Overcoming the chromatographic challenges when performing LC-MS/MS measurements of pyridoxal-5'-phosphate.

Pyridoxal-5'-phosphate (PLP), the active form of vitamin B6, is required for numerous enzymatic reactions. Vitamin B6 deficiency or exceptionally high levels of PLP have negative implications, making measurements of PLP imperative for diagnoses and monitoring in many clinical scenarios. Traditional assays are enzymatic, ELISA based, or employ HPLC with various detection modalities; all of these are prone to interferences and crossreactivity with other compounds. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) has been increasingly used to overcome these issues, but the high polarity of PLP raises chromatographic challenges. Using ion pairing reagents in the mobile phases is a possible solution, but these reagents often have deleterious effects on instrumentation. An alternative strategy is the addition of an ion pairing reagent after extraction, but prior to injection. To prove this, we used 1-octanesulfonic acid (OSA) without changing the LC method or column. With this technique, we observed a 2-4 fold increase in signal-to-noise ratio. Intraday and interday precision of replicate measurements also improved drastically compared to analyses without OSA, while also yielding a dramatic improvement in column life compared to our previous approach and to this point no deleterious effects on instrument hardware commonly associated with traditional ion pairing reagent techniques have been observed.

Milk metabolome reveals pyrimidine and its degradation products as the discriminant markers of different corn silage-based nutritional strategies.

The purpose of this study was to evaluate the effect of 6 different feeding systems (based on corn silage as the main ingredient) on the chemical composition of milk and to highlight the potential of untargeted metabolomics to find discriminant marker compounds of different nutritional strategies. Interestingly, the multivariate statistical analysis discriminated milk samples mainly according to the high-moisture ear corn (HMC) included in the diet formulation. Overall, the most discriminant compounds, identified as a function of the HMC, belonged to AA (10 compounds), peptides (71 compounds), pyrimidines (38 compounds), purines (15 compounds), and pyridines (14 compounds). The discriminant milk metabolites were found to significantly explain the metabolic pathways of pyrimidines and vitamin B6. Interestingly, pathway analyses revealed that the inclusion of HMC in the diet formulation strongly affected the pyrimidine metabolism in milk, determining a significant up-accumulation of pyrimidine degradation products, such as 3-ureidopropionic acid, 3-ureidoisobutyric acid, and 3-aminoisobutyric acid. Also, some pyrimidine intermediates (such as l-aspartic acid, N-carbamoyl-l-aspartic acid, and orotic acid) were found to possess a high discrimination degree. Additionally, our findings suggested that the inclusion of alfalfa silage in the diet formulation was potentially correlated with the vitamin B6 metabolism in milk, being 4-pyridoxic acid (a pyridoxal phosphate degradation product) the most significant and up-accumulated compound. Taken together, the accumulation trends of different marker compounds revealed that both pyrimidine intermediates and degradation products are potential marker compounds of HMC-based diets, likely involving a complex metabolism of microbial nitrogen based on total splanchnic fluxes from the rumen to mammary gland in dairy cows. Also, our findings highlight the potential of untargeted metabolomics in both foodomics and foodomics-based studies involving dairy products.

Clinical, biochemical and genetic findings in adult patients with chronic hypophosphatasemia.

