Measurement of immunoglobulin A in saliva by particle-enhanced nephelometric immunoassay: sample collection, limits of quantitation, precision, stability and reference range

Background: Total immunoglobulin A in saliva (s-IgA) is normally assayed using an enzyme-linked immunosorbent assay. We have investigated methodological issues relating to the use of particle-enhanced nephelometric immunoassay (PENIA)
to measure s-IgA in whole unstimulated saliva and determine its reference range.

Methods: Whole unstimulated resting saliva was collected to determine sample stability (temperature, time, effect of a protease inhibitor), limit of quantitation (LOQ), assay precision and analytical variation. The reference range for 134 healthy adults was determined.

Results: Linearity was excellent (4–10.3 mg L21, P, 0.001; R2 ¼ 0.997) and without significant bias (mean of 20.7%). The lowest intra- and inter-analytical coefficients of variation were 1.8% and 7.5% and LOQ was 1.4 mg L21. The concentration of s-IgA is stable at room temperature for up to 6 h, at 48C for 48 h, at 248C for two weeks and at 2808C for up to 1.3 yr. There is no evidence that a protease inhibitor increases the stability or that repeated freeze–thawing cycles degrade sample quality. The reference ranges for s-IgA concentration, s-IgA secretion, s-IgA:albumin and s-IgA:osmolality were 15.9–414.5 mg L21, 7.2–234.9 mg min21, 0.4–19 and 0.6–8.9, respectively.

Conclusion: Automated PENIA assay of s-IgA is precise and accurate. High stability of collected saliva samples and the ease and speed of the assay make this an ideal method for use in athletic and military training situations. The convenience of measuring albumin and IgA on the same analytical platform adds to the practicability of the test.

Correlation between extent of osteolytic damage and metastatic burden of human breast cancer metastasis in nude mice: Real-time PCR quantitation

Orthotopic or intracardiac injection of human breast cancer cell lines into immunocompromised mice allows study of the molecular basis of breast cancer metastasis. We have established a quantitative real-time PCR approach to analyze metastatic spread of human breast cancer cells inoculated into nude mice via these routes. We employed MDA-MB-231 human breast cancer cells genetically tagged with a bacterial β-galactosidase (Lac-Z) retroviral vector, enabling their detection by TaqMan® real-time PCR. PCR detection was linear, specific, more sensitive than conventional PCR, and could be used to directly quantitate metastatic burden in bone and soft organs. Attesting to the sensitivity and specificity of the PCR detection strategy, as few as several hundred metastatic MDA-MB-231 cells were detectable in 100 μm segments of paraffin-embedded lung tissue, and only in samples adjacent to sections that scored positive by histological detection. Moreover, the measured real-time PCR metastatic burden in the bone environment (mouse hind-limbs, n=48) displayed a high correlation to the degree of osteolytic damage observed by high resolution X-ray analysis (r 2=0.972). Such a direct linear relationship to tumor burden and bone damage substantiates the so-called `vicious cycle' hypothesis in which metastatic tumor cells promote the release of factors from the bone which continue to stimulate the tumor cells. The technique provides a useful tool for molecular and cellular analysis of human breast cancer metastasis to bone and soft organs, can easily be extended to other cell/marker/organ systems, and should also find application in preclinical assessment of anti-metastatic modalities.

Comprehensive profiling and quantitation of amine group containing metabolites

Primary and secondary amines, including amino acids, biogenic amines, hormones, neurotransmitters, and plant siderophores, are readily derivatized with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate using easily performed experimental methodology. Complex mixtures of these amine derivatives can be fractionated and quantified using liquid chromatography–electrospray ionization-mass spectrometry (LC–ESI-MS). Upon collision induced dissociation (CID) in a quadrupole collision cell, all derivatized compounds lose the aminoquinoline tag. With the use of untargeted fragmentation scan functions, such as precursor ion scanning, the loss of the aminoquinoline tag (Amq) can be monitored to identify derivatized species; and the use of targeted fragmentation scans, such as multiple reaction monitoring, can be exploited to quantitate amine-containing molecules. Further, with the use of accurate mass, charge state, and retention time, identification of unknown amines is facilitated. The stability of derivatized amines was found to be variable with oxidatively labile derivatives rapidly degrading. With the inclusion of antioxidant and reducing agents, tris(2-carboxyethyl)-phosphine (TCEP) and ascorbic acid, into both extraction solvents and reaction buffers, degradation was significantly decreased, allowing reproducible identification and quantification of amine compounds in large sample sets.

