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PubMed2003

Evaluation of an aqueous KOH digestion followed by hexane extraction for analysis of PCDD/Fs and dioxin-like PCBs in retailed fish.

Aqueous KOH digestion followed by hexane extraction has been employed in the extraction of dioxins (polychlorinated dibenzo- p -dioxins, dibenzofurans and dioxin-like polychlorinated biphenyls) for biological samples, but there are no reports on its evaluation. Therefore, we report here the evaluation of this extraction for the analysis of dioxins in retailed fish. The effect of the alkaline digestion on dioxins was evaluated by estimation of recoveries. The recoveries of dioxins after the alkaline digestion were good (79-106%) in various kinds of retailed fish except tuna. In tuna, loss of octachlorodibenzofuran (OCDF) was clearly observed, however, the loss was corrected by internal-standard quantification; (13)C12-labeled OCDF was added before the alkaline digestion. Comparative study showed that alkaline digestion followed by hexane extraction provides extraction efficiencies of dioxins equal to those of conventional Soxhlet extraction in fish. Additionally, in analysis of a certified reference fish sample with this extraction, the values obtained for certified isomers were almost equal to the certified values. Since the present method is very simple and inexpensive, it would be useful for analysis of dioxins in retailed fish.

Animals↗
PubMed2005

A systematic approach to quantitation of ephedra alkaloids in natural health products.

A method for accurate determination of ephedrine (E) alkaloids in natural health products (NHP) is described. The NIST dietary supplement standard reference materials (SRMs) were selected for these studies. These SRMs comprise ground Ma Huang herb (Ephedra sinica Stapf.), a spray dried extract of the former, and commercial formulations derived from gel caps and a protein drink. The efficiency of sonication-assisted extraction and Soxhlet extraction was studied using both ammonium formate and potassium phosphate in 3% methanol as extraction media. The efficiency of SPE clean-up of the extract deteriorated rapidly when increasing amounts of sample matrix or analyte were processed, because of limited cartridge capacity. Quantitation by the method of additions was required to ensure the highest accuracy using both LC-UV and ES-LC-MS-MS techniques. Whereas the LC-UV method is more convenient and precise, the results are more questionable than ES-LC-MS-MS, because species-specific detection is not possible.

Alkaloids↗
PubMed2006

Quantitative plasma determination of a novel antiretroviral derivative of zidovudine by solid-phase extraction and high-performance liquid chromatography.

A solid-phase extraction methodology, followed by high-performance liquid chromatography (HPLC) quantification with UV absorbance detection (lambda = 267 nm), was developed in order to study the stability of 3'-azido-3'-deoxy-5'-O-isonicotinoylthymidine (AZT-Iso), a novel derivative of the antiretroviral AZT, in different matrixes. The half-lives (t1/2) for AZT-Iso were 1.19, 1.13 and 0.30 h for human, rat and rabbit plasma, respectively, and 14.91 and 25.49 h for potassium phosphate buffer (pH 7.4) and human serum albumin solution, respectively. The HPLC method proved to be selective, sensitive and accurate. Good recovery, linearity and precision were achieved using p-fluorophenol as an internal standard. The validity of this method was tested using synthetic mixtures of the intact drug with its decomposition products. In conclusion, the method presented is applicable to in vivo pharmacokinetics studies of AZT-Iso in rats.

Animals↗
PubMed2000

Fluorimetric determination of methylmercury as an ion-association complex with rhodamine B in the presence of iodide.

A fluorimetric method for the determination of methylmercury was established. The method was based on the formation of an ionic pair between iodide-methylmercury-rhodamine B in hydrochloric acid, which can be extracted with benzene. The fluorescence emission was measured at lambda(ex)/lambda(em) 575/590 nm, and the experimental variables and possible interference were studied. The linear calibration range was 4 x 10(-8) mol/L to approximately 5 x 10(-7) mol/L with a correlation coefficient of 0.9992. The detection limit was 1 x 10(-8) mol/L. The method was used to determine methylmercury in human hair. The recovery was in the range of 91% to 105% and the relative standard deviation was 2.8%. The results agreed with those obtained by gas chromatography with electron capture detection.

