Aluminium in tea: SEC-ICP-MS speciation studies of infusions and simulated gastrointestinal digests

Abstract

The speciation of aluminium in tea infusions and in vitro gastrointestinal digests of tea infusions has been investigated using size exclusion chromatography coupled to inductively coupled plasma mass spectrometry (SEC-ICP-MS). At pH 2.5, following simulated gastric treatment, Al from tea eluted at a similar retention volume to that obtained for an aqueous Al standard. At pH 5.5, an aqueous Al standard was eluted from an SEC column in Tris buffer (Al recovery ≈ 100%) only in the presence of a complexing agent (NaF), and at a retention volume corresponding to a molecular mass greater than that expected for an ionic species. Aluminium associated with a tea infusion eluted in two fractions: a higher molecular weight fraction corresponding to Al strongly bound to ligands in the tea, and a lower molecular weight fraction probably comprised of labile Al eluting as Al-F complexes. Simulated gastrointestinal digestion produced three Al-bearing fractions, of which the two at higher molecular mass represented ligand-bound Al. The molarity of the Tris buffer strongly influenced the retention volume of the Al fractions, particularly for the ligand-bound Al. After simulated gastrointestinal digestion, the soluble labile fraction (15% of the Al from the tea infusion) was considered to be potentially available for absorption. The actual proportion of the fraction that might be absorbed would depend upon a number of physiological and nutritional factors.     

Behaviour of vanadate and vanadium-transferrin complex on different anion-exchange columns. Application to in vivo 48V-labelled rat serum

The behaviour of free [48V]vanadate and [48V]vanadium-transferrin complex was investigated on five different anion-exchange columns (Mono Q 5/5 HR, Hitrap Q HP, Sepharose Q FF, Sepharose DEAE FF and Hitrap Q XL). The recovery of both V-compounds was quantitative. The peak shape and retention time of vanadate varied according to the type of column. The vanadium-transferrin complex also showed different elution patterns depending on the type of column. Especially in case of the Sepharose Q FF, Mono Q 5/5 HR and Hitrap Q XL columns the vanadium-transferrin binding was degraded during elution on the column. The results clearly prove that care should be taken as to the choice of column for speciation purposes of vanadium compounds in order to prevent various artefacts showing up in the chromatograms. A Hitrap Q HP column was used to fractionate different vanadium compounds in rat serum.     

Variables in the high-pressure cation-exchange chromatography of proteins

The use of cation-exchange high-pressure liquid chromatography for the separation of proteins has been investigated. Several factors, including solvent composition, pH, flow rate, and temperature, were examined for their effects on the resolution of protein standards (insulin, β-lactoglobulin, and carbonic anhydrase B; molecular weight range, 6000 to 30,000 and pI range, 5.3 to 6.5). An initial comparison was made of the recovery of these proteins from three commercially available columns (Whatman Partisil SCX, Separation Industry CM silica, and MCB Reagents Lichrosorb KAT). In general, under the conditions employed, the SCX column gave the highest recovery of applied protein. Based on this recovery data, the Partisil SCX column was chosen for subsequent examination of chromatographic parameters that would optimize protein resolution. An increase in temperature decreased retention and resolution but increased recovery, with some proteins being affected more than others. A decrease in pH in the final eluant or an increase in pH in the initial eluant caused an increase in retention times. For some proteins, the decrease in pH resulted in a greater total recovery of protein. This information has been applied to the purification by cation-exchange high-pressure liquid chromatography of transforming growth factors from a human tumor cell line.     

The analysis of thiamin and its derivatives in whole blood samples under high pH conditions of the mobile phase

In this study a protocol for the analysis of thiamin and thiamin coenzymes in whole blood was developed. Thiamin and its coenzymes are analyzed by reversed phase liquid chromatography (RPLC), precolumn derivatisation with alkaline potassium ferricyanide and fluorescence detection, all at pH 10. Under these relatively high pH conditions the detectability of the analytes and the robustness of the method were substantially improved. The use of a high pH resistant RPLC column was a crucial step in developing this analysis method. Reproducibility, linearity, recovery, detection limit and column robustness were investigated. The within-batch CV was <2.5%, the between-batch CV <4.5%. The method was linear far above the physiological relevant concentration level. Recovery was almost 100% on an average. The limit of quantification was 1 nmol/l. The robustness of the RPLC column proved to be very high. Up to 1500 injections hardly any substantial changes in retention times and efficiency were observed. In summary: Using a high pH resistant RPLC column resulted in a robust, sensitive and precise method for the analysis of total Vitamin B1 and especially of TDP.     

