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Determination of vitamin B6 vitamers and pyridoxic acid in biological samples

Authors:
Sushil Sharma at American International School of Medicine
Sushil Sharma
Krishnamurti Dakshinamurti at University of Manitoba
Krishnamurti Dakshinamurti
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Abstract

For the determination of vitamin B6 vitamers (pyridoxal phosphate, pyridoxamine phosphate, pyridoxal, pyridoxine, pyridoxamine) and 4-pyridoxic acid in biological samples such as plasma, cerebrospinal fluid and rat brain regions, a sensitive micromethod using high-performance liquid chromatography (HPLC) with fluorescence detection in combination with post-column derivatization is described. Metaphosphoric acid tissue extracts with deoxypyridoxine as an internal standard were injected into the HPLC system with a binary gradient elution at a flow-rate of 1.2 ml/min. The excitation wavelength of the fluorescence detector was set at 328 nm and the emission wavelength at 393 nm with a 15-nm slit width for the photocell. This method allows the assay of vitamin B6 vitamers within 30 min in one chromatographic run. The present method has been applied extensively for the measurement of vitamin B6 vitamer levels in discrete brain regions of small animals, cells in culture and biopsy samples.
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Abstract
For the determination of vitamin B6 vitamers (pyridoxal phosphate, pyridoxamine
phosphate, pyridoxal, pyridoxine, pyridoxamine) and 4-pyridoxic acid in biological
samples such as plasma, cerebrospinal fluid and rat brain regions, a sensitive
micromethod using high-performance liquid chromatography (HPLC) with
fluorescence detection in combination with post-column derivatization is described.
Metaphosphoric acid tissue extracts with deoxypyridoxine as an internal standard were
injected into the HPLC system with a binary gradient elution at a flow-rate of 1.2
ml/min. The excitation wavelength of the fluorescence detector was set at 328 nm and
the emission wavelength at 393 nm with a 15-nm slit width for the photocell. This
method allows the assay of vitamin B6 vitamers within 30 min in one chromatographic
run. The present method has been applied extensively for the measurement of vitamin
B6 vitamer levels in discrete brain regions of small animals, cells in culture and biopsy
samples.
... The most frequently used method for analysis of TDP and PLP is a HPLC-based assay for each vitamin separately [11][12][13][14][15][16][17][18][19][20][21][22][23][24][25]. Indeed, the currently used HPLC-based methods in our laboratory for measuring TDP and PLP consist of two separate HPLC methods. ...
... Sample preparation may introduce human errors which are not noticed due to lack of proper internal standards in the HPLC methods. Moreover, sample preparation is time consuming [11][12][13][14][15][16][17][18][19][20][21][22][23][24][25]. ...
Simultaneous Determination of Underivatized Vitamin B1 and B6 in Whole Blood by Reversed Phase Ultra High Performance Liquid Chromatography Tandem Mass Spectrometry
Article
Full-text available
Background: Vitamin B1 (thiamine-diphosphate) and B6 (pyridoxal-5'phosphate) are micronutrients. Analysis of these micronutrients is important to diagnose potential deficiency which often occurs in elderly people due to malnutrition, in severe alcoholism and in gastrointestinal compromise due to bypass surgery or disease. Existing High Performance Liquid Chromatography (HPLC) based methods include the need for derivatization and long analysis time. We developed an Ultra High Performance Liquid Chromatography Tandem Mass spectrometry (UHPLC-MS/MS) assay with internal standards for simultaneous measurement of underivatized thiamine-diphosphate and pyridoxal-5'phosphate without use of ion pairing reagent. Methods: Whole blood, deproteinized with perchloric acid, containing deuterium labelled internal standards thiamine-diphosphate(thiazole-methyl-D3) and pyridoxal-5'phosphate(methyl-D3), was analyzed by UHPLC-MS/MS. The method was validated for imprecision, linearity, recovery and limit of quantification. Alternate (quantitative) method comparisons of the new versus currently