Micronutrient supplementation of lactating Guatemalan women acutely increases infants' intake of riboflavin, thiamin, pyridoxal, and cobalamin, but not niacin, in a randomized crossover trial.

BACKGROUND: Maternal supplementation during lactation could increase milk B-vitamin concentrations, but little is known about the kinetics of milk vitamin responses. OBJECTIVES: We compared acute effects of maternal lipid-based nutrient supplement (LNS) consumption (n = 22 nutrients, 175%-212% of the RDA intake for the nutrients examined), as a single dose or at spaced intervals during 8 h, on milk concentrations and infant intake from milk of B-vitamins. METHODS: This randomized crossover trial in Quetzaltenango, Guatemala included 26 mother-infant dyads 4-6 mo postpartum who were randomly assigned to receive 3 treatments in a random order: bolus 30-g dose of LNS (Bolus); 3 × 10-g doses of LNS (Divided); and no LNS (Control), with control meals. Mothers attended three 8-h visits during which infant milk consumption was measured and milk samples were collected at every feed. Infant intake was assessed as $\mathop \sum \nolimits_{i\ = \ 1}^n ( {{\rm{milk\ volum}}{{\rm{e}}_{{\rm{feed\ }}n}} \times \ {\rm{nutrient\ concentratio}}{{\rm{n}}_{{\rm{feed}}\ n}}} )$ over 8 h. RESULTS: Maternal supplementation with the Bolus or Divided dose increased least-squares mean (95% CI) milk and infant intakes of riboflavin [milk: Bolus: 154.4 (138.2, 172.5) µg · min-1 · mL-1; Control: 84.5 (75.8, 94.3) µg · min-1 · mL-1; infant: Bolus: 64.5 (56.1, 74.3) µg; Control: 34.5 (30.0, 39.6) µg], thiamin [milk: Bolus: 10.9 (10.1, 11.7) µg · min-1 · mL-1; Control: 7.7 (7.2, 8.3) µg · min-1 · mL-1; infant: Bolus: 5.1 (4.4, 6.0) µg; Control: 3.4 (2.9, 4.0) µg], and pyridoxal [milk: Bolus: 90.5 (82.8, 98.9) µg · min-1 · mL-1; Control: 60.8 (55.8, 66.3) µg · min-1 · mL-1; infant: Bolus: 39.4 (33.5, 46.4) µg; Control: 25.0 (21.4, 29.2) µg] (all P < 0.001). Only the Bolus dose increased cobalamin in milk [Bolus: 0.054 (0.047, 0.061) µg · min-1 · mL-1; Control: 0.041 (0.035, 0.048) µg · min-1 · mL-1, P = 0.039] and infant cobalamin intake [Bolus: 0.023 (0.020, 0.027) µg; Control: 0.015 (0.013, 0.018) µg, P = 0.001] compared with Control. Niacin was unaffected. CONCLUSIONS: Maternal supplementation with LNS as a Bolus or Divided dose was similarly effective at increasing milk riboflavin, thiamin, and pyridoxal and infant intakes, whereas only the Bolus dose increased cobalamin. Niacin was unaffected in 8 h. This trial was registered at clinicaltrials.gov as NCT02464111.

Comparisons of uptake and cell surface binding among pyridoxal, pyridoxine, and pyridoxamine in RAW264.7 cells.

OBJECTIVE: Vitamin B6 (B6) suppresses the expression of cyclooxygenase-2 stimulated by lipopolysaccharide in mouse macrophage RAW264.7 cells. The greatest effect is recognized for pyridoxal (PL) compared with pyridoxamine (PM), pyridoxine (PN), and pyridoxal 5'-phosphate (PLP). However, it has not been elucidated why PL has the strongest effect. We compared the uptakes and cell surface interactions among PL, PM, PN, and PLP in RAW264.7 cells. METHODS: Cyclo-oxygenase-2 mRNA expression was evaluated by real-time polymerase chain reaction. Intracellular B6 concentrations were measured by high-performance liquid chromatography. Interactions of B6s with the cell surface were analyzed using a surface plasmon resonance biosensor. B6 uptake speeds were measured using [(3)H]-PN. RESULTS: The intracellular PLP levels did not change significantly when cells were cultured in medium containing PL, PM, PN, or PLP. Only PL interacted with the cell surface. Although PM and PN were associated with the cell surface, their binding was only recognized during sample loading. After the change to phosphate buffered saline after sample loading, the binding resonances of PM and PN returned to baseline, whereas that of PL did not. Uptake of [(3)H]-PN was inhibited by non-labeled PN, PL, or PLP, but not PM, at 1 microM. The inhibition rate of PL was higher than those of PN and PLP. CONCLUSION: The inhibition of cyclo-oxygenase-2 mRNA expression by PL may be related to the cell surface interaction of PL, rather than the intracellular PLP level. The uptake mechanism for PN and PL may differ from that for PM.