Visualization and quantitation of GLUT4 translocation in human skeletal muscle following glucose ingestion and exercise

Insulin- and contraction-stimulated increases in glucose uptake into skeletal
muscle occur in part as a result of the translocation of glucose transporter 4
(GLUT4) from intracellular stores to the plasma membrane (PM). This study
aimed to use immunofluorescence microscopy in human skeletal muscle to
quantify GLUT4 redistribution from intracellular stores to the PM in response
to glucose feeding and exercise. Percutaneous muscle biopsy samples were
taken from the m. vastus lateralis of ten insulin-sensitive men in the basal
state and following 30 min of cycling exercise (65% VO2 max). Muscle biopsy
samples were also taken from a second cohort of ten age-, BMI- and VO2 maxmatched insulin-sensitive men in the basal state and 30 and 60 min following
glucose feeding (75 g glucose). GLUT4 and dystrophin colocalization, measured
using the Pearson’s correlation coefficient, was increased following
30 min of cycling exercise (baseline r = 0.47 0.01; post exercise
r = 0.58 0.02; P < 0.001) and 30 min after glucose ingestion (baseline
r = 0.42 0.02; 30 min r = 0.46 0.02; P < 0.05). Large and small GLUT4
clusters were partially depleted following 30 min cycling exercise, but not
30 min after glucose feeding. This study has, for the first time, used immunofluorescence microscopy in human skeletal muscle to quantify increases in
GLUT4 and dystrophin colocalization and depletion of GLUT4 from large
and smaller clusters as evidence of net GLUT4 translocation to the PM.

Quantitation of ascorbic acid in Arabidopsis thaliana reveals distinct differences between organs and growth phases

Optimal plant growth is the result of the interaction of a complex network of plant hormones and environmental signals. Ascorbic acid (AsA) is a crucial antioxidant in plants and is involved in the regulation of cell division, cell expansion, photosynthesis and hormone biosynthesis. Quantitative analysis of AsA in Arabidopsis thaliana organs was conducted using HPLC with d -isoascorbic acid (Iso-AsA) as an internal standard. Analysis revealed Àuctuations in the levels of AsA in different organs and growth phases when plants were grown under standard conditions. AsA concentrations increased in leaves in direct proportion to leaf size and age. Young siliques (seed set stage) and Àowering buds (open and unopened) showed the highest levels of AsA. A relationship was found between the level of AsA and indole acetic acid (IAA) in leaves, stems, Àowers, and siliques and the highest level of IAA and AsAwere found in the Àowers. In contrast, the lowest level of the plant hormone, salicylic acid, was found in the Àowers and the highest quantity measured in the leaves. Consequently, AsA has been found to be a multifunctional molecule that is involved as a key regulator of plant growth and development.

Simultaneous determination of irinotecan (CPT-11) and SN-38 in tissue culture media and cancer cells by high performance liquid chromatography: Application to cellular metabolism and accumulation studies

A simple and sensitive HPLC method was developed to simultaneously determine CPT-11 and its major metabolite SN-38 in culture media and cell lysates. Camptothecin (CPT) was used as internal standard (I.S.). Compounds were eluted with acetonitrile–50 mM disodium hydrogen phosphate buffer containing 10 mM sodium 1-heptane-sulfonate, with the pH adjusted to 3.0 using 85% (w/v) orthophosphoric acid (27/73, v/v) by a Hyperclon ODS (C18) column (200 mm × 4.6 mm i.d.), with detection at excitation and emission wavelengths of 380 and 540 nm, respectively. The average extraction efficiencies were 96.9–108.3% for CPT-11 in culture media and 94.3–107.2% for CPT-11 in cell lysates; and 87.7–106.8% for SN-38 in culture media and 90.1–105.6% for SN-38 in cell lysates. Within- and between-day precision and accuracy varied from 0.1 to 10.3%. The limit of quantitation (precision and accuracy <20%) was 5.0 and 2.0 ng/ml for CPT-11 and 1.0 and 0.5 ng/ml for SN-38 in culture media and cell lysates, respectively. This method was successfully applied to quantitate the cellular accumulation and metabolism of CPT-11 and SN-38 in H4-II-E, a rat hepatoma cell line.