Calibration↗
PubMed2000

Determination of nicotinamide (NA) using its polarographic catalytic wave in the presence of KIO3.

Nicotinamide (NA) yields a polarographic catalytic wave with a peak potential -1.38 V (vs. SCE) in 0.1 mol/L HAc-NaAc (pH 4.7)/4 x 10(-3) mol/L KIO3 buffer solution. The sensitivity of the catalytic wave increased in one order of magnitude as compared to that of the responding reduction wave without KIO3. Based on this observation, a new method for the determination of NA was recommended. The second order derivative peak current was proportional to the NA concentration in the range of 5 x 10(-8)- 6 x 10(-7) mol/L. 0.11-fold vitamin B1, 0.13-fold B2, 0.14-fold B6 and 8-fold nicotinic acid amounts do not interfere the determination of 1 x 10(-6) mol/L NA. The proposed method was used to determine the NA content in multivitamin tablets, with good agreement to the declared amount.

Hydrogen-Ion Concentration↗
PubMed1999

Effects of potassium bicarbonate supplementation on axial and peripheral bone mass in rats on strenuous treadmill training exercise.

We administered a potassium bicarbonate supplement to rats on strenuous treadmill training in order to determine the effect on bone mass and the metabolic acidosis seen with this type of training. A sample of 45 93-day-old female Wistar rats with a mean initial weight of 267 +/- 17 g were studied. The control group (15 rats) was not exercised or given potassium bicarbonate (Ex- PB-). The experimental group (30 rats) was randomly divided into two subgroups of 15 rats each, one that exercised and did not receive potassium bicarbonate (Ex+ PB-) and one that exercised and received potassium bicarbonate (Ex+ PB+), at a dose of 0.05 mg/kg/day administered by esophageal catheter on exercise days. Training consisted of treadmill running on 5 out of 7 days for a period of 11 weeks. Running time, treadmill speed, and the percent grade were gradually increased until week 7, then maintained until rats were sacrificed at the ened of 11 weeks. The bone mineral content (BMC) and bone mineral density (BMD) of the whole right femur and 5th lumbar vertebra were measured. Femoral and vertebral length were also measured. Femur length, weight, BMC, and BMD, and femur BMC/final weight ratio, and vertebral weight, BMD, and BMC, and vertebral BMC/final weight ratio were lower in the Ex+ PB- group than in either the controls or the Ex+ PB+ group (P < 0.01-P < 0.0001); the length of the 5th lumbar vertebra did not differ between groups.

Animals↗
PubMed2006

Regioselective carboxylation of 1,3-dihydroxybenzene by 2,6-dihydroxybenzoate decarboxylase of Pandoraea sp. 12B-2.

We found a bacterium, Pandoraea sp. 12B-2, of which whole cells catalyzed not only the decarboxylation of 2,6-dihydroxybenzoate but also the regioselective carboxylation of 1,3-dihydroxybenzene to 2,6-dihydroxybenzoate. The whole cells of Pandoraea sp. 12B-2 also catalyzed the regioselective carboxylation of phenol and 1,2-dihydroxybenzene to 4-hydroxybenzoate and 2,3-dihydroxybenzoate, respectively. The molar conversion ratio of the carboxylation reaction depended on the concentration of KHCO(3) in the reaction mixture. Only 5 or 48 % of 1,3-dihydroxybenzene added was converted into 2,6-dihydroxybenzoate in the presence of 0.1 M or 3 M KHCO(3), respectively. The addition of acetone to the reaction mixture increased the initial rate of the carboxylation reaction, but the final molar conversion yield reached almost the same value. When the efficient production of 2,6-dihydroxybenzoate was optimized using the whole cells of Pandoraea sp. 12B-2, the productivity of 2,6-dihydroxybenzoate topped out at 1.43 M, which was the highest value so far reported. No formation of any other products was observed after the carboxylation reaction.

Acetone↗
PubMed2000

Limited feeding of potassium nitrate for intracellular lipid and triglyceride accumulation of Nannochloris sp. UTEX LB1999.