Pharmacokinetic comparison of a delayed-release combination of doxylamine succinate and pyridoxine hydrocholoride (Diclectin) and oral solutions of these drugs in healthy women of childbearing age.

BACKGROUND: The delayed-release combination of doxylamine succinate and pyridoxine hydrochloride was the most commonly used antiemetic (Bendectin) approved by FDA for nausea and vomiting of pregnancy (NVP) until its removal of the market in 1983. The drug is widely used today in Canada (Diclectin). The pharmacokinetics of Diclectin has never been described in humans. OBJECTIVES: To compare the pharmacokinetics of Diclectin to oral solutions of its two components. SUBJECTS AND METHODS: A randomized, cross over, open label design, comparing the pharmacokinetics of Diclectin to those of the oral solutions of the two components in 18 healthy adult, non pregnant women of childbearing age. RESULTS: Diclectin exhibited similar oral bioavailability to those of the oral solutions. In contrast, the time-to-peak, (Tmax), reflecting the rate of absorption, was 3-6 times longer for the two components of the delayed-release drug confirming its delayed-release characteristics. CONCLUSION: The pharmacokinetic profile of Diclectin well explains its documented delayed efficacy.

HPLC determination of a novel aroylhydrazone iron chelator (o-108) in rabbit plasma and its application to a pilot pharmacokinetic study.

A high performance liquid chromatographic method for the determination of a biocompatible iron chelator, pyridoxal 2-chlorobenzoyl hydrazone (o-108), in rabbit plasma was developed and validated. The separation was achieved on a C18 column with the mobile phase composed of a mixture of 0.01 M phosphate buffer (pH 6) with the addition of EDTA (2 mM), methanol and acetonitrile (42:24:14; v/v/v). The method was validated with respect to selectivity, linearity (0.8-150 microg/mL), intra- and inter-day variability and stability. This method was successfully applied to the analysis of the samples obtained from a pilot pharmacokinetic experiment, in which the chelator was administered intravenously to rabbits.

Safety and tolerability of repeated administration of pyridoxal 2-chlorobenzoyl hydrazone in rabbits.

Recently, pyridoxal 2-chlorobenzoyl hydrazone (o-108) has been identified as an effective iron chelator [Link et al., Blood 2003; 101: 4172-79]. Since chronic treatment would be necessary in its potential indications, in the present study, the safety and tolerability of this agent after repeated administration was determined. Three doses of o-108 (25, 50, 100 mg/kg, in 10% Cremophor EL) were administered intraperitoneally, once weekly, for 10 weeks to three groups (n=5 each) of Chinchilla male rabbits. The effects on biochemical, haematological and cardiovascular parameters were examined during the experiment; histopathological examination was performed at the end of the experiment. Results were compared with control (saline 2 mL/kg, n=11) and vehicle groups (10% Cremophor EL, 2 mL/kg, n=12). No premature deaths occurred; the well-being of animals was evidenced by their body weight gain, although lower gain was observed with the highest dose (100 mg/kg). Significant elevations of cardiac troponin T plasma concentrations were observed with the highest dose of o-108, but no abnormalities were found in the cardiovascular function and only minor and inconsistent changes in haematological and biochemical parameters were observed. Histopathological examinations of selected organs revealed only weak and reversible changes through all studied groups. Thus, the data from this study suggest that o-108 remains a promising drug from the standpoint of the possibility of its repeated administration and warrants further investigation.

Spectral analysis applied to hepatobiliary dynamic scintigraphy with 99Tcm-N-pyridoxyl-5-methyltryptophan.