PURPOSE: The study aimed to define the clinical, biochemical and genetic features of adult patients with osteopenia/osteoporosis and/or bone fragility and low serum alkaline phosphatase (sALP). METHODS: Twenty-two patients with at least two sALP values below the reference range were retrospectively enrolled after exclusion of secondary causes. Data about clinical features, mineral and bone markers, serum pyridoxal-5'-phosphate (PLP), urine phosphoethanolamine (PEA), lumbar and femur bone densitometry, and column X-ray were collected. Peripheral blood DNA of each participant was analyzed to detect ALPL gene anomalies. RESULTS: Pathogenic ALPL variants (pALPL) occurred in 23% and benign variants in 36% of patients (bALPL), while nine patients harbored wild-type alleles (wtALPL). Fragility fractures and dental anomalies were more frequent in patients harboring pALPL and bALPL than in wtALPL patients. Of note, wtALPL patients comprised women treated with tamoxifen for hormone-sensitive breast cancer. Mineral and bone markers were similar in the three groups. Mean urine PEA levels were significantly higher in patients harboring pALPL than those detected in patients harboring bALPL and wtALPL; by contrast, serum PLP levels were similar in the three groups. A 6-points score, considering clinical and biochemical features, was predictive of pALPL detection [P = 0.060, OR 1.92 (95% CI 0.972, 3.794)], and more significantly of pALPL or bALPL [P = 0.025, OR 14.33 (95% CI 1.401, 14.605)]. CONCLUSION: In osteopenic/osteoporotic patients, single clinical or biochemical factors did not distinguish hypophosphatasemic patients harboring pALPL or bALPL from those harboring wtALPL. Occurrence of multiple clinical and biochemical features is predictive of ALPL anomalies, and, therefore, they should be carefully identified. Tamoxifen emerged as a hypophosphatasemic drug.

Effects of Pyridoxine Deficiency on Hippocampal Function and Its Possible Association with V-Type Proton ATPase Subunit B2 and Heat Shock Cognate Protein 70.

Pyridoxine, one of the vitamin B6 vitamers, plays a crucial role in amino acid metabolism and synthesis of monoamines as a cofactor. In the present study, we observed the effects of pyridoxine deficiency on novel object recognition memory. In addition, we examined the levels of 5-hydroxytryptamine (5-HT), 5-hydroxyindoleacetic acid (5-HIAA), 3,4-dihydroxyphenethylamine (DA), 3,4-dihydroxyphenylacetic acid, and homovanillic acid and the number of proliferating cells and neuroblasts in the hippocampus. We also examined the effects of pyridoxine deficiency on protein profiles applying a proteomic study. Five-week-old mice fed pyridoxine-deficient diets for 8 weeks and showed a significant decrease in the serum and brain (cerebral cortex, hippocampus, and thalamus) levels of pyridoxal 5'-phosphate, a catalytically active form of vitamin-B6, and decline in 5-HT and DA levels in the hippocampus compared to controls fed a normal chow. In addition, pyridoxine deficiency significantly decreased Ki67-positive proliferating cells and differentiated neuroblasts in the dentate gyrus compared to controls. A proteomic study demonstrated that a total of 41 spots were increased or decreased more than two-fold. Among the detected proteins, V-type proton ATPase subunit B2 (ATP6V1B2) and heat shock cognate protein 70 (HSC70) showed coverage and matching peptide scores. Validation by Western blot analysis showed that ATP6V1B2 and HSC70 levels were significantly decreased and increased, respectively, in pyridoxine-deficient mice compared to controls. These results suggest that pyridoxine is an important element of novel object recognition memory, monoamine levels, and hippocampal neurogenesis. Pyridoxine deficiency causes cognitive impairments and reduction in 5-HT and DA levels, which may be associated with a reduction of ATP6V1B2 and elevation of HSC70 levels in the hippocampus.

A fluorescent hydrazone exchange probe of pyridoxal phosphate for the assessment of vitamin B6 status.

Abnormal vitamin B6 status, marked by deficient intracellular concentrations of pyridoxal phosphate (PLP), is classified as a direct biomarker based on its biomedical significance. However, there exist no direct methods for measuring vitamin B6 status in intact cells. Here we describe the development of a fluorogenic probe, RAB6, which shows remarkable selectivity for PLP among the B6 vitamers and other cellular aldehydes.

Glutamate Decarboxylase (GAD) Extracted from Germinated Rice: Enzymatic Properties and Its Application in Soymilk.

