Graduation date: 1980

Thesis (Ph. D.)--University of Wisconsin--Madison, 19. / Typescript. Vita. Includes (as Section I): Preparation of samples for microbiological determination of riboflavin / F.M. Strong and L.E. Carpenter. Reprinted from Industrial and engineering chemistry, vol. 14 (15 Nov. 1942), p. 909-913. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.

Universiẗat, Diss., 2002--Bonn.

The primal aim of this thesis was to establish whether kinetic aspects of vitamin B6 metabolism predispose to earlier observed racial differences found in plasma pyridoxal-5'-phosphate (PLP). The active forms of vitamin B6 namely plasma PLP and pyridoxal (PL) as well as the three enzymes expressed in the erythrocyte involved in B6 metabolism, PL kinase, PLP phosphatase and pyridoxamine -5'- phosphate (pyridoxine -5'- phosphate) [PMP(PNP) ] oxidase were measured by high performance liquid chromatography. Phase one supported earlier experimental evidence and lower plasma PLP concentrations were found in blacks in a group of200 male volunteers recruited from the South African National Defence Force (SANDF). The respective enzyme activities involved in vitamin B6 metabolism, from the same test subjects, suggested similar PLP production from PMP and PL as well as PLP dephosphorylation which result in the release of PL into the circulating fluid. Since applied exclusion criteria eliminated the majority of biochemical, physiological, genetical - and disease related factors that influence vit B6 status, dietary factors and individual preferences regarding food intake, were most likely to be responsible for the significantly lower circulating plasma PLP encountered in blacks. Phase two compared pharmacokinetic parameters between 7 black - and 9 white test subjects recruited from the South African Police Services after a single 10 mg oral supplement ofpyridoxine hydrochloride. Statistical analysis of the parameters elimination half-life, elimination rate constant, clearance, volume of distribution, mean residence time, maximum peak concentration and time to maximum peak concentration failed to demonstrate any significant differences between the two groups. These results suggest consistent appearance rate, distribution and metabolism for the metabolites PLP and PL in the study population. A tendency in slower appearance rate, for both the metabolites PLP and PL, were observed in blacks and needs to be investigated further. The end product of vitamin B6 metabolism, 4-pyridoxic acid, which was expressed in terms of 24 hour urine volume, again failed to illustrate any significant differences between blacks and whites. These results suggested similar excretion properties in my population study. Furthermore, the pharmacokinetic parameters calculated for plasma PLP and PL respectively, were found to display one-compartment - and two-compartment pharmacokinetic model characteristics. This mono- and bi exponential elimination characteristics displayed by PLP and PL respectively could be of value in future research efforts in terms of sampling time. The distribution half-life can be determined by the calculation of two-compartment macro-rate constants. Fasting blood-samples should be collected when true baseline values are needed in the case of PL. Following vit B6 supplementation, one should allow at least 5 times the distribution half-life (5-6 hr in the case of PL) before blood-sampling in order to achieve true pharmacological response. Phase three of this study was conducted to illustrate the metabolic interplay ofthe enzymes PL kinase and PMP (PNP) oxidase involved in PLP production. The kinetic parameters, Michaelis- Menten constant and maximum velocity rate, at varying substrate concentrations, for the enzymes PL kinase and PMP (PNP) oxidase, were compared in 14 white - and 14 black male test subjects recruited from the SANDF. Both the average Michaelis-Menten constant and maximum velocity rate were higher in whites, but these differences were not statistically significant. The high individual variability for both parameters calculated, can possibly be ruled out if a crystalline enzyme form is used and should be investigated further. / Dissertation (MSc (Chemical Pathology))--University of Pretoria, 2001. / Chemical Pathology / unrestricted

