionine synthase (MS), on the cellular metabolism of MeCbl was tested in cultured fibroblasts from healthy persons and from a subject with an inherited defect in the synthesis of MeCbl. MeCbl bound to transcobalamin II (TCII) was taken up in larger amounts than cyanocobalamin (CN-Cbl), but was equal to the uptake of hydroxocobalamin (OH-Cbl). The form of Cbl in the lysosomes persisted as the same form, bound to TCII, to which the cells were exposed in the medium. Once released from the lysosomes, both MeCbl and OH-Cbl were converted in the same proportions to coenzyme forms, suggesting equivalent entry into common cellular pools of Cbl from which active forms are synthesized. Exogenous MeCbl enjoyed no advantage in binding to MS, in synthesis of MeCbl, and in supporting cell division in the absence of methionine. All evidence supported the concept that in human cells the active MeCbl on MS forms de novo on the enzyme. It appeared unlikely that therapeutic MeCbl would have any advantage over OH-Cbl in the treatment of MeCbl deficiency or Cbl deficiency in general.
Asunto(s)
Fibroblastos/citología , Fibroblastos/metabolismo , Vitamina B 12/análogos & derivados , 5-Metiltetrahidrofolato-Homocisteína S-Metiltransferasa/metabolismo , División Celular , Línea Celular , Citosol/química , Citosol/metabolismo , Humanos , Pulmón/citología , Lisosomas/metabolismo , Metionina/metabolismo , Vitamina B 12/sangre , Vitamina B 12/química , Vitamina B 12/metabolismoRESUMEN
The pathogenesis of superficial siderosis of the central nervous system (CNS) may be examined by the repeated intracisternal injection of washed autologous red blood cells (RBC). In rabbits, the injections cause the accumulation of iron in the cytoplasm of microglial cells and astrocytes of cerebellar and cerebral cortices. Immunocytochemistry for ferritin reveals enhanced reaction product mainly in microglia but hemosiderin occurs only after extending the injections to 6 months. In an effort to determine the biochemical correlates of these morphological changes, iron, ferritin, ferritin subunits and the ferritin repressor protein (FRP) were quantitated. There was no increase of total iron or ferritin in the exposed cortical areas. However, the injections of RBC caused dramatic shifts of the relative contributions by heavy (H-) and light (L-) ferritin subunits. The initial response was a prompt increase of the H/L ratio to over 4.0 from the normal ratio near 1.0. Extended injections caused the ratio to drop to below unity, and the predominance of L-ferritin at 6 months coincided with the appearance of granular hemosiderin. This investigation also confirmed the presence of FRP in rabbit brain cytosols but the induction of experimental superficial siderosis did not change its levels or in vitro affinity for the iron-responsive element in ferritin messenger ribonucleic acid. It is proposed that the incrustation by hemosiderin which characterizes superficial siderosis of the CNS in humans occurs when prolonged exposure to hemoglobin produces persistent shifts of the H/L-ratios by accumulation of L-ferritin.
Asunto(s)
Enfermedades del Sistema Nervioso Central/metabolismo , Siderosis/metabolismo , Animales , Química Encefálica/efectos de los fármacos , Enfermedades del Sistema Nervioso Central/patología , Cerebelo/efectos de los fármacos , Cerebelo/metabolismo , Corteza Cerebral/efectos de los fármacos , Corteza Cerebral/metabolismo , Citosol/efectos de los fármacos , Citosol/metabolismo , Electroforesis en Gel de Poliacrilamida , Ensayo de Inmunoadsorción Enzimática , Ferritinas/biosíntesis , Ferritinas/metabolismo , Hemosiderina/biosíntesis , Histocitoquímica , Hierro/metabolismo , Proteína 1 Reguladora de Hierro , Proteínas Reguladoras del Hierro , Cinética , Proteínas de Unión al ARN/metabolismo , Conejos , Siderosis/patologíaRESUMEN
Total serum homocysteine (Hcy) was measured in patients with either low serum folate, low serum vitamin B12 (B12), or potential metabolic defects, in order to evaluate Hcy as an indicator of the tissue status of the two vitamins. An increased Hcy supported the diagnosis of frank tissue deficiency in those patients in whom tissue deficiency was evident by other means. A Hcy within the reference interval enabled the clinician to identify those patients whose low serum vitamin level and symptoms did not reflect a tissue deficiency of B12 or folate of clinical consequence. Children with inherited disturbances of B12 metabolism, and whose serum B12 was within the reference interval, were correctly identified by an increased Hcy. A declining Hcy was evidence of correction of a deficiency even when other laboratory or clinical measurements of a response were obscured.