Glutamate decarboxylase (GAD) is an important enzyme in biological metabolisms acting on catalyzing the irreversible α-decarboxylation of L-glutamic acid to γ-aminobutyric acid (GABA) and CO2, which was focused in this study. Three rice varieties different in color were germinated at different times and used for crude GAD extraction. Crude GADs with an optimal germination time from germinated black (GBR), red (GRR), and white (GWR) rice were evaluated for enzymatic properties, including the effect of pHs, temperatures, and concentrations of both L-glutamic acid and pyridoxal 5'-phosphate (PLP). Crude GAD with optimum enzymatic properties was selected to be partially purified using ammonium sulfate (AMS) precipitation. The obtained GAD was supplemented to soymilk and determined for GABA content. All crude GADs from germinated rice at 10 germination days presented the highest enzyme activity. For enzymatic properties, crude GADs showed the highest activity at pH in a range of 5.6-6.0 at 60ºC. The Km values of crude GADs were in the range of 7.68-8.06 mM for L-glutamic acid and 0.15-0.20 µM for PLP and were the lowest in crude GAD from GBR. GAD from GBR presented the highest enzyme activity in the fraction with 50% saturation (v/v) after AMS precipitation and it was purified for 14.61 folds. The addition of this GAD (1.0%, v/v) resulted in the increasing of GABA content in soymilk to 53.79 mg/100 mL, accounted for 1.23 times compared with control.

Pharmaceutical compounding of orphan active ingredients in Belgium: how community and hospital pharmacists can address the needs of patients with rare diseases.

BACKGROUND: Pharmaceutical compounding of orphan active ingredients can offer cost-effective treatment to patients when no other drug product is available for a rare disease or during periods of drug product shortages. Additionally, it allows customized therapy for patients with rare diseases. However, standardized compounding formulas and procedures, and monographs are required to ensure the patients' safety. RESULTS: Standardized formulas and compounding procedures were developed for seven orphan active ingredients (L-arginine, sodium benzoate, sodium phenylbutyrate, L-carnitine, chenodesoxycholic acid, primaquine phosphate, pyridoxal phosphate) and one non-orphan molecule (sodium perchlorate) regularly compounded by hospital pharmacists for extemporaneous use. The stability of these formulations was evaluated over 3 months at refrigerated (5 °C) and standard storage conditions (25 °C/60%RH) using HPLC-based assays and a suitable shelf life was assigned to the formulations. Additionally, suitable analytical methods for quality control of formulations of pyridoxal phosphate and sodium perchlorate were developed as monographs for these components were not available in the European Pharmacopeia or United States Pharmacopeia. CONCLUSIONS: Availability of compounding formulas and protocols, as well as stability information, for orphan active ingredients can improve patients' access to treatment for rare diseases. Such data were collected for seven orphan active ingredients to treat patients with rare diseases when no other treatment is available. More efforts are needed to develop standardized formulas and compounding procedures for additional orphan active ingredients whose clinical efficacy is well-known but which are not available as products with a marketing authorization. Additionally, a legal framework at EU level is required to enable the full potential of pharmaceutical compounding for orphan active ingredients.

Stability-indicating HPLC-PDA assay for simultaneous determination of paracetamol, thiamine and pyridoxal phosphate in tablet formulations.

With the increased number of multi-drug formulations, there is a need to develop new methods for simultaneous determinations of drugs. A precise, accurate and reliable liquid chromatographic method was developed for simultaneous determination of paracetamol, thiamine, and pyridoxal phosphate in pharmaceutical formulations. Separation of analytes was carried out with an Agilent Poroshell C18 column. A mixture of ammonium phosphate buffer (pH = 3.0), acetonitrile and methanol in the ratio of 86:7:7 (V/V/V) was used as the mobile phase pumped at a flow rate of 1.8 mL min-1. Detection of all three components, impurities and degradation products was performed at the selected wavelength of 270 nm. The developed method was validated in terms of linearity, specificity, precision, accuracy, LOD and LOQ as per ICH guidelines. Linearity of the developed method was found in the range 17.5-30 µg mL-1 for thiamine, 35-60 µg mL-1 for pyridoxal phosphate and 87.5-150 µg mL-1 for paracetamol. The coefficient of determination was ≥ 0.9981 for all three analytes. The proposed HPLC method was found to be simple and reliable for the routine simultaneous analysis of paracetamol, thiamine and pyridoxal phosphate in tablet formulations. Complete separation of analytes in the presence of degradation products indicated selectivity of the method.

A kinetic spectrophotometric method for the determination of pyridoxal-5'-phosphate based on coenzyme activation of apo-d-phenylglycine aminotransferase.