Implementation of an HPLC polystyrene divinylbenzene column for separation of activated sludge exopolymers

A high-pressure size-exclusion chromatography procedure for separation of activated sludge exopolymers was investigated and implemented in order to achieve a documented and faster separation procedure than the conventional low-pressure size-exclusion chromatography methods previously suggested in studies of activated sludges from a traditional and an advanced from activated sludges from a traditional and an advanced activated sludge treatment plant performing biological nitrogen and phosphorus removal were used. For both types of exopolymers the separation was largely dependent on the mobile-phase. Using NaCl and ortho-phosphate in the molar proportion 10:1 it was shown that for a mobile-phase ionic strength of 0.011 and pH in the range 7.0–10.0 no irreversible column adsorption occurred. For a standard procedure a mobile-phase pH of 7.0 was selected in order to separate the exopolymers into the maximal number of peaks. Alterations in the mobile-phase, i.e. using a pH below 7.0 or a mobile-phase ionic strength above 0.011, changed the separation for both types of exopolymers and caused irreversible column adsorption. Similarly, using deionized water as the mobile-phase irreversible column adsorption was introduced and the separation was strongly affected. The method applicability for qualitative characterization of exopolymers was demonstrated. The method was found to be successful in showing differences and similarities between exopolymers from two different activated sludge treatment plants, showing degradation of exopolymer compounds due to exoenzymes in the exopolymers and showing that snow melting and subsequent high conductivity in the inlet to the waste-water treatment plant had an impact on the chromatographic fingerprint of the extracted exopolymers.     

Purification and analysis of a 5kDa component of enamel matrix derivative

High performance liquid chromatography (HPLC) methods were used to analyse a 5kDa component purified from enamel matrix derivative (EMD), the active ingredient in Emdogain, a commercial product for periodontal tissue regeneration. After initial purification by size-exclusion chromatography (SEC) on a 100 cm x 5 cm column (Bio-Gel P-30 Fine, 280 nm), collected fractions were analysed by size-exclusion HPLC (SE HPLC; TSK-Gel Super SW2000, 220 nm). The fractions containing only the 5kDa component were analysed by reversed-phase high-pressure chromatography (RP HPLC; YMC-Pack ODS-A, 200 nm), revealing four peaks of the 5kDa component. From 1200 mg of EMD (of which 9% is the 5kDa component), approximately 65 mg of lyophilised 5kDa component were obtained, corresponding to a recovery of 60%. The SE HPLC method was mainly suitable for qualitative analysis, whereas the RP HPLC method was appropriate for both qualitative and quantitative analysis.     

Analysis of rat uterine estrogen receptors by high-pressure liquid chromatography methods

Cytosolic (ERc) and nuclear (ERn) estrogen receptors prepared from rat uteri were characterized by size-exclusion and ion-exchange HPLC. The oligomeric ERc eluted as a single, sharp peak near the exclusion volume of the gel column; ERn eluted as a broad peak. When salt-extracted ERn was partially purified sequentially by Sephadex G-200, DEAE-cellulose chromatography and polyacrylamide gel electrophoresis, the partially purified receptor moieties were not distinguishable by the sucrose gradient method, but showed characteristic retention times in the size-exclusion HPLC column. Further distinction in net surface charges was observed between ERc and ERn moieties by ion-exchange high-pressure liquid chromatography (HPLC). Molybdate-stabilized ERc was eluted as sharp peak at 0.27 M salt gradient. In contrast, fresh extracts of ERn emerged as a broad peak in the region of 0.1-0.2 M salt gradient. In the absence of molybdate, ERc dissociated into several 4-5 S molecules, which were well resolved in the DEAE column. This report, therefore, demonstrates the usefulness of size-exclusion and ion-exchange HPLC for steroid receptor analysis.     

Characterization of cadmium- and lead- phytochelatin complexes formed in a marine microalga in response to metal exposure

