Pyridoxamine Dihydrochloride Research Articles

A sensitive UPLC-MS/MS method for simultaneous quantification of one-carbon metabolites & co-factors in human plasma

One-carbon metabolism is an important metabolic pathway involved in many diseases, such as congenital malformations, tumours, cardiovascular diseases, anaemia, depression, cognitive diseases and liver disease. However, the current methods have specific defects in detecting and qualifying the related compounds of one-carbon metabolism. In this study, a validated method was established to simultaneously quantify 22 one-carbon metabolites & co-factors in human plasma and applied to the study of correlation between one-carbon metabolism and colorectal cancer in human plasma samples, which were from 44 healthy subjects and 55 colorectal cancer patients. The method used ultra-high performance liquid chromatography coupled with triple quadrupole mass spectrometry (UPLC-MS/MS), and the analytes included betaine, L-carnitine, L-cystathionine, L-cysteine, dimethylglycine, DL-homocysteic acid, homocysteine, methionine, pyridoxal hydrochloride, pyridoxamine dihydrochloride, pyridoxine dihydrochloride, S-(5′-Adenosyl)-L-homocysteine, serine, choline chloride, folic acid, glycine, pyridoxal phosphate monohydrate, riboflavin, taurine, 5-methyltetrahydrofolate, S-(5′-adenosyl)-L-methionine disulfate salt, trimethylamine oxide. The developed method was successfully applied to the quantification of 22 one-carbon metabolites & co-factors in human plasma from colorectal cancer patients and healthy individuals. The plasma concentrations of dimethylglycine was significantly decreased in the patients compared with the healthy individuals, while L-cystathionine was increased.

Determination of Vitamin B3 Vitamer (Nicotinamide) and Vitamin B6 Vitamers in Human Hair Using LC-MS/MS.

Water-soluble B vitamins participate in numerous crucial metabolic reactions and are critical for maintaining our health. Vitamin B deficiencies cause many different types of diseases, such as dementia, anaemia, cardiovascular disease, neural tube defects, Crohn’s disease, celiac disease, and HIV. Vitamin B3 deficiency is linked to pellagra and cancer, while niacin (or nicotinic acid) lowers low-density lipoprotein (LDL) and triglycerides in the blood and increases high-density lipoprotein (HDL). A highly sensitive and robust liquid chromatography–tandem mass spectroscopy (LC/MS-MS) method was developed to detect and quantify a vitamin B3 vitamer (nicotinamide) and vitamin B6 vitamers (pyridoxial 5′-phosphate (PLP), pyridoxal hydrochloride (PL), pyridoxamine dihydrochloride (PM), pridoxamine-5′-phosphate (PMP), and pyridoxine hydrochloride (PN)) in human hair samples of the UAE population. Forty students’ volunteers took part in the study and donated their hair samples. The analytes were extracted and then separated using a reversed-phase Poroshell EC-C18 column, eluted using two mobile phases, and quantified using LC/MS-MS system. The method was validated in human hair using parameters such as linearity, intra- and inter-day accuracy, and precision and recovery. The method was then used to detect vitamin B3 and B6 vitamers in the human hair samples. Of all the vitamin B3 and B6 vitamers tested, only nicotinamide was detected and quantified in human hair. Of the 40 samples analysed, 12 were in the range 100–200 pg/mg, 15 in the range 200–500 pg/mg, 9 in the range of 500–4000 pg/mg. The LC/MS-MS method is effective, sensitive, and robust for the detection of vitamin B3 and its vitamer nicotinamide in human hair samples. This developed hair test can be used in clinical examination to complement blood and urine tests for the long-term deficiency, detection, and quantification of nicotinamide.

Analytical Method Development and Validation of Rp-Hplc Method for Simultaneous Estimation of Pyridoxamine Dihydrochloride and Acetylcysteine in Tablet Dosage Form.

