Sex differences in amino acids lost via sweating could lead to differential susceptibilities to disturbances in nitrogen balance and collagen turnover.

Fluid collected during sweating is enriched with amino acids derived from the skin's natural moisturising factors and has been termed "faux" sweat. Little is known about sex differences in sweat amino acid composition or whether faux sweat amino acid losses affect nitrogen balance. Faux sweat collected by healthy adults (n = 47) after exercise, and at rest by chronic fatigue patients, was analysed for amino acid composition. Healthy females had higher total amino acid concentrations in sweat (10.5 ± 1.2 mM) compared with healthy males (6.9 ± 0.9 mM). Females had higher levels of 13 amino acids in sweat including serine, alanine and glycine. Higher hydroxyproline and proline levels suggested greater collagen turnover in females. Modelling indicated that with conservative levels of exercise, amino acid losses in females via faux sweat were triple than those predicted for urine, whereas in males they were double. It was concluded that females were more susceptible to key amino acid loss during exercise and/or hot conditions. Females reporting chronic fatigue had higher levels of methionine in faux sweat than healthy females. Males reporting chronic fatigue had higher levels of numerous amino acids in faux sweat compared to healthy males. Higher amino acid loss in faux sweat associated with chronic fatigue could contribute to a hypometabolic state. Depending on activity levels, climatic conditions and gender, amino acid losses in sweat and skin leachate could influence daily protein turnover where periods of continuously high turnover could lead to a negative net nitrogen balance.

Diverse characteristics of the urinary excretion of amino acids in humans and the use of amino acid supplementation to reduce fatigue and sub-health in adults.

BACKGROUND: The excretion of amino acids in urine represents an important avenue for the loss of key nutrients. Some amino acids such as glycine and histidine are lost in higher abundance than others. These two amino acids perform important physiological functions and are required for the synthesis of key proteins such as haemoglobin and collagen. METHODS: Stage 1 of this study involved healthy subjects (n = 151) who provided first of the morning urine samples and completed symptom questionnaires. Urine was analysed for amino acid composition by gas chromatography. Stage 2 involved a subset of the initial cohort (n = 37) who completed a 30 day trial of an amino acid supplement and subsequent symptom profile evaluation. RESULTS: Analyses of urinary amino acid profiles revealed that three groups could be objectively defined from the 151 participants using k-means clustering. The amino acid profiles were significantly different between each of the clusters (Wilks' Lambda = 0.13, p < 0.0001). Cluster 1 had the highest loss of amino acids with histidine being the most abundant component. Cluster 2 had glycine present as the most abundant urinary amino acid and cluster 3 had equivalent abundances of glycine and histidine. Strong associations were observed between urinary proline concentrations and fatigue/pain scores (r = .56 to .83) for females in cluster 1, with several other differential sets of associations observed for the other clusters. CONCLUSIONS: Different phenotypic subsets exist in the population based on amino acid excretion characteristics found in urine. Provision of the supplement resulted in significant improvements in reported fatigue and sleep for 81% of the trial cohort with all females reporting improvements in fatigue. TRIAL REGISTRATION: The study was registered on the 18th April 2011 with the Australian New Zealand Clinical Trials Registry ( ACTRN12611000403932 ).

Ceramides are associated with inflammatory processes in human mediastinal adipose tissue.

BACKGROUND AND AIM: Ceramides are poorly characterized in human adipose tissue. The aim of this study was to investigate concentrations of different ceramide species in human subcutaneous and visceral adipose tissue depots and to determine associations between ceramides and global gene expression profiles. METHODS AND RESULTS: Concentrations of six ceramide species were determined in plasma and in subcutaneous and mediastinal adipose tissue from 10 overweight subjects (BMI 29.4 ± 4.9 kg/m(2)). In the adipose tissue biopsies gene expression arrays were performed and relationships between ceramides and gene expression analyzed. Immunostaining of the two adipose tissue depots was performed in an independent group of 10 patients. Mediastinal adipose tissue contained significantly higher concentrations (p < 0.05) of all six ceramide species than the subcutaneous depot. Of the six ceramides in plasma, concentrations of only two (Cer d18:1/18:0 and Cer d18:1/22:0) correlated significantly (p < 0.05) with the corresponding species in mediastinal adipose tissue, but there were no significant correlations between ceramides in plasma and subcutaneous adipose tissue. Multivariate analysis identified significant correlations between the total ceramide concentration and global gene expression within mediastinal, but not subcutaneous adipose tissue, according to cross-validation. Gene ontology analysis of genes related to ceramides in the mediastinal depot revealed that genes positively correlated with ceramides were associated mainly with immune and inflammatory categories, while genes negatively correlated with ceramides were associated mainly with lipid and carbohydrate metabolism. CONCLUSIONS: Ceramides in human mediastinal adipose tissue may be involved in inflammation and lipid and carbohydrate metabolism.

