Design, development and performance of a novel multidose dry-powder inhaler.

The authors describe the design and development of a breath-actuated multidose dry-powder inhaler and summarize the in vitro and in vivo data demonstrating its robustness and performance in the laboratory and during clinical use. Drugs for the treatment of asthma--including budesonide, beclomethasone dipropionate and salbutamol--when formulated with lactose powder as a carrier and dispensed via this device, have exhibited clinical efficacy and safety profiles comparable with standard pressurized metered-dose inhalers and dry-powder formulations.

Compliance with nedocromil sodium and a nedocromil sodium/salbutamol combination. Compliance Working Group.

Nonadherence to prescribed preventive medication is common in asthma. We wanted to assess whether the combination of a beta 2-bronchodilator with an anti-inflammatory treatment in the same metered-dose inhaler (MDI) with a regular dosing schedule might improve compliance. A double-blind study was used to compare use (two actuations four times daily for 12 weeks) of 2 mg nedocromil sodium (n = 101) with a combination of 2 mg nedocromil sodium and 100 micrograms salbutamol (n = 100) in mild-to-moderate asthma patients (mean age 42 +/- 14 yrs; 98 males and 103 females). Compliance was measured using the electronic Nebulizer Chronolog (NC) device, change in MDI canister weight, patient questionnaire and physician assessment. The mean +/- SD number of actuations per day for nedocromil sodium and the combination during the primary period of assessment (Weeks 11-12) was 4.2 +/- 2.6 and 4.6 +/- 2.5 (NC), 5.3 +/- 2.1 and 5.3 +/- 2.0 (canister weight), and 7.5 +/- 1.3 and 7.4 +/- 1.3 (questionnaire), respectively. Physician assessment rated compliance as "good" to "excellent". The first and final days of the period were not used in the NC analysis to exclude part-days of treatment and drug-dumping (repeated actuations without inhalation), and may account for the difference between NC and canister weight results. The mean number of two-actuation doses per day determined from the NC was 2.1 +/- 1.3 for nedocromil sodium and 2.4 +/- 2.1 for the combination. Thirty five percent (nedocromil sodium) and 34% (combination) of the patients were compliant (6-10 actuations per day for > or = 60% of the days). We conclude that compliance is poor in asthma, electronic recording revealed the dumping phenomenon and, in this study, the combination of an inhaled beta 2-bronchodilator with a preventive treatment did not improve compliance over a three month period in patients with mild-to-moderate asthma.

Dietary guidelines in perspective.

To assess whether dietary guidelines for Americans are appropriate for young children, the evolution of dietary guidance, the nature of the guidelines, evidence used to support the concept of diet modification to prevent heart disease and the rationale for extending application of the guidelines to children have been examined. As health improved during this century, life expectancy lengthened, and diseases associated with aging became major causes of death. As a consequence, emphasis on dietary advice for selecting a nutritionally adequate diet--the primary need of children--declined, whereas emphasis on dietary advice for preventing chronic and degenerative diseases increased. It is clear from reading the text accompanying the guidelines that they were proposed to prevent diseases of aging by reducing consumption of animal products. Critical evaluation of evidence bearing on the concept of the guidelines reveals that there are grounds for skepticism about claims for the effectiveness of diet modification as a measure for reducing the incidence of heart disease. Also, the rationale for extending the guidelines to young children is based on inferences from observations on adults, not on direct evidence that children will benefit from following them. There is, thus, ample justification for proposing separate dietary guidelines for children.

Leucine affects the metabolism of valine by isolated perfused rat hearts: relation to branched-chain amino acid antagonism.