A reverse-phase high-performance liquid chromatography (RP-HPLC) method for the simultaneous estimation of Pyridoxamine dihydrochloride and Acetylcysteine in the marketed formulation is developed. Chromatography carried out at 30oc temperature on Agilent Zorbax Bonus-RP (250 x 4.6 mm, 5 µ) coloum. Coloum using a mobile phase 0.1% trifluroacetic acid in water: acetonitrile (80:20v/v) with flow rate 1ml/min (DAD scan at 210nm). Validation parameters such as system suitability, linearity, precision, accuracy are considered as reported International Conference on Harmonization guidelines. The retention times for Pyridoxamine dihydrochloride and Acetylcysteine are 2 min and 3.4 min. The linearity range for Pyridoxamine dihydrochloride and Acetylcysteine is 30-70 µg/ml and 180-420 µg/ml. The %RSD for accuracy was found to be less than 2%. Hence the proposed method was found to be accurate, precise, reproducible, and specific and can be used for simultaneous analysis of these drugs in tablet formulation.

The Effects of Metformin, Aminoguanidine, and Pyridoxamine on Methylglyoxal Induced Cardiac Myocytes Injury

In diabetics, formation of advanced glycation end (AGE) products has been closely associated with cardiovascular complications. In order to mitigate the injury caused by AGEs, it is critical to understand the influence of the AGE precursor methylglyoxal. Methylglyoxal is a glycation intermediate and it has been found to be elevated in the blood of diabetic patients. The impact of methylglyoxal on cardiac cells is not well elucidated. In this study, the effects of methylglyoxal on H9C2 myoblast cell viability were evaluated. Thereafter, methylglyoxal induced cell injury was investigated by co‐treatment of methylglyoxal with metformin, aminoguanidine hydrochloride, or pyridoxamine dihydrochloride. Cell viability was evaluated after incubation of drugs for 24 hours by measuring absorbance at 450 nm by using tetrazolium to differentiate metabolically active and inactive cells (e.g., CCK‐8 kit). We found that methylglyoxal (1200 μM) significantly reduced cell viability by 72±6% when compared to the untreated control (n=4, p<0.05). By contrast, co‐treatment of metformin (1–80 mM) significantly reduced methylglyoxal‐induced cell injury and increased cell viability to 69% – 122± 6% (n=4, p<0.05 vs. methylglyoxal) when compared to the untreated control. Similarly, aminoguanidine hydrochloride (250–1000 μM) treated cells significantly increased cell viability to 77% – 109±10% (n=4, p<0.05 vs. methylglyoxal) when compared to the untreated control. Additionally, pyridoxamine dihydrochloride (0.1–15 μM, n=4) showed dose‐dependent protection to increase cell viability. Highest pyridoxamine (15 μM) treated cells completely abolished methylglyoxal induced cell injury by increasing cell viability to 99± 4% (p<0.05 vs. methylglyoxal) when compared to the untreated control. However, when the three drugs were washed out from the medium prior to the addition of the methylglyoxal, the protective effects were lost and cell viability was reduced similarly as methylglyoxal (n=1). Administration of all three drugs alone did not affect cell viability after incubation for 24 hours. The preliminary data suggests that metformin, aminoguanidine hydrochloride, or pyridoxamine dihydrochloride, when incubated concurrently with a high concentration of methylglyoxal, protect cardiac cells from methylglyoxal‐induced cell injury. The mechanism underlying the protective effects will be further investigated.Support or Funding InformationThis study was supported by the Center for Chronic Disorders of Aging, the Division of Research and the Department of Bio‐Medical Sciences at Philadelphia College of Osteopathic Medicine.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Effect of UV Irradiation on the Nutritional Quality and Cytotoxicity of Apple Juice.

UV-C irradiation operating at 254 nm wavelength on the polyphenolic and vitamin contents of apple juice including cytotoxicity analysis was studied. UV doses ranging from 0 to 150 mJ·cm-2 were selected for the treatments. Polyphenols (catechin, epicatechin, chlorogenic acid, and phloridzin) and vitamins (riboflavin, thiamine hydrochloride, pyridoxal hydrochloride, pyridoxine, pyridoxamine dihydrochloride, cyanocobalamin, choline chloride, biotin, niacin, and niacinamide) were chemically profiled. It was observed that UV treatment of apple juice at disinfection doses caused minor reductions (p < 0.05) in the concentrations of two main polyphenols (i.e., chlorogenic acid and epicatechin). In contrast, significant (p < 0.05) decreases in vitamin concentrations were observed (p < 0.05). The irradiated juice was evaluated for cytotoxic effects. The irradiated apple juice showed no cytotoxic effects on normal intestinal cells, and both irradiated and nonirradiated samples are significantly comparable in inhibiting the growth of human colon cancer cells. Overall, these results indicated that UV-C treatment of apple juice neither significantly degraded polyphenols nor generated cytotoxic compounds.