This study was performed to evaluate the application of spectral analysis (SA) to hepatobiliary dynamic scintigraphy with 99Tcm-N-pyridoxyl-5-methyltryptophan (99Tcm-PMT) in 82 patients with a wide range of liver function, and to compare it with compartment analysis (CA) and deconvolution analysis (DA). The rate of uptake of PMT by the liver from the blood (k1) obtained by SA (y, min-1) agreed well with the k1 value obtained using CA (x, min-1) (y = 1.079x + 0.000, r = 0.993, standard error of the estimate (S.E.E.) = 0.042 min-1). The mean residence time (MRT) of PMT in the liver obtained by SA (y, min) also agreed well with the MRT value obtained by DA (x, min) (y = 1.036x - 0.759, r = 0.967, S.E.E.-1.014 min) and that obtained by CA (x, min) (y = 0.859x + 1.006, r = 0.931, S.E.E. = 1.428 min). The fraction of the measured blood activity superimposed on the true liver activity (f) obtained by SA (y) correlated well with the f value obtained by CA (x) (y = 1.168x - 0.004, r = 0.924, S.E.E. = 0.043). We conclude that the application of SA to hepatobiliary dynamic scintigraphy with PMT appears to be useful in evaluating the functional status of the liver, since it facilitates the interpretation of the kinetic behaviour of PMT in the liver and allows us to extract quantitative parameters corresponding to those obtained by CA or DA.

Determination of total pyridoxal in human plasma following oral administration of vitamin B6 by high-performance liquid chromatography with post-column derivatization.

An HPLC method for determining total pyridoxal from plasma was developed for a relative bioavailability comparison of two oral vitamin B6 (pyridoxine HCl) preparations. After cleavage of the pyridoxal-5-phosphate with the acid phosphatase enzyme, the total pyridoxal was determined by HPLC. Pyridoxal was separated on a reversed-phase column, post-column derivatized to pyridoxal-semicarbazide, and then detected by fluorescence and quantitated. The limit of detection was 2 ng/mL and interday variation (3 days) over the whole concentration range (13-215 ng/mL spiked) was < 4.1%. In the relative bioavailability study, 16 human subjects were put on a low vitamin B6 diet for a period of 3 days. On the 2nd and 3rd days, 14 blood samples were taken per subject at the same times each day. The drug was administered on the 3rd day. Total endogenous pyridoxal detected on the 2nd day varied in plasma between 13 and 17 ng/mL. Pharmacokinetic parameters corrected for background are reported for two vitamin B6 (40 mg) preparations. Briefly, the pharmacokinetic results for the Ratiopharm preparation compared with the Hoffmann-La Roche preparation are, respectively: AUC0-24, 369.2 and 352.6 ng.h/mL; AUC24-48, 1638.2 and 1662.3 ng.h/mL; net Cmax, 193.0 and 197.1 ng/mL; tmax, 1.25 and 1.44 h; and relative bioavailability, 97.9% (Westlake, 88-112%).

Liposome-mediated delivery of gallium to macrophage-like cells in vitro: demonstration of a transferrin-independent route for intracellular delivery of metal ions.

Gallium (Ga) prevents the activation of macrophages and might be useful as an immunosuppressive agent. It is taken up by the malignant cells through the transferrin (Tf) receptor pathway, but this pathway may be insufficient in the case of non-malignant cells. We studied the Tf-independent, liposome-mediated delivery of Ga to macrophage-like cells in vitro by a growth inhibition assay. The growth inhibitory properties of Ga for other types of cells was also evaluated. Ga complexed with nitrilotriacetate (GaNTA) and encapsulated in DSPG-liposomes was 16 and 48 times more potent for RAW 264 cells than free GaNTA and Ga-nitrate, respectively. CV1-P cells were also somewhat sensitive to liposomal Ga, but other cell lines with lower endocytotic capacity were insensitive. The inhibition of RAW 264 cell growth induced by liposomal or free GaNTA was partially reversed with iron-loading of the cells, indicating that this form of Ga causes an intracellular iron deficiency similar to that produced by Tf-bound Ga. Our results indicate that encapsulation of Ga in negatively charged liposomes provides a transferrin independent route for intracellular delivery of the compound to macrophages, which is of special interest in the treatment of autoimmune diseases, such as rheumatoid arthritis.