A new PLP assay method based on the coenzyme activation of apo-d-phenylglycine aminotransferase (apo-d-PhgAT) is reported. The assay process is comprised of two steps. First, PLP present in plasma samples is allowed to reconstitute apo-d-PhgAT, forming active holo-d-PhgAT. In the second step, the enzymatic activity of reconstituted d-PhgAT is determined using d-4-OH-phenylglycine as the amino donor substrate with 4-OH-benzoylformate (OH-BZF) as the reaction product. OH-BZF absorbs UV light strongly at 334 nm (molar absorption coefficient = 25.4 × 103 M-1cm-1) and its rate of formation is monitored spectrophotometrically. The rate of the transamination reaction catalyzed by the reconstituted d-PhgAT is directly proportional to the amount of PLP in the sample. The method is applicable for determining PLP in the concentration range from 5.2 to 250 nM and requires 50 µL of plasma sample. The mean within- and between-run coefficient of variations (CVs) were 8.1% and 12.4%, respectively. Analytical recoveries ranged from 98 to 108%. The assay was specific and showed good correlation with the established method (CDC, Method No: 4002.05). The assay requires one reaction catalyzed by a single enzyme, does not require a radioactive substrate, and a derivatization reagent is not needed. This PLP determination process is relatively simple to perform and can be completed using common laboratory equipment.

Pyridoxal 5'-phosphate assay based on lucigenin chemiluminescence.

The authors describe the first chemiluminescence (CL) based method for determination of pyridoxal 5'-phosphate (PLP). PLP is found to generate intense CL with lucigenin higher than that of the conventional lucigenin-H2O2 system by a factor of about 9.0. This new finding is used to be in a detection method for PLP via flow injection analysis (FIA). Response is linear in the 50 nM to 200 µM PLP concentration range with a correlation coefficient of 0.998, and the detection limit (at an S/N of 3) is 6.9 nM. The assay is highly selective over various amino acids, vitamins, sugars, coenzymes and metal ions cofactors. It exhibits advantages over the commonly employed HPLC methods in that it is rapid, more economic, eco-friendly and high throughput FIA detection of PLP without the need for toxic derivatization reagents, organic solvents, and HPLC instrumentation. The method was successfully applied to the determination of PLP in (spiked) human blood samples with recoveries in the range from 96.2-101.6% with % RSD < 4.0. The new system is also employed to determine lucigenin in the linear range of 0.3 to 100.0 µM with a correlation coefficient of 0.994 and the limit of detection is 0.04 µM. Graphical abstract Schematic of the chemiluminescent assay for pyridoxal 5'-phosphate (PLP). Lucigenin-PLP demonstrates 9-fold stronger chemiluminescence intensity than the lucigenin-H2O2 system. The detection limit of PLP is 6.9 nM. The method can detect PLP in human serum with good recoveries.

Development of a reversed phase liquid chromatographic method for analysis of pyridoxal-5'-phosphate and its impurities.

Since no proper method is available in literature for the analysis of pyridoxal-5'-phosphate, a reversed phase liquid chromatographic method was developed and validated for specificity, sensitivity, linearity, precision and accuracy. Nine potential related substances and forced degradation products could be successfully separated from the main peak. The separation was achieved on a Polaris C18 column (250 × 4.6 mm i.d., 5 µm) using a mobile phase consisting of 20 mM ammonium formate in 0.65% formic acid - acetonitrile (98.8:1.2, v/v). Isocratic elution was performed at a flow rate of 1.0 mL/min and the analytes were detected by UV at 240 nm. The volatile mobile phase allowed also direct coupling to an ion-trap mass spectrometer with a positive electrospray ionization source to characterize unknown peaks in the chromatogram. The method can be used for quality control purposes as required by regulatory authorities to ensure the product's safety and efficacy.

Rapid determination of the various native forms of vitamin B6 and B2 in cow's milk using ultra-high performance liquid chromatography.