Creatine transporters: a reappraisal

Creatine (Cr) plays a key role in cellular energy metabolism and is found at high concentrations in metabolically active cells such as skeletal muscle and neurons. These, and a variety of other cells, take up Cr from the extra cellular fluid by a high affinity Na⁺/Cl^--dependent creatine transporter (CrT). Mutations in the crt gene, found in several patients, lead to severe retardation of speech and mental development, accompanied by the absence of Cr in the brain.
In order to characterize CrT protein(s) on a biochemical level, antibodies were raised against synthetic peptides derived from the N- and C-terminal cDNA sequences of the putative CrT-1 protein. In total homogenates of various tissues, both antibodies, directed against these different epitopes, recognize the same two major polypetides on Western blots with apparent Mr of 70 and 55 kDa. The C-terminal CrT antibody (α-CrTCOOH) immunologically reacts with proteins located at the inner membrane of mitochondria as determined by immuno-electron microscopy, as well as by subfractionation of mitochondria. Cr-uptake experiments with isolated mitochondria showed these organelles were able to transport Cr via a sulfhydryl-reagent-sensitive transporter that could be blocked by anti-CrT antibodies when the outer mitochondrial membrane was permeabilized. We concluded that mitochondria are able to specifically take-up Cr from the cytosol, via a low-affinity CrT, and that the above polypeptides would likely represent mitochondrial CrT(s). However, by mass spectrometry techniques, the immunologically reactive proteins, detected by our anti-CrT antibodies, were identified as E2 components of the agr-keto acid dehydrogenase multi enzyme complexes, namely pyruvate dehydrogenase (PDH), branched chain keto acid dehydrogenase (BC-KADH) and α-ketoglutarate dehydrogenase (α-KGDH). The E2 components of PDH are membrane associated, whilst it would be expected that a mitochondrial CrT would be a transmembrane protein. Results of phase partitioning by Triton X-114, as well as washing of mitochondrial membranes at basic pH, support that these immunologically cross-reactive proteins are, as expected for E2 components, membrane associated rather than transmembrane. On the other hand, the fact that mitochondrial Cr uptake into intact mitoplast could be blocked by our α-CrTCOOH antibodies, indicate that our antisera contain antibodies reactive to proteins involved in mitochondrial transport of Cr. The presence of specific antibodies against CrT is also supported by results from plasma membrane vesicles isolated from human and rat skeletal muscle, where both 55 and 70 kDa polypeptides disappeared and a single polypeptide with an apparent electrophoretic mobility of ~ 60 kDa was enriched This latter is most likely representing the genuine plasma membrane CrT.
Due to the fact that all anti-CrT antibodies that were independently prepared by several laboratories seem to cross-react with non-CrT polypeptides, specifically with E2 components of mitochondrial dehydrogenases, further research is required to characterise on a biochemical/biophysical level the CrT polypeptides, e.g. to determine whether the ~ 60 kDa polypeptide is indeed a bona-fide CrT and to identify the mitochondrial transporter that is able to facilitate Cr-uptake into these organelles. Therefore, the anti-CrT antibodies available so far should only be used with these precautions in mind. This holds especially true for quantitation of CrT polypeptides by Western blots, e.g. when trying to answer whether CrT's are up- or down-regulated by certain experimental interventions or under pathological conditions.
In conclusion, we still hold to the scheme that besides the high-affinity and high-efficiency plasmalemma CrT there exists an additional low affinity high Km Cr uptake mechanism in mitochondria. However, the exact biochemical nature of this mitochondrial creatine transport, still remains elusive. Finally, similar to the creatine kinase (CK) isoenzymes, which are specifically located at different cellular compartments, also the substrates of CK are compartmentalized in cytosolic and mitochondrial pools. This is in line with ¹⁴C-Cr-isotope tracer studies and a number of [³¹P]-NMR magnetization transfer studies, as well as with recent [¹H]-NMR spectroscopy data.

Simultaneous determination of the lactone and carboxylate forms of irintecan (CPT-11) and its actie metabolite SN-38 by high-performance liquid chromatography: Application to plasma pharmacokinetic studies in the rat.

Irinotecan (CPT-11) and its main metabolite SN-38 are potent anticancer derivatives of camptothecin (CPT), with active lactone and inactive carboxylate forms coexisting. A simple and sensitive HPLC method using the ion-pairing reagent tetrabutylammonium hydrogen sulfate (TBAHS) was developed to simultaneously determine all four analytes in rat plasma samples. Camptothecin (CPT) was used as internal standard. The mobile phase was 0.1 M potassium dihydrogen phosphate containing 0.01 M TBAHS (pH 6.4)–acetonitrile (75:25, v/v). Separation of the compounds was carried out on a Hypersil C18 column, monitored at 540 nm (excitation wavelength at 380 nm). All four compounds gave linear response as a function of concentration over 0.01–10 μM. The limit of quantitation in rat plasma was 0.01, 0.008, 0.005 and 0.005 μM for CPT-11 lactone, CPT-11 carboxylate, SN-38 lactone and SN-38 carboxylate, respectively. The method was successfully used in the study on the effect of coadministered thalidomide on the plasma pharmacokinetics of CPT-11 and SN-38 in rats. Coadministered thalidomide (100 mg/kg body weight by intraperitoneal injection) significantly increased the AUC_0–10h values of CPT-11 lactone and CPT-11 carboxylate by 32.6% and 30.3 %, respectively, (P < 0.01), but decreased the values by 19.2% and 32.4% for SN-38 lactone and carboxylate, respectively, (P < 0.05). Accordingly, the value of total body clearance (CL) of CPT-11 lactone was significantly lower in combination group compared to the control (1.329 versus 1.837 L/h/kg, P = 0.0002). Plasma t_1/2β values for SN-38 lactone and carboxylate were significantly (P < 0.01) smaller in rats with coadministered thalidomide, as compared to rats receiving CPT-11 alone. Further studies are needed to explore the underlying mechanisms for the observed kinetic interaction between CPT-11 and thalidomide.