A 7-day intravenous toxicity study and neurotoxicity assessment of pyridorin in Sprague-Dawley rats.

Pyridorin®, a naturally occurring metabolite of vitamin B6 that inhibits and scavenges reactive oxygen species, is being developed as a potential therapeutic for acute kidney injury. An investigational new drug application (IND) was opened for Pyridorin in support of its ongoing oral drug clinical development program. Currently, a Pyridorin intravenous (IV) formulation is being developed for use in surgical patients. To support the IND for Pyridorin, a full battery of nonclinical Good Laboratory Practice compliant studies was performed with no neurological or behavioral signs of toxicity seen following oral or IV administration of pyridoxine dihydrochloride (the active ingredient in Pyridorin). However, excessive ingestion of vitamin B6 has been reported to cause neurotoxic syndrome in humans. Therefore, under Food and Drug Administration recommendation, a 7-day IV study in rats was conducted to further evaluate the drug's potential to cause neurotoxicity. Blood plasma samples indicated that exposure to pyridoxamine dihydrochloride and its metabolites, pyridoxal, pyridoxine, and 4-pyridoxic acid was linearly dose proportional and independent of gender. At doses of up to 200 mg/kg/day pyridoxine dihydrochloride, no treatment-related effects were seen in rats, providing further evidence for the absence of pyridoxine dihydrochloride-related changes in the nervous system. A no observed adverse effect level of 200 mg/kg/day was identified for this study.

Involvement of advanced glycation end-products in 'hyperglycemic memory'.

The series of reports by the DCCT Skin Collagen Ancillary Study Group are very interesting and informative. The hypothesis from these reports clearly indicates that AGEs are a strong candidate for the mechanism of the 'hyperglycemic memory.'

Synthesis and cytotoxic activity of dibutyltin complexes derived from pyridoxamine and salicylaldehydes

The synthesis of monomeric pentacoordinated diorganotin complexes derived from pyridoxamine dihydrochloride and substituted salicylaldehydes is described. The complexes were characterized using UV/Vis, IR, MS, as well as 1H, 13C, and 119Sn NMR techniques; the molecular structure of the 1e complex was established by X-ray diffraction, which showed a distorted trigonal bipyramidal geometry, in which the basal plane is defined by the butyl groups and the azomethine nitrogen atom, whereas the oxygen atoms from the aromatic ring occupy axial positions. The cytotoxic activity of the complexes against human cell lines U-251 (glioblastoma), PC-3 (prostate), K-562 (chronic myelogenous leukemia), HCT-15 (human colorectal), MCF-7 (human breast), SKLU-1 (non-small cell lung), and MDA-MB-251 (human breast) was evaluated, and the inhibitory percentage values indicated higher activities than the reference standard, cis-platin, and non-cytotoxic activity was observed in culture of mononuclear cells from peripheral blood on healthy Wistar rats. Oral toxicity studies were performed by the OECD test guide line 423 and reveals that LD50 of complexes 1a, 1b, 1c–1g is in the range of 200–300 mg/Kg body weight and can be classified under category 3, the complexes 1c and 1h resulted to be the less toxic.

