The supportive effect of supplementation with α-keto acids on physical training in type 2 diabetes mellitus.

The maintenance of physical activity is crucial for the prevention and management of type 2 diabetes (T2D), and exercise induced changes including production of metabolites like ammonia can result in fatigue and exercise intolerance. Nutritional supplements may serve as an effective measure in supporting patients undergoing physical training by acting on their metabolism. This study investigates the effects of supplementation with α-keto acids (KAS) on exercise tolerance and glucose control in T2D patients. In a double-blind, placebo-controlled, randomized study 28 T2D patients underwent 6 weeks training on a cycle ergometer while they were supplemented with either a placebo or KAS (0.2 g kg(-1) body weight each day). The weekly training volume, power output at maximum and lactic threshold, leg muscle torque, the plasma concentration and 8 h urinary discharge of glucose, ammonia and urea were determined before and after the training as well as after one week of recovery. With KAS the patients did significantly more voluntary exercise (213 vs. 62 min, P < 0.01), reached a higher VO2max (27.3 vs. 24.8 ml min(-1) kg(-1)), higher power output (224 vs. 193 watts, P < 0.05) and greater endurance capacity (108 vs. 96 watts at lactic threshold, P < 0.05). Although the patients without KAS improved their glucose control after the training (P < 0.05), this effect could not be maintained after recovery as it was in the KAS group, where there was a prolonged benefit in glucose control. KAS also affected the ammonia and urea metabolism. KAS delivered supportive effects on the physical training along with a prolonged benefit in glucose control in T2D patients.

Field-expedient screening and injury risk algorithm categories as predictors of noncontact lower extremity injury.

In athletics, efficient screening tools are sought to curb the rising number of noncontact injuries and associated health care costs. The authors hypothesized that an injury prediction algorithm that incorporates movement screening performance, demographic information, and injury history can accurately categorize risk of noncontact lower extremity (LE) injury. One hundred eighty-three collegiate athletes were screened during the preseason. The test scores and demographic information were entered into an injury prediction algorithm that weighted the evidence-based risk factors. Athletes were then prospectively followed for noncontact LE injury. Subsequent analysis collapsed the groupings into two risk categories: Low (normal and slight) and High (moderate and substantial). Using these groups and noncontact LE injuries, relative risk (RR), sensitivity, specificity, and likelihood ratios were calculated. Forty-two subjects sustained a noncontact LE injury over the course of the study. Athletes identified as High Risk (n = 63) were at a greater risk of noncontact LE injury (27/63) during the season [RR: 3.4 95% confidence interval 2.0 to 6.0]. These results suggest that an injury prediction algorithm composed of performance on efficient, low-cost, field-ready tests can help identify individuals at elevated risk of noncontact LE injury.

Inhibition of benzalkonium chloride-induced skin inflammation in mice by an indol-1-ylpropan-2-one inhibitor of cytosolic phospholipase A2 α.

BACKGROUND: Irritant contact dermatitis (ICD) is a frequent and often underrated problem for which the major efficacious therapy is still local glucocorticoids, although they have known adverse effects due to their wide spectrum of action. A more focused therapeutic strategy would be the inhibition of a key enzyme for biosynthesis of the lipid mediators, cytosolic phospholipase A(2) α (cPLA(2) α), in ICD. We are analysing the pharmacological and biological effects of a selective cPLA(2) α inhibitor. OBJECTIVES: To examine the usefulness of the potent and selective cPLA(2) α inhibitor 3-(5-carboxypentanoyl)-1-[3-(4-octylphenoxy)-2-oxopropyl]indole-5-carboxylic acid (compound 1) for therapy of inflammatory skin disorders. METHODS: We examined clinical and cellular effects of a selective cPLA(2) α inhibitor (compound 1) on ICD in mice. RESULTS: Topical application of the compound significantly reduced ear swelling after induction by the irritant benzalkonium chloride. Concomitantly, compound 1 inhibited the accumulation of granulocytes as well as the expression of inflammatory proteins such as tumour necrosis factor-α, interleukin-1ß and macrophage inflammatory proteins 1α and 1ß in the ear tissue. In primary murine keratinocytes, the benzalkonium chloride-induced expression of these proteins was also downregulated after treatment with compound 1 in vitro. CONCLUSIONS: Compound 1 is a well-aimed agent for the treatment of nonspecific skin inflammation as it selectively inhibits cPLA(2) α and as it acts on an early stage of skin inflammation after its elicitation.

