B vitamins are of interest in preventative and protective strategies in cardiovascular disease. However, the safety and efficacy of B vitamins has been questioned. Previous research from this group has demonstrated that B6 supplementation alone or in combination with folic acid and B12 reduces angiogenic response. This study determined the effect of vitamin B6 deficiency on the angiogenic response after ischemia in vivo and in vitro using a rodent model. Results indicated that vitamin B6 deficiency enhanced the early angiogenic response by increasing blood flow in vivo after an ischemic event. In vitro measurements demonstrated that vitamin B6 deficiency influenced endothelial progenitor cell (EPC) function and angiogenic growth factor release early after ischemia. In conclusion, B6 deficiency appears to have a modest effect on increasing blood flow and angiogenic markers after ischemia. Additional research is needed to further characterize the impact of lowered vitamin B6 on angiogenesis and its mechanisms.

B vitamins are of interest in preventative and protective strategies in cardiovascular disease. However, the safety and efficacy of B vitamins has been questioned. Previous research from this group has demonstrated that B6 supplementation alone or in combination with folic acid and B12 reduces angiogenic response. This study determined the effect of vitamin B6 deficiency on the angiogenic response after ischemia in vivo and in vitro using a rodent model. Results indicated that vitamin B6 deficiency enhanced the early angiogenic response by increasing blood flow in vivo after an ischemic event. In vitro measurements demonstrated that vitamin B6 deficiency influenced endothelial progenitor cell (EPC) function and angiogenic growth factor release early after ischemia. In conclusion, B6 deficiency appears to have a modest effect on increasing blood flow and angiogenic markers after ischemia. Additional research is needed to further characterize the impact of lowered vitamin B6 on angiogenesis and its mechanisms.

Graduation date: 1980

Thesis--Auburn University, 2005. / Abstract. Vita. Includes bibliographical references (leaves 32-35).

Thesis (Ph. D.)--University of Wisconsin--Madison, 1941. / Typescript. Includes abstract and vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references (leaves 50-53).

Thesis (M.S.)--University of Wisconsin--Madison, 1964. / eContent provider-neutral record in process. Description based on print version record. Bibliography: l. 67-73.

Vitamin B6 was discovered almost 90 years ago, and since then it has received a lot of interest from the scientific community due to its role in human health and its impact on several biochemical processes. One of the most interesting aspect of vitamin B6 studied in the past decade is its role in cancer. From the research on this subject so far, the following can be suggested: early-stage cancer cells have a higher vitamin B6 content than normal cells due to its role in metabolic processes. As the cancer makes progress, there is a change in vitamin B6 activation and trapping in the cell, decreasing the amount of active vitamin B6 in the cell in order to resist cell death. From these conclusions, we can see that vitamin B6 could potentially be an interesting radiotracer to use for diagnosis and staging of cancers. One of the most predominant form of imaging which is done nowadays to detect and diagnose cancers is Positron Emission Tomography (PET) imaging. Research on PET imaging is driven by the potential of new radiotracers which can be added to the current arsenal of tools for the fight against cancer. Therefore, this project focuses on the attempted synthesis of two potential radiotracers derived from vitamin B6 based on the insertion of fluorine-18. None of the two proposed radiotracers were successfully synthesized but we successfully synthesized one cold standard and difluorinated pyridoxine with cold conditions similar to radiochemistry. The main issues which were faced were the degradation of the potential precursors when attempting fluorination, the lack of reactivity of intermediates for the formation of precursors and an acetyl migration leading to the wrong precursors. By using a milder fluorination strategy to avoid degradation (room temperature, no free fluoride source: AgF2 as fluorinating agent), the 6-Fluoropyridoxine cold standard was synthesized. By changing the protection strategy (not using any acetyl groups), acetyl migration was avoided which led to the synthesis of a difluorinated pyridoxine using mild conditions (room temperature). The difluorination was also successful using harsher conditions (heat). There is still a lot of work to do to synthesize a radiotracer derived from vitamin B6 but there are some signs that this may be possible with additional work.

