Effects of Pyridoxal-5'-Phosphate on Aminotransferase Activity Assay

BACKGROUND: Pyridoxal-5'-phosphate (P5P), a coenzyme of the aspartate aminotransferase (AST) and alanine aminotransferase (ALT) reactions, is required to measure aminotransferase levels (IFCC method). However, a modified IFCC method that uses a reagent devoid of P5P is commonly used in laboratories in Korea. To determine the differences between the two methods, we compared aminotransferase levels measured by using the IFCC method and modified IFCC method. METHODS: Serum levels of AST and ALT, with and without P5P, were measured in 2,318 patients. Based on the allowable limits of performance set by the Royal College of Pathologists of Australasia (RCPA), differences between the two methods were analyzed under various conditions. RESULTS: Higher AST and ALT values were obtained by the IFCC method compared to modified IFCC method, showing significant differences between the two methods (AST, 5.8±14.2 IU/L; ALT, 2.8±6.9 IU/L) (P<0.001). Values exceeding RCPA criteria were more frequently observed in emergency orders (AST, 65.8%; ALT, 14.4%) than in routine orders (AST, 3.2%; ALT, 9.6%), as well as in inpatient wards (AST, 70.4%; ALT, 18.5%) compared to outpatient clinics (AST, 56.6%; ALT, 10.0%). However, the differences between the two methods were not significant among the disease groups, except for the acute myocardial infarction group. CONCLUSIONS: The method using reagents without P5P underestimated aminotransferase activity. The effect of P5P was more significant in patients with acute myocardial infarction, considered as P5P-deficient. In conclusion, the IFCC method with P5P should be applied for measuring AST and ALT serum levels.

Theoretical studies on the pyridoxal-5'-phosphate dependent enzyme dopa decarboxylase: Effect of Thr 246 residue on the co-factor-enzyme binding and reaction mechanism.

Decarboxylation of amino acid is a key step for biosynthesis of several important cellular metabolites in the biological systems. This process is catalyzed by amino acid decarboxylases and most of them use pyridoxal-5'-phosphate (PLP) as a co-factor. PLP is bound to the active site of the enzyme by various interactions with the neighboring amino acid residues. In the present investigation, density functional theory (DFT) and real-time dynamics studies on both ligand-free and ligand-bound dopa decarboxylases (DDC) have been carried out in order to elucidate the factors responsible for facile decarboxylation and also for proper binding of PLP in the active site of the enzyme. It has been found that in the crystal structure Asp271 interacts with the pyridine nitrogen atom of PLP through H-bonding in both native and substrate-bound DDC. On the contrary, Thr246 is in close proximity to the oxygen of 3-OH of PLP pyridine ring only in the substrate-bound DDC. In the ligand-free enzyme, the distance between the oxygen atom of 3-OH group of PLP pyridine ring and oxygen atom of Thr246 hydroxyl group is not favorable for hydrogen bonding. Thus, present study reveals that hydrogen bonding with O3 of PLP with a hydrogen bond donor residue provided by the enzyme plays an important role in the decarboxylation process.

Generalized Convulsions Caused by Overconsumption of Ginkgo Nuts in 6 Year-old Male / 대한소아신경학회지

The ginkgo nuts are commonly eaten because of its nutritive value. It also taken as an herbal medicine for its antitussive and expectorant properties throughout Korea, Japan, and China. A range of symptoms of ginkgo nut intoxication including vomiting, abdominal pain, irritability, headaches, and seizures have been reported. It can be fetal in severe cases if emergency care was not available. It was discovered that the toxicity of ginkgo nuts was due to 4-O-methoxypyridoxine. The convulsion can be prevented by administration of anticonvulsants and pyridoxal phosphate. We report a 6-year-old male, without any past or family histories of epilepsy, who presented with generalized tonic clonic seizures. About 7 hours before the seizure, he had consumed approximately 40 roasted ginkgo nuts. He exhibited vomiting and abdominal pain 5 hours after ingestion. After treatment with diazepam and pyridoxal phosphate, his symptoms were resolved and electroencephalogram was normalized. We report on the patient and emphasize pediatricians and parents who have children should be recognizing that neurotoxicity of ginkgo nuts, particularity convulsion inducing effect, can occur occasionally.