A high performance liquid chromatographic (HPLC) system consisting of an acetonitrile/phosphate buffer, a Spherisorb ODS column, and UV detector was used to separate the B₆ compounds pyridoxol, pyridoxal, pyridoxamine, and 4'-deoxypyridoxine. A gas chromatography (GC) equipped with a ⁶³Ni electron capture (EC) detector was used to separate the N-methyl-bis-trif luoroacetamide derivatives of the B₆ compounds on a l.54m x 2mm i.d. glass column packed with 10% SP2100 on Supelcoport 90-100 mesh at 125°C and an inlet pressure of 40 psig. Clean and successful separations of all the B₆ forms were obtained by HPLC and GC-EC. Total B₆ values as determined by HPLC and GC-EC for all 3 foods were higher than the corresponding total B₆ values as determined by S. uvarum assay. Several of the B₆ vitamer values for foods obtained by GC-EC agree with corresponding values obtained by HPLC. The HPLC method seemed to be the most satisfactory of the 3 methods for the quantitation of B₆ vitamers in foods and has the following advantages: increased sensitivity, method simplicity, and good precision. / Ph. D.

In vitro data from our laboratory demonstrate that vitamin B6 (B6) supplementation of estrogen receptor - positive and - negative breast cancer cells is growth inhibitory. Others have reported that dietary B6 supplementation resulted in increased fibrosarcoma pyridoxal phosphate (PLP) concentrations and a significant inverse relationship between tumor PLP concentration and tumor volume in mice. This suggests that, in contrast to data reported for normal cells, tumor cells are capable of accumulating supplemental B6. In the current study, we investigated the effects of dietary B6 supplementation on 7,12-dimethylbenz(a)anthracene (DMBA)-induced mammary carcinoma in rats. Specifically, we aimed to identify the effect of pyridoxine (PN) supplementation on tumor growth and vitamin uptake by tumor cells. To accomplish this, 50 d old female Sprague Dawley rats were gavaged with 15 mg DMBA and fed a diet containing either 7, 350, or 1050 mg PN-HCl/kg diet, which is the equivalent of 1, 50, or 150x the National Research Council's B6 requirement for rats, respectively. These levels of PN have previously been shown to produce no overt signs of toxicity in rats. Throughout the experiment, the percent of rats with tumors and the average number of tumors per rat remained similar between groups. Mammary tumor growth rates were significantly increased in response to dietary B6 supplementation (P < 0.05). Liver PLP and pyridoxal (PL) concentrations did not differ between dietary treatment groups. Plasma PL and PLP concentrations were significantly higher in the group fed the 150x diet compared with the 1x diet (P < 0.001, P < 0.05). Mammary tissue PL concentrations of the 150x group were significantly higher (P < 0.05) than the 1x group, but no differences were observed in mammary PLP concentrations. Similarly to mammary tissue, no differences between groups were observed in tumor PLP concentration. However, tumor PL concentrations in both the 50x and 150x dietary treatment groups were significantly higher than those from the rats fed the 1x diet (P < 0.002). These data demonstrate that previously reported inhibitory effects of supplemental B6 on breast cancer growth in vitro do not occur in response to dietary supplementation at 50 or 150 times the B6 requirement in vivo. In fact, dietary B6 at 150x the requirement may actually promote mammary tumor growth. In light of these results, investigation of the effects of supplemental B6 on cancer growth in humans is warranted. Supported by American Cancer Society Grant # IRG-99-225-01. / Master of Science

Two studies were conducted to determine the effect of varying vitamin B-6 (B- 6) status on lymphocyte mitogenic response and pyridoxal 5'-phosphate (PLP) concentration in peripheral blood mononuclear cells (PBMC) in young women. In the first study, women were fed 1 mg/d for the first week and 1.5, 2.1 and 2.7 mg/d during 2 weeks of each of the subsequent 3 experimental periods. Plasma PLP and urinary 4: pyridoxic acid (4-PA) were increased with increasing B-6 intake. B-6 intake > 2.1 mg significantly enhanced lymphocyte proliferation, and non-significantly increased plasma interleukin-2 concentration. Lymphocyte proliferation was significantly correlated with B-6 intake, erythrocyte aminotransferase activity coefficients and plasma PLP. PBMC PLP tended to increase after 2 weeks of 2.7 mg B-6 intake, and was significantly correlated with plasma PLP. In the second study, women consumed their normal diets whose estimated mean dietary B-6 intake was 0.9 mg for 27 d. For the last 20 d, all subjects were given a multivitamin supplement containing 1.8 mg B- 6, and half of the subjects were given an additional 50 mg of B-6 supplement. Plasma PLP and urinary 4-PA were significantly higher in the group with 50 mg B-6, but lymphocyte proliferation did not significantly differ between the groups. After 10 d of supplementation, lymphocyte proliferation was significantly higher than the other time points. A significant increase in PBMC PLP was observed after 3 days and 20 days following 50 mg and multivitamin supplementation only, respectively. After 20 days of supplementation, there was no significant difference of mean PBMC PLP between the groups. PBMC PLP was significantly correlated with plasma PLP, PL and 4-PA. In both studies, no strong relationship was found between PBMC PLP and lymphocyte proliferation. The findings from these studies demonstrate no further benefit of a higher B-6 intake than 2.1 mg on lymphocyte mitogenic response, once the response was significantly enhanced with B-6 intake 0.8 mg higher than the current recommendation. Finally, results from two studies suggest that the current recommendation of vitamin B-6 for young women may not be adequate to maximize lymphocyte mitogenic response and PLP concentration in PBMC. / Graduation date: 2002

