Circulating Glucocorticoid Bioactivity and Serum Glucocorticoid-Responsive Biomarkers during Steroid Therapy in Children and Adolescents with Inflammatory Bowel Disease

Objective: Glucocorticoid therapy is used worldwide to treat various inflammatory and immune conditions, including inflammatory bowel disease (IBD). In IBD, 80% of the patients obtain a positive response to the therapy; however the development of glucocorticoid-related side-effects is common. Our aim was therefore to study the possibility of optimizing glucocorticoid therapy in children and adolescents with IBD by measuring circulating glucocorticoid bioactivity (GBA) and serum glucocorticoid-responsive biomarkers in patients receiving steroid treatment for active disease. Methods: A total of sixty-nine paediatric IBD patients from the Paediatric Outpatient Clinics of the University Hospitals of Helsinki and Tampere participated in the studies. Control patients included 101 non-IBD patients and 41 disease controls in remission. In patients with active disease, blood samples were withdrawn before the glucocorticoid therapy was started, at 2-4 weeks after the initiation of the steroid and at 1-month intervals thereafter. Clinical response to glucocorticoid treatment and the development of steroid adverse events was carefully registered. GBA was analyzed with a COS-1 cell bioassay. The measured glucocorticoid therapy-responsive biomarkers included adipocyte-derived adiponectin and leptin, bone turnover-related collagen markers amino-terminal type I procollagen propeptide (PINP) and carboxyterminal telopeptide of type I collagen (ICTP) as well as insulin-like growth factor 1 (IGF-1) and sex hormone-binding globulin (SHBG), and inflammatory marker high-sensitivity C-reactive protein (hs-CRP). Results: The most promising marker for glucocorticoid sensitivity was serum adiponectin that associated with steroid therapy–related adverse events. Serum leptin indicated a similar trend. In contrast, circulating GBA rose in all subjects receiving glucocorticoid treatment but did not associate with the clinical response to steroids or with glucocorticoid therapy-related side-effects. Of notice, young patients (<10 years) showed similar GBA levels than older patients, despite receiving higher weight-adjusted doses of glucocorticoid. Markers of bone formation were lower in children with active IBD than in the control patients, probably reflecting the suppressive effect of the active inflammation. The onset of the glucocorticoid therapy further suppressed bone turnover. Inflammatory marker hs-CRP decreased readily after the initiation of the steroid, however the decrease did not associate with the clinical response to glucocorticoids. Conclusions: This is the first study to show that adipocyte-derived adiponectin associates with steroid therapy-induced side-effects. Further studies are needed, but it is possible that the adiponectin measurement could aid the recognition of glucocorticoid-sensitive patients in the future. GBA and the other markers reflecting glucocorticoid activity in different tissues changed during the treatment, however their change did not correlate with the therapeutic response to steroids or with the development of glucocorticoid-related side effects and therefore cannot guide the therapy in these patients. Studies such as as the present one that combine clinical data with newly developed biomolecular technology are needed to step-by-step build a general picture of the glucocorticoid actions in different tissues.

A bile acid derived potassium ion sensor

A chenodeoxycholic acid based K+ ion sensor has been designed using a modular approach in which a fluorophore and a cation receptor are attached to the bile acid backbone. In the absence of K+ the fluorescence of the molecule is quenched because of through-space, photo-induced electron-transfer from the aza-crown unit. Fluorescence enhancement was observed upon titration with K+ (and other alkali metal ions too). In methanol, good selectivity towards the sensing of K+ has been observed.

Characterization of the interaction of lipid A and lipopolysaccharide with human serum albumin: implications for an endotoxin carrier function for albumin

The interactions of lipid A and lipopolysaccharide (LPS) with human serum albumin (HSA) were examined using fluorescence methods. Lipid A binds HSA with a stoichiometry of 2:1 with dissociation constants of 1.0 µM and 6.0 µM for the high- and low-affinity interactions, respectively. Lipid A displaces HSA-bound dansylsarcosine competitively, but not HSA-bound warfarin, suggesting that domain III-A, and not domain 11-A, is a lipid A binding site. Domain I does not contribute a site for lipid A. Based on these data, and the structural similarity between subdomains III-A and III-B, it is proposed that these two regions of HSA represent the high- and low-affinity sites of interaction of lipid A. Whole LPS also binds HSA, displacing dansylsarcosine, and its lipid A moiety appears to be the interaction site. However, there are differences between LPS and free lipid A. Polymyxin B forms ternary complexes with LPS bound to HSA, suggesting that the regions on LPS recognized by HSA and polymyxin B are different. The observed affinity of lipid A for HSA and mass action effects due to its abundance in the circulation would imply a major LPS carrier function for HSA.