Determination of thalidomide by high performance liquid chromatography: plasma pharmacokinetic studies in the rat

A sensitive and simple high performance liquid chromatography (HPLC) method was developed and validated for the determination of thalidomide in rat plasma. Chromatography was accomplished with a reversed-phase Hypersil C18 column. Mobile phase consisted of acetonitrile-10 mM ammonium acetate buffer (pH 5.50) (28:72, v/v), at a flow rate of 0.8 ml/min. Thalidomide was monitored by ultraviolet detector at 220 nm and it gave a linear response as a function of concentration over 0.02–50 μM. The limit of quantitation in rat plasma was 0.50 ng (0.02 μM plasma concentration) with an aliquot of 20 μl. Results from a 3-day validation study indicated that this method allows for simple and rapid quantitation of thalidomide with excellent accuracy and reliability. Using this validated assay, the effect of coadministered irinotecan (CPT-11) on the plasma pharmacokinetics of thalidomide in rats was determined. Coadministration of CPT-11 (intravenously, 60 mg/kg) increased the maximum plasma concentration (C_max) and area under the plasma concentration–time curve (AUC_{0–10 h}) of thalidomide by 32.29 and 11.66%, respectively, as compared to the control, but none of the effect of CPT-11 was of statistical significance (P > 0.05). Concomitant CPT-11 also caused a 10.04% decrease in plasma clearance (CL) and 14.51% decrease in volume of distribution (V_d) (P > 0.05). These results suggest that coadministered CPT-11 did not significantly alter the plasma pharmacokinetics of thalidomide in rats. Further studies are warranted to explore the pharmacokinetic and pharmacodynamic interactions between CPT-11 and thalidomide.

Determination of rate constants using microelectrodes

An understanding of the rate and the mechanism of reaction is of fundamental importance in the many facets of chemistry. Electrochemical systems are further complicated by the heterogeneous boundary, between the solution and the electrode, that the electron passes through before any electrochemical reaction can take place. This thesis concerns the development of methods for analysing electrode kinetics. One part involves the further development of the Global Analysis procedure to include electrodes with a spherical geometry which are traditionally the most popular form of electrodes. Simulated data is analysed to ascertain the accuracy of the procedure and then the known artifacts of uncompensated solution resistance and charging current are added to the simulated data so that the effects can be observed. The experimental analysis of 2-methyl-2-nitropropane is undertaken and comparisons are made with the Marcus-Hush electrochemical theories concerning electrode kinetics. A related section explores procedures for the kinetic analysis of steady state voltammetric data obtained at microdisc electrodes. A method is proposed under the name of Normalised Steady State Voltammetry and is tested using data obtained from a Fast Quasi-Explicit Finite Difference simulation of diffusion to a microdisc electrode. In a second area of work using microelectrodes, the electrochemical behaviour of compounds of the general formula M(CO)3(η3 - P2P1) where M is either Cr, Mo or W and P2P' is bis(2-diphenylphosphinoethyl)phenylphosphine) is elucidated. The development of instrumentation and mathematical procedures relevant to the measurement and quantitation of these systems is also investigated. The tungsten compound represents the first examples where the 17-electronfac+ and mer+ isomers are of comparable stability.