As the formation of pyridoxal phosphate, the active cofactor of vitamin B6, is dependent on riboflavin 5-phosphate, we propose a fast and simple ultra-high performance liquid chromatography method for the simultaneous determination of the native B6 vitamers pyridoxal, pyridoxine, pyridoxamine, their mono phosphorus esters and 4-pyridoxic acid as well as vitamin B2 as riboflavin and its phosphorus ester riboflavin 5-phosphate in milk. Separation was achieved under 6.0min by reversed-phase and pH gradient elution. Sample preparation was optimized regarding various acids and pH levels. Changes in those parameters led to significant deviations of sample matrix breakdown efficiency. The optimized method was then validated regarding specificity, accuracy, precision, linearity, range, detection and quantification limits. As the method performed satisfactory, is was used to study commercial liquid cow's milk (n=31), regarding effects of the employed preservation technique (pasteurization, extended shelf-life, ultra-high temperature) on the composition and content of B6 and B2 vitamers. In cow's milk, vitamin B6 mostly consists of pyridoxal and its phosphate ester, with pyridoxal phosphate being the bulk component. The catabolite of the vitamin B6 metabolism, 4-pyridoxic acid was present in significant amounts in all studied samples, with up to 2.69µmolL-1. Vitamin B2 was present as riboflavin and its phosphate ester up to 12.86µmolL-1.

RETRACTED: Bimetallic magnetic nanoparticle as a new platform for fabrication of pyridoxine and pyridoxal-5'-phosphate imprinted polymer modified high throughput electrochemical sensor.

This article has been retracted: please see Elsevier Policy on Article Withdrawal (https://www.elsevier.com/about/our-business/policies/article-withdrawal). This article has been retracted at the request of the Editor-in-Chief following concerns raised by a reader. The article uses an electron micrograph identical to another publication. Fig. 2B is identical to Fig. 1C published in Biosensors and Bioelectronics Volume 78, 15 April 2016, Pages 454-463, https://doi.org/10.1016/j.bios.2015.11.092. In addition, the extraordinary similarities observed between the data presented in Fig. 1D and in Fig. 3C in ACS Biomater. Sci. Eng., 2017, 3 (9), pp 2120­2135, https://doi.org/10.1021/acsbiomaterials.7b00089, Fig. 4A in Colloids and Surfaces B: Biointerfaces, Volume 142, 1 June 2016, Pages 248­258 https://doi.org/10.1016/j.colsurfb.2016.02.053 and Fig. 4C in Biosensors and Bioelectronics, Volume 97, 15 November 2017, Pages 208­217, https://doi.org/10.1016/j.bios.2017.06.003 are highly unlikely. This problem with the data casts doubt on all the data, and accordingly also the conclusions based on that data, in this publication. As such this article represents a severe abuse of the scientific publishing system. The scientific community takes a very strong view on this matter and apologies are offered to readers of the journal that this was not detected during the submission process.

Contents of all forms of vitamin B6, pyridoxine-ß-glucoside and 4-pyridoxic acid in mature milk of Japanese women according to 4-pyridoxolactone-conversion high performance liquid chromatography.

The contents of six vitamin B6 forms, pyridoxine-ß-glucoside, and 4-pyridoxic acid in mature milk of 20 Japanese lactating women consuming ordinary Japanese foods were determined by a 4-pyridoxolactone-conversion HPLC method. These compounds were determined with the average recovery rate of 83.9% or more. The average total content of vitamin B6 forms was 1.01 ± 0.32 (µmol/L). Pyridoxal and pyridoxal 5'-phosphate were found in all of the samples, and their average contents were 0.71 ± 0.28 (µmol/L) and 0.16 ± 0.07 (µmol/L), respectively. Pyridoxamine, pyridoxine, pyridoxamine 5'-phosphate, pyridoxine 5'-phosphate, and pyridoxine-ß-glucoside were found in 15, 14, 13, 9, and 7 samples, respectively. The presence of pyridoxine 5'-phosphate was for the first time found in human milk. A method for the determination of 4-pyridoxic acid, which is the excretion form of vitamin B6, was modified to quantitate it by isocratic HPLC. 4-Pyridoxic acid was found in all samples, and its average content was 0.094 ± 0.040 (µmol/L), which was only 12% of its content in cow (Holstein) milk. The total content of vitamin B6 forms, and predominant presence of pyridoxal among other vitamin B6 forms in the Japanese women's milk samples shared similar characteristics with American women's milk samples.