This study examined the effect of vitamin B-6 supplementation and exhaustive submaximal exercise on plasma catecholamine concentrations, and the relationship between plasma catecholamines and fuel use, heart rate and oxygen consumption. Five trained men (age= 18-35 years; V0₂max=53 ml 0₂/kg/min.) participated in two controlled dietary periods that were identical except for the addition of 20 mg/d pyridoxine (PN) supplementation during the second period. On the seventh morning of each period, fasted subjects exercised to exhaustion on a cycle ergometer at 74.5% ± 7.8 V0₂max. Blood was drawn pre-exercise (twice), 60 minutes into exercise, immediately post-exercise and 60 minutes post-exercise. Plasma was analyzed for norepinephrine, epinephrine, glucose, pyridoxal 5'-phosphate (PLP), lactic acid, glycerol and free fatty acids (FFA). Heart rate and oxygen consumption were measured pre-exercise and at 10-minute intervals during exercise. Mean plasma PLP concentration was significantly higher during the supplemented versus the nonsupplemented trial at all time points. There were no statistically significant differences in mean plasma catecholamine concentrations or mean plasma fuel concentrations between the nonsupplemented and supplemented trials at any of the time points examined. There were significant changes in the mean plasma concentrations of norepinephrine, lactic acid, glycerol and FFA over time in both trials. Respiratory exchange ratios (R) were higher during the supplemented trial compared to the nonsupplemented trial, but the differences did not attain statistical significance. There were no significant differences in mean exercise times to exhaustion or mean heart rates between the trials. The overall mean oxygen consumption during exercise was consistently higher during the supplemented versus the nonsupplemented trial and the difference attained significance (p=0.016) at one time point (10 min.). The mean oxygen consumption during rest was lower during supplementation versus nonsupplementation, but the difference was not statistically significant. The percent plasma volume change (PVC) was significantly greater at post-exercise, relative to pre-exercise, during the supplemented versus the nonsupplemented trial. The percent PVC also increased significantly over time during the supplemented but not the nonsupplemented trial. These results suggest that 20 mg/d of vitamin B-6 supplementation does not effect plasma catecholamine concentrations, fuel utilization or heart rate at rest or during submaximal exercise to exhaustion. The results may suggest a higher oxygen consumption during exhaustive exercise after PN supplementation. / Graduation date: 1996