Statistical molecular design, parallel synthesis, and biological evaluation of a library of thrombin inhibitors.

A library of thrombin inhibitors has been designed using statistical molecular design. An aromatic scaffold was used, with three varied positions corresponding to three pockets at the active site of thrombin (the S-, P-, and D-pockets). The selection was performed in the building block space, and previously acquired data were included in the design procedure. The design resulted in six, four, and six building blocks for the first (S), second (P), and third (D) pockets, respectively. A second round of selection applied to the combined selected building blocks resulted in a subset of 18 compounds. The selected library was synthesized in parallel and biologically evaluated. The compounds were analyzed with respect to their inhibition (pIC(50)) of thrombin; membrane permeability, estimated by migration behavior in micellar media (CE log k') and pK(a); and specificity with respect to inhibition (K(i)) of trypsin. Multivariate QSAR studies of the responses yielded valuable results and information that could only be found using statistical molecular design in combination with multivariate analysis.

One-carbon metabolism and other biochemical correlates of cognitive impairment as visualized by principal component analysis.

In the present report, 101 ambulatory elderly patients complaining about cognitive disturbances were investigated using the Mini-Mental State Examination (MMSE). Laboratory investigations, brain imaging, and electroencephalography were performed. Twelve patients were diagnosed with subjective memory complaints (SMC), 32 with mild cognitive impairment (MCI), 43 with dementia of the Alzheimer type (DAT), and 14 with vascular dementia (VAD). Thirty-three percent of the SMC group, 31% of the MCI group, 45% of the DAT group, and 62% of the VAD group had increased serum homocysteine (s-HCY). Principal component analysis of 19 variables showed 3 significant principal components by cross-validation. The cognitive impairment in the patients (MMSE) was explained to 50%. According to the principal component analysis, the population followed two different routes to cognitive impairment: one correlated with disturbance of one-carbon metabolism (cerebrospinal fluid vitamin B12, plasma B12, plasma folate, and s-HCY) and the other correlated with more classic dementia, as marked by cerebrospinal fluid tau, vascular risk factors, atrophy on brain imaging, possession of the apolipoprotein E4 allele, and age. There was poor discrimination between DAT and VAD.

Lack of replication of association findings in complex disease: an analysis of 15 polymorphisms in prior candidate genes for sporadic Alzheimer's disease.

There is considerable enthusiasm for the prospect of using common polymorphisms (primarily single nucleotide polymorphisms; SNPs) in candidate genes to unravel the genetics of complex disease. This approach has generated a number of findings of loci which are significantly associated with sporadic Alzheimer's disease (AD). In the present study, a total of 15 genes of interest were chosen from among the previously published reports of significant association in AD. Genotyping was performed on polymorphisms within those genes (14 SNPs and one deletion) using Dynamic Allele Specific Hybridization (DASH) in 204 Swedish patients with sporadic late-onset AD and 186 Swedish control subjects. The genes chosen for analysis were; low-density lipoprotein receptor-related protein (LRP1), angiotensin converting enzyme (DCP1), alpha-2-macroglobulin (A2M), bleomycin hydrolase (BLMH), dihydrolipoyl S-succinyltransferase (DLST), tumour necrosis factor receptor superfamily member 6 (TNFRSF6), nitric oxide synthase (NOS3), presenilin 1 (PSEN1), presenilin 2 (PSEN2), butyrylcholinesterase (BCHE), Fe65 (APBB1), oestrogen receptor alpha (ESR1), cathepsin D (CTSD), methylenetetrahydrofolate reductase (MTHFR), and interleukin 1A (IL1A). We found no strong evidence of association for any of these loci with AD in this population. While the possibility exists that the genes analysed are involved in AD (ie they have weak effects and/or are population specific), results reinforce the need for extensive replication studies if we are to be successful in defining true risk factors in complex diseases.