Determination of quality of pyridoxal-5'-phosphate enzyme preparations by spectroscopic methods.

The present study evaluates purified aspartate transaminase (AST, EC 2.6.1.1) preparations from three commercial sources. The enzyme molecule contains pyridoxal-5'-phosphate coenzyme (PLP), which provides AST characteristic absorption spectra in the wavelength range of 300-500 nm. The coenzyme bound in the active site also shows circular dichroism (CD) spectra in the same range. Besides, AST like other proteins may be modified in vitro or in vivo by reactions with other molecules, e.g. reactive sugars, and may form fluorescent products (advanced glycation end products, AGE). Spectroscopic methods were used to assess the quality of AST preparations from three different sources, Serva, Roche, and Sigma. Absorption spectra showed that the peak 360 nm characteristic of the active PLP form of AST prevailed in the Serva and Sigma preparations, while 330 nm was the major peak in the Roche preparation. CD spectra demonstrated the major maximum at 360 nm in the Serva and Roche samples, thus suggesting the predominance of the active PLP form in both preparations. The Sigma sample showed a CD profile less characteristic of AST. Fluorescence measurements revealed formation of AGE in the case of the Roche preparation, while fluorescence of the other two preparations was low. In general, the Serva sample presented the most convenient properties of purified AST among the preparations tested. The results will be used for the selection of a commercial enzyme preparation applicable in our future spectroscopic studies of glycation of AST as a model protein and in our research of the influence of antioxidants on this process.

Plasma folate as marker of folate status in epidemiological studies: the European Investigation into Cancer and Nutrition (EPIC)-Potsdam study.

Folate deficiency is often discussed as a potential risk factor for CVD and some cancers. Reliable assessment of folate status in large-scale epidemiological studies is therefore of major importance. The present study assessed the value of plasma folate (PF) compared with erythrocyte folate (EF) as a marker of folate status in 363 participants in the European Investigation into Cancer and Nutrition (EPIC)-Potsdam cohort. EF and PF, total homocysteine (tHcy), pyridoxine, cobalamin, creatinine, total protein and packed cell volume were determined; glutamate carboxypeptidase (GCP) C1561T, reduced folate carrier (RFC) G80A and methylenetetrahydrofolate (MTHFR) C677T polymorphisms were analysed. Anthropometric measurements were taken and dietary intake was assessed with the EPIC-Potsdam food-frequency questionnaire. Comparison of EF and PF with factors that may modulate their concentrations was performed. Cross-classification of blood folates in quintile categories resulted in correct classification into the same or adjacent category of 75.5 % of all subjects. Age, BMI, pyridoxine and cobalamin, fruit and vegetable intake, and vitamin supplementation 24 h before blood draw were positively associated with EF and with PF. For tHcy an inverse association was found. Participants with the MTHFR 677TT genotype showed significantly elevated EF concentrations compared with those with 677CT genotype; EF and PF were more strongly correlated (r 0.78, P<0.0001) for participants with MTHFR 677TT genotype than for those with the 677CC or 677CT genotype. In summary, our present results indicate that plasma folate seems to be a suitable marker for assessment of folate status for use in large-scale epidemiological studies.

Kinetic and structural characterization of Slr0077/SufS, the essential cysteine desulfurase from Synechocystis sp. PCC 6803.