Phytochelatins (PC_n) are thiol-containing peptides with general structure (-Glu-Cys)_n-Gly enzymatically synthesized by plants and algae in response to metal exposure. They are involved in the cellular detoxification mechanism for their capability to form stable metal-phytochelatin complexes. The speciation of Cd and Pb complexes with phytochelatins has been studied in laboratory cultures of the marine diatom Phaeodactylum tricornutum. An approach based on size-exclusion chromatography (SEC) with off-line detection of phytochelatins, by reverse-phase HPLC, and metal ion, by atomic absorption spectrometry, has been used. The formation of Cd- and Pb-PC_n complexes with n-value from 3 to 6 was demonstrated. The metal-PC_n complexes formed with Cd appear to be different from those formed with Pb for the number of molecules of peptide involved in the complex and for the amount of the metal ion bound. The chromatographic behaviour of metal-PC_n complexes is consistent with Pb-PC_n complexes in which only a molecule of peptide binds the metal ion, and with Cd-PC_n complexes containing two or more molecules of peptide. The metal/peptide molar ratio in Cd-PC_n complexes was higher that in Pb-PC_n complexes. The formation of Cd- or Pb-PC₂ complexes was not demonstrated, probably for a dissociation during the cellular extract preparation. The effectiveness of phytochelatins in the detoxification of these two metal ions in this alga is discussed.     

Method for isolation of MMP-7-ase isoenzymes from rat muscle tissue extract

Two MMP-7-ase isoenzymes were purified 100-fold from rat muscle extract to apparent homogeneity, with an overall yield of 10%, using homogenization, ultracentrifugation, high-performance aqueous size-exclusion and high-performance anion exchange chromatography methods. When using a TSK G-2000SW column, the separation resulted in a 6-fold purification and 30% recovery of isoenzymes B and C. This concentrated enzyme extract was then passed through a TSK-DEAE-2SW column, using salt gradient at pH 7.5, with an additional 25-fold purification and 90% recovery of the isoenzymes. Two symmetrical enzyme peaks, representing isoenzymes B and C, were detected when performing purity tests of the active enzymes on the anion exchanger and reversed-phase HPLC columns. The procedures involved are extraction, ultracentrifugation, chromatographies and enzyme assays and require less than five hours.     

A Review of: “Biological Recognition and Assembly,D. S. Eisenberg,J. A. Lake and C. F. Fox,Eds. Alan R. Liss,New York, 1980; hardbound, 355 pages, $52.00”

Two MMP-7-ase isoenzymes were purified 100-fold from rat muscle extract to apparent homogeneity, with an overall yield of 10%, using homogenization, ultracentrifugation, high-performance aqueous size-exclusion and high-performance anion exchange chromatography methods. When using a TSK G-2000SW column, the separation resulted in a 6-fold purification and 30% recovery of isoenzymes B and C. This concentrated enzyme extract was then passed through a TSK-DEAE-2SW column, using salt gradient at pH 7.5, with an additional 25-fold purification and 90% recovery of the isoenzymes. Two symmetrical enzyme peaks, representing isoenzymes B and C, were detected when performing purity tests of the active enzymes on the anion exchanger and reversed-phase HFLC columns. The procedures involved are extraction, ultracentri-fugation, chromatographies and enzyme assays and require less than five hours.     

Purification of Growth-Promoting Peptides and Proteins,and of Histones,by High Pressure Silica gel Chromatography

A rapid method for the purification of histones and a variety of growth-promoting proteins and peptides by chromatography on silica gel has been developed. The isolation of the growth-promoting components of serum has been hampered by excessive losses associated with the use of water-based purification methods. The solubility of many growth-promoting serum proteins in acidic methanol-H₂O solutions (eg. insulin, albumin, the somatomedins) provides a basis for purification on high-pressure silica gel columns, while peptides and histones can be purified in similar solvents. After column chromatography, the solvent is removed by flash-evaporation, or the protein may be precipitated directly from the solvent by neutralization of the pH and the addition of ethanol. The retention of biological activity (eg. somatomedin-C binding to insulin receptors and cell-growth stimulation) and recovery are excellent.     

Monolithic chromatography on conjoint liquid chromatography columns for speciation of platinum-based chemotherapeutics in serum of cancer patients