Antidipsogenic effects of central adenosine-5'-triphosphate

Besides other physiological functions, adenosine-5'-triphosphate (ATP) is also a neurotransmitter that acts on purinergic receptors. In spite of the presence of purinergic receptors in forebrain areas involved with fluid-electrolyte balance, the effect of ATP on water intake has not been investigated. Therefore, we studied the effects of intracerebroventricular (icv) injections of ATP (100, 200 and 300 nmol/µL) alone or combined with DPCPX or PPADS (P1 and P2 purinergic antagonists, respectively, 25 nmol/µL) on water intake induced by water deprivation. In addition, the effect of icv ATP was also tested on water intake induced by intragastric load of 12 percent NaCl (2 mL/rat), acute treatment with the diuretic/natriuretic furosemide (20 mg/kg), icv angiotensin II (50 ng/µL) or icv carbachol (a cholinergic agonist, 4 nmol/µL), on sodium depletion-induced 1.8 percent NaCl intake, and on food intake induced by food deprivation. Male Holtzman rats (280-320 g, N = 7-11) had cannulas implanted into the lateral ventricle. Icv ATP (300 nmol/µL) reduced water intake induced by water deprivation (13.1 ± 1.9 vs saline: 19.0 ± 1.4 mL/2 h; P < 0.05), an effect blocked by pre-treatment with PPADS, but not DPCPX. Icv ATP also reduced water intake induced by NaCl intragastric load (5.6 ± 0.9 vs saline: 10.3 ± 1.4 mL/2 h; P < 0.05), acute furosemide treatment (0.5 ± 0.2 vs saline: 2.3 ± 0.6 mL/15 min; P < 0.05), and icv angiotensin II (2.2 ± 0.8 vs saline: 10.4 ± 2.0 mL/2 h; P < 0.05), without changing icv carbachol-induced water intake, sodium depletion-induced 1.8 percent NaCl intake and food deprivation-induced food intake. These data suggest that central ATP, acting on purinergic P2 receptors, reduces water intake induced by intracellular and extracellular dehydration.

Folic acid and vitamin B[6] deficiencies related hyperhomocysteinemia in apparently healthy Pakistani adults; is mass micronutrient supplementation indicated in this population?

To determine the plasma/serum levels of homocysteine, and vitamins folate, B6 and B12, in Pakistani healthy adults. Cross-sectional study. The Aga Khan University, from October 2006 to April 2008. Fasting levels of plasma/serum folic acid, pyridoxal phosphate [PLP], vitamin B12 and homocysteine were determined in 290 apparently healthy hospital personnel from institutions in two cities of Pakistan. Spearman correlation test and linear regression analysis was conducted. There were 219 males and 71 females with mean age of 46 +/- 10.5 years and mean body mass index of 23.5 +/- 3.8. Mean plasma homocysteine levels in Pakistani normal adults were found to be 17.95 +/- 8.4 mol/l. Mean concentrations of plasma/serum folate, vitamin B12 and PLP were found to be 5 +/- 3.9 ng/ml, 522 +/- 296 pg/ml and 21.6 +/- 14 nmol/l, respectively. Serum/plasma levels of folate, vitamin B12 and PLP were negatively correlated with plasma homocysteine [rho coefficient=-0.367, p < 0.001; -0.173, p=0.004; -0.185, p=0.002, respectively]. Serum folate and plasma PLP levels were inversely related with plasma homocysteine, adjusted for gender, age, smoking and body mass index [p < 0.001 and p=0.003, respectively]. Percent deficiency values of folate, vitamin B6 and vitamin B12 were 39.7%, 52.8% and 6.6% respectively. The high levels of plasma homocysteine could indicate a reason for mass micronutrient supplementation to prevent the high incidence of cardiovascular disease observed in Pakistani population

On the modified process of human hemoglobin based blood substitutes / 生物医学工程学杂志

Purified hemoglobin was modified with pyridoxal 5-phosphate(PLP) and polymerized with glutaric dialdehyde(GDA) to get the products. By comparison of the physical, chemical and biological properties of different procedures for modification before and after polymerization, there is no significant difference in molecular distribution, methemoglobin(MetHb) concentration, oxygen carrier capacity, P50 and spectra. Furthermore, the procedure of modification after polymerization can save PLP greatly and decrease cost greatly. So the procedure of modification after polymerization is a better way in research and production. The addition of GDA could control the increasing of MetHb. By comparison on the physical, chemical and biological properties of different procedures, there is no significant difference in molecular distribution, MetHb concentration, oxygen carrier capacity and spectra between the procedure of adding GDA before PLP and that after PLP. But the P50 of adding GDA before PLP is much lower than that after PLP. So the procedure of adding GDA after PLP is a better way.