Salt in the desert: A comparison of clinical versus serum-calculated osmolality determination of dehydration

Background: Clinicians frequently use their own judgement to assess patient’s hydration status although there is limited evidence for the diagnostic utility of any individual clinical symptom. Hence, the aim of this study was to compare the diagnostic accuracy of clinically assessed dehydration in older hospital patients (using multiple symptoms), versus dehydration measured using serum-calculated osmolality (CO) as the reference standard. Method: Participants were 44 hospital patients aged ≥ 60 years. Dehydration was assessed clinically and pathologically (CO) within 24 hours of admission and at study exit. Indicators of diagnostic accuracy were calculated. Results: Clinicians identified 27% of patients as dehydrated at admission, and 19% at exit, compared to 19% and 16% using CO. Agreement between the measures was fair at admission and poor at exit. Clinical assessment showed poor sensitivity for predicting dehydration with reasonable specificity. Conclusions: Compared to the use of CO, clinical assessment of dehydration in older patients was poor. Given that failure to identify dehydration in this population may have serious consequences, we recommend that clinicians do not rely upon their own assessments without also using the reference standard.

Raman and infra-red spectra of crystalline potassium sulphamate

Raman spectrum of a single crystal of potassium sulphamate has been recorded for the first time using λ 2536 radiation of mercury as the exciter. Thirty-three Raman lines have been observed of which nine belong to the lattice oscillations. The infra-red absorption spectrum of the substance was taken in the powder form in potassium bromide disc using Carl Zeiss UR 10 IR spectrometer. Thirty-six absorption maxima could be identified of which twenty-five have been recorded for the first time. The analysis clearly shows that the N-H bond in the crystalline potassium sulphamate is not hydrogen-bonded to any appreciable extent.

Effect of anti-luteinizing hormone serum on the ovulation of rats

IN the cyclic female albino rat, a release of pituitary luteinizing hormone (LH) occurs on the afternoon of proestrus1-5. This apparently induces ovulation, for ova are seen in the Fallopian tube 12 h later. Similarly, it is well known that in immature rats primed with pregnant mare serum gonadotrophin (PMS), ovulation can be induced by the administration of human chorionic gonadotrophin (HCG) or LH, the ova being seen in the Fallopian tube 12 h later. No information is available, however, about the mode of action of LH, released or administered, in bringing about ovulation. We have approached this problem by blocking the action of the ovulating hormone (LH) at various times after administration. © 1970 Nature Publishing Group.

Polarisation studies on the Raman spectra of cubic crystals - Part II: Calcium fluoride and potassium aluminium sulphate

Polarisation characters of the Raman lines of calcium fluoride (fluorspar) and potassium aluminium sulphate (alum) were investigated under the following conditions. Unpolarised light was incident normally on a face of the crystal making an angle 22.5° with a cubic face and the light scattered transversely along a cubic axis was analysed by a double image prism kept with its principal axes inclined at 45° to the vertical. Under these conditions the depolarisation factors of the Raman lines belonging to the totally symmetric (A), the doubly degenerate (E) and the triply degenerate (F) modes should be respectively =1, >1 and <1. The characteristic Raman line of CaF2 at 322 cm-1 exhibited a depolarisation value less than 1, showing thereby that the corresponding mode is a triply degenerate one (F). The Raman lines observed in the spectrum of K-alum were also classified and the results were compared with those given by previous investigators using standard crystal orientations.

Potassium phosphate as a reagent for the gravimetric estimation of lithium

Previous attempts for the quantitative estimation of lithium as orthophosphate, employing an alkali metal phosphate, have not been successful. A method, is described for the estimation of lithium as trilithium phosphate from 60% ethyl alcohol solution at 65° to 70° C., employing potassium phosphate reagent, at pH 9.5. The method is applicable in the presence of varying amounts of sodium and/or potassium cations and chloride, sulfate, nitrate, and phosphate anions.