LC-MS assay for quantitative determination of cardio glycoside in human blood samples.

A method is described for liquid chromatography-mass spectrometry analysis of the cardio glycosides digoxin and digitoxin in biological samples. The method was optimized for use in the forensic field and, therefore, comprises the determination from whole blood and tissue samples. Sample cleanup by solid phase extraction (SPE) on a functionalized polymeric phase was sufficient to limit matrix suppression to <10% for all analytes. Chromatographic separation was achieved using an RP-8 column. Detection of the cardio glycosides was performed with electrospray ionization in the positive mode. The system was run in single ion monitoring mode, measuring the sodium adducts (M + Na)+ of the analyte and of the internal standard, respectively. The method was fully validated for the analysis of blood samples and was also successfully applied in forensic cases. The method was accurate and precise over a linear concentration range up to 50 ng/g blood. Lower limit of quantitation was 0.2 ng/g for digoxin and 2 ng/g for digitoxin, respectively. As deuterated analyte was used as internal standard, we also present a new microwave-enhanced method for the fast preparation of the labelled analyte within 20 min.

Quantitative determination of taxine B in body fluids by LC-MS-MS.

A new specific and sensitive LC-MS-MS method for the detection of taxine B and isotaxine B, the main toxic pseudo-alkaloids from yew (Taxus sp.), in biological samples (blood, urine, gastric content) was developed. Biological samples were prepared for LC-MS-MS by means of solid-phase extraction (SPE) procedure and yielded a recovery of 86%. Chromatographic separation was achieved using an RP(18) column. Detection of taxine B and isotaxine B was performed using multiple reaction monitoring with m/z 584.2 as precursor ion, i.e. [M+H](+), of both isomers and m/z 194.3 and m/z 107.1 as product ions after collision-induced dissociation. Docetaxel was applied as internal standard. The method was fully validated for the analysis of blood samples. Linearity was proven in the range from 0.1-500 ng/g. The limit of detection and the limit of quantitation are 0.4 and 2 ng/g, respectively. The method was applied to the determination of taxine B and isotaxine B in four fatal cases (two humans, two horses) with suspected yew intoxication. Blood levels were 105, 168, 174 and 212 ng/g.

Cryptic Species in the Anopheles (Nyssorhynchus) albitarsis (Diptera: Culicidae) Complex: Incongruence Between Random Amplified Polymorphic DNA-Polymerase Chain Reaction Identification and Analysis of Mitochondrial DNA COI Gene Sequences.

Random amplified polymorphic DNA (RAPD) diagnostic bands are one tool used to differentiate cryptic mosquito species in the Anopheles albitarsis Complex. Monophyly of four species (A. albitarsis Lynch-Arribálzaga, A. albitarsis B, A. deaneorum Rosa-Freitas, and A. marajoara Galvão & Damasceno) currently identified with the RAPD technique was assessed using sequences of the cytochrome oxidase I (COI) mitochondrial DNA (mtDNA) gene. Maximum parsimony, maximum likelihood, and Bayesian analyses support monophyly for A. albitarsis s.s., A. albitarsis B, and A. deaneorum. Anopheles marajoara, as identified by RAPD banding patterns, was either polyphyletic or paraphyletic in all phylogenetic analyses. The phylogenetic pattern and within-species genetic distances observed in A. marajoara suggest the existence of a previously unidentified species (species E) in northern Brazil and Venezuela. Diagnostic RAPD bands were unable to distinguish between A. marajoara and species E, probably because of the low number of correlated bands used to identify species and weaknesses of the RAPD technique, in particular, violations of the untested assumption of homology of comigrating bands. A. marajoara (even without species E) is paraphyletic with respect to A. deaneorum; if A. deaneorum is a separate species from A. marajoara, then A. marajoara may consist of two or more species in Amazonian Brazil. Based on mtDNA COI sequences, there are at least four phylogenetic species within the Albitarsis Complex: A. albitarsis s.s., A. albitarsis B, A. marajoara, and species E; the species status of A. deaneorum is ambiguous.

Development of a psilocin immunoassay for serum and blood samples.