Study of polymerizing hemoglobin on cation exchange chromatography / 生物医学工程学杂志

Poly-hemoglobin is the active component of hemoglobin-based blood substitutes. The excess reactivity of glutaraldehyde with hemoglobin in solution leads to poly-hemoglobin of a wide molecular weight distribution and a high average molecular weight. A new polymerization method has been tested to decrease the molecular weight distribution and the average molecular weight. The poly-hemoglobin with lower degree of modification (polymerization) was found enriched on the cation exchange columns and further polymerized with glutaraldehyde. The poly-hemoglobin of narrower molecular weight distribution has been prepared in this way.

ATP released from beta-amyloid-stimulated microglia induces reactive oxygen species production in an autocrine fashion

Present study demonstrated that fibrillar beta-amyloid peptide (fAbeta(1-42)) induced ATP release, which in turn activated NADPH oxidase via the P2X(7) receptor (P2X(7)R). Reactive oxygen species (ROS) production in fAbeta(1-42)-treated microglia appeared to require Ca2+ influx from extracellular sources, because ROS generation was abolished to control levels in the absence of extracellular Ca2+. Considering previous observation of superoxide generation by Ca2+ influx through P2X(7)R in microglia, we hypothesized that ROS production in fAbeta-stimulated microglia might be mediated by ATP released from the microglia. We therefore examined whether fAbeta(1-42)-induced Ca2+ influx was mediated through P2X(7)R activation. In serial experiments, we found that microglial pretreatment with the P2X(7)R antagonists Pyridoxal-phosphate-6-azophenyl-2',4'- disulfonate (100 micrometer) or oxidized ATP (100 micrometer) inhibited fAbeta-induced Ca2+ influx and reduced ROS generation to basal levels. Furthermore, ATP efflux from fAbeta(1-42)-stimulated microglia was observed, and apyrase treatment decreased the generation of ROS. These findings provide conclusive evidence that fAbeta-stimulated ROS generation in microglial cells is regulated by ATP released from the microglia in an autocrine manner.

role of pyridoxal-5'-phosphate with simple sugars on cardiovascular diseases prediction in rats

The objective of the present study is to assess the effect of different dietary simple sugars [sucrose, glucose and fructose] on the prediction of cardiovascular disease [CVD] risk factors, with regard to supplementation with high dose of vitamin B6 [500 mg/100 g diet] in the form of pyridoxal-5'-phosphate [PLP]. It was concluded that, long term consumption of high carbohydrate diets increase the prediction of CVD by increasing plasma TG, total cholesterol and LDL-C levels. Dietary glucose was the best sugar used for substitution of starch, while fructose is considered as a risk factor for CVD. Plasma albumin showed little association with CVD. Pyridoxal-5'-phosphate and glucose in combination act in a synergistic manner through highly significant reduction in plasma LDL-C and significantly increase HDL-C levels throughout the first and second periods of the experiment. Supplementation with pyridoxal-5'-phosphate did not affect the plasma glucose levels on 21 days, but improves and normalizes the platelets count, and reduces the elevated TG levels via several mechanisms, and hence, reduces the risk factors leading to CVD