Asunto(s)
Deficiencia de Ácido Fólico/diagnóstico , Homocisteína/sangre , Deficiencia de Vitamina B 12/diagnóstico , Adulto , Índices de Eritrocitos , Eritrocitos/metabolismo , Femenino , Ácido Fólico/sangre , Deficiencia de Ácido Fólico/sangre , Humanos , Masculino , Valores de Referencia , Deficiencia de Vitamina B 12/sangreRESUMEN
Age-, sex-, and other risk factor-specific recommendations, published last year by the U.S. Preventive Services Task Force, for preventive clinical care have raised questions concerning the costs of providing health insurance for these kinds of services. Our study provides estimates of the costs to health insurance programs of covering the Task Force recommendations for the general population (not including high-risk populations). We estimate the average increase in the 1990 cost per month in a self-insured plan without cost-sharing to be $4.35 to $6.89 for an employee with family coverage and $1.21 to $2.41 for an employee with individual coverage. Preventive services would cost the Medicare program $3.99 to $5.10 per enrollee per month.
Asunto(s)
Seguro de Salud/economía , Servicios Preventivos de Salud/economía , Adolescente , Adulto , Anciano , Niño , Preescolar , Costos y Análisis de Costo , Deducibles y Coseguros , Honorarios y Precios , Femenino , Humanos , Lactante , Masculino , Persona de Mediana Edad , Servicios Preventivos de Salud/estadística & datos numéricos , Estados UnidosRESUMEN
This study was designed to study the effects of small bowel resection on daily urinary excretion patterns, plasma and bone levels of magnesium, phosphorus and calcium in rats on long-term total parenteral nutrition (TPN). Male Sprague-Dawley rats weighing 300 to 350 g were randomly divided into two groups with six rats in each group. Control consists of rats whose small intestines were transected but anastomosed. Resected rats had 70% of their small intestine removed. After intestinal resection and transection, rats were infused with a balanced TPN solution for 17 days. Resected rats excreted significantly more calcium than transected rats during the first 10 days of TPN infusion. Peak excretion occurred between day 3 and 4 followed by a trend toward a slightly higher than normal level of calcium excretion between days 10 and 17. Urinary losses of phosphorus and magnesium were not influenced by bowel resection. Plasma and tibia calcium, phosphorus and magnesium levels were not altered. The effects of small bowel resection on urinary calcium loss is specific and our data demonstrate the involvement of gut in regulating urinary calcium excretion and suggest that gut may play a significant role in TPN induced metabolic bone disease.
Asunto(s)
Calcio/orina , Intestino Delgado/metabolismo , Magnesio/orina , Nutrición Parenteral Total , Fósforo/orina , Animales , Enfermedades Óseas Metabólicas/etiología , Huesos/metabolismo , Calcio/metabolismo , Intestino Delgado/cirugía , Magnesio/metabolismo , Masculino , Nutrición Parenteral Total/efectos adversos , Fósforo/metabolismo , Distribución Aleatoria , Ratas , Ratas Endogámicas , Factores de TiempoRESUMEN
Cobalamin G mutation (cblG) typically presents as a severe megaloblastic anemia during the first few weeks of life. The anemia responds completely to treatment with high doses of Cbl but the neurologic manifestations respond more slowly and not always completely. Cultured fibroblasts from two affected infants and virus-transformed lymphoblasts from one of the infants expressed the following: poor growth in the absence of methionine, the ability to take up and internalize Cbl bound to transcobalamin II, impaired synthesis of methionine from homocysteine, the ability to bind incoming Cbl to the respective coenzymes, but an inability to synthesize methylcobalamin in spite of a normal capacity to synthesize adenosylcobalamin. The in vitro activity of the methyltransferase dependent on methylcobalamin of cell extracts varied from low to high depending on the conditions of culture and assay. The cblG cells were unusually sensitive to the concentration of adenosylmethionine in the assay. More adenosylmethionine was required by cblG cells to achieve the same level of enzyme activity as control cells and high concentrations of adenosylmethionine did not suppress activity as in control cells. It was postulated that the defect in cblG is in the metabolism of adenosylmethionine, an essential substance for the synthesis of methionine from homocysteine.