The cerebrospinal fluid levels of tau, growth-associated protein-43 and soluble amyloid precursor protein correlate in Alzheimer's disease, reflecting a common pathophysiological process.

Cerebrospinal fluid (CSF) levels of tau (total tau), growth-associated protein-43 (GAP-43), soluble amyloid precursor protein (sAPP; i.e. total sAPP), and beta-amyloid(42) (Abeta(42)) were studied in patients with frontotemporal dementia (FTD; n = 14), Alzheimer's disease (AD; n = 47) and vascular dementia (VAD; n = 16), and in age-matched controls (n = 12). CSF-tau was increased in AD compared to controls and FTD (p < 0.001 for both). CSF-GAP-43 was increased in AD compared to controls (p < 0.05), and both CSF-GAP-43 and CSF-sAPP were increased in AD compared to FTD (p < 0.01). Positive and highly significant correlations were found between CSF-tau and CSF-GAP-43 in all groups and between CSF-tau, CSF-GAP-43 and CSF-sAPP in AD. The correlations found may reflect a common pathophysiologic process such as axonal degeneration.

Chemography: the art of navigating in chemical space.

Combinatorial chemistry needs focused molecular diversity applied to the druglike chemical space (drugspace). A drugspace map can be obtained by systematically applying the same conventions when examining the chemical space, in a manner similar to the Mercator convention in geography: Rules are equivalent to dimensions (e.g., longitude and latitude), while structures are equivalent to objects (e.g., cities and countries). Selected rules include size, lipophilicity, polarizability, charge, flexibility, rigidity, and hydrogen bond capacity. For these, extreme values were set, e.g., maximum molecular weight 1500, calculated negative logarithm of the octanol/water partition between -10 and 20, and up to 30 nonterminal rotatable bonds. Only S, N, O, P, and halogens were considered as elements besides C and H. Selected objects include a set of "satellite" structures and a set of representative drugs ("core" structures). Satellites, intentionally placed outside drugspace, have extreme values in one or several of the desired properties, while containing druglike chemical fragments. ChemGPS (chemical global positioning system) is a tool that combines these predefined rules and objects to provide a global drugspace map. The ChemGPS drugspace map coordinates are t-scores extracted via principal component analysis (PCA) from 72 descriptors that evaluate the above-mentioned rules on a total set of 423 satellite and core structures. Global ChemGPS scores describe well the latent structures extracted with PCA for a set of 8599 monocarboxylates, a set of 45 heteroaromatic compounds, and for 87 alpha-amino acids. ChemGPS positions novel structures in drugspace via PCA-score prediction, providing a unique mapping device for the druglike chemical space. ChemGPS scores are comparable across a large number of chemicals and do not change as new structures are predicted, making this tool a well-suited reference system for comparing multiple libraries and for keeping track of previously explored regions of the chemical space.

Quantitative analysis of film coating in a fluidized bed process by in-line NIR spectrometry and multivariate batch calibration.

A method is described which enables real-time analysis of film coating on pharmaceutical pellets during an industrial manufacturing process. Measurements were conducted on the solid particulate material by near-infrared (NIR) spectrometry utilizing a diffuse reflectance fiber-optic probe positioned inside a fluidized bed process vessel. Time series of NIR spectra from 11 batches generated a three-way data matrix that was unfolded and modeled by partial least squares (PLS) in a multivariate batch calibration. The process conditions were deliberately varied according to an experimental design. This yielded good predictability of the coating thickness with a best model fit, R2 = 0.97, for one PLS-projection, and a root-mean-square error of calibration = 2.2 microm (range tested 0-50 microm). The regression vector was shown to be highly influenced by responses that are both direct (aliphatic C-H stretch overtones) and indirect (aromatic C-H stretch overtones), from film component and core material, respectively. The impact of different data pre-treatment methods on the normalization of the regression vector is reported. Justification of the process calibration approach is emphasized by good correlation between values predicted from NIR data and reference image analysis data on dissected pellets and a theoretical nonlinear coating thickness growth model. General aspects of in-line NIR on solids and multivariate batch calibration are discussed.