Trained male cyclists (6 in study 1, 5 in study 2) cycled to exhaustion (EXH) at 75% of VO₂ max twice; once in the non-supplemented (NS) state and once in the vitamin B-6 (B-6)(20 mg PN) supplemented (S) state. The diet contained 2.3 mg B-6 in study 1 and 1.9 mg B-6 in study 2. Urine was collected during each dietary period. During each exercise (EX) test, blood was drawn prior to (PRE), one hour during (DX), immediately after (POST) and one hour after (POST 60) EX and sweat was collected. Compared to baseline (PRE) levels, plasma pyridoxal 5'-phosphate (PLP) and vitamin B-6 (PB-6) concentrations increased at DX, decreased at POST, and decreased below PRE at POST 60 in the NS and S states. EX to EXH in the S state resulted in a greater increase in PLP DX in study 1 (31% increase vs. 16%) and PB-6 in study 2 (25% increase vs. 11%) as compared to the NS state. Red blood cell (RBC) PLP significantly increased from POST to POST 60 in the S state in study 2. The excretion of urinary 4-pyridoxic acid (4-PA) and urinary B-6 (UB-6) was not significantly altered by EX to EXH. The mean excretion of 4-PA was significantly greater in the NS state in study 2 (7.98 ±1.83 mmol/d) as compared to the excretion in study 1 (6.20 ±0.93 mmol/d), whereas the excretion was significantly greater in the S state in study 1 (92.2 ±8.69 mmol/d) compared to the excretion in study 2 (82.7 ±6.16 mmol/d). The percent of B-6 intake excreted as UB-6 (6% in study 1 and 10% in study 2) was significantly different between the studies in the NS state. Vitamin B-6 supplementation did not significantly alter the rise in growth hormone (hGH) concentration seen with EX to EXH. The loss of B-6 in sweat with EX to EXH was not altered by B-6 supplementation. The loss of B-6 in sweat ranged from 0.0011 mmol to 0.0039 mmol. Therefore, EX to EXH in the B-6 S state resulted in a greater increase in plasma PLP and PB-6 DX as compared to the NS state. The decrease in PB-6 and PLP at POST 60 in the S state coincided with a significant increase in RBC PLP, suggesting the movement of B-6 from the plasma into the RBC at POST 60. EX to EXH and B-6 supplementation did not alter the excretion of 4-PA or UB-6 suggesting that B-6 metabolism was unchanged. The loss of B-6 in sweat was comparable to previously reported values and was not altered by B-6 supplementation. B-6 supplementation did not alter the changes in hGH resulting from EX to EXH alone. / Graduation date: 1995

Vitamin B6, or more specifically the active form pyridoxal 5ʹ-phosphate (PLP), plays a crucial role as a cofactor for numerous enzymes linked to carbohydrate, fatty acid, and amino acid metabolism. There is a high prevalence of moderate vitamin B6 deficiency in the population that may be further exacerbated through the ingestion of vitamin B6 antagonists present in the food supply. For example, flaxseed contains the anti-pyridoxine factor 1-amino D-proline (1ADP) in the form of a dipeptide called linatine. In order to address these issues, the current study was designed to: 1) characterize and quantify the total amount of anti-pyridoxine factors present in flaxseed through the use of UPLC/ESI-MS analysis, 2) investigate the in vivo effects of synthetic and flaxseed-derived 1ADP on amino acid metabolism using a rat model of moderate B6 deficiency, and 3) identify novel biomarkers of vitamin B6 inadequacy using a LC-Qtof-MS based non-targeted metabolomics approach. The total anti-pyridoxine content, measured as 1ADP equivalents, in the flaxseed extract was found to be 177-437 μg/g of whole flaxseed, depending on the variety tested. Plasma biochemical analyses revealed that B6 vitamers, particularly PLP concentrations were reduced (P≤0.001), due to 1ADP ingestion (10 mg/kg diet) irrespective of the sources. Oral ingestion of flaxseed-derived 1ADP in moderately vitamin B6-deficient rats increased plasma cystathionine (P≤0.001), and decreased plasma α-aminobutyric acid (P≤0.001) and glutamic acid (P=0.017) concentrations compared to the controls. However, the ingestion of synthetic 1ADP elicited greater perturbations in amino acid profile compared to the flaxseed-derived 1ADP, which was predominantly in the form of the dipeptide linatine. Additionally, oral ingestion of the synthetic as well as the flaxseed-derived 1ADP significantly (P≤0.05) inhibited the activities of hepatic PLP-dependent enzymes involved in transsulphuration reactions of methionine metabolism. The use of a non-targeted metabolomics approach identified ten potential lipophilic markers of vitamin B6-insufficiency: glycocholic acid, glycoursodeoxycholic acid, murocholic acid, N-docosahexaenoyl GABA, N-arachidonoyl GABA, lumula, nandrolone, orthothymotinic acid, cystamine and 3-methyleneoxindole. These data serve to highlight potential deleterious effects of anti-pyridoxine factors linked to flaxseed in a population at risk for moderate vitamin B6 deficiency. / October 2015