Cysteine desulfurases, designated NifS, IscS, and SufS, cleave L-cysteine to form alanine and an enzyme cysteinyl persulfide intermediate. Genetic studies on the photosynthetic cyanobacterium Synechocystis sp. PCC 6803 have shown that of the three Nif/Isc/SufS-like proteins encoded in its genome only the sequence group II protein, Slr0077/SufS, is essential. This protein has been overexpressed in Escherichia coli, purified to homogeneity, shown to bind pyridoxal-5'-phosphate (PLP) and to catalyze cysteine desulfuration, and characterized in terms of its structure and kinetics. The results suggest that catalysis in the absence of accessory factors has two constituent pathways, one involving nucleophilic attack by C372 to form the Slr0077/SufS-bound cysteinyl persulfide intermediate and the second involving intermolecular attack by the sulfur of a second molecule of the substrate on the initial l-cysteine-PLP complex to form free l-cysteine persulfide. The second pathway is operant in the C372A variant protein, explaining why it retains significant activity, which is proportional to the concentration of l-cysteine (i.e., does not saturate). C-S bond cleavage by the first (normal) pathway is considerably less efficient than the equivalent step in a group I desulfurase (Slr0387) from the same organism (characterized in the accompanying paper). The 1.8 A crystal structure of the protein, which is very similar to that previously reported for E. coli SufS, shows that the loop on which C372 resides is well-ordered and shorter by 11 residues than the corresponding disordered loop of the group I NifS-like protein from Thermotoga maritima. Sequence comparisons establish that the T. maritima and Slr0387 proteins have loops of similar length. The combined structural and kinetic data imply that the modest activity of Slr0077/SufS and other SufS proteins in comparison to their sequence group I (NifS/IscS-like) paralogues results from inefficiency in the nucleophilic attack step associated with differences in the structure or dynamics of this loop. The recent reports that SufS proteins can be activated manyfold by binding to SufE thus implies that the accessory protein either accelerates nucleophilic attack by the conserved cysteine residue of SufS by a conformational mechanism or itself contributes a nucleophilic cysteine for more efficient intermolecular attack.

Effects of different dietary amounts of LCPUFA n3 and vitamin B6 on lipid composition and antioxidant defences in rat kidney.

Our previous report demonstrated that, when vitamin deficiency is associated with high contents of long chain polyunsaturated fatty acids (LCPUFA) n3, lipid peroxidation susceptibility in rat heart and liver increases. In this paper, we evaluated the effect of the same dietary administration on lipid composition and antioxidant defenses of rat kidney. Results showed that vitamin B(6) deficiency, when associated with a fish oil diet, as compared to vegetable oil condition, increased relative kidney weight and decreased pyridoxal-5P contents. The different LCPUFA n3 dietary contents produced, on kidney phospholipids, effects interlaced with those of vitamin B(6) deficiency; in particular fish oil and vitamin B(6) deficient diet caused a significant decrease of arachidonic acid showing that the processes of elongation and desaturation of linoleic acid were slowed. Also, peroxidation susceptibility was higher, as demonstrated both by increased TBARS formation and glutathione peroxidase activity, and by decreased vitamin E contents and reduced glutathione/oxidized glutathione ratio.

Determination of mangafodipir trisodium and related impurities in bulk substance and pharmaceutical formulation by ion-pair high-performance liquid chromatography.

The development of an ion-pair liquid chromatographic method for determination of mangafodipir trisodium and related impurities is described. Good resolution was obtained when using a polymeric reverse-phase column and a mobile phase of pH* 10.5 composed by borate buffer, acetonitrile, and tetrabutylammonium hydrogensulphate as ion pair agent. Validation of the method showed good selectivity, precision, accuracy and linearity, and detection limits of 0.1--0.2 microg/ml.

Determination of vitamin B(6) in cooked sausages.

A reverse-phase high-performance liquid chromatography (HPLC) method has been described for the determination of various active forms of vitamin B(6) in meat products. Different extracting agents were tested to solubilize fully the analyte for quantification. The best data were obtained by extracting the samples with 5% (w/v) metaphosphoric acid. Separation by HPLC was performed with fluorescence detection (excitation, 290 nm; emission, 395 nm), on a 10 cm x 0.46 cm i.d. Hypersil BDS C(18) 5 microm column using a mixture of 50 mM phosphate buffer (pH 3.2) and acetonitrile (99:1, v/v) as mobile phase. Precision of the method was 0.5% (within a day) and 4.3% (between days). The detection limits were 0.020 mg/100 g for pyridoxal and pyridoxamine, 0.017 mg/100 g for pyridoxamine phosphate, 0.500 mg/100 g for pyridoxal phosphate, and 0.033 mg/100 g for pyridoxol, with a signal-to-noise ratio of 3. The recovery ranged from 92.0 to 100.0%.