Chemical information management in drug discovery: optimizing the computational and combinatorial chemistry interfaces.

Structure-property relationships, central to many of today's drug discovery strategies, are not straightforward to deal with when trying to predict drug efficacy, that is, the combined outcome of target affinity, pharmacodynamic behavior, pharmacokinetic properties, and metabolic fate. In this article, we discuss the handling of chemical property information in reagents-for-synthesis selection, enumeration, and virtual library construction. We describe the use of diversity assessment and/or experimental design in selection of compound-libraries-to-be-synthesized. Our overall objective was to identify good-quality drug candidates through reliable structure-activity relationship data, with the minimum number of compounds synthesized and tested. Chemical filters, property filters, scoring functions, and utilization of interactive visualization tools are discussed. The concept of chemical diversity and aspects of chemical space navigation employing a proprietary tool, Chemical Global Positioning System (ChemGPS), for mapping the drug-related chemical space are examined. Guidelines and workflow recommendations for the practicing medicinal chemist are proposed.

Statistical molecular design of building blocks for combinatorial chemistry.

The reduction of the size of a combinatorial library can be made in two ways, either base the selection on the building blocks (BB's) or base it on the full set of virtually constructed products. In this paper we have investigated the effects of applying statistical designs to BB sets compared to selections based on the final products. The two sets of BB's and the virtually constructed library were described by structural parameters, and the correlation between the two characterizations was investigated. Three different selection approaches were used both for the BB sets and for the products. In the first two the selection algorithms were applied directly to the data sets (D-optimal design and space-filling design), while for the third a cluster analysis preceded the selection (cluster-based design). The selections were compared using visual inspection, the Tanimoto coefficient, the Euclidean distance, the condition number, and the determinant of the resulting data matrix. No difference in efficiency was found between selections made in the BB space and in the product space. However, it is of critical importance to investigate the BB space carefully and to select an appropriate number of BB's to result in an adequate diversity. An example from the pharmaceutical industry is then presented, where selection via BB's was made using a cluster-based design.

The synaptic-vesicle-specific proteins rab3a and synaptophysin are reduced in thalamus and related cortical brain regions in schizophrenic brains.

Two synaptic-vesicle proteins, rab3a and synaptophysin, have been studied on post-mortem brain tissues of schizophrenics and healthy controls. We found significantly reduced levels of rab3a in thalamus (p<0.001); for both proteins in gyrus cinguli and hippocampus (p<0.0001); for rab3a in frontal and parietal cortex (p<0.05); and no differences in temporal cortex or cerebellum in schizophrenics compared with controls. Reduced synaptic density may be a prominent feature of the molecular neuropathology of schizophrenia.

Sensitivity, specificity, and stability of CSF-tau in AD in a community-based patient sample.

OBJECTIVE: To evaluate the sensitivity and specificity of CSF-tau in clinical practice as a diagnostic marker for AD compared with normal aging and depression, to study the stability of CSF-tau in longitudinal samples, and to determine whether CSF-tau levels are influenced by different covariates such as gender, age, duration or severity of disease, or possession of the APOE-epsilon4 allele. METHODS: Consecutive AD patients from a community-based sample were studied, including 407 patients with AD (274 with probable AD and 133 with possible AD), 28 patients with depression, and 65 healthy elderly control subjects. A follow-up lumbar puncture was performed in 192 AD patients after approximately 1 year. CSF-tau was determined using a sandwich ELISA, which was run as a routine clinical neurochemical analysis. RESULTS: CSF-tau was increased in probable (690+/-341 pg/mL; p < 0.0001) and possible (661+/-447 pg/mL; p < 0.0001) AD, but not in depression (231+/-110 pg/mL) compared with control subjects (227+/-101 pg/mL). Receiver operating characteristics analysis showed that a cutoff level of 302 pg/mL resulted in a sensitivity of 93% (95% CI, 90-96%) and a specificity of 86% (95% CI, 75-94%), with an area under the curve of 0.95 to discriminate AD from control subjects. Within the AD group, CSF-tau did not differ significantly between baseline and follow-up investigations, and was relatively stable between baseline and 1-year follow-up levels, with a coefficient of variation of 21.0%. High CSF-tau levels were also found in most AD patients with very short duration of dementia, and with Mini-Mental State Examination scores >23 (n = 205). In total, 193 of 205 patients (sensitivity, 94%) had a CSF-tau level higher than 302 pg/mL. CONCLUSIONS: CSF-tau has a high sensitivity and specificity to differentiate AD from normal aging and depression, as demonstrated in a large community-based series of consecutive AD patients during which analyses were run continually in a clinical neurochemical laboratory. The increase in CSF-tau is found very early in the disease process in AD, is stable over time, and has a low interindividual variation on repeated sampling. Although high CSF-tau is found in some neurologic conditions (e.g., stroke), these findings suggest that CSF-tau may be of use to help in differentiating AD from normal aging and depression, especially early in the course of the disease, when the symptoms are vague and the diagnosis is especially difficult.