After the immunisation of rabbits with a psilocin-specific immunogen, polyclonal antisera were obtained. With these antisera a competitive, heterogeneous radioimmunoassay for the detection of psilocin was developed. As tracer a derivative of psilocin was synthesised, which contained a tritiated CH(3) group. The antisera showed a specific reaction with psilocin. The cross-reactivity of structurally related endogenous substances like serotonin, tryptophan and tyrosine was below 0.01%. Also common drugs of abuse (Delta(9)-tetrahydrocannabinol, cocaine, morphine, amphetamine) showed negligible cross-reactivity (0.01-2%). Only tricyclic neuroleptics with a (dimethylamino)ethyl side-chain showed some cross-reactivity (20%). Spiked serum and blood samples were analysed with this new immunoassay and the results obtained were compared with the values measured with a validated GC-MS method.

Novel 3-dodecanoylindole-2-carboxylic acid inhibitors of cytosolic phospholipase A(2).

Derivatives of 1-[2-(4-carboxyphenoxy)ethyl]-3-dodecanoylindole-2-carboxylic acid (4) with modified substituents at the indole-1-position were synthesized and evaluated for their ability to inhibit the arachidonic acid release in human platelets mediated by the cytosolic phospholipase A(2). One of the most active compounds obtained was 26 with an IC(50) of 0.44 microM.

Comparison of the inhibition of the cytosolic phospholipase A2-mediated arachidonic acid release by several indole-2-carboxylic acids and 3-(pyrrol-2-yl)propionic acids in bovine and in human platelets.

The inhibition of the cytosolic phospholipase A2 (cPLA2)-mediated arachidonic acid release by several indole-2-carboxylic acids and 3-(pyrrol-2-yl)propionic acids was measured in intact human platelets using calcium ionophore A23187 as stimulant. The comparison of the obtained data with the inhibition data evaluated with bovine platelets showed that analogous results were obtained with both cell types.

Involvement of different protein kinases and phospholipases A2 in phorbol ester (TPA)-induced arachidonic acid liberation in bovine platelets.

The effect of various phospholipase A2 and protein kinase inhibitors on the arachidonic acid liberation in bovine platelets induced by the protein kinase activator 12-O-tetradecanoylphorbol-13-acetate (TPA) was studied. TPA stimulates arachidonic acid release mainly by activating group IV cytosolic PLA2 (cPLA2), since inhibitors of this enzyme markedly inhibited arachidonic acid formation. However, group VI Ca2+-independent PLA2 (iPLA2) seems to contribute to the arachidonic acid liberation too, since the relatively specific iPLA2 inhibitor bromoenol lactone (BEL) decreased arachidonic acid generation in part. The pronounced inhibition of the TPA-induced arachidonic acid release by the protein kinase C (PKC) inhibitors GF 109203X and Ro 31-82220, respectively, and by the p38 MAP kinase inhibitor SB 202190 suggests that the activation of the PLA2s by TPA is mediated via PKC and p38 MAP kinase.

Bilobalide, a constituent of Ginkgo biloba, inhibits NMDA-induced phospholipase A2 activation and phospholipid breakdown in rat hippocampus.

In rat hippocampal slices superfused with magnesium-free buffer, glutamate (1 mM) caused the release of large amounts of choline due to phospholipid breakdown. This phenomenon was mimicked by N-methyl-D-aspartate (NMDA) in a calcium-sensitive manner and was blocked by NMDA receptor antagonists such as MK-801 and 7-chlorokynurenate. The NMDA-induced release of choline was not caused by activation of phospholipase D but was mediated by phospholipase A2 (PLA2) activation as the release of choline was accompanied by the formation of lyso-phosphatidylcholine (lyso-PC) and glycerophospho-choline (GPCh) and was blocked by 5-[2-(2-carboxyethyl)-4-dodecanoyl-3,5-dimethylpyrrol-1-yl]pentano ic acid, a PLA2 inhibitor. Bilobalide, a constituent of Ginkgo biloba, inhibited the NMDA-induced efflux of choline with an IC50 value of 2.3 microM and also prevented the formation of lyso-PC and GPCh. NMDA also caused a release of choline in vivo when infused into the hippocampus of freely moving rats by retrograde dialysis. Again, the effect was completely inhibited by bilobalide which was administered systemically (20 mg/kg i.p.). Interestingly, convulsions which were observed in the NMDA-treated rats were almost totally suppressed by bilobalide. We conclude that release of choline is a sensitive marker for NMDA-induced phospholipase A2 activation and phospholipid breakdown. Bilobalide inhibited the glutamatergic excitotoxic membrane breakdown both in vitro and in vivo, an effect which may be beneficial in the treatment of brain hypoxia and/or neuronal hyperactivity.