Limited feeding of nitrate during culture of Nannochloris sp. UTEX LB1999 for intracellular lipid and triglyceride accumulation was investigated with the aim of obtaining cells superior for liquefaction into a fuel oil. The intracellular lipid contents and the percentage of triglycerides in the lipids of cells grown in a nitrogen-limited medium (0.9 mM KNO3) were 1.3 times as high as those grown in a modified NORO medium containing 2.0-9.9 mM KNO3. However, the cell concentration was too low for the practical production of fuel oil by high-pressure liquefaction of the cell mass. A single feeding of 0.9 mM nitrate after nitrate depletion during cultivation in a nitrate-limited medium increased the cell concentration to twice that obtained without such feeding, and the lipid content was maintained at a high level. The timing of nitrate feeding, i.e., whether it was given during the log phase (before nitrate depletion), the constant growth phase (just after the depletion), or the stationary phase (after the depletion), had negligible effect on the intracellular lipid content and percentage of triglycerides in the lipids. When 0.9 mM nitrate was intermittently fed ten times during the log phase in addition to the initial nitrate feed (0.9 mM), the cell concentration reached almost the same (2.16 g/l) and the intracellular lipid content and the percentage of triglycerides in the lipids increased from 31.0 to 50.9% and 26.0 to 47.6%, respectively, compared with those of cells cultured in a modified NORO medium containing 9.9 mM KNO3 without additional nitrate feeding.

Chlorophyta↗
PubMed2002

The continuing importance of thyroid scintigraphy in the era of high-resolution ultrasound.

At the molecular level, the uptake of radioiodine and pertechnetate is proportional to the expression of the thyroidal sodium/iodine symporter (NIS). Qualitative and quantitative scintigraphic evaluation of the thyroid is performed with a gamma camera fitted with an on-line computer system and enables determination of the iodine uptake or the technetium uptake (TCTU) as an iodine clearance equivalent. Despite new molecular genetic insights into congenital hypothyroidism, the iodine-123 or pertechnetate scan remains the most accurate test for the detection of ectopic thyroid tissue. Following the identification of specific mutations of the genes coding for the NIS, thyroid peroxidase and pendrin, the discharge test has lost its role in establishing the diagnosis of inherited dyshormonogenesis, but it is still of value in the assessment of defect severity. In PDS mutations the test can be used to establish the diagnosis of syndromic disease. Quantitative pertechnetate scintigraphy is the most sensitive and specific technique for the diagnosis and quantification of thyroid autonomy. The method has proved to be valuable in risk stratification of spontaneous or iodine-induced hyperthyroidism, in the estimation of the target volume prior to radioiodine therapy and in the evaluation of therapeutic success after definitive treatment. In iodine deficiency areas the thyroid scan remains indispensable for the functional characterisation of a thyroid nodule and is still a first-line diagnostic procedure in cases of suspected thyroid malignancy. This is especially of importance in patients with Graves' disease, among whom a relatively high prevalence of cancer has been found in cold thyroid nodules. While determination of the TCTU is without any value in the differentiation between autoimmune thyroiditis and Graves' disease in most cases, it is of substantial importance in the differentiation between hyperthyroid autoimmune thyroiditis and Graves' disease.

Adult↗
PubMed2003

Evidence that 99mTc-(V)-DMSA uptake is mediated by NaPi cotransporter type III in tumour cell lines.

In vivo studies have demonstrated that pentavalent technetium-99m dimercaptosuccinic acid [(99m)Tc-(V)-DMSA] may be a useful tumour imaging agent. Several studies have suggested that (99m)Tc-(V)-DMSA uptake may be related to the structural similarity between the (99m)Tc-(V)-DMSA core and the PO(4)(3-) anion. As phosphate ions enter cells via NaPi cotransporters, we investigated whether (99m)Tc-(V)-DMSA uptake is mediated by NaPi cotransporters. (99m)Tc-(V)-DMSA and phosphate uptake kinetics were compared in three cancer cell lines (MCF-7, G152 and MG-63) under several conditions (with and without sodium and NaPi cotransporter inhibitor and at different pH). Determination of molecular NaPi cotransporter mRNA expression was performed by reverse-transcriptase polymerase chain reaction (Rt-PCR) assay. Results obtained in the presence of NaPi inhibitor, in sodium-free medium and at alkaline pH showed that (99m)Tc-(V)-DMSA accumulation is linked to NaPi cotransporter functionality. MCF-7 and G152 exhibited the same tracer uptake, whereas MG-63 showed the highest phosphate accumulation and the lowest (99m)Tc-(V)-DMSA uptake. These results were in accordance with mRNA NaPi expression, i.e. all cell lines expressed NaPi type III but MG-63 also co-expressed NaPi type I. The total level of NaPi cotransporter was highly correlated with phosphate accumulation, while the level of type III was related to (99m)Tc-(V)-DMSA uptake. We have demonstrated that (99m)Tc-(V)-DMSA uptake is specifically mediated by NaPi type III in cancer cells.