Toward minimalistic modeling of oral drug absorption.

Poor intestinal permeability of drugs constitutes a major bottleneck in the successful development of candidate drugs. Fast computational tools to help in designing compounds with increased probability of oral absorption are required, since both medicinal and combinatorial chemists are under pressure to consider increasing numbers of virtual and existing compounds. The QSAR paradigm for drug absorption is expressed as a function of molecular size, hydrogen-bonding capacity, and lipophilicity. A nonlinear PLS model that can be achieved with minimal computational efforts is described. The QSAR model correlates human intestinal absorption (%HIA) data, and apparent Caco-2 cell permeability data, to parameters calculated from molecular structures. Two properties were found to be relevant for absorption predictions, namely H-bonding capacity, and hydrophobic transferability. The parsimony principle was applied in several aspects: single conformers were used to compute molecular surface areas; the definitions of "polar" and "nonpolar" surfaces were done in a simplistic fashion; simple and fast 2D descriptors were used to estimate other properties; the 1 PLS component model was selected. These choices result in a minimalistic model for oral absorption. The use of both %HIA and Caco-2 permeability data was found to stabilize and improve the model. This QSAR model can serve as a simple, quantitative extension of the "rule of five" scheme (Lipinski, C.A., Lombardo, F., Dominy, B.W., and Feeney, P.J. Adv. Drug Deliv. Rev. 1997, 23, 3-25), in a manner that can prove beneficial to the drug discovery process.

Vibrational spectrometry for the assessment of active substance in metoprolol tablets: a comparison between transmission and diffuse reflectance near-infrared spectrometry.

Near-infrared spectrometry (NIR) was used to quantify metroprolol succinate in controlled release tablets. Metoprolol tablets were made according to an experimental design using different strengths around a central strength of 47.5 mg per tablet. A comparison was made between NIR in the diffuse reflectance mode and the transmission mode. This showed that, although a narrower wavelength range was available in the transmission mode, predictions were much better for models based on transmission spectra than for models based on diffuse reflectance spectra. The main reason for this is that in the reflectance mode NIR spectrometry is very sensitive to the inhomogeneity of the material, while in the transmission mode this problem is less severe. This is due to the larger volume of the material scanned in the transmission mode compared to that in diffuse reflectance. Spectra were taken before and after the tablets were stored under humid conditions. This allowed the final calibration models to be made more robust towards variations in the amount of water in the tablet. Different batches of metoprolol pellets and microcrystalline cellulose were used during the production of the tablets. this resulted in models that were more robust towards possible batch-to-batch differences in the main constituents.

Gastrointestinal transit of amoxicillin modified-release tablets and a placebo tablet including pharmacokinetic assessments of amoxicillin.

BACKGROUND: We have investigated the gastrointestinal transit time of, the influence of food intake on, the disintegration of, and the pharmacokinetics of amoxicillin in a modified-release form. METHODS: Radiolabelled modified-release tablets of amoxicillin and placebo tablets were administered, in an open three-way, randomized, crossover design, as single doses during omeprazole treatment, to six male healthy subjects during fasting and non-fasting conditions. Radioscintigraphic images and plasma samples were obtained. RESULTS: The estimated mean (and range) gastric emptying time of the modified-release tablet after drug administration was 0.3 h (0.1-1.0 h) during fasting conditions, 4.3 h (1.7-5.0 h) after a light breakfast, and 4.9 h (1.9-18.0 h) after a heavy breakfast. The small-intestinal transit time during fasting conditions was 4.7 h (2.9-6.9 h) and was not significantly changed after light or heavy breakfast intake. The relative bioavailability of the modified-release tablet was 55%, compared with a commercially available amoxicillin immediate-release tablet. CONCLUSION: The modified-release tablet of amoxicillin administered postprandially apparently increases the amoxicillin release time in the stomach. The relevance of its use for anti-H. pylori treatment can be questioned.