Red cell aspartate aminotransferase saturation with oral pyridoxine intake

CONTEXTO E OBJETIVO: A enzima aspartato aminotransferase apresenta o piridoxal fosfato como coenzima, oriunda da piridoxina existente em alimentos vegetais frescos. A anemia sideroblástica responsiva à vitamina B6, mielofibrose e síndrome de Peyronie respondem a altas doses de piridoxina. O objetivo foi investigar a máxima resposta da aspartato aminotransferase à suplementação oral com piridoxina. TIPO DE ESTUDO E LOCAL: Experimento controlado, na Seção de Hematologia, Instituto Adolfo Lutz. MÉTODOS: A atividade da aspartato aminotransferase eritrocitária foi determinada (antes e após) em voluntários que receberam suplementação por 15-18 dias (30 mg, 100 mg e 200 mg diariamente). Estudo in vitro também foi realizado, com sangue de sete indivíduos. As atividades enzimáticas antes e após a incubação foram determinadas, seguindo o mesmo protocolo do estudo in vivo. RESULTADOS: O estudo in vivo revelou um aumento gradativo da saturação da aspartato aminotransferase com doses crescentes de piridoxina. 83% de saturação foi alcançada com 30 mg diariamente, 88% com 100 mg e 93% com 200 mg. O estudo in vitro não revelou saturação de 100%.CONCLUSÕES: Tanto in vivo quanto in vitro, não se revelou saturação completa da aspartato aminotransferase por sua coenzima piridoxal-5-fosfato nos eritrócitos. Entretanto, a dose de 200 mg diariamente poderia ser empregada com segurança no tratamento da anemia sideroblástica, mielofibrose e síndrome de Peyronie. Embora a saturação máxima nos eritrócitos não seja atingida, os eritroblastos e outras células nucleadas que contenham as organelas citoplasmáticas certamente atingirão a saturação completa, possivelmente à razão dos resultados obtidos nas doenças citadas.

Gadobenate Dimeglumine as an Intrabiliary Contrast Agent: Comparison with Mangafodipir Trisodium with Respect to Non-dilated Biliary Tree Depiction

OBJECTIVE: To compare the efficacy of Mangafodipir trisodium (Mn-DPDP) -enhanced MR cholangiogrphy (MRC) and Gadobenate dimeglumine (Gd-BOPTA) -enhanced MRC in visualizing a non-dilated biliary system. MATERIALS AND METHODS: Eighty-eight healthy liver donor candidates underwent contrast-enhanced T1-weighted MRC. Mn-DPDP and Gd-BOPTA was used in 36 and 52 patients, respectively. Two radiologists reviewed the MR images and rated the visualization of the common duct, the right and left hepatic ducts, and the second-order branches using a 4-point scale. The contrast-to-noise ratio (CNR) of the common duct to the liver in the two groups was also compared. RESULTS: Mn-DPDP MRC and Gd-BOPTA MRC both showed similar visualization grades in the common duct (p = .380, Mann-Whitney U test). In the case of the proximal bile ducts, the median visualization grade was significantly higher with Gd-BOPTA MRC than with Mn-DPDP MRC (right hepatic duct: p = 0.016, left hepatic duct: p = 0.014, right secondary order branches: p = 0.006, left secondary order branches, p = 0.003). The common duct-to-liver CNR of the Gd-BOPTA MRC group was significantly higher (38.90+/-24.50) than that of the Mn-DPDP MRC group (24.14+/-17.98) (p = .003, Student's t test). CONCLUSION: Gd-BOPTA, as a biliary contrast agent, is a potential substitute for Mn-DPDP.

assay for phosphoinositide-specific phospholipase C and the effect of pyridoxal-5-phosphate on the assay

In platelets, the major stimulatory second messengers are inositol 1, 4, 5 triphosphate [IP3] and 1,2-diacyiglycerol [DAG] produced upon the hydrolysis of phosphoinositides by phosphoinositide-speciftc phospholpase C [P1-PLC]. Pyridoxal-5-phosphate [PLP] is well known as a potent inhibitor of platelet aggregation. The mechanism[s] of its inhibitory action remains to be elucidated. P1-PLC was assayed and the effect of PLP on the assay was examined in an attempt to explain the nature of the inhibitory effect of PLP on platelet function. The assay was satisfactory and was inhibited by PLP with full inhibition at 10mMPLP

Effect of Endurance Training on the Plasma Homocysteine and B Vitamin Levels in Male Adolescent Field Hockey Players

