The effect of vitamin B-6 (B-6) status on plasma fatty acids (FA) levels and lipid metabolism was investigated in this metabolic study. Eight female subjects were fed for 28 days. For the first 7 days, they were fed a constant diet containing 2.10 mg of B-6. For the rest of the period (21 days), they were differentiated in terms of B-6 intake; 4 of them were fed a low (0.93 mg/day) and 4 a high (2.60 mg/day) B-6 diet. B-6 status indices, plasma FA concentration and lipid profile were determined. Plasma pyridoxal 5'-phosphate and total B-6 concentration (P<0.01), urinary 4- pyridoxic acid and total B-6 concentration (P<0.001) showed a significant difference between the two groups at the end of the study. Erythrocyte PLP failed to show any significant difference between the two groups throughout the diet study. There was no significant difference in the plasma FA or lipid profile between the two groups. Plasma total cholesterol (TC) of the low B-6 group decreased slightly (7 %), but was not statistically significant. When comparing day 7 and day 28 values, plasma triglycerides increased (9 %) for the high and decreased for the low B-6 group. LDL-C decreased (5 %) for the high B-6 group but did not change in the low B-6 group. HDL-C decreased slightly in both groups (~8 %). There was no clear evidence that a low intake of vitamin B-6 affects the fatty acid and lipid metabolism. Further studies are required to identify the relationship between vitamin B-6 and fatty acid and lipid metabolism in humans. / Graduation date: 1997

Research suggests that, in individuals with carpal tunnel syndrome (CTS), low plasma pyridoxal 5'-phosphate (PLP) concentrations are related to an increased incidence and severity of symptoms associated with CTS. This study was designed to determine the relationship between plasma and red blood cell PLP concentrations and the severity and incidence of CTS symptoms. Thirty people with CTS were selected for a 9 month exercise study. Subjects were divided into either vitamin users or non-vitamin users based on supplement use data gathered at the beginning of the study. Blood was drawn at 1, 6 and 9 months. CTS symptoms questionnaires and health questionnaires were also administered at these intervals. The symptoms questionnaires were used to gather data on the frequency and nature of hand and wrist symptoms. Health questionnaires focused on vitamin supplement usage including frequency, amount and length of use. Mean plasma PLP, total plasma vitamin B6 and erythrocyte PLP concentrations were significantly higher in the sixteen vitamin users when compared to the fourteen non-vitamin users (p<0.001). While there was variation in plasma PLP and total plasma vitamin B6 over time, within each group, there were no significant changes in any of the status measures over the nine month period. Mean erythrocyte PLP concentration, in particular, was stable over time. In vitamin users, the intensity of pain, numbness and tingling was significantly higher when compared to non-vitamin users. In both groups, plasma PLP was negatively correlated with pain. This correlation reached statistical significance in vitamin users at month one and nine (p<0.01), but not at month six; a statistically significant correlation between these two variables was not found in non-vitamin users at any time point. Pain was also negatively and significantly correlated with plasma total vitamin B6 and erythrocyte PLP in vitamin users. No other symptoms were significantly correlated with the status measures. These results indicate that a higher vitamin B6 status may be related to a decrease in the severity of pain experienced by some individuals with CTS. / Graduation date: 1998

The purpose of this study was to examine the effect of extreme exercise on vitamin B-6 metabolism and urea nitrogen. Nine men and five women completed two 5-day trials; Trial 1 (T1) included a 50-km ultramarathon on day 4 and during Trial 2 (T2) subjects were "inactive" on day 4. During both trials, subjects consumed a diet providing men 2.0 and women 1.5 mg of vitamin B-6. With the exception of the ultramarathon, T1 activity was replicated during T2. Twenty four-hour urine collections were completed and blood was drawn pre-race (pre), mid-race (mid), post-race (post) and 60 minutes post race (P-60). On the inactive, day blood was drawn at the same intervals. Plasma was analyzed for pyridoxal 5'-phosphate (PLP), pyridoxal, 4-pyridoxic acid (4-PA), urea nitrogen (PUN), creatinine, albumin, glucose, and lactate concentration and alkaline phosphatase activity. Urine was analyzed for 4PA, creatinine, and total urinary nitrogen (TUN). During T1, compared to pre, plasma PLP concentration increased 17% at mid, decreased 5% by post, and 19% by P-60. During T2, plasma PLP concentration decreased 13% pre to P-60. During T1, plasma 4-PA concentration increased 135% and the percent dietary vitamin B-6 that was excreted as urinary 4-PA the day of the ultramarathon was higher than that excreted the day before and the day after. During T1, from pre to post mean PUN concentration increased 36.9%, and the average rate ofincrease from pre to mid, mid to post, and post to P60 was 0.5, 1.75, and 2 mg/dL/hour, respectively. During T1 on days 3, 4, and 5,88%, 100%, and 95% of nitrogen intake was excreted in the urine compared to 86%, 83%, and 84% for the same days during T2. The day of the ultramarathon, 24-hour TUN excretion was 2 g higher than the previous day. Extreme exercise of greater than six hours initially increases the plasma concentration of PLP but ultimately results in a significant decrease in plasma PLP, an increase in plasma 4-PA, and an increase in percent of dietary vitamin B-6 (as 4-PA) excreted in the urine. Additionally, the rate of change in PUN inoeases as duration increases. / Graduation date: 2001