Asunto(s)
Anemia Megaloblástica/genética , Mutación , Vitamina B 12/genética , 5-Metiltetrahidrofolato-Homocisteína S-Metiltransferasa/metabolismo , Anemia Megaloblástica/tratamiento farmacológico , División Celular/efectos de los fármacos , Células Cultivadas , Femenino , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Fibroblastos/patología , Humanos , Lactante , Masculino , Metionina/biosíntesis , Metionina/farmacología , S-Adenosilmetionina/farmacología , Transcobalaminas/metabolismo , Vitamina B 12/análogos & derivados , Vitamina B 12/biosíntesis , Vitamina B 12/metabolismo , Vitamina B 12/uso terapéuticoRESUMEN
Neither normal human B lymphoblasts (RPMI 6410) transformed by the EB virus nor human peripheral blood lymphocytes (PBL) stimulated by a mitogen replicated well when the methionine (Met) of the medium was replaced with homocysteine (Hcy). Cbl bound to human transcobalamin II (TC II) substantially increased cell division over that observed when the Cbl of the medium was in the free form. Although, as expected, the TC II enhanced the cell entry of Cbl 1000-fold, this was not the basis of the TC II effect. Through adjustment of the respective concentrations of free Cbl and TC II-Cbl in the medium, equal amounts of Cbl entered the cell, yet the TC II effect persisted. TC II-Cbl did not restore cell division in the absence of Met by virus-transformed lymphoblasts from a child with defective Met synthesis from Hcy. The TC II did not act by enhanced induction of the Cbl-dependent methionine synthase activity of cell extracts but the ability of intact cells to produce Met from Hcy by the Cbl-dependent process appeared to have a role in the TC II effect.
Asunto(s)
Linfocitos/metabolismo , Metionina/metabolismo , Transcobalaminas/fisiología , Células Cultivadas , Homocisteína/metabolismo , Humanos , Activación de Linfocitos , Vitamina B 12/metabolismoRESUMEN
Presented is a modification of an assay for total serum homocysteine (Hcy) in which the Hcy plus radioactive adenosine is converted enzymatically to labeled S-adenosylhomocysteine (AdoHcy). The modifications included a commerical source for the AdoHcy hydrolase, adenosine labeled with either 14C or 3H, and separation of the AdoHcy by thin layer chromatography. The assay was sensitive to 25 pmol. Hcy levels in sera from 18 controls ranged from 6.9 to 12.1 mumol/L with a mean of 9.1 and a SD of 1.5 mumol/L. The total serum Hcy was increased in vitamin B12 and folate deficiency. The level was high in congenital defects of vitamin B12 metabolism, blocking the methylation of Hcy regardless of the serum vitamin B12 levels, but was normal in the absence of tissue deficiency even if the serum vitamin B12 levels were low. The procedure has been found practical in two years of use and requires only 0.1 mL of serum.