Elevated plasma homocysteine is an independent risk factor for the development of cardiovascular disease. Exercise is generally believed to reduce the plasma homocysteine levels and therefore, being beneficial for cardiovascular disease(CVD). However, there is a possibility that athletes undergoing strenuous training and competition which increase oxidative stress may suffer from increased plasma homocysteine levels. The purpose of this study was to investigate the influence of endurance training on the plasma concentrations of B vitamins and homocysteine in 23 male adolescent field hockey players. Data collection and blood sampling was performed during the training period and non-training period. Following the training period, significant changes in energy and vitamin B6 intakes were observed in these subjects. Plasma vitamin B2, pyridoxal phosphate (PLP) and homocysteine levels were significantly higher during the training period than non-training period, whereas no difference was observed in plasma folate and vitamin B12 levels. Positive correlation was observed between plasma folate and folic acid intakes. When energy, B vitamin intakes were adjusted there was a significant negative correlation between plasma homocysteine levels and plasma riboflavin, folate and vitamin B12 levels. In conclusion, it is suggested that athletes with oxidative stress by strenuous exercise may need B vitamins since riboflavin, folic acid and vitamin B12 were shown to be negatively correlated with plasma homocysteine in athletes during the training period.

Role of exocrine cells in pancreatic enhancement using Mn-DPDP-enhanced MR imaging / 中华医学杂志(英文版)

<p><b>OBJECTIVE</b>To investigate role of exocrine cells in the pancreatic enhancement images at Manganese (II) N, N'-dipyridoxylethlenediamine-N, N'-diacetate 5, 5'-bisc (Mn-DPDP)-enhanced magnetic resonance (MR) imaging.</p><p><b>METHODS</b>Artificial pancreatic leakage was constructed in six dogs using a fistula tube inserted into the duodenum papillae. Pancreatic juice was collected before and after intravenous infusion of 2 ml/kg of Mn-DPDP at a rate of 2 - 3 ml/min. The Mn content of pancreatic juice was measured by atomic absorption spectroscopy. T(1)-weighted spin-echo images and T(1)-weighted spoiled phase gradient-echo (SPGR) images were obtained prior and approximately 30 min after the administration of Mn-DPDP at 1.5T.</p><p><b>RESULTS</b>The Mn content of pancreatic secretion increased 60.47 +/- 21.83 micro g/dl after the administration of Mn-DPDP (t = 6.785, P < 0.01). The signal/noise ratio (S/N) of the pancreas increased 53 percent +/- 49 percent and 62 percent +/- 44% on T(1)W spin echo images and SPGR images, respectively.</p><p><b>CONCLUSIONS</b>Exocrine cells of the pancreas can absorb manganese and excrete it through the pancreatic juice. Exocrine cells play an important role in the enhancement of the pancreas in MR imaging with Mn-DPDP.</p>

Vitamin B6 status in children with bronchial asthma

This study was carried out to assess vitamin B6 status in children with bronchial asthma to justify its use as an adjunct to theophylline therapy. It included 30 asthmatics and 20 healthy children as controls. Patients were 17 males and 13 females with ages ranging from 6 to 12 years [median: 10 years]. Diagnosis was based on clinical criteria of asthma, pulmonary function tests and the exclusion of other chest diseases by chest X-ray, complete hemogram and ESR. Patients were divided into two distinct clinical groups: 17 mild and 13 moderate asthmatics, while severe asthmatics and those who received theophylline [or one of its derivatives] four weeks prior to the study were all excluded. Blood samples were taken from all children at diagnosis. Levels of PLP in mild asthmatics did not show any significant difference from controls, while in moderate asthmatics, these levels were significantly lower than the controls. It was concluded that potentially depressed vitamin B6 status in bronchial asthma might be aggravated by theophylline therapy. Vitamin B6 supplementation to asthmatic children treated by theophylline is suggested

Reversible inhibition by pyridoxal 5'-phosphate and irreversible inactivation by urea and guanidine hydrochloride of thymidylate synthase from Lactobacillus leichmannii.

Pyridoxal 5'-phosphate reversibly inhibited thymidylate synthase from Lactobacillus leichmannii. The inhibition was competitive with dUMP (Ki = 1 microM) and non-competitive with 5,10-CH2-THF (Ki = 0.08 microM). Treatment of native or pCMB-treated enzyme with urea (5 M) or guanidine hydrochloride (4 M) resulted in inactivation and dissociation of the homodimer (74 kDa) into monomer (37 kDa).

effect of pyridoxal - 5 - phoaphate on the activity of myokinase enzyme

Pyridoxal-5-phosphate [PLP] inhibits platelet aggregation. It was suggested that PLP may exert its inhibitory effect by binding to ADP receptors. An ADP binding site in another system was considered to provide an analogy from which deductions may be made in regard to ADP binding sites on the platelet surface. The enzyme myokinase was believed to provide a suitably analogous ADP binding site. It has been shown that PLP did not inhibit the activity of myokinase from which it was inferred that PLP is unlikely to exert its inhibitory activity on platelet aggregation by binding to ADP receptor on the platelet surface

Inactivation of a beta-glucosidase from Arthrobotrys conoides by diethyl pyrocarbonate: evidence of histidine at the active site.