used routine HPLC methods were established with Deming regression. Results: Thiamine-diphosphate and pyridoxal-5'phosphate were measured within 2.5 minutes instrumental run time. Limits of detection were 2.8 nmol/L and 7.8 nmol/L for thiamine-diphosphate and pyridoxal-5'phosphate respectively. Limit of quantification was 9.4 nmol/L for thiamine-diphosphate and 25.9 nmol/L for pyridoxal-5'phosphate. The total imprecision ranged from 3.5-7.7% for thiamine-diphosphate (44-157 nmol/L) and 6.0-10.4% for pyridoxal-5'phosphate (30-130 nmol/L). Extraction recoveries were 101-102% ± 2.5% (thiamine-diphosphate) and 98-100% ± 5% (pyridoxal-5'phosphate). Deming regression yielded slopes of 0.926 and 0.990 in patient samples (n = 282) and national proficiency testing samples (n = 12) respectively, intercepts of +3.5 and +3 for thiamine-diphosphate (n = 282 and n = 12) and slopes of 1.04 and 0.84, intercepts of -2.9 and +20 for pyridoxal-5'phosphate (n = 376 and n = 12). Conclusion: The described UHPLC-MS/MS method allows simultaneous determination of underivatized thiamine-diphosphate and pyridoxal-5'phosphate in whole blood without intensive sample preparation.
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... The most frequently used method for analysis of TDP and PLP is a HPLC-based assay for each vitamin separately [11][12][13][14][15][16][17][18][19][20][21][22][23][24][25]. Indeed, the currently used HPLC-based methods in our laboratory for measuring TDP and PLP consist of two separate HPLC methods. ...
... Sample preparation may introduce human errors which are not noticed due to lack of proper internal standards in the HPLC methods. Moreover, sample preparation is time consuming [11][12][13][14][15][16][17][18][19][20][21][22][23][24][25]. ...
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... These activities were measured by spectrophotometry using paraoxon and phenylacetate, respectively, as substrates [28]. Vitamin B 6 in serum was quantified using high performance liquid chromatography with ultraviolet detection and isocratic elution [29]. Serum levels of vitamin B 12 and folate were measured by chemiluminescent immunoassay with paramagnetic particles using the Access Immunoassay System (Beckman Coulter Inc., Brea, California, USA). ...
Sleepiness, inflammation and oxidative stress markers in middle-aged males with obstructive sleep apnea without metabolic syndrome: A cross-sectional study
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  • Jan 2015
Background The simultaneous occurrence of metabolic syndrome and excessive daytime sleepiness are very common in obstructive sleep apnea (OSA) patients. Both conditions, if present in OSA, have been reported to be associated with inflammation and disruption of oxidative stress balance that impair the cardiovascular system. To verify the impact of daytime sleepiness on inflammatory and oxidative stress markers, we evaluated OSA patients without significant metabolic disturbance.Methods Thirty-five male subjects without diagnostic criteria for metabolic syndrome (Adult Treatment Panel III) were distributed into a control group (n¿=¿10) (43¿±¿10.56 years, apnea-hypopnea index - AHI 2.71¿±¿1.48/hour), a non-sleepy OSA group (n¿=¿11) (42.36¿±¿9.48 years, AHI 29.48¿±¿22.83/hour) and a sleepy OSA group (n¿=¿14) (45.43¿±¿10.06 years, AHI 38.20¿±¿25.54/hour). Excessive daytime sleepiness was considered when Epworth sleepiness scale score was¿¿¿10. Levels of high-sensitivity C-reactive protein, homocysteine and cysteine, and paraoxonase-1 activity and arylesterase activity of paraoxonase-1 were evaluated.ResultsPatients with OSA and excessive daytime sleepiness presented increased high-sensitivity C-reactive protein levels even after controlling for confounders. No significant differences were found among the groups in paraoxonase-1 activity nor arylesterase activity of paraoxonase-1. AHI was independently associated and excessive daytime sleepiness tended to have an association with high-sensitivity C-reactive protein.Conclusions In the absence of metabolic syndrome, increased inflammatory response was associated with AHI and daytime sleepiness, while OSA was not associated with abnormalities in oxidative stress markers.