Structure-activity relationships of (4-acylpyrrol-2-yl)alkanoic acids as inhibitors of the cytosolic phospholipase A2: variation of the substituents in positions 1, 3, and 5.

Derivatives of 3-(1,3,5-trimethyl-4-octadecanoylpyrrol-2-yl)propionic acid (1) and (1,3,5-trimethyl-4-octadecanoylpyrrol-2-yl)acetic acid (4) were prepared and evaluated for their ability to inhibit the cytosolic phospholipase A2 of intact bovine platelets. While replacement of one of the methyl groups in position 1, 3, or 5 of the acetic acid 4 by a benzyl residue did not influence the inhibitory potency significantly, the introduction of a dodecyl chain led to compounds which even enhanced the enzymatic activity. Stepwise elongation of the alkyl substituent in position 1 showed that the ability to inhibit the enzyme was lost when the alkyl chain exceeded a length of five carbons in case of compound 1 or six carbons in case of compound 4. Introduction of a polar functional group at the end of the 1-alkyl chain of these inactive pyrroles, however, restored or even elevated inhibitory potency. The most preferable of the polar terminal functions investigated was the carboxylic acid moiety. 6-[2-(2-Carboxyethyl)-4-dodecanoyl-3,5-dimethylpyrrol-1-yl]hexanoi c acid (65c) and 6-[2-(carboxymethyl)-4-dodecanoyl-3,5-dimethylpyrrol-1-yl]nonanoic acid (66f) were the synthesized inhibitors with the greatest potency. With IC50 values of 3.4 and 3.3 microM, respectively, they were about 3-fold more active than the standard cPLA2 inhibitor arachidonyl trifluoromethyl ketone (IC50: 11 microM).

Synthesis, biological evaluation, and structure-activity relationships of 3-acylindole-2-carboxylic acids as inhibitors of the cytosolic phospholipase A2.

3-Acylindole-2-carboxylic acid derivatives were prepared and evaluated for their ability to inhibit the cytosolic phospholipase A2 of intact bovine platelets. To define the structural requirements for enzyme inhibition, the carboxylic acid group, the acyl residue, and the moiety in position 1 were systematically modified. Furthermore, different substituents were introduced into the phenyl part of the indole. Replacement of the carboxylic acid group in position 2 of the indole with an acetic or propionic acid substituent led to a decrease of inhibitory potency. Enzyme inhibition was optimal when the acyl residue in position 3 had a length of 12 or more carbons. Conformational restriction of the acyl residue did not influence activity. Introduction of alkyl chains at position 1 of the indole with 8 or more carbons resulted in a loss of activity. However, replacing the omega-methyl group of such compounds with a carboxylic acid moiety was found to increase inhibitory potency significantly. Among the tested indole derivatives, 1-[2-(4-carboxyphenoxy)ethyl]-3-dodecanoylindole-2-carboxyli c acid (29b) had the highest potency. With an IC50 of 0.5 microM it was about 20-fold more active than the standard cPLA2 inhibitor arachidonyl trifluoromethyl ketone (IC50: 11 microM).

3-(3,5-Dimethyl-4-octadecanoylpyrrol-2-yl)propionic acids as inhibitors of 85 kDa cytosolic phospholipase A2.

3-(1,4-Diacylpyrrol-2-yl)propionic acids were designed as inhibitors of cytosolic phospholipase A2. Enzyme inhibition was assayed by evaluation of calcium ionophore A23187-induced arachidonic acid release from bovine platelets. While the synthesized bisacyl compound 3-[3,5-dimethyl-4-octadecanoyl-1-(3-phenylpropionyl)pyrrol-2-yl] propionic acid was inactive at 33 microM, the related monoacylated 3-(3,5-dimethyl-4-octadecanoylpyrrol-2-yl)-propionic acid and 3-(1,3,5-trimethyl-4-octadecanoylpyrrol-2-yl)-propionic acid proved to be inhibitors of cytosolic phospholipase A2(IC50: 24 microM and 13 microM, respectively).

3-(Octadecanoylaminomethyl)indole-2-carboxylic acid derivatives and 1-methyl-3-octadecanoylindole-2-carboxylic acid as inhibitors of cytosolic phospholipase A2.