Biomarkers, Tumor↗
PubMed2002

Statistical media optimization and production of ITS alpha-amylase from Aspergillus oryzae in a bioreactor.

The production of an intermediate temperature-stable (ITS) alpha-amylase from Aspergillus oryzae was studied by using a central composite design with three independent variables, viz., starch, yeast extract, and K(2)HPO(4). The model equation provided a suitable model for the response surface for alpha-amylase production, and, from the optimal concentrations of the medium components, a model was predicted, which was then used for enzyme production in a 150-L bioreactor. In the bioreactor studies, the enzyme yields (161 U/ml) were similar to that of the shake flask (133 U/ml); however, the time required for maximum alpha-amylase production in the bioreactor was reduced to 48 h compared with 120 h in shake flask cultures. An increased level of phosphate in the medium and low inoculum size were necessary to control the excessive foaming in the bioreactor; however, control of the pO(2) level and agitation was not mandatory for enzyme production. The peak enzyme production coincided with the increase in pH of the fermentation broth and was maximal when the pH of the system was above 7.5. Thus, in the present study, pH acted as an indicator of the initiation or end of the enzyme synthesis or of the fermentation cycle.

Aspergillus oryzae↗
PubMed2006

Organic potassium salts or fibers effects on mineral balance and digestive fermentations in rats adapted to an acidogenic diet.

BACKGROUND: Fibers and potassium (K) organic salts in plant foods are liable to affect Ca and Mg balance at digestive and renal levels, respectively. K organic salts could counteract the acidifying effects of western diets and consequences of excess NaCl. AIM OF THE STUDY: To study this question, male rats were adapted to a basal acidifying low-K (LK) diet, or to diets supplemented with a fiber mix (LK/F), or K citrate (HK) or both (HK/F). RESULTS: HK and HK/F diets displayed a marked alkalinizing effect in urine and promoted citraturia, but this effect was not modulated by fibers. The effect of fibers on Ca digestive absorption was more potent than K citrate effect on Ca renal excretion. In contrast, K citrate effect on kidney Mg excretion was more effective than that of fibers on Mg digestive absorption, a maximal effect on Mg balance was observed in rats fed the HK/F diet. Digestive fermentations in rats fed the LK/F diet were characterized by high-propionic acid fermentations and succinate accumulation. In rats adapted to the HK/F diet, K citrate supplementation depressed succinate and increased butyrate concentrations. CONCLUSION: Organic anions arising from digestive fermentations seem to be not directly involved in the alkalinizing effects of plant foods. Fibers and organic K salts exert distinct effects on Ca and Mg metabolism, but with interesting interactions as to Mg balance, digestive fermentations and urine pH.

Acid-Base Equilibrium↗
PubMed1998

Growth requirements and nitrogen metabolism of Malassezia furfur.

Investigations on the nutrient requirements of Malassezia yeasts led to the preparation of a new minimal medium consisting of an amino nitrogen and a lipid source, which only allowed the growth of the species M. furfur. Carbohydrates, electrolytes, vitamins and trace elements were not required. Using the basal medium, a nitrogen auxanogram for M. furfur was developed, which allowed investigation of the assimilation of 22 amino acids and 9 further nitrogen sources. With the exception of cysteine, all amino acids were metabolized, as were ammonium salts, urea, creatine, creatinine, uric acid and allantoin. KNO3, however, failed to support growth. Depending on the source used, the yeast cells changed micromorphologically: both oval and round forms were induced. Assimilation of several amino acids resulted in dimorphism, especially in the case of glycine and serine. The cell yield differed significantly depending on the nitrogen source; short-chain unbranched amino acids were utilized most readily. Thus, M. furfur can be characterized as a relatively undemanding yeast species which is optimally adapted to the superficial skin environment. All other lipid-dependent Malassezia species seem to require more complex media.