Asunto(s)
Ácido Fólico/sangre , Homocisteína/sangre , Vitamina B 12/sangre , Adenosina/sangre , Adenosilhomocisteinasa , Adulto , Anciano , Radioisótopos de Carbono , Femenino , Homocisteína/normas , Humanos , Hidrolasas/sangre , Lactante , Masculino , Persona de Mediana Edad , Estándares de Referencia , TritioRESUMEN
The activity of the enzyme methionine synthetase (MS) (methyltetrahydrofolate:homocysteine methyltransferase) (EC 2.1.1.13) was measured in human lymphocytes of various types and cobalamin (vitamin B12) status. Total and holo MS activity was low in unstimulated peripheral blood lymphocytes from persons with tissue deficiency of cobalamin, but not in cells from those with low serum cobalamin levels for other reasons. The MS activity of the lymphocyte was increased by treatment of the patients with vitamin B12. The number of lymphocytes was often low or low normal in the circulation of those deficient in cobalamin. Holo MS activity was low in an established line of human B cells, RPMI 6410 cells, depleted of cobalamin. The total and holo MS activity of both RPMI 6410 cells, replete or depleted, and lymphocytes stimulated in culture was increased by cobalamin in vitro; 222 nmol/L free cobalamin was roughly the equivalent of 0.22 nmol/L cobalamin bound to transcobalamin II. Both lymphocytes and RPMI 6410 cells required folate for growth and could meet these needs via methylfolate, homocysteine, and the cobalamin-dependent MS reaction. Depleted RPMI 6410 cells, however, used cobalamin in some way in addition to the provision of available folate from methylfolate. The consequences of the reduced MS activity in deficient cells could include a reduction in available folate with diminished capacity for clonal expansion of lymphocytes in reaction to infection and impairment of essential methylations including those of protein synthesis. The prompt induction of MS activity by cobalamin, especially in the in vitro model, suggests an effect of therapeutic vitamin B12 well in advance of the numerical increase in cells of the blood.
Asunto(s)
5-Metiltetrahidrofolato-Homocisteína S-Metiltransferasa/sangre , Linfocitos/enzimología , Metiltransferasas/sangre , Deficiencia de Vitamina B 12/enzimología , Vitamina B 12/fisiología , Línea Celular , Ácido Fólico/fisiología , Humanos , Activación de Linfocitos , Vitamina B 12/uso terapéutico , Deficiencia de Vitamina B 12/tratamiento farmacológicoRESUMEN
Human peripheral blood lymphocytes stimulated with phytohemagglutinin and a lymphocyte model consisting of the RPMI 6410 cell, a human virus-transformed B cell, required added methionine (Met) for growth of the cultures. This failure to meet all needs for Met via endogenous synthesis, which is characteristic of oncogenic transformation, occurred even in the presence of adequate homocysteine, methylfolate (5-CH3-H4PteGlu) and cobalamin (Cbl)-dependent methionine synthetase activity. Folinic acid (5-CHO-H4PteGlu), which provides available folate independently of Cbl, improved growth only slightly in the absence of Met. Free Cbl at 222 nM, an amount great enough to alter other intracellular events, failed to increase growth in the absence of Met, but 0.22 nM Cbl bound to transcobalamin II did, however, enhance growth.
Asunto(s)
Linfocitos/fisiología , Metionina/metabolismo , 5-Metiltetrahidrofolato-Homocisteína S-Metiltransferasa/metabolismo , Ciclo Celular , Línea Celular , Células Cultivadas , Medios de Cultivo , Humanos , Activación de Linfocitos , Transcobalaminas/metabolismo , Vitamina B 12/metabolismoRESUMEN
We studied the use of stat (ie, statim--as soon as possible) laboratory tests at a university teaching hospital. For the 20 most frequently performed tests, 35.7% of the determinations were performed on a stat basis. The frequency with which a test was ordered on a stat basis ranged from 6.0% (for ESR) to 61.5% (amylase level). The proportion of laboratory test requests from a hospital service that were ordered stat ranged from 3.8% (orthopedic surgery) to 100% (emergency services). The daily number of regular laboratory test requests decreased significantly over the weekend, but the daily number of stat requests fell less sharply. The number of requests for routine tests decreased after 6 pm, but the number of requests for stat tests remained relatively constant until about midnight.