Serine transhydroxymethylase (STHM), a pyridoxal 5'- phosphate requiring enzyme is indirectly involved in pyrimidine deoxynucleotide metabolism. A decrease in the activity of this enzyme could lead to altered deoxycytidine (dC) metabolism. This study was undertaken to determine if a vitamin B-6 deficiency affects dC metabolism. The effect of a vitamin B-6 deficiency on the activity of STHM in liver, thymus, spleen and bone marrow was examined. In addition, the effect of a vitamin B-6 deficiency on urinary excretion of dC was examined. The effect of a vitamin B-6 deficiency on the urinary excretion and tissue retention of ³H label from ip injected ³H-dC was monitored. Rats were assigned in groups of six to one of four treatment groups: ad libitum control (ALC), pair fed control (PFC), ad libitum deficient (ALD) or meal fed deficient (MFD). At the end of weeks 2 and 6, rats from each treatment group received an ip injection of ³H-dC. Urines were collected for 24 hours following the ip inhibited due to lack of cofactor, then dTMP levels would fall. In an attempt to increase the concentration of dTMP, enzymes active in the conversion of dC and dCMP to dUMP would be expected to increase. Thus, dC salvage pathways would increase and dC synthesis would decrease as metabolism shifts toward production of deoxythymidine triphosphate (dTTP). The result would be lower urinary dC excretion. The present study was undertaken to explore the relationship between vitamin B-6 and pyrimidine deoxynucleotide metabolism. There were four hypothesis tested: Vitamin B-6 deficient rats will excrete less urinary dC than either ad libitum or pair fed controls; vitamin B-6 deficient rats will excrete a lower percentage of labeled dC in urine than control rats; vitamin B-6 deficient rats will incorporate less labeled dC into DNA than control rats but may retain more label in tissues as dC metabolites; activity of STHM from tissues of vitamin B-6 deficient rats will be lower than that from the control rats. / Graduation date: 1990

In two studies we tested the effect of vitamin B-6 (B-6) intake on in vitro lymphocyte, proliferation and IL-2 production in healthy young women. In Study I, 6 women were fed a constant diet containing 0.84 mg (4.96 μmols) of B-6 for 12 d, and 1.24 mg (7.33 μmols) and 2.44 mg (14.42 μmols) of B-6 during two subsequent 10-d periods. Lymphocyte proliferation in response to the mitogens concanavalin A (Con A) and pokeweed mitogen (PWM) was significantly higher (p < 0.05) at 2.44 mg than at 0.84 mg intake and the pre-study value. In Study II, 10 women who consumed their self-selected diets were randomly divided into a PN and a placebo group of five each. Following a 5-d baseline period, the PN group received a daily supplement of 1.5 mg (7.29 μmols) and 50 mg (243 μmols) pyridoxine.HC1 (PN.HC1) for 7 and 6 d, respectively. This was followed by a 28-d washout period during which no supplement or placebo was administered. After daily 1.5 mg supplementary PN.HC1 for seven days, lymphocyte proliferation in response to phytohemagglutinin (PHA), Con A and PWM, and IL-2 production were significantly higher than the baseline (p < 0.05) and that of the placebo group (p < 0.05). The 50 mg PN.HC1 supplement increased IL-2 production, but not lymphocyte proliferation as compared with the 1.5 mg PN.HC1 supplement period. Lymphocyte proliferation and IL-2 production were significantly correlated with lymphocyte pyridoxal 5'- phosphate concentrations (p < 0.01). We conclude that a B-6 intake of 1.5 to 2 times the RDA improves lymphocyte proliferation and IL-2 production in healthy young women and this effect of B-6 on immunocompetence is transitory. To explore the basis for the effect of B-6 on immune function, putrescine was added to the culture medium (2 - 200 μmol/L). In vitro lymphocyte proliferation and IL-2 production were not changed by the addition of putrescine under our experimental conditions. / Graduation date: 1995