Synthesis of N-(4'-pyridoxyl)sphingosine and its uptake and metabolism by isolated cells.

N-(4'-pyridoxyl)sphingosine was synthesized and characterized as a stable compound for specialized delivery of a bioactive lipid. It was found to be facilely taken up by hepatocytes although by a mechanism more typical for lipids than the one used by natural vitamin B6. Some of the N-(4'-pyridoxyl)sphingosine was metabolically acted upon inside the cell to release pyridoxal 5'-phosphate and sphingosine, but formation of pyridoxal 5'-phosphate from the synthetic compound was poor compared with natural vitamin forms of B6, which may partly be due to entrapment within cell membranes and to constraints at the level of cytosolic pyridoxal kinase which is responsible for phosphorylation of the vitamin. Unlike the parent long-chain base, the B6 conjugate was not particularly cytotoxic. Furthermore, the compound was neither an activator nor inhibitor of the respiratory burst of human neutrophils. These findings identify N-(4'-pyridoxyl)sphingosine as an interesting tool for studies of the cellular transport, metabolism, and functions of both vitamin B6 and sphingosine.

Metabolism of [14C]- and [32P]pyridoxal 5'-phosphate and [3H]pyridoxal administered intravenously to pigs and goats.

To gain more information about the kinetics of vitamin B-6 metabolism in vivo, the metabolism of tracer was examined after the simultaneous intravenous administration of [32P] and [14C]pyridoxal phosphate and [3H]pyridoxal in two 93-kg pigs and two 60-kg goats. In the pigs, [14C] removal was monophasic with T1/2 of 16 and 18 min and clearance of 165 and 248 mL/min. In the goats, [14C] removal was biphasic with T1/2 of 49 and 114 min for 0-30 min and 209 and 227 min for 0.5-6 h (clearance 20 and 17 mL/min). Uptake of pyridoxal phosphate by liver and resecretion into the plasma were too small to cause a detectable decrease in the [32P]:[14C] ratio. Pyridoxal removal from plasma was similar in both species, with a half-life of approximately 12 min from 0-30 min and approximately 50 min for 0.5-3 h. Clearance of [3H]pyridoxal in the four animals ranged from 412 to 2258 mL/min. Little [14C] entered the erythrocytes. The [3H] entered readily but was converted to pyridoxal phosphate faster in the pigs than in the goats. [14C] and [3H] were excreted as pyridoxic acid at the same rate. However, during the 54 h after injection the goats excreted approximately 60% of the [14C] doses in the urine compared with approximately 30% in the pigs. About 5-10% of the [14C] and [3H] doses were recovered in goat milk over 54 h. Pyridoxal kinase activity was higher in lactating mammary tissue than in liver, kidney or muscle of goats.

Pyridoxal-5'-phosphate and pyridoxal biokinetics in male Wistar rats fed graded levels of vitamin B-6.

Biokinetic parameters of plasma pyridoxal-5'-phosphate (PLP) and pyridoxal (PL) disposition were studied in male Wistar rats (age 8 mo) fed a purified diet containing less than 0.5, approximately 3 or approximately 6 mg pyridoxine.HCl/kg diet from weaning, with animals fed the 6 mg/kg diet serving as the control group. Basal plasma PLP concentration was lower in both the less than 0.5 and 3 mg/kg diet groups than in control animals (98 +/- 12, 314 +/- 40 and 514 +/- 56 nmol/L, respectively). Basal plasma PL concentration was lower in the less than 0.5 mg/kg diet group only [60 nmol/L (measured in pooled samples), 190 +/- 73 and 235 +/- 63 nmol/L for less than 0.5, 3 and 6 mg/kg diet groups, respectively]. In both the less than 0.5 and 3 mg/kg diet groups, PLP clearance was lower than in control rats (0.158 +/- 0.025, 0.131 +/- 0.040 and 0.240 +/- 0.051 L.h-1.kg body weight-1, respectively). In the less than 0.5 mg/kg diet group, PLP synthesis was more efficient than in control animals (34.7 +/- 9.3, 12.1 +/- 2.5 and 16.7 +/- 11.4% for less than 0.5, 3 and 6 mg/kg diet groups, respectively). In both the less than 0.5 and 3 mg/kg diet groups, volume of distribution of PLP as well as of PL was larger than in controls. It is concluded that B-6 vitamer metabolism is influenced by vitamin B-6 status. The metabolic pathway involved (PLP synthesis and/or PLP degradation) was observed to depend on degree of vitamin B-6 deficiency.