3-(1-Acylaminooctadecyl)indole-2-carboxylic acids and 3-(1-acylaminooctadecyl)-1-methylindole-2-carboxylic acids were designed and synthesized as inhibitors of cytosolic phospholipase A2. Enzyme inhibition was assayed by evaluation of calcium ionophore A23187-induced arachidonic acid release from bovine platelets. While compounds with 1-octadecanoylaminooctadecyl groups in position 3 of the indole were inactive inhibition data for 3-[1-(3-phenylpropionylamino)octadecyl]indole-2-carboxylic acids could not be evaluated because of lysis of the platelets. However 3-(octadecanoylaminomethyl)indole-2-carboxylic acid derivatives and 1-methyl-3-octadecanoylindole-2-carboxylic acid proved to be inhibitors of cytosolic phospholipase A2. The most active inhibitor was the latter compound with an IC50 of 8 microM.

Respiratory syncytial virus-enriched globulin for the prevention of acute otitis media in high risk children.

Acute otitis media (AOM) has been associated with respiratory syncytial virus (RSV) infection; AOM develops in up to one third of children with RSV illness. A masked multicenter trial used an immune globulin enriched with RSV-neutralizing antibodies (RSVIG) to prevent RSV infection of the lower respiratory tract in 249 children with either bronchopulmonary dysplasia, congenital heart disease, or prematurity. To determine whether monthly RSVIG therapy might decrease the incidence of AOM, we retrospectively analyzed the records of 109 children in two of the centers. RSVIG was administered during RSV season of a high dose of 750 mg/kg monthly or a low dose of 150 mg/kg monthly; control children received no RSVIG. Children were examined for AOM by masked observers using pneumatic otoscopy. No difference in sex, race, underlying diagnosis, number of persons in the home, exposure to smoking, or atopy was found between groups studied. In recipients of high doses of RSVIG, significantly less AOM developed per season than in control children (mean episodes, 0.15 vs 0.78; p = 0.003), and fewer episodes of RSV-related AOM occurred (0 vs 5; p = 0.047). Low doses of RSVIG did not have a clinically significant impact. High doses of RSVIG appeared to have a significant impact on preventing AOM (both RSV- and non-RSV-related AOM) in these-high risk populations. This finding may have important implications in the development of improved preventive modalities for AOM.

Safety and bioequivalency of three formulations of respiratory syncytial virus-enriched immunoglobulin.

Respiratory syncytial virus (RSV) causes serious illness (lower respiratory illness) in preterm infants. RSV antibody-enriched immunoglobulin (RSVIG) that was lyophilized (LYO) protected against RSV lower respiratory illness. The Food and Drug Administration now requires an additional viral inactivation step (VI). We compared LYO, LYO-VI, and a more convenient liquid RSVIG (LIQ-VI) in 30 preterm infants (median age, 7 months; median weight, 5.4 kg). Infants were randomized to receive LYO (n = 10), LYO-VI (n = 10), or LIQ-VI (n = 10) in monthly infusions of 750 mg/kg of body weight per dose (December to March). Children were monitored closely for adverse reactions to RSVIG and for RSV illness.

Safety and immunogenicity of a purified haemagglutinin antigen in very young high-risk children.

Forty-three high-risk preterm children received either one of three doses of purified hemagglutinin antigen (HA) (7.5 micrograms/0.25 ml, 22.5 micrograms/0.25 ml or 67.5 micrograms/0.25 ml) or standard split product vaccine (ST) (22.5 micrograms/ml dose) over the 1990-1991 influenza season. Components for all vaccines included A/Shanghai 16/89, A/Taiwan 1/86 and B/Yamagata 16.88. Sera for antibody was drawn before, 6 weeks and 4 months after the first vaccine dose. The study was randomized and blinded. All children received two 0.25 ml doses of vaccine 4 weeks apart. No significant local or systemic reactions occurred. Six weeks after the first dose, children receiving ST vaccine had significantly higher seroconversion rates to A/Shanghai (p = 0.03) and to A/Taiwan (p = 0.01) than did those receiving equivalent HA vaccine. However, seroconversion rates were significantly higher for those children receiving the highest HA dose. All four vaccine groups responded poorly to B/Yamagata. Geometric mean titres were low for all groups and declined over 4 months. These results suggest that the equivalent dose of HA vaccine offers no advantage over ST vaccine in the immunization of high-risk preterm children.