Pyridoxamine Dihydrochloride in Diabetic Nephropathy (PIONEER-CSG-17): Lessons Learned from a Pilot Study

Background/Aims: Pyridoxamine dihydrochloride (Pyridorin™) blocks pathogenic oxidative pathways in the progression of diabetic nephropathy. The pyridoxamine pilot study was designed to test entry criteria and outcomes. Subjects had SCr 1.3-3.5 mg/dl, protein-to-creatinine ≥1,200 mg/g and used a surrogate outcome of ΔSCr over 52 weeks. Subjects had to be on a maximally tolerated dose of ACE/ARB for 3 months; stable other antihypertensive doses for 2 months; stable diuretic dose for 2 weeks, and BP ≤160/90 mm Hg; or enter a Pharmaco-Stabilization Phase (PSP). This pilot failed to detect an effect on ΔSCr in intent-to-treat analysis. Methods: We queried the locked clinical trial database for subgroups in which there was a treatment effect. Results: Subjects not requiring PSP and those with entry SCr <2.0 mg/dl had a treatment effect. Subjects entering PSP required more changes in antihypertensive medications and experienced larger ΔSCr over 52 weeks. PSP subjects with BP >140/90 mm Hg had no treatment effect, but those ≤140/90 mm Hg did. Conclusion: Time required for acute effects of ACE/ARB to stabilize is unknown, but these data suggest >3 months. Thus, subjects in the pivotal trial must be on ACE/ARB for 6 months. Frequent antihypertensive adjustment could engender SCr changes unrelated to CKD progression. Thus, we will require subjects to have BP ≤150/90 mm Hg and on stable antihypertensives for 26 weeks, or ≤140/90 mm Hg and on stable antihypertensives for 13 weeks. Since ΔSCr over 52 weeks is limited as a surrogate outcome, the pivotal trial uses a time-to-event analysis of baseline SCr to at least a 50% increase in SCr or ESRD as the primary outcome. This substantial ΔSCr is protected from noise and is clinically relevant. The pyridoxamine pilot provided critical information to inform the design of PIONEER-CSG-17, which we conducted under the SPA agreement with FDA.

The role of vitamin B6 as an antioxidant in the presence of vitamin B2-photogenerated reactive oxygen species. A kinetic and mechanistic study

We report on the photostability of a mixture of vitamins B6 and B2 (riboflavin, Rf) upon visible light irradiation and on the possible role of the vitamin B6 family (B6D) as deactivators of reactive oxygen species (ROS). The work is a systematic kinetic and mechanistic study under conditions in which only Rf absorbs photoirradiation. Pyridoxine, pyridoxal hydrochloride, pyridoxal phosphate and pyridoxamine dihydrochloride were studied as representative members of the vitamin B6 family. The visible light irradiation of dissolved Rf and B6D in pH 7.4 aqueous medium under aerobic conditions induces photoprocesses that mainly produce B6D degradation. The overall oxidative mechanism involves the participation of ROS. Photogenerated (3)Rf* is quenched either by oxygen, giving rise to O(2)((1)Δ(g)) by electronic energy transfer to dissolved ground state oxygen, or by B6D yielding, through an electron transfer process, the neutral radical RfH˙, and O(2)˙(-) in an subsequent step. B6D act as quenchers of O(2)((1)Δ(g)) and O(2)˙(-), the former in a totally reactive event that also inhibits Rf photoconsumption. The common chromophoric moiety of B6D represented by 3-hydroxypyridine, constitutes an excellent model that mimics the kinetic behavior of the vitamin as an antioxidant towards Rf-generated ROS. The protein lysozyme, taken as an O(2)((1)Δ(g))-mediated oxidizable biological target, is photoprotected by B6D from Rf-sensitized photodegradation through the quenching of electronically excited triplet state of the pigment, in a process that competes with O(2)((1)Δ(g)) generation.