Computer-assisted image analysis of liver collagen: relationship to Ishak scoring and hepatic venous pressure gradient

Histopathological scoring of disease stage uses descriptive categories without measuring the amount of fibrosis. Collagen, the major component of fibrous tissue, can be quantified by computer-assisted digital image analysis (DIA) using histological sections. We determined relationships between DIA, Ishak stage, and hepatic venous pressure gradient (HVPG) reflecting severity of fibrosis. One hundred fifteen patients with hepatitis C virus (HCV) who had undergone transplantation had 250 consecutive transjugular liver biopsies combined with HVPG (median length, 22 mm; median total portal tracts, 12), evaluated using the Ishak system and stained with Sirus red for DIA. Liver collagen was expressed as collagen proportionate area (CPA). Median CPA was 6% (0.2-45), correlating with Ishak stage (stage 6 range, 13%-45%), and with HVPG (r = 0.62; P < 0.001). Median CPA was 4.1% when HVPG was less than 6 mm Hg and 13.8% when HVPG was 6 mm Hg or more (P < 0.0001) and 6% when HVPG was less than 10 mm Hg and 17.3% when HVPG was 10 mm Hg or higher (P < 0.0001). Only CPA, not Ishak stage/grade, was independently associated by logistic regression, with HVPG of 6 mm Hg or more [odds ratio, 1.206; 95% confidence interval (CI), 1.094-1.331; P < 0.001], or HVPG of 10 mm Hg or more (odds ratio, 1.105; 95% CI, 1.026-1.191; P = 0.009). CPA increased by 50% (3.6%) compared with 20% in HVPG (1 mm Hg) in 38 patients with repeated biopsies. Conclusion: CPA assessed by DIA correlated with Ishak stage scores and HVPG measured contemporaneously. CPA was a better histological correlate with HVPG than Ishak stage, had a greater numerical change when HVPG was low, and resulted in further quantitation of fibrosis in cirrhosis.

Application of dried blood spots to determine vitamin D status in a large nutritional study with unsupervised sampling: the Food4Me project

An efficient and robust method to measure vitamin D (25-hydroxy vitamin D3 (25(OH)D3) and 25-hydroxy vitamin D2 in dried blood spots (DBS) has been developed and applied in the pan-European multi-centre, internet-based, personalised nutrition intervention study Food4Me. The method includes calibration with blood containing endogenous 25(OH)D3, spotted as DBS and corrected for haematocrit content. The methodology was validated following international standards. The performance characteristics did not reach those of the current gold standard liquid chromatography-MS/MS in plasma for all parameters, but were found to be very suitable for status-level determination under field conditions. DBS sample quality was very high, and 3778 measurements of 25(OH)D3 were obtained from 1465 participants. The study centre and the season within the study centre were very good predictors of 25(OH)D3 levels (P<0·001 for each case). Seasonal effects were modelled by fitting a sine function with a minimum 25(OH)D3 level on 20 January and a maximum on 21 July. The seasonal amplitude varied from centre to centre. The largest difference between winter and summer levels was found in Germany and the smallest in Poland. The model was cross-validated to determine the consistency of the predictions and the performance of the DBS method. The Pearson's correlation between the measured values and the predicted values was r 0·65, and the sd of their differences was 21·2 nmol/l. This includes the analytical variation and the biological variation within subjects. Overall, DBS obtained by unsupervised sampling of the participants at home was a viable methodology for obtaining vitamin D status information in a large nutritional study.

Immunofluorescence microscopy of SNAP23 in human skeletal muscle reveals colocalization with plasma membrane, lipid droplets, and mitochondria

Synaptosomal-associated protein 23 (SNAP23) is a SNARE protein expressed abundantly in human skeletal muscle. Its established role is to mediate insulin-stimulated docking and fusion of glucose transporter 4 (GLUT4) with the plasma membrane. Recent in vitro research has proposed that SNAP23 may also play a role in the fusion of growing lipid droplets (LDs) and the channeling of LD-derived fatty acids (FAs) into neighboring mitochondria for β-oxidation. This study investigates the subcellular distribution of SNAP23 in human skeletal muscle using immunofluorescence microscopy to confirm that SNAP23 localization supports the three proposed metabolic roles. Percutaneous biopsies were obtained from the m. vastus lateralis of six lean, healthy males in the rested, overnight fasted state. Cryosections were stained with antibodies targeting SNAP23, the mitochondrial marker cytochrome c oxidase and the plasma membrane marker dystrophin, whereas intramuscular LDs were stained using the neutral lipid dye oil red O. SNAP23 displayed areas of intense punctate staining in the intracellular regions of all muscle fibers and continuous intense staining in peripheral regions of the cell. Quantitation of confocal microscopy images showed colocalization of SNAP23 with the plasma membrane marker dystrophin (Pearson's correlation coefficient r = 0.50 ± 0.01). The intense punctate intracellular staining colocalized primarily with the mitochondrial marker cytochrome C oxidase (r = 0.50 ± 0.012) and to a lesser extent with LDs (r = 0.21 ± 0.01) visualized with oil red O. We conclude that the observed subcellular distribution of SNAP23 in human skeletal muscle supports the three aforementioned metabolic roles.