Previous studies have found that smoking may have a negative effect on vitamin B-6 indices and have demonstrated a possible association between smoking and depressed plasma pyridoxal-5'-phosphate (PLP) concentration. Individuals with plasma PLP values below the adequate level of 30 nmoles/L might benefit from consumption of vitamin B-6 supplements, but no data are available on vitamin B-6 status in smokers consuming a controlled vitamin B-6 intake and receiving a vitamin B-6 supplement. The objectives of this research were to assess vitamin B-6 status in smokers as compared to non-smokers receiving a controlled diet and to evaluate the effect of an oral vitamin B-6 supplementation in these subjects. The vitamin B-6 (B-6) status of 5 (four males / one female) smokers (S) and 4 (three males / one female) non-smokers (NS) was assessed. A constant diet was fed for 20 days and provided 1.95 mg of B-6 or 1.65 mg of B-6 for males and females, respectively. For the last 10 days, an additional 2-mg of pyridoxine (PN) was given daily. Blood samples were collected on days 1.7, 11.14 and 21; and 24 hour urine samples were collected daily. Urinary 4-pyridoxic acid (4-PA) and total B-6 (UB6) excretion, plasma B-6 vitamers (PLP, PN, pyridoxal and 4-PA) and red blood cell PLP (RBC PLP) concentrations, as well as plasma alkaline phosphatase activity (APA) were determined. Mean plasma PLP, 4-PA, and RBC PLP concentrations were significantly lower (P [less than or equal to] 0.05) at all time points in S compared to NS. With a daily supplement of 2-mg vitamin B-6, the mean plasma PLP concentration of S increased 85.8% but was 48.5% lower than that of NS consuming 1.65-1.95 mg/d of B-6. Mean plasma pyridoxal concentrations were not different between S and NS before and after supplementation. Excretion of 4-PA was not significantly different between S and NS, but the mean values of 4-PA excretion were consistently greater in NS compared to that of S throughout the 20-day study. The percent of ingested B-6 excreted as 4-PA for the S and NS was 38 and 49 in the non-supplemented period, and 47 and 53 in the supplemented period, respectively, indicating that non-smokers excreted more 4-PA than smokers. However, the difference in 4-PA excretion between S and NS was not significantly different both before and after supplementation (P>0.05). In addition, there was no significant difference between S and NS for plasma PN concentration, AP, and UB6 excretion for both periods. Results suggested an adverse effect of smoking on B-6 metabolism, thus an increased requirement of vitamin B-6 in smokers. A 2-mg PN supplement was sufficient to bring the concentration of plasma PLP in smokers to the level suggested as adequate, but it didn't bring it to the level of non-smokers. / Graduation date: 1999