Allantoin↗
PubMed2000

Studies on water transport through the sweet cherry fruit surface: characterizing conductance of the cuticular membrane using pericarp segments.

Water conductance of the cuticular membrane (CM) of mature sweet cherry fruit (Prunus avium L. cv. Sam) was investigated by monitoring water loss from segments of the outer pericarp excised from the cheek of the fruit. Segments consisted of epidermis, hypodermis and several cell layers of the mesocarp. Segments were mounted in stainless-steel diffusion cells with the mesocarp surface in contact with water, while the outer cuticular surface was exposed to dry silica (22 +/- 1 degrees C). Conductance was calculated by dividing the amount of water transpired per unit area and time by the difference in water vapour concentration across the segment. Conductance values had a log normal distribution with a median of 1.15 x 10(-4) m s(-1) (n=357). Transpiration increased linearly with time. Conductance remained constant and was not affected by metabolic inhibitors (1 mM NaN3 or 0.1 mM carbonylcyanide m-chlorophenylhydrazone) or thickness of segments (range 0.8-2.8 mm). Storing fruit (up to 42 d, 1 degrees C) used as a source of segments had no consistent effect on conductance. Conductance of the CM increased from cheek (1.16 +/- 0.10 x 10(-4) m s(-1)) to ventral suture (1.32 +/- 0.07 x 10(-4) m s(-1)) and to stylar end (2.53 +/- 0.17 x 10(-4) m s(-1)). There was a positive relationship (r2=0.066**; n=108) between conductance and stomatal density. From this relationship the cuticular conductance of a hypothetical astomatous CM was estimated to be 0.97 +/- 0.09 x 10(-4) m s(-1). Removal of epicuticular wax by stripping with cellulose acetate or extracting epicuticular plus cuticular wax by dipping in CHCl3/methanol increased conductance 3.6- and 48.6-fold, respectively. Water fluxes increased with increasing temperature (range 10-39 degrees C) and energies of activation, calculated for the temperature range from 10 to 30 degrees C, were 64.8 +/- 5.8 and 22.2 +/- 5.0 kJ mol(-1) for flux and vapour-concentration-based conductance, respectively.

Biological Transport↗
PubMed2001

Changes in the structure of xyloglucan during cell elongation.

Xyloglucans were isolated by sequential extraction of the cell walls of pea (Pisum sativum L. cv. Alaska) with a xyloglucan-specific endoglucanase and KOH. The xyloglucan content and xyloglucan-oligosaccharide composition were determined for fractions obtained from the elongating and non-elongating segments of pea stems grown in the light and in darkness. The results were consistent with the hypothesis that regulated growth of the cell wall depends on xyloglucan metabolism. Furthermore, the characterization of xyloglucan extracted from leaves of light-grown pea plants indicates that xyloglucan metabolism is tissue specific. Changes in xyloglucan subunit structure observed in elongating stems are consistent with the in muro realization of a metabolic pathway that was previously proposed solely on the basis of the in vitro activities of plant glycosyl hydrolases.

2,4-Dichlorophenoxyacetic Acid↗
PubMed2000

Discrepancy between nitrate reduction rates in intact leaves and nitrate reductase activity in leaf extracts: what limits nitrate reduction in situ?