This investigation was conducted to determine the effect of estrogen replacement therapy (ERT) on vitamin B-6 status of postmenopausal women. Nineteen postmenopausal women served as subjects. Nine (54.7 + 4.7 years) were taking ERT (experimental group); ten (56.8 + 2.3 years) were not (control group). For three consecutive days, subjects recorded their dietary intake and collected their 24-hour urine specimens. On the fourth day, a fasting blood sample was drawn from the subjects. The dietary intake of vitamin B-6, as well as the concentration of total vitamin B-6 in plasma (PB6; and urine (UB6) were measured. PB6 and UB6 were determined by a microbiological method with Saccharomyces uvarum as the assay organism. The mean age, height, hematocrit and hemoglobin values were similar for the two groups. The experimental group was significantly heavier than the control group (p<0.05). The experimental group had a lower mean PB6 than the control group: 47.7 ± 19.7 nmol/L vs. 56.2 + 20.6 nmol/L. These means were not significantly different (p=0.05). PB6 was positively correlated with dietary vitamin B-6 intake (p=0.0001) and vitamin B-6 to protein ratio (p=0.0021). When the means were adjusted for dietary vitamin B-6 and the vitamin B-6 to protein ratio, the mean PB6 of the experimental group (42.7 nmol/L) was significantly lower than that of the control group (60.6 nmol/L) (p<0.05). PB6 was not positively correlated with either age (r=0.20) or the vitamin B-6 dietary history score (r=0.15). UB6 was similar for the two groups. UB6 correlated positively with daily dietary intake of vitamin B-6 (r=0.51, p<0.05) and the ratio of vitamin B-6 to protein (r=0.47, p<0.05), UB6 was not significantly correlated to urine volume (r=0.05). The mean daily intakes of vitamin B-6 and protein were similar for the two groups. One of the 19 subjects had a vitamin B-6 intake that was less than 67 percent of the RDA. Most subjects' (89%) intake of vitamin B-6 was adequate when the ratio of 0.016 mg of vitamin B-6 per g of protein was used as the standard. / Graduation date: 1990