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Vitamin B6
Chapter
  • Apr 2000
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Effect of gene polymorphisms on the concentration of serum folate, plasma total homocysteine and serum nitrate in healthy subjects after folic acid supplementation
Thesis
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  • Jun 2018
Background: Folic acid supplementation can increase the concentration of serum folate and decrease the concentration of plasma total homocysteine (p-tHcy), which is considered a risk factor for cardiovascular diseases. The aim of this study was to investigate whether genetic polymorphisms involved in folate metabolism and endothelial nitric oxide synthase (eNOS) affect the concentration of serum folate, plasma total homocysteine and serum nitrate in healthy subjects after folic acid supplementation. Method: It was a randomized, double blind, cross over study. Half of the participants were given folic acid 800 µg/day and the other half of the participants received placebo for 2 weeks. Results: The polymorphisms in folate gene and eNOS gene had a significant increase in the concentration of serum folate and decrease in p-tHcy after folic acid supplementation. However, these gene polymorphisms did not affect the concentration of serum nitrate. Conclusion: Polymorphisms in folate gene and eNOS gene affect the concentration of serum folate and p-tHcy but do not have any effect on the concentration of serum nitrate in healthy individuals after folic acid supplementation.
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Vitamin B6: Effects of Deficiency, and Metabolic and Therapeutic Functions
Chapter
  • Jan 2017
The vitamin B6 vitamers include pyridoxine, pyridoxal, and pyridoxamine, as well as their phosphorylated forms such as pyridoxal phosphate , which is a key coenzyme for a surprising variety of enzymes involved in myriad aspects of metabolism. Vitamin B6 also contributes to the synthesis of many neurotransmitters. Given this widespread role, it is not surprising that vitamin B6 deficiency can induce many negative effects including convulsive seizures in infants, developmental delay, hypertension, and susceptibility to atherosclerosis. Conversely, the administration of vitamin B6 vitamers, or the manipulation of vitamer-bound enzymes, has shown promise against cancer, parasitic diseases such as malaria, and Parkinson’s disease. In this chapter, we examine the critical and broad role played by vitamin B6 vitamers and their coenzymes in metabolism, with a focus on the detrimental effects of deficiency and their therapeutic potential.
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Evaluation of timing and rEsponsEs to physiothErapEutic trEatmEnt
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  • Jan 2008
In several treatment regimens, the recognition of chronobiology contributes to the therapeutic process through the effective use of temporization protocols. The purpose of the present study was to evaluate the relationship between the response to physiotherapeutic treatment and the time of day when such treatment was performed, as well as the chronotype of orthopedic and rheumatologic patients in a clinical physiotherapy school. The population studied was treated in the morning and evening periods. The patients were divided into three groups of pathologies with similar treatments, which were as follows: syndrome of shoulder impact (n=33), knee artrosis (n=17), and lombalgia (n=23). At the end of ten treatment sessions, data concerning pain, percentage of subjective improvement, chronotype, and age were compared. At the end of the study, it was observed that the time of day when treatment was performed infuenced the results of individuals treated in the evenings but had no infuence on the individuals treated in the morning. In addition, the evening schedule was the most well suited for intermediate individuals.
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Analysis of vitamin B6 derivatives in biological samples with high performance liquid chromatography
Article
  • Apr 2004
  • CHEM J CHINESE U
For the determination of vitamin B-6 derivatives, a high performance liquid chromatography (HPLC) system composed of a single reversed-phase octadecylsilane column and fluorescence detector with an isocratic elution was proposed. This HPLC system perfectly analyzed pyridoxine, pyridoxal, pyridoxamine, pyridoxine 5'-phosphate and pyridoxamine 5'-phosphate together with pyridoxic acid in biological samples. In this method, relatively low fluorescence intensity of pyridoxal 5'-phasphate was improved by converting it to pyridoxic 5'-phosphate with KCN. The analytical procedure is very simple and economical, it is a useful tool for studying vitamin B-6 in animal body.