This study was conducted to determine the effects of different concentrations of leucine on the transport, transamination and oxidation of valine and on incorporation of valine into heart proteins in the isolated perfused rat heart. Valine metabolism was studied in rat hearts perfused with medium containing glucose and graded levels of L-leucine. In transport studies L-phenylalanine was also tested. Uptake of L-[1-14C]valine (0.2 mmol/L) was significantly reduced (-50%) by inclusion of 0.2 mmol/L phenylalanine or leucine, and by -70% by inclusion of 1.0 mmol/L phenylalanine or leucine in the perfusate. Transamination of valine decreased by 37 and 48%, and oxidation of valine by 53 and 71%, respectively, when 0.2 or 1.0 mmol/L leucine was included in the perfusate. Tissue concentrations of valine decreased by 43, 48 and 62% in the presence of 0.2, 0.5 and 1.0 mmol/L leucine, respectively; tissue concentrations of leucine, glutamate and alanine increased approximately 11-fold, 1.2-fold and 0.5-fold, respectively, when 1.0 mmol/L leucine was present in the perfusate. Addition of 0.2-1.0 mmol/L leucine did not affect incorporation of valine into heart proteins. We conclude that 1) competition among large neutral amino acids for transport into heart occurs at physiological concentrations of these amino acids in plasma; 2) inhibition of valine uptake by leucine can limit the rate of valine catabolism in heart; and 3) depletion of tissue valine concentration by an excess of leucine did not affect the rate of protein synthesis.

Protein quality, amino acid balance, utilization, and evaluation of diets containing amino acids as therapeutic agents.

Basic concepts of amino acid nutrition are summarized and quantitative amino acid requirements of different age-groups based on current knowledge are presented. The newer concepts of "conditionally indispensable" amino acids and organ-specific requirements for amino acids are discussed. The concepts of nitrogen balance, limiting amino acid, protein utilization, protein efficiency ratio, and amino acid score for determining protein quality are reviewed, and examples of low-, intermediate-, and high-quality proteins are provided. Problems in assessing efficiency of nitrogen utilization when single amino acids or amino acids in combination with balanced diets are used as therapeutic agents are discussed in relation to the potential roles of the branched-chain amino acids, arginine, and glutamine in trauma and as immunostimulators.

Transport of amino acids into rat erythrocytes and its potential physiological role.

Previous studies have proposed the possibility that erythrocytes (RBC) are involved in the interorgan transport of amino acids; however, this role has not been confirmed. In order to study the likelihood that erythrocytes are involved in the interorgan transport, rates of influx and efflux of glycine, threonine, lysine, histidine and leucine were measured in rat red blood cells. Time course of influx of leucine, a large neutral amino acid, was very rapid (319 mumoles/L RBC. min), and a steady state was reached between 5 to 10 min of incubation, whereas glycine influx was very slow (5.04 mumoles/L RBC. min). Threonine influx was similar to leucine although the rate was slower (41.4 mumoles/L RBC. min); however, the steady state was reached in 30 minutes and its uptake showed less capacity. Histidine and lysine showed a continuous influx, and did not reach a steady state after 60 min of incubation. Efflux of leucine was extremely rapid indicating a rapid equilibration between the incubation medium and the intracellular space of the erythrocytes. Threonine efflux had a half life (t1/2) of between two to three min, independently of the medium used. Histidine showed a t1/2 of around six min, whereas for the small neutral amino acid glycine it was of 14 to 17 min. These results indicate that some large neutral amino acids are not involved in the potential interorgan transport by red blood cells due to the rapid equilibration of the concentration of amino acid between cells and the medium.

Competition for transport of amino acids into rat heart: effect of competitors on protein synthesis and degradation.

Transport of the neutral amino acids, 2-(methylamino)isobutyrate (MeAIB) and Phe, was examined in isolated rat hearts perfused by the Langendorff method. Hearts were perfused by recirculating for various time periods buffer containing [14C]-MeAIB or [14C]-Phe plus desired additions. Uptake of MeAIB was linear for approximately 30 minutes; Phe uptake was linear for a maximum of 5 minutes, and reached a steady state after 15 minutes. Km and Vmax for MeAIB were 1.1 +/- 0.03 mmol/L and 37.7 +/- 0.4 pmol/microL intracellular fluid (ICF)/min; values for Phe were 1.8 +/- 0.02 mmol/L and 364 +/- 5 pmol/microL ICF/minute. Uptake of MeAIB (0.2 mmol/L) was reduced 95% in the presence of Ser (10 mmol/L), and less severely by large neutral amino acids ([LNAA], 10 mmol/L) such as Phe and Leu (by 46% and 54%, respectively). Uptake of Phe (0.2 mmol/L) was reduced by LNAA such as Val, Leu, and Ile (by 51%, 78%, and 81%, respectively), or by commercial preparations used in parenteral nutrition, eg, Travasol or Travasol plus extra branched-chain amino acids (BCAA) (Branchamin); Ser had little effect (8% reduction). Insulin in the perfusion medium increased the fractional rate of protein synthesis. Individual BCAA at physiological concentrations (0.2 mmol/L) did not alter the rate of protein synthesis. Branchamin or Travasol plus Branchamin also had no effect on the rate of protein synthesis in heart, but did depress the rate of degradation. These studies suggest that amino acid transport into heart may be affected by normal levels of plasma amino acids, whereas protein synthesis is not.