Studies on interaction of colloidal Ag nanoparticles with Bovine Serum Albumin (BSA)

Biofunctionalization of noble metal nanoparticles like Ag, Au is essential to obtain biocompatibility for specific biomedical applications. Silver nanciparticles are being increasingly used in bio-sensing applications owing to excellent optoelectronic properties. Among the serum albumins, the most abundant proteins in plasma, a wide range of physiological functions of Bovine Serum Albumin (BSA) has made it a model system for biofunctionalization. In absence of adequate prior reports, this study aims to investigate the interaction between silver nanoparticles and BSA. The interaction of BSA [0.05-0.85% concentrations] with Ag nanoparticles [50 ppm concentration] in aqueous dispersion was Studied through UV-vis spectral changes, morphological and surface structural changes. At pH 7, which is More than the isoelectric point of BSA, a decrease in absorbance at plasmon peak of uninteracted nanciparticles (425 mn) was noted till 0.45% BSA, beyond that a blue shift towards 410 urn was observed. The blue shift may be attributed to enhanced electron density on the particle surfaces. Increasing pH to 12 enhanced the blue shift further to 400 rim. The conformational changes in BSA at alkaline pH ranges and consequent hydrophobic interactions also played an important role. The equilibrium adsorption data fitted better to Freundlich isotherm compared to Langmuir Curve. The X-ray diffraction study revealed complete coverage of Ag nanoparticles by BSA. The scanning electron microscopic study of the interacted nanoparticles was also carried Out to decipher morphological changes. This study established that tailoring the concentration of BSA and pH of the interaction it was possible to reduce aggregation of nanoparticles. Biofunctionalized Ag nanoparticles with reduced aggregation will be more amenable towards bio-sensing applications. (C) 2009 Elsevier B.V. All rights reserved.

Studies on non-specific interference due to serum in the avidin-biotin microELISA for monkey chorionic gonadotropin and a method for its elimination

A study has been carried out on the non-specific interference due to serum in the avidin biotin micro-ELISA for monkey chorionic gonadotropin. Results suggest that it is not due to any proteolytic activity in the serum, but immunoglobulin or associated factors interfering at the level of antigen-antibody interaction. This interference was eliminated by heating samples at 60°C for 30 min.

Selective para metalation of unprotected 3-methoxy and 3,5-dimethoxy benzoic acids with n-butyl lithium-potassium tert-butoxide (LIC-KOR): synthesis of 3,5-dimethoxy-4-methyl benzoic acid

The potassium salt of 3-methoxy and 3,5-dimethoxy benzoic acids undergoes deprotonation at the position para to the carboxylate group selectively when treated with LIC-KOR in THF at -78 degrees C and it has been extended to the synthesis of 3,5-dimethoxy-4-methyl benzoic acid. (C) 2000 Elsevier Science Ltd. All rights reserved.

A spectroscopic study of the interaction of isoflavones with human serum albumin

Genistein and daidzein, the major isoflavones present in soybeans, possess a wide spectrum of physiological and pharmacological functions. The binding of genistein to human serum albumin (HSA) has been investigated by equilibrium dialysis, fluorescence measurements, CD and molecular visualization. One mole of genistein is bound per mole of HSA with a binding constant of 1.5 +/- 0.2 X 10(5) m(-1). Binding of genistein to HSA precludes the attachment of daidzein. The ability of HSA to bind genistein is found to be lost when the tryptophan residue of albumin is modified with N-bromosuccinimide. At 27 degrees C (pH 7.4), van't Hoff's enthalpy, entropy and free energy changes that accompany the binding are found to be -13.16 kcal.mol(-1), -21 cal.mol(-1)K(-1) and -6.86 kcal.mol(-1), respectively. Temperature and ionic strength dependence and competitive binding measurements of genistein with HSA in the presence of fatty acids and 8-anilino-1-naphthalene sulfonic acid have suggested the involvement of both hydrophobic and ionic interactions in the genistein-HSA binding. Binding measurements of genistein with BSA and HSA, and those in the presence of warfarin and 2,3,5-tri-iodobenzoic acid and Forster energy transfer measurements have been used for deducing the binding pocket on HSA. Fluorescence anisotropy measurements of daidzein bound and then displaced with warfarin, 2,3,5-tri-iodobenzoic acid or diazepam confirm the binding of daidzein and genistein to subdomain IIA of HSA. The ability of HSA to form ternery complexes with other neutral molecules such as warfarin, which also binds within the subdomain IIA pocket, increases our understanding of the binding dynamics of exogenous drugs to HSA.

A.C. electrical conductivity of pure and doped potassium perchlorate

A.C. electrical conductivity of potassium perchlorate (KP) has been measured in the temperature range 25�325°C at frequencies ranging from 50�500 Hz using an automated technique. The results are interpreted in terms of a novel mechanism involving Schottky defects in the anion sublattice and Frenkel defects in the cation sublattice. Theconductivity behavior of KP is compared with literature data on similar low-symmetry systems containing polyatomic ions.