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A multicenter comparison of whole blood vitamin B6 assays
Article
Full-text available
  • Oct 2015
Background: The aim of this study was to compare different analytical methods that are currently in use in the Netherlands for the measurement of whole blood vitamin B6. Methods: This method comparison study consisted of two separate parts. (1) Four laboratories participated in a pilot study in which the commercial Chromsystems and INstruchemie method, and a laboratory developed liquid chromatography-tandem mass spectrometry (LC-MS/MS) method and HPLC method were compared. Sixty-nine frozen whole blood samples and six lyophilized whole blood samples were used for comparison. (2) In the nationwide part of the study, 49 laboratories participated in the analysis of three identical sets of two whole blood samples of which one set was freshly analyzed, one set was analyzed after storage at -20 °C and one set was analyzed after lyophilization. Results: In both parts of the study, the HPLC and LC-MS/MS methods showed equivalent results for all sample types tested. The Chromsystems method showed a positive bias of 45% (pilot study) and 30% (nationwide study) towards the LC-MS/MS method when fresh or frozen samples were used. The measurement of lyophilized samples showed no differences between the methods. The results of the INstruchemie method were inconclusive due to the low number of participants. Conclusions: The different analytical methods for measuring vitamin B6 produce different results when whole blood patient samples are measured. The recognition of a reference method or the development of suitable reference materials and quality control materials might serve as a first step towards improved standardization or harmonization of the whole blood vitamin B6 assay.
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Recommendations for Status Assessment of Vitamin B-6
Chapter
  • Jan 1981
Since the discovery of vitamin B-6 and the subsequent elucidation of the various roles that vitamin B-6 plays in the body, numerous methods for assessing vitamin B-6 status have been developed. The previous paper points out the available methods and future developments in methodology. A number of direct and indirect methods have been utilized. Just as there are a variety of methods available for determining a specific vitamin B-6 metabolite, so there are an equal variety of approaches to assessing vitamin B-6 status. Both the discussions following each of the papers presented at this Workshop and the follow-up indepth discussion of the papers dealing with status assessment pointed out the importance of recognizing that there is not one “best” method for status assessment./Also, as stressed in the discussion session of the Workshop, it is one thing to develop good methodology; it's another matter to put them into real practical use. The utilization of the methods for status assessment can be applied to studies of healthy individuals, evaluation of individuals with specific pathological conditions, and population studies. However, the choice of the method to be used must take into account the particular situation being studied. The use of urinary vitamin B-6 metabolite excretion in a large population study may be more advantageous than collection of blood samples.
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Determination of vitamin B6 in foods and other biological materials by paired-ion high-performance liquid chomatography
Article
  • May 1985
A paired-ion reverse-phase high-performance liquid chromatographic (HPLC) method was developed for the determination of the individual B-6 vitamers in foods and other biological materials. Samples were extracted with sulfosalicyclic acid and extracts purified by preparative anion exchange chromatography. The analytical separation of the B-6 vitamers and the internal standard 4′-deoxypyridoxine was achieved by gradient elution with an octadecylsilyl column within 23 min. The use of a post-column bisulfite reagent permitted sensitive fluorometric detection of all B-6 vitamers. High recovery and precision were obtained. The results compared favorably with previously reported data for a variety of samples. This procedure provides a convenient alternative to previous methods for the determination of vitamin B-6.