A feather-sexed strain of laying hens was more responsive to dietary supplements of choline and methionine than a vent-sexed strain.

A response surface design was used to study Cho and Met interactions with corn and soybean diets, using two strains of hens. The strains were a feather-sexed line (FS strain), and a vent-sexed line (SS strain). The diets contained 3% meat and bone meal and, on chemical analysis, 15.1% crude protein, .29% Met, .225% Cys, and 1,041 ppm of Cho. Nine diets were fed from 20 to 68 wk of age, using added Met levels ranging from 0 to 500 ppm and added Cho levels ranging from 0 to 1,500 ppm, to fix the design points. The FS strain consumed significantly more feed per day (117 versus 108 g) than the SS strain, but there were no significant differences for the 24 to 68 wk period in egg production, egg weight, or feed per dozen eggs. Three and five combinations of Met and Cho were significant in improving egg production (P less than .05) out of the eight combinations for the SS and FS strains, respectively. The best egg production for the FS strain for the period 24 to 68 wk was observed at 250 ppm Met and 1,500 ppm Cho, or 427 ppm Met and 220 ppm added Cho. The SS strain showed no significant (P greater than .05) dietary responses in egg production between 250 ppm Met and no Cho, or 427 ppm Met and either 220 or 1,280 ppm Cho. The SS strain showed no significant (P greater than .05) dietary response in egg weight to either Cho or Met.(ABSTRACT TRUNCATED AT 250 WORDS)

Protein selection by rats adapted to high or moderately low levels of dietary protein.

After preliminary studies on flavor acceptability, patterns and indices of subsequent feeding behavior were monitored by computer in young rats which were adapted to 15% or 70% casein diets before being offered, sequentially, choices between flavored diet pairs in which the proportions of percentage casein were 5/65, 5/55, 5/45, 5/35 and 5/25. Similarly adapted rats received these choices in the reverse sequence. Rats adapted to 15% casein usually ate randomly from the diet pairs and selected approximately 15-30% casein; individual behaviors were prominent. The 70% casein groups avoided the higher casein diet, often within minutes (except for the first-offered 5/25 choice), and seldom selected more than 10% casein; individual differences were infrequent. Such rats also distinguished between flavored 70% and 65% casein diets. Sizes and numbers of meals and rates of eating differed for the paired diets, especially for rats adapted to 70% casein. A flavor added to the 70% casein adaptation diet was not avoided when present only in the 5% casein diet of a 5/65 choice. Rats adapted to 70% soy protein before receiving flavored 5/65 to 5/25 choices selected 20-28% soy protein, a level far above those of casein selections by rats adapted to 70% casein. Dietary adaptation and type of protein thus affect subsequent diet selection and feeding patterns and indices.

Three-day dietary intake of incarcerated and nonincarcerated adolescent males.

We collected 3-day dietary intake information from both incarcerated (n = 137) and nonincarcerated (n = 42) adolescent males to provide an objective assessment of diets of residents of a juvenile correctional facility. Average daily intake of energy, protein, 13 indicator micronutrients and of macronutrients as percent of energy were calculated. Average intakes of the total group of adolescents (n = 179) were greater than 90% of the 1980 recommended dietary allowance (RDA) for energy, protein, and 10 micronutrients. There were no statistically significant differences between black (n = 63) and white (n = 63) incarcerated subgroups and white (n = 41) nonincarcerated subjects on average measures of energy, protein, macronutrients as percent of energy, and seven micronutrients. Cumulative intakes (as a proportion of the 1980 RDA) of nonincarcerated subjects and at least one group of incarcerated subjects were significantly different for vitamin B6, folate, vitamin C, iron, sodium, and thiamin. However, average intakes of vitamin C, sodium, and iron by all groups exceeded the RDA. Overall, the total group of adolescents did not appear to be at significant nutritional risk. Folate, copper, and magnesium intakes were consistently below RDA in all groups. We suggest that these findings are not indicative of nutritional inadequacy but are, rather, cues that some RDAs may be inappropriately high.