Relationship of uptake of technetium-99m(Sn)-N-pyridoxyl-5-methyltryptophan by hepatocellular carcinoma to prognosis.

The relationship of technetium-99m(Sn)-N-pyridoxyl-5-methyltryptophan (99mTc-PMT) uptake by hepatic tumors to survival was studied in 162 cases of hepatocellular carcinoma (HCC). The median survival of 82 patients in whom hepatic tumors showed increased uptake in delayed 99mTc-PMT imaging was 1013 days, which was significantly longer than the survival time of 398.5 days of 80 patients in whom hepatic tumors did not show increased uptake of radioactivity (p less than 0.002). The relationship between the ability of hepatic tumors to take up 99mTc-PMT and survival was also analyzed in patients with HCC showing filling defects in 99mTc-colloid liver images and, in relation to the therapy, serum values of bilirubin and alpha-fetoprotein. Results indicated that the degree of 99mTc-PMT uptake by hepatic tumors is closely correlated with the prognosis of patients with HCC.

Intestinal absorption of pyridoxal 5'-phosphate at physiological levels in rats.

The intestinal absorption of pyridoxal 5'-phosphate (PLP) at physiological levels (10(-7) -10(-6) M) was studied in comparison with that of pyridoxal (PL) in rat, using in vitro everted sac and an intestinal preparation that permitted continuous in situ collection of mesenteric venous blood. After PLP administration (10(-6) -10(-3) M) in situ, larger amounts of PLP were found in the mesenteric venous plasma than after PL administration at the same dose. The amount of PLP found in the mesenteric venous plasma was dependent on its dose at lower concentrations up to 10(-4) M but became independent at higher concentrations. After PL administration at various doses, the amount of PL found in the mesenteric venous blood increased linearly with the dose. When various concentrations of PLP were added to the mucosal side, under the in vitro condition with protection from alkaline phosphate hydrolysis, PLP was detected in the serosal side and the extent of PLP transport was dependent on the initial concentration of PLP in the mucosal side. When various concentrations of PL were added to the mucosal side, the extent of PL transport was independent of the initial concentration of PL in the mucosal side. In rat pretreated with actinomycin D, PLP transport in vitro was inhibited but not that of PL. N2-induced anoxia and pyridoxamine 5'-phosphate and anion transport inhibitor (4,4'-diisothiocyanostilben-2,2'-disulfonic acid disodium salt) showed no effect on PLP transport. These results suggest that PLP can be absorbed in the phosphorylated form and imply the presence of a saturable process for direct absorption of PLP itself and a diffusive process for PL absorption. In addition, the result of the in vivo neonatal experiment suggests that the neonatal intestine also can transport PLP in phosphorylated form.

A new synthetic method for 99mTc labeled N-pyridoxyl-5-methyltryptophan as a hepatoma imaging agent.

N-Pyridoxyl-5-methyltryptophan (5-PMT) was synthesized by a simplified method using sodium borohydride for the reduction of a Schiff base of pyridoxal and 5-methyltryptophan. Lyophilized kits containing 5-PMT, stannous chloride and L-(+)-ascorbic acid were prepared and labeled to afford 99mTc-5-PMT with 96% or higher radiochemical purity analysed by two thin-layer chromatographic solvent systems. 99mTc-5-PMT showed a rapid blood clearance, a faster hepatobiliary transit and a lower renal retention in comparison with 99mTc-5-EHIDA in rats. Eleven (61%) of 18 patients with histologically confirmed hepatocellular carcinoma showed positive images at 2 to 5 h after i.v. injection. The smallest tumor that could be identified was 2 cm in diameter with the best tumor/liver ratio of 4. In conclusion, 99mTc-5-PMT synthesized by sodium borohydride reduction shows great promise as a useful hepatoma imaging agent.