Die Phosphatase PDXP (auch bekannt als Chronophin) gehört zur Familie der HAD Phosphatasen, einer ubiquitär exprimierten Enzymklasse mit wichtigen physiologischen Funktionen. PDXP zeigt Phosphatase-Aktivität gegenüber seinem Substrat Pyridoxal 5´-Phosphat (PLP), der aktivierten Form von Vitamin B6. PDXP-defiziente Mäuse (Knockout-Mäuse) weisen im Vergleich zu Wildtypen verdoppelte PLP-Konzentrationen in Erythrozyten sowie im Gesamthirn auf. Vermutlich kommt PDXP daher eine wichtige Funktion in Erythrozyten und im Hirn zu. Ziel dieser Arbeit war es, erste Einblicke in diese Funktion(en) von PDXP zu erlangen. Hierzu wurden HPLC-basierte Analysen der erythrozytären PLP-Konzentrationen in Wildtyp- sowie PDXP-defizienten Mäusen durchgeführt. Dabei ließen sich die rund doppelt so hohen erythrozytären PLP-Level in den KO-Mäusen bestätigen. Zudem ist es gelungen, eine Methode zur Messung der endogenen Phosphatase-Aktivität von PDXP in Erythrozytenlysaten zu etablieren. So konnte im Wildtyp anhand der Verringerung der PLP-Konzentrationen pro Zeiteinheit eine erythrozytäre PDXP-Aktivität nachgewiesen werden. Dazu waren die Inkubation mit Pyridoxin, sowie die Anwendung eines Inhibitors der PDXK notwendig. Eine bis dato vermutete Funktion der PDXP, zur Mobilisation von erythrozytärem PLP während Fastenzeiten, konnte ausgeschlossen werden. So zeigte der Vergleich der erythrozytären PLP-Konzentrationen aus gefasteten mit normal gefütterten Tieren in beiden Genotypen exakt dieselbe prozentuale PLP-Verringerung. Während Nahrungszufuhr ließ sich jedoch eine Funktion der Phosphatase PDXP als „Converter“ von Pyridoxin zu Pyridoxal erkennen. Ausgehend von PN konnte im Wildtyp (über die Zwischenprodukte PNP und PLP) eine PDXP-abhängige Dephosphorylierung von PLP zu PL erfolgen. So wies der Wildtyp eine rund vierfach höhere PL-Produktion auf, verglichen mit der PDXP-defizienten Maus. Die Phosphatase PDXP erwies sich als essenziell für die erythrozytäre Konversion von Pyridoxin zu Pyridoxal. Dadurch erreicht der Organismus eine metabolische Flexibilität, die ihn bis zu einem gewissen Grad unabhängig von der Nahrungsauswahl macht. Zudem können Zellen oder Organe, denen durch das Fehlen der PNPO, die Konversion zu PLP nicht möglich ist, mit PL versorgt werden. Aus der hohen Reaktivität von PLP mit umliegenden Nucleophilen ergibt sich eine gewisse Problematik für die Zelle im Umgang mit freiem PLP. So liegt der Großteil des erythrozytären PLPs gebunden an Proteine (vor allem Hämoglobin) vor. Anhand von Filtern (MWCO, 3000) ließ sich zwischen der hier definiert als „freien“ und der „gebundenen“ Form von PLP differenzieren. So konnten erste Erkenntnisse zur Rolle von PDXP als Determinator freier PLP-Konzentrationen in Erythrozyten und insbesondere im Hippocampus erlangt werden. Im Hippocampus ergaben sich insgesamt deutlich höhere Konzentrationen an freiem PLP als in den Erythrozyten und es bestand zudem ein Unterschied zwischen den Genotypen. So wiesen die KO-Mäuse ~1/3 höhere freie PLP-Konzentrationen im Vergleich zu den Wildtypen auf. Schließlich konnte ein Effekt des Tieralters auf den PLP-Metabolismus festgestellt werden. Sowohl in den Erythrozyten als auch im Hippocampus ergaben sich alterskorrelierte Änderungen ihrer PLP-Konzentrationen. Zudem zeigten Western Blot Analysen altersbedingte Unterschiede ihrer Vitamin B6-Enzymexpressionen. So wiesen ältere Wildtypen im Hippocampus eine fünffach erhöhte PDXP-Expression verglichen mit jüngeren Tieren auf. In den Erythrozytenlysaten hingegen zeigten ältere Tiere beider Genotypen eine rund vierfach geringere PNPO-Expression gegenüber jüngeren Tieren. Die mit dem Alter eintretende physiologische Verringerung der erythrozytären PNPO-Expression würde somit für den Organismus einen Verlust seiner metabolischen Flexibilität bedeuten, die mit der Konversion von PN zu PL einhergeht. / The phosphatase PDXP, also called Chronophin, is a member of the ubiquitously expressed HAD-phosphatases, which have some important physiological functions in the organism. Its substrate pyridoxal 5´-phosphate (PLP) is the active form of vita-min B6, an important cofactor of several reactions. PDXP-deficient mice (KO-mice) have PLP-concentrations in erythrocytes and in the whole brain twice as high as wildtype mice. It is assumed that PDXP therefore has an important function in erythrocytes and in the brain. The aim of the study was to gain initial insights into these functions of PDXP. For this purpose, HPLC-based analyses of the PLP-concentrations in erythrocytes from WT- and KO-mice were carried out. The doubled PLP-levels in the RBCs of KO-mice could be confirmed. In addition, a method for measuring the endogenous phosphatase activity of PDXP in red cell lysates was established. The activity of PDXP could be measured by the reduction of its substrate PLP over time. This required the incubation with pyridoxine and the inhibition of PDXK by ginkgotoxine. An assumed function of PDXP in mobilization of PL(P) from the erythrocytes in fasting conditions could be ruled out. Therefore, a comparison between the PLP-concentrations in RBCs of fasted mice with normal fed ones was done. Surprisingly the fasted KO-mice showed the same percentaged decrease of cellular PLP-level as the fasted WT-mice. During vitamin B6 intake however, a function of PDXP as being a “converter” of pyridoxine to pyridoxal was found. Starting with PN, a PDXP-mediated dephosphorylation from PLP to PL could take place in the wildtype mice (via the intermediate steps PNP and PLP). Consequently, the WT´s production of PL quadrupled compared to the KO´s. PDXP turned out to be essential for the conversion of pyridoxine to pyridoxal in erythrocytes. This conversion confers some metabolic flexibility to the organism and to a certain extent makes it independent of the choice of food. Moreover, cells and organs, that due to the absence of PNPO cannot produce PL(P) themselves, can be provided via erythrocytes. The high reactivity of PLP with surrounding nucleophiles poses a certain problem for the cell in dealing with free PLP. The majority of the PLP in RBCs is bound to proteins (primarily hemoglobin). It was distinguished between the here termed “free” PLP and the bound PLP by using filter devices with a MWCO at 3 kDa. First insights could be gained about PDXP as a determinant of free PLP-levels in erythrocytes and hippocampus. The amount of free PLP in the hippocampus was significantly higher than in the RBCs. Additionally, the hippocampus showed some differences in the con¬centration of free PLP between WT- and KO-mice. The level of free PLP in PDXP deficient mice was one third higher than in wildtype mice. Finally, some correlation between the age of the mice and their PLP-metabolism was found. The results revealed changes of the PLP-concentrations with age in the RBCs and the hippocampus. Moreover, western blot analyses showed some age-related differences in the expression of vitamin B6 enzymes. In the hippocampus older wildtype mice showed a quintupled expression of PDXP compared to younger ones. However, western blot analyses of red blood cell lysates from older animals revealed a lower expression of PNPO by a factor of four. For the organism this physiological reduction of its PNPO expression with age would mean a loss of metabolic flexibility, that is accompanied by the conversion from PN to PL.