Relationship between protein intake and hepatic protein synthesis in rats.

Protein synthesis has been measured in vitro in postmitochondrial extracts from livers of rats fed levels of casein ranging from 0 to 40% by weight. The maximal capacity for protein synthesis per milligram of RNA, measured with each amino acid added at 250 mumol/L, was 40-60% higher in rats fed a protein-free diet than in those fed 6 or 15% casein. Our results suggest that the livers of rats fed a protein-free diet are primed for the synthesis of tissue proteins and, given an adequate supply of amino acids, the rate of protein synthesis would be as high as or higher than the rate in protein-replete animals. When amino acids were added to the in vitro system at concentrations found in plasma of rats fed 0, 6, 15 or 40% casein the rate of protein synthesis increased by three- to fourfold over this range, with the highest rate observed for the 15% dietary casein level. We conclude that when protein intake is below the requirement level, the rate of liver protein synthesis may be limited by amino acid supply, by the capacity of the system for protein synthesis or by both.

Thiamin status of incarcerated and nonincarcerated adolescent males: dietary intake and thiamin pyrophosphate response.

We measured thiamin status in 137 incarcerated and 42 nonincarcerated adolescent males by use of both dietary intake data and a standard biochemical assay, thiamin pyrophosphate (TPP) response. Average thiamin intake of the total group was greater than 120% of the age-specific recommended dietary allowance (RDA). Ninety-two percent of incarcerated subjects and 93% of nonincarcerated subjects were consuming greater than or equal to 70% of RDA. Although average daily thiamin intake of nonincarcerated subjects was significantly higher than that of incarcerated subjects, both groups appeared to be at minimal risk for marginal thiamin status. Comparison of TPP response values indicated that there was no significant difference between groups. However, approximately 24% of the total population appeared to have less than adequate RBC thiamin on the basis of current standards for TPP response. Neither dietary intake nor reported previous alcohol intake was correlated with TPP response. These discrepant findings raise questions about the usefulness of the TPP response as the sole indicator of marginal thiamin status.

High levels of dietary amino and branched-chain alpha-keto acids alter plasma and brain amino acid concentrations in rats.

Plasma and brain amino acid and plasma branched-chain alpha-keto acid (BCKA) concentrations were measured in rats fed diets containing high levels of individual amino and alpha-keto acids. Consumption of a low-protein (9% casein) diet high in leucine or alpha-ketoisocaproate depressed plasma concentrations of isoleucine and valine and their respective keto acids, alpha-keto-beta-methylvalerate and alpha-ketoisovalerate. High dietary levels of alpha-keto-beta-methylvalerate or alpha-ketoisovalerate (but not of isoleucine or valine) depressed plasma concentrations of the other BCKA and their respective branched-chain amino acids (BCAA). Consumption of a low protein, high phenylalanine diet depressed plasma concentrations of both BCAA and BCKA. Brain large neutral amino acid pools of rats fed all low-protein, high-amino acid diets were depleted. Consumption of diets high in individual BCKA increased brain concentrations of aromatic amino acids. In this study of rats allowed to feed for only 6 h/d, elevated brain phenylalanine concentration was associated with a significant depression of food intake, whereas elevated brain BCAA concentrations were not. Also, elevated plasma BCKA concentrations, comparable with those observed in maple syrup urine disease, were accompanied by elevations in concentrations of aromatic amino acids in brain but not in plasma.

Neutral amino acid transport into rat skeletal muscle: competition, adaptive regulation, and effects of insulin.