Pyridorin in Type 2 Diabetic Nephropathy

Pyridoxamine dihydrochloride (Pyridorin, NephroGenex) inhibits formation of advanced glycation end products and scavenges reactive oxygen species and toxic carbonyls, but whether these actions translate into renoprotective effects is unknown. In this double-blind, randomized, placebo-controlled trial, we randomly assigned 317 patients with proteinuric type 2 diabetic nephropathy to twice-daily placebo; Pyridorin, 150 mg twice daily; or Pyridorin, 300 mg twice daily, for 52 weeks. At baseline, the mean age ± SD was 63.9±9.5 years, and the mean duration of diabetes was 17.6±8.5 years; the mean serum creatinine level was 2.2±0.6 mg/dl, and the mean protein-to-creatinine ratio was 2973±1932 mg/g. Regarding the primary end point, a statistically significant change in serum creatinine from baseline to 52 weeks was not evident in either Pyridorin group compared with placebo. However, analysis of covariance suggested that the magnitude of the treatment effect differed by baseline renal function. Among patients in the lowest tertile of baseline serum creatinine concentration, treatment with Pyridorin associated with a lower average change in serum creatinine concentration at 52 weeks (0.28, 0.07, and 0.14 mg/dl for placebo, Pyridorin 150 mg, and Pyridorin 300 mg, respectively; P=0.05 for either Pyridorin dose versus placebo); there was no evidence of a significant treatment effect in the middle or upper tertiles. In conclusion, this trial failed to detect an effect of Pyridorin on the progression of serum creatinine at 1 year, although it suggests that patients with less renal impairment might benefit.

Delayed intervention with AGE inhibitors attenuates the progression of diabetes-accelerated atherosclerosis in diabetic apolipoprotein E knockout mice

Formation of AGEs is increased in the diabetic milieu, which contributes to accelerated atherogenesis. We studied whether delayed treatment with AGE-inhibiting compounds, alagebrium chloride and pyridoxamine dihydrochloride, affected established atherosclerosis in experimental diabetes in comparison with the angiotensin-converting enzyme inhibitor, quinapril. Streptozotocin-induced diabetic male Apoe (-/-) mice (n = 24 per group) received, by oral gavage, from week10 to 20 of diabetes: no treatment; alagebrium (1mgkg(-1)day(-1)); pyridoxamine (1g/l in drinking water); or quinapril (30mgkg(-1)day(-1)). Atherosclerotic lesion area (en face analysis) was evaluated for all groups. Delayed intervention with alagebrium decreased plaque area in the diabetic Apoe (-/-) mice compared with untreated mice (total plaque area: alagebrium 10.6 ± 1.6%, untreated, 15.1 ± 1.5%, p < 0.05). This anti-atherosclerotic effect was comparable with that achieved with quinapril (quinapril 8.4 ± 1.4%, vs untreated, p < 0.05). Pyridoxamine also attenuated plaque development in diabetic mice (5.7 ± 1.2% vs untreated 11.9 ± 1.1%, p < 0.05). The anti-atherosclerotic effect conferred by alagebrium and quinapril was associated with a significant reduction in vascular oxidative stress and circulating AGEs and methylglyoxal, although preformed AGEs were not removed from the vascular wall with either delayed intervention. Inhibition of AGE accumulation, using a delayed intervention with alagebrium or pyridoxamine, significantly attenuated the progression of established diabetes-associated atherosclerosis, similar to results obtained with quinapril. These findings provide further evidence that blockade of AGE-mediated pathways may present a novel therapy for the prevention of atherosclerosis in diabetes.

Biosynthesis of Vitamin B6 in Yeast. Incorporation Pattern of Trioses.

Two yeasts, Saccharomyces cerevisiae ATCC 7752 and Candida utilis ATCC 9256, were incubated in the presence of variously multiply 13C-labeled samples of d-glucose. The 13C incorporation pattern within pyridoxamine dihydrochloride, established by 13C NMR spectroscopy, differed from that which had previously been found within pyridoxine, isolated from Escherichia coli. Thus, the origin of the carbon skeleton of vitamin B6 in yeast differs substantially from its origin in E. coli. In particular, in yeast the distribution of 13C within the C5 chain C-2‘,2,3,4,4‘ of pyridoxamine corresponds to the distribution of 13C within the C5 chain C-1,2,3,4,5 of the ribose component of cytidine. It follows that the C5 chains of pyridoxamine and of ribose originate from a common glucose-derived pentulose or pentose intermediate. By contrast, in E. coli the C5 chain of pyridoxine is derived from 1-deoxy-d-xylulose 5-phosphate which, in turn, originates by condensation of pyruvic acid with glyceraldehyde 3-phosphate.

2'-hydroxypyridoxol, a biosynthetic precursor of vitamins B(6) and B(1) in yeast.