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Analysis of B-6 vitamers and pyridoxic acid in plasma, tissues and urine using high performance liquid chromatography
Article
  • Mar 1989
  • NUTR RES
We developed an HPLC (high performance liquid chromatography) method for analysis of vitamin B-6 metabolites, including the excretory form, that is suitable for plasma, tissues and urine. The method is based on modifications of the reverse-phase ion-pairing procedure of Gregory and Feldstein (1), and can be used to separate pyridoxal phosphate, pyridoxamine phosphate, pyridoxal, pyridoxine, pyridoxamine and pyridoxic acid in samples from laboratory animals and humans. Samples were extracted with metaphosphoric acid and analyzed using binary linear gradient elution to separate B-6 vitamers. Recoveries were near 100% with analytical precision of 7% or less for all vitamers. Detection limits ranged from 0.5–2.0 pmol (0.1–0.4 ng)/injection, which will permit accurate analysis of samples that contain low concentrations of B-6 vitamers, such as plasma from humans and animals in marginal or deficient B-6 nutritional status. Vitamer concentrations in rat liver obtained with this method agreed closely with values obtained with the established HPLC method of Coburn and Mahuren (2) for vitamin B-6.
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Pyridoxine status as assessed by the concentration of B6-aldehyde vitamers
Article
  • Feb 1989
Forty-five male Lohmann chicks were randomly assigned to an adequate (30 mumol PN/kg) or a B6-deficient diet. Chicks were grown to 6 weeks of age and the vitamin B6 status was assessed according to the level of B6-aldehyde vitamers in plasma and erythrocytes. Chicks fed a limited amount of pyridoxine showed no nervous signs, but significant metabolic changes. It was found that PL was the major metabolite in plasma and only a trace of PLP was detected, suggesting a different metabolic pathway from those of other animal species or healthy humans. This particular metabolism parallels an elevated ALP concentration. The measurement of plasma PLP level routinely used for the assessment of vitamin B6 status in humans is misleading in a situation with raised ALP.
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Assay of pyridoxal-5'-phosphate, pyridoxal and pyridoxic acid in biological material
Article
  • Feb 1989
The two vitamin B6-vitamers having an aldehyde function are oxidised to the corresponding acids and subjected to an HPLC separation on an RP 18 phase with a solvent consisting of 5% methanol in phosphate buffer at pH 3.5. The detection is carried out by fluorometry with excitation at 318 nm and emission at 418 nm. The peaks obtained correspond to pyridoxic acid 5'-phosphate and pyridoxic acid. Pyridoxal-5'-phosphate is determined as pyridoxic acid 5'-phosphate. Pyridoxal is determined as pyridoxic acid by subtracting the amount of pyridoxic acid already existing before oxidation.
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Determination of Vitamin B6 Derivatives in Foods and Biological Materials by Reversed-phase HPLC
Article
  • Jul 1989
Through use of a simplified analyzing system, seven vitamin B6 derivatives were determined with a satisfactory sensitivity and precision. This system consisted of a single reversed-phase ODS column with a fluorescence detector employing an isocratic solvent system. Each vitamin B6 derivative in some foods and biological materials was determined, based on the measurement of the integrated peak area. The data obtained by this method were compared with those obtained from a bioassay by Saccharomyces uvarum ATCC 9080, after acid hydrolysis of these materials.
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Plasma B6 vitamer and 4-pyridoxic acid concentrations of men fed controlled diets
Article
  • Jul 1988
  • J CHROMATOGR A
A rapid and sensitive procedure is described for the analysis of all the B6 vitamers and 4-pyridoxic acid in human plasma utilizing reversed-phase high-performance liquid chromatography with ultraviolet and fluorometric detection. Pyridoxal phosphate values obtained by radiometric and chromatographic, ultraviolet and fluorometric, assays were highly correlated as were pyridoxine phosphate values determined using both detectors. Plasma B6 vitamer and 4-pyridoxic acid concentrations of 22 men fed diets containing 0.75-0.98 mg of vitamin B6 daily for eight weeks were in the range of reported values; pyridoxal phosphate was their predominant plasma B6 vitamer. This methodology should be useful in the assessment of vitamin B6 requirements.
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