This study was designed to evaluate the effect of glucose on plasma pyridoxal 5'- phosphate (PLP) concentration. The objective was to determine whether there was a negative relationship between glucose ingestion and plasma PLP concentration and to evaluate the possible mechanism of decreased PLP after acute glucose ingestion. Seven healthy subjects (three males and four females) completed the oral glucose tolerance test (OGTT) on three separate occasions over a period of three weeks. Each week, subjects ingested the assigned solutions (a water solution with artificial sweetener equivalent to 25g glucose, a 25g glucose or a 75g glucose load) in a randomized order. Plasma PLP, pyridoxal (PL), 4-pyridoxic acid (4-PA), pyridoxine (PN), glucose, insulin, alkaline phosphatase (AP) activity and red blood cell PLP concentrations were measured at 0 (fasting) (TO), 1 (T1), 2 (T2) and 3 (T3) hours. The mean vitamin B-6 intake based on two 3-day dietary records was 1.57 ± 0.34 mg/day. All subjects had normal glucose tolerance. There were gender differences among the three solutions. Both the water solution and the 75g glucose load showed a significant decrease in the mean plasma PLP concentration was observed at T3 for males and at T2 for females (p<0.05). An overall mean decrease of 20% (9nmol/L) and 15% (7 nmol/L) was observed for males and females, respectively, after the 75g glucose load. The 25g glucose load resulted in a lower decrease in the mean plasma PLP concentration at each time point compared with the 75g glucose load, but no significant difference was found in the level of decrease between the two glucose loads. Both genders had a non-significant increase in the mean plasma PL and PN concentrations for the three solutions. Mean plasma 4-PA concentration was decreased at T1 with the three solutions. There was no significant change in the plasma AP activity at any time points after the three solutions. In addition, no significant increase in mean red blood cell PLP concentration was observed at all time points after the three solutions. This study found a negative relationship between glucose ingestion and plasma PLP concentration. However, it did not provide clear evidence for the hypothesized mechanism of the decreased plasma PLP concentration after acute glucose load. Further studies are required to determine the mechanism by which glucose decreases plasma PLP concentration. / Graduation date: 2000