In separate experiments cultures of the yeast Saccharomyces cerevisiae ATTC 7752 were grown in the presence of [5',5'-2H2]- or of [2',2',5',5'-2H4]-3-hydroxy-2,4,5-tri(hydroxymethyl)pyridine (i.e., 2'-hydroxypyridoxol). The 2H NMR spectra of the samples of pyridoxamine dihydrochloride and of thiamin chloride hydrochloride that were isolated from the two experiments showed the presence of deuterium at the corresponding sites. Entry of deuterium from the specifically 2H-labeled samples of 2'-hydroxypyridoxol into the predicted sites of pyridoxamine and of the pyrimidine unit of thiamin provides the first unequivocal evidence that, in yeast, 2'-hydroxypyridoxol is an intermediate on the route from a C5-sugar into vitamin B6, and adds to the evidence that pyridoxol serves as a precursor of the pyrimidine unit of thiamin, supplying the C5N unit, C-2',2,N-1,C-6,5,5' as an intact unit.

Biosynthesis of vitamin B(6) in yeast: incorporation pattern of glucose.

Two yeasts, Saccharomyces cerevisiae ATCC 7752 and Candida utilis ATCC 9256, were incubated in the presence of variously multiply (13)C-labeled samples of D-glucose. The (13)C incorporation pattern within pyridoxamine dihydrochloride, established by (13)C NMR spectroscopy, differed from that which had previously been found within pyridoxine, isolated from Escherichia coli. Thus, the origin of the carbon skeleton of vitamin B(6) in yeast differs substantially from its origin in E. coli. In particular, in yeast the distribution of (13)C within the C(5) chain C-2',2,3,4,4' of pyridoxamine corresponds to the distribution of (13)C within the C(5) chain C-1,2,3,4,5 of the ribose component of cytidine. It follows that the C(5) chains of pyridoxamine and of ribose originate from a common glucose-derived pentulose or pentose intermediate. By contrast, in E. coli the C(5) chain of pyridoxine is derived from 1-deoxy-D-xylulose 5-phosphate which, in turn, originates by condensation of pyruvic acid with glyceraldehyde 3-phosphate.

Chlorination of Pyridinium Compounds: POSSIBLE ROLE OF HYPOCHLORITE, N-CHLORAMINES, AND CHLORINE IN THE OXIDATION OF PYRIDINOLINE CROSS-LINKS OF ARTICULAR CARTILAGE COLLAGEN TYPE II DURING ACUTE INFLAMMATION

Reactive oxygen species produced by activated neutrophils and monocytes are thought to be involved in mediating the loss of collagen and other matrix proteins at sites of inflammation. To evaluate their potential to oxidize the pyridinoline (Pyd) cross-links found in collagen types I and II, we reacted hydrogen peroxide (H(2)O(2)), hypochlorous acid/hypochlorite (HOCl/OCl(-)), and singlet oxygen (O(2)((1)delta g)) with the Pyd substitutes, pyridoxamine dihydrochloride and vitamin B(6), which share the same chemical structure and spectral properties of Pyd cross-links. Neither H(2)O(2) (125-500 microm) nor O(2)((1)delta g) (10-25 microm) significantly changed the spectral properties of pyridoxamine or vitamin B(6). Reaction of HOCl/OCl(-) (12.5-50 microm) with pyridoxamine at pH 7.2 resulted in a concentration-dependent appearance of two new absorbance peaks and a decrease in fluorescence at 400 nm (excitation 325 nm). The new absorbance peaks correlated with the formation of an N-chloramine and the product of its subsequent reaction with pyridoxamine. In contrast, the extent to which HOCl reacted with vitamin B(6), which lacks a primary amine group, was variable at this pH. At lysosomal pH 5.5, Cl(2)/HOCl/OCl(-) reacted with both pyridoxamine and vitamin B(6). Four of the chlorinated products of this reaction were identified by gas chromatography-mass spectrometry and included 3-chloropyridinium, an aldehyde, and several chlorinated products with disrupted rings. To evaluate the effects of Cl(2)/HOCl/OCl(-) on Pyd cross-links in collagen, we exposed bone collagen type I and articular cartilage type II to HOCl. Treatment of either collagen type with HOCl at pH 5. 0 or 7.2 resulted in the oxidation of amine groups and, for collagen type II, the specific decrease in Pyd cross-link fluorescence, suggesting that during inflammation both oxidations may be used by neutrophils and monocytes to promote the loss of matrix integrity.