Nitrate reductase (NR) activity in spinach leaf extracts prepared in the presence of a protein phosphatase inhibitor (50 microM cantharidine) was measured in the presence of Mg2+ (NRact) or EDTA (NRmax), under substrate saturation. These in-vitro activities were compared with nitrate reduction rates in leaves from nitrate-sufficient plants. Spinach leaves containing up to 60 micromol nitrate per g fresh weight were illuminated in air with their petiole in water. Their nitrate content decreased with time, permitting an estimation of nitrate reduction in situ. The initial rates (1-2 h) of nitrate consumption were usually lower than NRact, and with longer illumination time (4 h) the discrepancy grew even larger. When leaves were fed through their petiole with 30 mM nitrate, initial in-situ reduction rates calculated from nitrate uptake and consumption were still lower than NRact. However, nitrate feeding through the petiole maintained the in situ-nitrate reduction rate for a longer time. Initial rates of nitrate reduction in situ only matched NRact when leaves were illuminated in 5% CO2. In CO2-free air or in the dark, both NRact and in-situ nitrate reduction decreased, but NRact still exceeded in-situ reduction. More extremely, under anoxia or after feeding 5-amino-4-imidazole carboxyamide ribonucleoside in the dark, NR was activated to the high light level; yet in spite of that, nitrate reduction in the leaf remained very low. It was examined whether the standard assay for NRact would overestimate the in-situ rates due to a dissociation of the inactive phospho-NR-14-3-3 complex after extraction and dilution, but no evidence for that was found. In-situ NR obviously operates below substrate saturation, except in the light at high ambient CO2. It is suggested that in the short term (2 h), nitrate reduction in situ is mainly limited by cytosolic NADH, and cytosolic nitrate becomes limiting only after the vacuolar nitrate pool has been partially emptied.

14-3-3 Proteins↗
PubMed2000

Isolation and characterisation of cell wall polysaccharides from cocoa (Theobroma cacao L.) beans.

Cell wall material (CWM) was prepared from sun-dried cocoa (Theobroma cacao L.) bean cotyledons before and after fermentation. The monosaccharide composition of the CWM was identical for unfermented and fermented beans. Polysaccharides of the CWM were solubilised by sequential extraction with 0.05 M trans-1,2-diaminocyclohexane-N,N,N',N'-tetraacetic acid (CDTA), 0.05 M Na2CO3, and 1 M, 4 M and 8 M KOH. The non-cellulosic sugar composition for each fraction was similar for unfermented and fermented samples, indicating that fermentation caused no significant modification of the structural features of individual cell wall polysaccharides. Pectic polysaccharides accounted for 60% of the cell wall polysaccharides but only small amounts could be solubilised in solutions of CDTA, Na2CO3, and 1 M and 4 M KOH. The bulk of the pectic polysaccharides were solubilised in 8 M KOH and were characterised by a rhamnogalacturonan backbone heavily substituted with side-chains of 5-linked arabinose and 4-linked galactose. Linkage analysis indicated the presence of additional acidic polysaccharides, including a xylogalacturonan and a glucuronoxylan. Cellulose, xyloglucan and a galactoglucomannan accounted for 28%, 8% and 3% of the cell wall polysaccharides, respectively. It is concluded that the types and structural features of cell wall polysaccharides in cocoa beans resemble those found in the parenchymatous tissue of many fruits and vegetables rather than those reported for many seed storage polysaccharides.

Cacao↗
PubMed2002

The effect of physical and chemical treatment on the mechanical properties of the cell wall of the alga Chara corallina.

Single large internode cells of the charophyte (giant alga) Chara corallina were dissected to give sheets of cell wall, which were then notched and their mechanical properties in tension determined. The cells were subjected to a thermal treatment in excess water (cf. cooking), which had little effect on strength but increased the stiffness, contrasting with the behaviour of higher-plant tissues. Extraction in CDTA (cyclohexane-trans-1,2-diamine-N,N,N',N'-tetraacetate) or 4 M KOH reduced the strength from 17 MPa to 10 MPa, although sequential extraction in CDTA and 4 M KOH reduced the strength further to 4 MPa. The stiffness decreased from 500 MPa to 300 MPa on extraction in CDTA or 4 M KOH, while falling to 70 MPa after extraction in CDTA followed by 4 M KOH. Conventional sequential extraction in CDTA, Na2CO3 at 1 degrees C and 20 degrees C, and KOH at 0.5 M, 1 M, 2 M and 4 M caused a gradual decrease in stiffness and strength after the CDTA treatment to the same lower values. This result is in keeping with mechanical properties for plant tissues, but in contrast to the removal of pectic polysaccharides from model cell wall systems, which does not reduce the stiffness.

Algorithms↗