Analysis of procainamide-derivatised heparan sulphate disaccharides in biological samples using hydrophilic interaction liquid chromatography mass spectrometry

Glycosaminoglycans (GAGs) are a family of linear heteropolysaccharides made up of repeating disaccharide units that are found on the surface and extracellular matrix of animal cells. They are known to play a critical role in a wide range of cellular processes including proliferation, differentiation and invasion. To elucidate the mechanism of action of these molecules, it is essential to quantify their disaccharide composition. Analytical methods that have been reported involve either chemical or enzymatic depolymerisation of GAGs followed by separation of non-derivatised (native) or derivatised disaccharide subunits and detection by either UV/fluorescence or MS. However, the measurement of these disaccharides is challenging due to their hydrophilic and labile nature. Here we report a pre-column LC-MS method for the quantification of GAG disaccharide subunits. Heparan sulphate (HS) was extracted from cell lines using a combination of molecular weight cutoff and anion exchange spin filters and digested using a mixture of heparinases I, II and III. The resulting subunits were derivatised with procainamide, separated using hydrophilic interaction liquid chromatography and detected using electrospray ionisation operated in positive ion mode. Eight HS disaccharides were separated and detected together with an internal standard. The limit of detection was found to be in the range 0.6–4.9 ng/mL. Analysis of HS extracted from all cell lines tested in this study revealed a significant variation in their composition with the most abundant disaccharide being the non-sulphated ∆UA–GlcNAc. Some structural functional relationships are discussed demonstrating the viability of the pre-column method for studying GAG biolog

Heparan sulfate disaccharide measurement from biological samples using pre-column derivatization, UPLC-MS and single ion monitoring

Glycosaminoglycans are a heterogeneous family of linear polysaccharides comprised of repeating disaccharide subunits that mediate many effects at the cellular level. There is increasing evidence that the nature of these effects is determined by differences in disaccharide composition. However, the determination of GAG disaccharide composition in biological samples remains challenging and time-consuming. We have developed a method that uses derivatization and selected ion recording and RP-UPLCMS resulting in rapid separation and quantification of twelve heparin/heparin sulfate disaccharides from 5 μg GAG. Limits of detection and quantitation were 0.02–0.15 and 0.07–0.31 μg/ml respectively. We have applied this method to the novel analysis of disaccharide levels extracted from heparan sulfate and human cancer cell lines. Heparan sulfate disaccharides extracted from biological samples following actinase and heparinase incubation and derivatized using reductive amination with 2-aminoacridone. Derivatized disaccharides were analyzed used UPLC-MS with single ion monitoring. Eight HS disaccharide subunits were separated and quantified from HS and cell lines in eleven minutes per sample. In all samples the most abundant subunits present were the unsulfated ΔUA-GlcNAc, ΔUA-GlcNAc,6S and ΔUA,2S-GlcNS,6S. There was considerable variation in the proportions and concentrations of disaccharides between different cell lines. Further studies are needed to examine the significance of these differences

Can quantification of Serum Glycans predict Pre-Eclampsia?

Objectives: To determine if concentrations of placental glycans and glycan components are altered in pre-eclamspia and to determine if serum levels can predict pre-eclampsia. Methods: Serum samples were collected from women in the third trimester of singleton pregnancy but before the onset of pre-eclampsia and also from women during unaffected pregnancies at the samegestational age. Tissues were collected from the basal plate of placentas collected at delivery following uncomplicated singleton pregnancy (term and preterm) and from pregnancies complicated by preeclampsia. Pre-eclampsia was diagnosed according to International Society for the Study of Hypertension in Pregnancy criteria. Glycan components were isolated using a combination of enzyme digestion, molecular weight filtration and ion exchange chromatography, and then derivatised prior to separation using hydrophilic interaction liquid chromatography. Components were detected using electrospray ionisation operated in positive ion mode with single ion monitoring. Results: Specific glycan components (designated glycan 1, 2 and 3) were significantly altered in the serum from women who went on to have preeclampsia compared to those who had an unaffected pregnancy. Interestingly, levels of the same biomarkers were also elevated in nulliparous versus multiparous pregnancy. Biomarkers were also significantly altered in placental tissues from pregnancies complicated by preeclampsia Conclusion: This study suggests that altered glycan levels may contribute to impaired placental development and that the glycome is a potential diagnostic target for pre-eclampsia, and possibly other disorders of pregnancy

Thyroid uptake studies in infectious hepatitis

Thyroid-function studies were done in 43 cases of infectious hepatitis with varying degree of liver damage as judged by serum bilirubin levels. A different pattern of thyroid uptakes was seen in patients with moderate liver damage and those with severe liver damage. A good correlation was observed between thyroid uptake and degree of liver damage