Stereoselective synthesis of a pyridoxamine coenzyme-amino acid chimera: Assembly of a polypeptide incorporating the pyridoxamine moiety

The stereoselective synthesis of ( S)-2-amino- N α -(9-fluorenylmethoxycarbonyl)-3-( α 4,3- O- bis- tert-butoxycarbonylpyridoxamin-5-yl)propanoic acid ( 4) was accomplished from pyridoxamine dihydrochloride. A key step is the stereoselective alkylation of (−)pseudoephedrine glycinamide with the pyridoxamine bromide 7. Amenability of the amino acid derivative 4 to solid phase methodology was demonstrated by its incorporation into a synthetic hexapeptide. Critical to the orthogonal protection of the pyridoxamine moiety is the in situ acetylation of the C3-hydroxyl group during peptide assembly.

Determination of water-soluble vitamins by liquid chromatography with a parallel dual-electrode electrochemical detector

A method for the determination of water-soluble vitamins (ascorbic acid, pyridoxine hydrochloride, pyridoxal hydrochloride, pyridoxamine dihydrochloride, p-aminobenzoic acid, folic acid) by liquid chromatography, with a parallel dual-electrode electrochemical detector, is described. One electrode was controlled at +0.80 V ( vs. SCE), the other at +1.20 V ( vs. SCE). The possibility of interference by eight other water-soluble vitamins (riboflavin, nicotinamide, cyanocobalamin, menadione, dextro calcium pantothenate, thiamine, nicotinic acid, dextro biotin) was studied. These vitamins did not interfere when a parallel dual-electrode detector system was used. The estimation of five of the vitamins was studied in detail. The linear ranges found were 10 ng—1.2 μg for pyridoxine hydrochloride, 2 ng—2μg for pyridoxal hydrochloride, 10 ng-3 μg for pyridoxamine dihydrochloride, 5–200 ng for folic acid and 0.6–200 ng for p-aminobenzoic acid, the limits of detection being 3, 0.6, 1, 2 and 0.06 ng respectively. Application of the technique to the estimation of vitamin B 6 in tablets is illustrated. The results indicate that the vitamin B 6 in these tablets existed in the pyridoxine hydrochloride form and the B 6 content agreed well with liquid chromatograph by spectrophotometric analysis.

Effect of pyridoxal on growth of nutritionally variant streptococci and other bacteria on sheep blood agar

Supplementation of sheep blood agar with pyridoxal hydrochloride or pyridoxamine dihydrochloride aided recovery of nutritionally variant streptococci; however, it was found to prevent recovery of 6 of 173 (3%) primary isolates of Streptococcus pyogenes and to adversely affect laboratory recognition of other streptococci. Pyridoxal hydrochloride or pyridoxamine dihydrochloride should not be a routine supplement in all sheep blood agar.

Viridans streptococcal endocarditis: the role of various species, including pyridoxal-dependent streptococci.

The viridans streptococci are a heterogeneous group of organisms frequently associated with microbial endocarditis. Isolates from consecutive patients with endocarditis due to viridans streptococci who were seen at The New York Hospital from 1944 to 1955 and from 1970 to 1978 were speciated, and the relative frequencies and patterns of sus ceptibility to penicillin were determined. Vitamin B6-dependent streptococci, classified as Streptococcus mitior, accounted for 5%-6% of microbial endocarditis in both time periods. Since these nutritional variants require the active forms of vitamin B6 for growth in routein media, they can be responsible for culture-negative endocarditis. Media must be supplemented with either pyridoxal hydrochloride or pyridoxamine dihydrochloride (the active forms of vitamin B6) for isolation, identification, and subsequent testing of susceptibility to antimirobial agents. Pyridoxine hydrochloride does not support the growth of these organisms in vitro.