Diagnosis of dementias using partial least squares discriminant analysis.

Twenty patients with Alzheimer's disease (AD), 34 with senile dementia of the Alzheimer type (SDAT) and 12 with vascular dementia (VAD) were carefully investigated. The diagnoses were made according to the DSM-III-R criteria and for AD/SDAT also according to the NINCDS-ADRDA criteria. The data set consisted of 29 descriptor variables obtained at the examination, and the data were fitted to a partial least squares model using dummy variables for the response variables (i.e. clinical diagnoses). The distribution of the scores over the descriptor variable space revealed adequate classification regarding all three diagnoses, with some overlap between SDAT and AD and between VAD and SDAT. The study demonstrates a clear difference in symptomatology between AD and SDAT with no influence of age per se or the severity of dementia.

Gangliosides in human fetal brain.

The ganglioside concentration and composition were determined in 42 human fetal brains from gestational week 10 to 22, a period that is morphologically characterized by rapid neuroblast proliferation and migration. The ganglioside concentration was constant during this period, approximately 1 mumol of ganglioside sialic acid/g of fresh tissue weight. At gestational week 10 the ganglioside pattern was dominated by gangliosides of the ganglio b series, with the major ganglioside being GT1b, contributing 40% of total ganglioside sialic acid, whereas GD1b and GD3 contributed only 15 and 10%, respectively. The proportion of b series ganglioside decreased to gestational week 22, with the most pronounced relative reduction affecting GD3, but also GT1b and GD1b to a lesser extent. The ganglioside GQ1b increased in content from gestational week 10 and peaked around week 16. The proportion of GD1a increased markedly between gestational week 12 and 14 and slowly between week 14 and 18 and then increased rapidly from week 20. Ganglioside GM1 underwent a similar change. Gangliosides of the lacto series contributed 6-10% of ganglioside sialic acid between gestational week 10 and 15, and thereafter the proportion slowly decreased. 3'-isoLM1 decreased rapidly in content from gestational week 10 (20 nmol/g of fresh weight) to week 22 (less than 0.5 nmol/g of fresh weight), whereas the gangliosides of the neolacto series (3'-LM1 and 3',8'-LD1) showed a slower and less marked decline in level. The biological significance of the ganglioside changes is discussed.

Glycosphingolipid glycosyltransferases in human fetal brain.

The developmental pattern of gangliosides in human fetal brain should reflect the activities of the respective glycosyltransferases. LA2-synthase activity, along with that of GM3-, GD3-, GM2-, and GM1-synthases, was determined in human fetal brain at 10-22 weeks of gestation. LA2-synthase is the pivotal enzyme in lacto series ganglioside formation. LA2-synthase activity decreased during the study period, mirroring a similar temporal decline in levels of the lacto series gangliosides, particularly 3'-isoLM1. The developmental profiles of the ganglio series glycosyltransferase activities demonstrate distinct changes that correspond to the ganglioside pattern between fetal weeks 10 and 22. In particular, the marked increase in GM2-synthase activity at 20 and 22 weeks of gestation and the decline in GD3-synthase activity after 15 weeks could explain the prominent expression of the a series gangliosides in this period of rapid neuronal outgrowth. However, a similar decline (two- to 2.5-fold) in GM3-synthase activity suggests a more likely conclusion, namely, that the two sialyltransferase activities are derived mainly from astroglial cells, which show a marked proliferation during the 10-15th fetal weeks. The data do not negate the hypothesis that GM3- and GD3-synthase are the critical enzymes in the regulation of ganglioside biosynthesis but do indicate a need to reevaluate the significance of GM2-synthase in expression of the a series gangliosides.