Solar Cycle Related Changes at the Base of the Convection Zone

The frequencies of solar oscillations are known to change with solar activity. We use Principal Component Analysis to examine these changes with high precision. In addition to the well-documented changes in solar normal mode oscillations with activity as a function of frequency, which originate in the surface layers of the Sun, we find a small but statistically significant change in frequencies with an origin at and below the base of the convection zone. We find that at r=(0.712^{+0.0097}_{-0.0029})R_sun, the change in sound speed is \delta c^2 / c^2 = (7.23 +/- 2.08) x 10^{-5} between high and low activity. This change is very tightly correlated with solar activity. In addition, we use the splitting coefficients to examine the latitudinal structure of these changes. We find changes in sound speed correlated with surface activity for r >~ 0.9R_sun.Comment: 29 pages, 11 figures, accepted for publication in Ap

Can Naked Singularities Yield Gamma Ray Bursts?

Gamma-ray bursts are believed to be the most luminous objects in the Universe. There has been some suggestion that these arise from quantum processes around naked singularities. The main problem with this suggestion is that all known examples of naked singularities are massless and hence there is effectively no source of energy. It is argued that a globally naked singularity coupled with quantum processes operating within a distance of the order of Planck length of the singularity will probably yield energy burst of the order of M_pc^2\approx2\times 10^{16} ergs, where M_p is the Planck mass.Comment: 4 pages, TeX, no figure

Diagnosis of hyperthyroidism by external liver counting : correlation between external scintillation counting of the liver and plasma protein-bound iodine<SUP>131</SUP>

External liver counting clearly distinguishes between euthyroid and hyperthyroid patients (t = 10.2); hyperthyroid and nontoxic goitre patients (t = 11.9). Individual values for these patients show a good separation between the groups. Correlation coefficient between liver counts and PBI131 in euthyroid and hyperthyroid patients was found to be 0.84 and correlation coefficient for hyperthyroid and nontoxic goitre patients was 0.76. The advantages of external liver counting as an alternative method to plasma PBI131 estimations are: (a) simplicity of the techniques; (b) no additional well scintillation assembly needed, and (c) no errors in results due to contamination of glassware, chemicals, etc(a) simplicity of the techniques; (b) no additional well scintillation assembly needed, and (c) no errors in results due to contamination of glassware, chemicals, et

A Comparison of Solar Cycle Variations in the Equatorial Rotation Rates of the Sun's Subsurface, Surface, Corona, and Sunspot Groups

Using the Solar Optical Observing Network (SOON) sunspot-group data for the period 1985-2010, the variations in the annual mean equatorial-rotation rates of the sunspot groups are determined and compared with the known variations in the solar equatorial-rotation rates determined from the following data: i) the plasma rotation rates at 0.94Rsun, 0.95Rsun,...,1.0Rsun measured by Global Oscillation Network Group (GONG) during the period 1995-2010, ii) the data on the soft X-ray corona determined from Yohkoh/SXT full disk images for the years 1992-2001, iii) the data on small bright coronal structures (SBCS) which were traced in Solar and Heliospheric Observatory (SOHO)/EIT images during the period 1998-2006, and iv) the Mount Wilson Doppler-velocity measurements during the period 1986-2007. A large portion (up to approximate 30 deg latitude) of the mean differential-rotation profile of the sunspot groups lies between those of the internal differential-rotation rates at 0.94Rsun and 0.98Rsun.The variation in the yearly mean equatorial-rotation rate of the sunspot groups seems to be lagging that of the equatorial-rotation rate determined from the GONG measurements by one to two years.The amplitude of the latter is very small.The solar-cycle variation in the equatorial-rotation rate of the solar corona closely matches that determined from the sunspot-group data.The variation in the equatorial-rotation rate determined from the Mount Wilson Doppler-velocity data closely resembles the corresponding variation in the equatorial-rotation rate determined from the sunspot-group data that included the values of the abnormal angular motions (> 3 deg per day) of the sunspot groups. Implications of these results are pointed out.Comment: 22 pages, 10 figures, accepted by Solar Physic

Non-LTE Model Atmospheres for Late-Type Stars II. Restricted NLTE Calculations for a Solar-Like Atmosphere

We test our knowledge of the atomic opacity in the solar UV spectrum. Using the atomic data compiled in Paper I from modern, publicly available, databases, we perform calculations that are confronted with space-based observations of the Sun. At wavelengths longer than about 260 nm, LTE modeling can reproduce quite closely the observed fluxes; uncertainties in the atomic line data account fully for the differences between calculated and observed fluxes. At shorter wavelengths, departures from LTE appear to be important, as our LTE and restricted NLTE calculations differ. Analysis of visible-near infrared Na I and O I lines, two species that produce a negligible absorption in the UV, shows that observed departures from LTE for theses species can be reproduced very accurately with restricted (fixed atmospheric structure) NLTE calculations.Comment: 13 pages, 11 figures, to appear in Ap