Modification of A. conoides beta-glucosidase by diethylpyrocarbonate caused rapid inactivation of the enzyme. The kinetic analyses showed that the inactivation by diethylpyrocarbonate resulted from the modification of an average of one histidine residue per mole of enzyme. The modified enzyme showed an increase in absorbance at 240 nm. Sulphydryl, lysine and tyrosine residues were not modified by diethylpyrocarbonate treatment. The substrate offered significant protection against diethylpyrocarbonates modification. The results indicate that diethylpyrocarbonate was interacting with the enzyme at or near the active site.

Interactions of methoxyamine with pyridoxal-5'-phosphate-Schiff's base at the active site of sheep liver serine hydroxymethyltransferase.

The mechanism of interaction of methoxyamine with sheep liver serine hydroxymethyltransferase (EC 2.1.2.1) (SHMT) was established by measuring changes in enzyme activity, visible absorption spectra, circular dichroism and fluorescence, and by evaluating the rate constant by stopped-flow spectrophotometry. Methoxyamine can be considered as the smallest substituted aminooxy derivative of hydroxylamine. It was a reversible noncompetitive inhibitor (Ki = 25 microM) of SHMT similar to O-amino-D-serine. Like in the interaction of O-amino-D-serine and aminooxyacetic acid, the first step in the reaction was very fast. This was evident by the rapid disappearance of the enzyme-Schiff base absorbance at 425 nm with a rate constant of 1.3 x 10(3) M-1 sec-1 and CD intensity at 430 nm. Concomitantly, there was an increase in absorbance at 388 nm (intermediate I). The next step in the reaction was the unimolecular conversion (1.1 x 10(-3) sec-1) of this intermediate to the final oxime absorbing at 325 nm. The identity of the oxime was established by its characteristic fluorescence emission at 460 nm when excited at 360 nm and by high performance liquid chromatography. These results highlight the specificity in interactions of aminooxy compounds with sheep liver serine hydroxymethyltransferase and that the carboxyl group of the inhibitors enhances the rate of the initial interaction with the enzyme.

Circular dichroism studies of the coenzyme environment in the active sites of mutant forms of the beta-subunit in the tryptophan synthase alpha 2 beta 2 complex.

The circular dichroism has been used to evaluate the effect of mutation on the environment of the pyridoxal phosphate coenzyme in the active site of the beta-subunit in the tryptophan synthase alpha 2 beta 2 complex from Salmonella typhimurium. Seven mutant forms of the alpha 2 beta 2-complex with single amino acid replacements at residues 87, 109, 188, 306, and 350 of the beta-subunit have been prepared by site-directed mutagenesis, purified to homogeneity, and characterized by absorption and circular dichroism spectroscopy. Since the wild type and mutant alpha 2 beta 2 complexes all exhibit positive circular dichroism in the coenzyme absorption band, pyridoxal phosphate must bind asymmetrically in the active site of these enzymes. However, the coenzyme may have an altered orientation or active site environment in five of the mutant enzymes that display less intense ellipticity bands. The mutant enzyme in which lysine 87 is replaced by threonine has very weak ellipticity at 400 nm. Since lysine 87 forms a Schiff base with pyridoxal phosphate in the wild type enzyme, our results demonstrate the importance of the Schiff base linkage for rigid or asymmetric binding. Although the mutant enzymes display spectra in the presence of L-serine that differ from that of the wild type enzyme, addition of alpha-glycerol 3-phosphate converts the spectra of two of the mutant enzymes to that of the wild type enzyme. We conclude that this alpha-subunit ligand may produce a conformational change in the alpha-subunit that is transmitted to the mutant beta-subunits and partially corrects conformational alterations in the mutant enzymes.