BackgroundMonolithic chromatography using convective interaction media (CIM) disks or columns can be used in the separation step of speciation analysis. When different monolithic disks are placed in one housing, forming conjoint liquid chromatography (CLC) monolithic column, two-dimensional separation is achieved in a single chromatographic run.MethodsHere, we assembled low-pressure (maximum 50 bar) CLC monolithic column, which consists of two 0.34 mL shallow CIM monolithic disks and high-pressure CLC column (maximum 150 bar) from 0.1 mL analytical high performance short bed CIMac monolithic disks. Both the CLC columns constructed from affinity Protein G and weak anion exchange diethylamine (DEAE) disks, were applied for the speciation of cisplatin, oxaliplatin and carboplatin in spiked standard serum proteins, spiked human serum and serum of cancer patients. The analytical performances of the CLC columns used were evaluated by comparing their robustness, selectivity, repeatability and reproducibility. The separated serum proteins were detected on-line by ultraviolet (UV) and eluted Pt species by inductively coupled plasma mass spectrometry (ICP-MS). For accurate quantification of the separated Pt species (unbound Pt-based chemotherapeutic from species associated to transferrin (Tf), human serum albumin (HSA) and Immunoglobulin G (IgG)), post column isotope dilution (ID)-ICP-MS was used.ResultsThe data from analyses showed that both tested CLC monolithic columns gave statistically comparable results, with the low-pressure CLC column exhibiting better resolving power and robustness. It also enables more effective cleaning of monolithic disks and to analyse larger series of serum samples than the high-pressure CLC column. Analyses of serum samples of cancer patients treated with cisplatin or carboplatin showed that Pt-chemotherapeutics were bound preferentially to HSA (around 80%). The portion of unbound Pt in general did not exceed 2%, up to 5% of Pt was associated with Tf and approximately 20% with IgG. Column recoveries, calculated as a ratio between the sum of concentrations of Pt species eluted and concentration of total Pt in serum samples, were close to 100%.ConclusionsLow-pressure CLC column exhibited greater potential than high-pressure CLC column, and can be thus recommended for its intended use in speciation analysis of metal-based biomolecules.     

Optimization of Lipase Purification by Rotating Annular Size-exclusion Chromatography

Crude porcine lipase was purified by continuous rotating annular size-exclusion chromatography. Sephadex G-75 was used as the size-exclusion packing material. Initial studies by this group on a similar unit have been reported [Genest et al. (1998) "Continuous purification of porcine lipase by rotating annular size-exclusion chromatography", Appl. Biochem. Biotechnol. 73, 215-230]. This article presents the results of optimization studies carried out on a modified unit. These modifications resulted in a better performance of the column and a higher throughput. Purification fold values of around 11 were achieved in most runs. The activity recovered was around 99% and the productivity was around 3 mg lipase/mg gel h.     

Characterization of biotechnological processes and products using high-performance liquid chromatography (HPLC) IV. SEC,HIC, and IEC separations of proteins

Proteins are separated by means of size-exclusion (SEC), hydrophobic-interaction (HIC) and ion-exchange chromatography (IEC). Analytical and semipreparative HPLC glass columns are the basis of the chromatographic analyses. Using a short (100 × 3.8 mm i.d.) column packed with Si 200 Polyol standard of different molecular weights (ovalbumin, MW = 43 000; chymotrypsinogen A, MW = 25 000; ribonuclease, MW = 13 700 and contrycal, MW = 6512) could be distinguished. Basic proteins (e.g., chymotrypsinogen A, cytochrome C) are separated on aluminium oxide (Li-Chrosorb Alox T) by cation-exchange chromatography. Correlations between the retention times of proteins and their isoelectric points or the buffer concentration of the mobile phase are investigated. Furthermore, two examples of liquid chromatographic purification procedures for enzymes of biotechnological interest are demonstrated. One enzyme extract (thermostable protease) is separated by hydrophobic-interaction chromatography, another one (β-galactosidase from a thermophilic microorganism) is purified on a weakly basic anion-exchange resin (pore size: 130 nm) based on a styrene-divinylbenzene copolymer.     

Identification of Zn-nicotianamine and Fe-2'-Deoxymugineic acid in the phloem sap from rice plants (Oryza sativa L.)

In higher plants, the supply of metals such as Zn and Fe via phloem is important for the growth and physiology of young organs. However, little information is available on the speciation (chemical forms) of these metals in the phloem fluids. Because the pH of phloem fluids is slightly alkaline and the concentration of phosphate, which may bind to metals, is high, Zn and Fe in phloem fluids could be precipitated if these metals do not form complexes with some ligand compounds. In the present experiment, we examined the chemical forms of Zn and Fe in phloem sap collected from rice (Oryza sativa L.) by separating the phloem sap using size-exclusion and anion-exchange chromatography, and identifying the contents using electrospray ionization time-of-flight mass spectrometry. The low molecular weight chemical forms of Zn and Fe were identified as Zn-nicotianamine and Fe(III)-2'-deoxymugineic acid complexes, respectively. This report is the first to identify metal-chelate complexes in rice phloem sap.     