Amino acid (AA) transport systems A and L, which transfer preferentially small neutral AA (SNAA) and large neutral AA (LNAA), respectively, were studied in the isolated soleus muscle with the specific models, 2-(methylamino)isobutyrate (MeAIB) and 2-aminobicyclo[2,2,1]heptane-2-carboxylate (BCH). Affinity for MeAIB was greater than for BCH (Km = 3.2 +/- 0.2 and 8.7 +/- 0.2 mm, respectively). Rate of transport of MeAIB (Vmax = 104 +/- 3 pmol/microL/min) was slower than for BCH (970 +/- 12 pmol/microL/min), but accumulation was far more concentrative; transport of BCH, but not MeAIB, rapidly reached a steady-state level. MeAIB transport was reduced in the presence of SNAA; BCH transport was reduced to a lesser extent only by LNAA. Mixtures of AA at concentrations resembling those in plasmas of rats fed either a 6% or 50% casein diet reduced transport of MeAIB, whereas BCH transport was low only with the latter mixture. Only MeAIB transport was stimulated by insulin. Preincubation of muscles for 5 hours in a AA-free medium stimulated subsequent MeAIB uptake by about twofold to fourfold; this effect was suppressed by inhibitors of protein synthesis. Selective differences were thus observed in transport by skeletal muscle of model AA for the A and L systems: increased transport resulting from various stimuli was limited to the model for the A system, and transport of either model was depressed with mixtures containing physiological levels of AA. Changes in dietary protein or AA intake may thus alter transport of certain neutral AA into skeletal muscle via changes in plasma AA pools.

Branched-chain and other amino acids in tissues of rats fed leucine-limiting amino acid diets containing norleucine.

Amino acid concentrations were measured in plasma, brain, muscle and liver from rats fed leucine-limiting diets containing varying proportions of other indispensable amino acids (IAA), the branched-chain amino acids (BCAA) and norleucine, a BCAA analog known to compete with large neutral amino acids (LNAA) for transport into tissues. Leucine was low and other IAA were high when dietary IAA were 125% and leucine was 65% of requirements; higher leucine and lower IAA concentrations occurred when dietary IAA were 75% of requirements. Tissue leucine was high and isoleucine and valine were low in rats fed excess leucine. Norleucine induced dose-dependent reductions in BCAA, especially in brain and muscle in which isoleucine or valine were sometimes undetectable. Leucine was not depressed further when control values were low as in the rats fed 125% IAA. Norleucine frequently prevented the high BCAA found after feeding additional BCAA. Other LNAA tended to be low in the brain and muscle of rats fed norleucine. Lysine was high only in the tissues of rats fed 75% IAA and norleucine; this effect was prevented when added leucine was given. Brain tryptophan, but not always serotonin, was low in rats fed norleucine. The results show transport-related, selective and usually marked depletions of tissue BCAA in rats fed norleucine; this suggests norleucine may be an aid in the treatment of clinical conditions involving excesses of BCAA.

Altered phosphorylation state of branched-chain 2-oxo acid dehydrogenase in a branched-chain acyltransferase deficient human fibroblast cell line.

The abundance and phosphorylation state of the polypeptide constituents of the human branched-chain 2-oxo acid dehydrogenase complex were examined in mitochondria from normal and maple syrup urine disease (MSUD) fibroblasts. In normal fibroblast mitochondria two forms of the E1 alpha subunit were observed: non-phosphorylated (E1 alpha) and phosphorylated (E1 alpha-P). About 40-50% of E1 alpha was present as E1 alpha-P. The ability to quantitate the two forms of E1 alpha permitted examination of the association between decreased capacity of oxidize branched-chain 2-oxo acids and the phosphorylation state of E1 alpha. Changes in phosphorylation state of E1 alpha were observed in MSUD fibroblasts as compared to control cells. Of particular interest was the absence of E1 alpha-P in an MSUD fibroblast line which lacked the dihydrolipoyl acyltransferase (E2) subunit of the dehydrogenase complex. In two MSUD cell lines deficient in E1 alpha, the abundance of E1 alpha-P appeared to be preferentially reduced. A fourth MSUD cell line contained normal quantities of E3, E2 and both forms of the E1 alpha polypeptide. Our results indicate that alterations in the abundance of dehydrogenase complex polypeptides in MSUD fibroblasts may influence the phosphorylation state of the E1 alpha polypeptide. They demonstrate the potential for examining simultaneously mutations which affect both the catalytic and regulatory components of the dehydrogenase complex.