Regulation,purification and partial characterization of glutamine synthetase fromStreptomyces aureofaciens

The activity of glutamine synthetase (GS) fromStreptomyces aureofaciens was regulated by the availability of the nitrogen source. Rich nitrogen sources repressed GS synthesis and increased GS adenylylation. The enzyme was purified 270-fold to virtual homogeneity with 37% recovery. The molar mass of the native enzyme and its subunits was determined to be 620 and 55 kDa, respectively, indicating that GS is composed of 12 identical subunits. The enzyme has a hexagonal-bilayered structure as observed by electron microscopy. The isoelectric point of the purified GS was at pH 4.2. The enzyme was stable for 1 h at 50°C but lost activity rapidly when incubated at 65 and 70°C. Mg²⁺ supported relative synthetic activity of 100 and 72%, respectively, with the corresponding pH optima of 7.3 and 7.0. Mn²⁺ ions activated transferase activity at a pH optimum of 7.0. The temperature optimum for all GS activities was 50°C. Intermediates of the citric acid cycle exerted insignificant effects on the synthetic activities. There was no SH-group essential for the GS activity.     

HPLC analysis of E. coli rRNA on ASAHIPAK GS-520 and nucleobases, nucleosides and nucleotides on ASAHIPAK GS-320

High-performance gel filtration chromatography using packed column Asahipak GS-520 was employed to separate E. coli 23S, 16S and 5S rRNAs. Low-molecular-weight components of nucleic acid were separated with Asahipak GS-320. Concurrent, rapid analysis of nucleobases, nucleosides and nucleotides was obtained isocratically. The elution of these substances is also described.     

Association and dissociation properties of natural human interferon γ

The properties of natural human interferon γ (IFN-γ) molecules dissolved in protein-denaturing and non-denaturing solvents were examined by high-performance size-exclusion chromatography on a gel permeation column. IFN-γ and tritium-labeled IFN-γ molecules formed either dimers (>90.5%) with the molecular mass of 60 kDa or probably tetramers (<9.5%) with the molecular mass of approximately 100 kDa in non-denaturing solvents, and no monomer was detected. These oligomers were dissociated in protein-denaturing solvents such as 6 M guanidine hydrochloride, and IFN-γ existed as monomers. There is no effect on formation of the monomer based on the dissociation of oligomers by acid treatment at pH 4.0. The monomers in protein-denaturing solvents formed dimers by association when applied to a column equilibrated with a non-denaturing solvent of phosphate buffer, pH 7.0. In conclusion, natural human IFN-γ forms oligomers, particularly dimers, in non-denaturing solution, and this oligomer formation is a reversible reaction.     

A simple progesterone radioimmunoassay without column chromatography

A radioimmunoassay for plasma progesterone without Chromatographie purification was developed. The 11-hemi-succinate of 11 -hydroxy-progesterone conjugated to bovine serum albumin was injected into rabbits to stimulate antibody production. The resulting antisera was used at a final dilution of 1:3500. The mean recovery of labeled progesterone added to 100 samples after ether extraction (88.9 ± 9.1%) was higher than the recovery obtained when column chromatography followed ether extraction (84.8 ± 7.5%). For comparison, plasma pools were assayed for progesterone with and without the use of columns. A female plasma pool (luteal phase) gave a mean of 546.3 ± 26.5 (SD) ng/100 mls (n = 5) without column chromatography and 557.2 ± 20.8 (SD) ng/100 mls (n = 5) with column chromatography. Another female plasma pool (follicular phase) gave a mean of 87.9 ± 9.6 (SD) ng/100 mls (n = 24) with column chromatography and 93.3 ± 8.6 (SD) ng/100 mls (n = 7) without column chromatography. A male plasma pool gave a mean of 22.8 ± 4.4 (SD) ng/100 mls (n = 13) with column chromatography and 21.8 ± 7.7 (SD) ng/100 mls (n = 3) without column chromatography. The intra assay and inter assay precision gave a coefficient of variation of 3.7 for six samples and 10.9 for 24 samples, respectively. The specificity of the antibody was determined by checking cross reactivity with 26 steroids. The sensitivity (25 pg) and accuracy were proven to be highly satisfactory.     

Study of humoral hemolytic factors in hemorrhage using chromatographic methods]

The kidney-dependent increase of the haemolytic activity of blood serum after acute decompensated blood loss was demonstrated in the experiments on rats. The preparative ion-exchange chromatography on DEAE-Toyoperal was used to separate the haemolytic active component of the posthaemorrhagic blood serum with the max value of the specific activity of 6.86 A/micrograms protein. The analysis of the separated component by size-exclusion chromatography on TSK-G3000-SW column indicated a molecular mass of 80-100 kDa. In injection in the circulatory system in in vivo experiments the dose-dependent effect of the action of separated component was demonstrated.