Call number: LD2668 .T4 1986 M37 / Master of Science / Human Nutrition

Malaria ist eine schwerwiegende Krankheit, die jährlich über eine Million Menschen tötet. Die zunehmende Resistenzbildung gegenüber den verwendeten Medikamenten macht die Entwicklung neuer Antimalariamittel dringend notwendig. Daher sind die Vitamin B6 Synthese und der Shikimatweg von besonderem Interesse, da diese beiden Synthesewege nur im Parasiten und nicht im Menschen vorkommen. Unter der Voraussetzung, dass diese essentiell für den Parasiten sind, böten sie ideale Ansatzpunkte zur Entwicklung neuer Antimalariamittel. Voraus gegangene Studien haben gezeigt, dass Plasmodium falciparum in der Lage ist, PLP de novo mittels eines bifunktionalen Enzymkomplex, bestehend aus den Proteinen Pdx1 und Pdx2, zu synthetisieren. Pdx1 stellt dabei die eigentliche Synthase dar, während Pdx2 als Glutaminase-Partner das benötigte Ammoniumion für den heterocyclen Ring bereitstellt. Zusätzlich dazu verfügt der Parasit auch über einen salvage pathway um PLP zu „recyclen“, in dem der Pyridoxalkinase PdxK eine Schlüsselfunktion zufällt. Knockout Studien der pdx1 im Mausmalariasystem P. berghei haben gezeigt, dass PbPdx1 für eine optimale Entwicklung der Blutstadien benötigt wird, nicht jedoch für deren Überleben. Im Rahmen dieser Arbeit habe ich die Effekte eines pbpdxK(-) Knockouts in demselben System untersucht. Es konnte eine monoklonale Knockoutlinie generiert werden, was zeigte, dass PbPdxK nicht essentiell für das Überleben des Parasiten in den Blutstadien ist. Die Entwicklung während des Blutstadiums war von dem pbpdxK(-) Knockout nicht betroffen. Allerdings zeigte sich im Moskitostadium eine drastische Reduktion der Sporozoitenzahl sowohl in den Mitteldärmen als auch in den Speicheldrüsen. Dieses Ergebnis legt nahe, dass PbPdxK essentiell für das Überleben der Sporozoiten ist. Daneben wurde versucht, die Gene pfpdx1, pfpdx2 sowie pfpdxK in P. falciparum 3D7 durch Verwendung der single cross over Strategie auszuschalten. Es konnte jedoch für keines der genannten Konstrukte eine Integration in die jeweiligen Genloci anhand von PCR-Analysen nachgewiesen werden. Ebenso scheiterte der Versuch, durch Rekombination eines komplementären Genabschnitts die Funktion des Gens zu rekonstituieren. Daher bleibt es unklar, ob pfpdx1, pfpdx2 und pfpdxK durch Knockout Strategien auszuschalten sind oder nur für Genmanipulationen nicht zugänglich sind. Die Kultivierung von P. falciparum 3D7 Parasiten in Vitamin B6 depletiertem Medium hatte keinen Effekt auf deren Wachstum. Eine anschließende Analyse der Proteinextrakte zeigte eine erhöhte Expression der PfPdxK, während sich das Expressionslevel der PfPdx1 nicht veränderte. Es scheint, dass der Parasit in der Lage ist Vitamin B6 Mangel durch vermehrte Nutzung des salvage pathways vollständig zu kompensieren. Frühere Arbeiten zeigten, dass der C-Terminus der Pdx1 in die Aktivität des PLP Synthasekomplexes involviert ist. Aus diesem Grund wurden verschiedene C-terminale Deletionsmutanten der PfPdx1 konstruiert und dabei bis zu 30 Aminosäuren entfernt. Diese Analysen ergaben, dass der C-Terminus vier verschiedene Funktionen besitzt: das Assembly der Pdx1 Untereinheiten zum Dodekamer, die Bindung des Pentosesubstrats Ribose 5-Phosphat, die Bildung des Intermediats I320 und schließlich die PLP Synthese. Diese unterschiedlichen Funktionen wurden durch verschiedene Deletionsvarianten identifiziert. Darüber hinaus waren alle Deletionsvarianten in der Lage, die Glutaminase Pdx2 zu aktivieren, was zeigt, dass das Dodekamer nicht Vorraussetzung für die Glutaminaseaktivität ist. Aufgrund der geringen PLP Syntheseaktivität in vitro wurde vermutet, dass der PfPdx1/PfPdx2 Komplex durch einen zusätzlichen Faktor aktiviert wird. Daher wurde versucht, mittels Yeast 2-Hybrid, basierend auf einer PCR-amplifizierten P. falciparum 3D7 cDNA-Bibliothek als bait und PfPdx1 als prey, einen Interaktionspartner zu identifizieren. Mehrere Klone wurden gewonnen, die alle einen Bereich des Mal13P1.540, einem putativen Hsp70 Proteins, enthielten. Jedoch scheiterten alle Versuche, die Protein-Protein-Interaktion mit rekombinant exprimierten Protein zu bestätigen. Ebenso war es nicht möglich, das vollständige Mal13P1.540 rekombinant zu exprimieren sowie dessen Lokalisation in vivo zu bestimmen. Daher bleibt die Interaktion von PfPdx1 und Mal13P1.540 ungeklärt. Neben der Vitamin B6 Biosynthese konnten auch einige Gene des Shikimatweges in Plasmodium identifiziert werden. In P. berghei konnten der C-terminale Teil der 3-Dehydroquinatsynthase (2) sowie die Shikimatkinase (5) und die 5-Enoylpyruvylshikimat 3-Phosphatsynthase (6) in einem open reading frame (ORF) identifiziert werden, der dieselbe genetische Organisation aufweisen wie der Arom-Komplex der Hefen. Mit Hilfe eines Komplementationsassay wurde die Funktionalität dieses ORFs überprüft. Dazu wurden S. cerevisiae BY4741Δaro1, ein Hefestamm ohne funktionalen Arom-Komplex, mit dem Pb2_6_5_ABC Fragment transformiert. Die so transformierten Hefen waren nicht in der Lage, auf Mangelplatten ohne aromatische Aminosäuren zu wachsen, was zeigte, dass das Pb2_6_5_ABC Konstrukt den BY4741Δaro1 Phänotyp nicht komplementieren konnte. Der Versuch, mit Hilfe des Baculovirussytems rekombiant exprimiertes Protein zu erhalten, verlief erfolglos. Ebenso war es nicht möglich, Teile des Proteins für Immunisierungen zu exprimieren. Daher bleibt die Funktionalität des Pb2_6_5_ABC Konstruktes ungeklärt. / Malaria is a serious burden of mankind causing over one million deaths a year. In view of the raising number of resistances to common drugs there is an urgent need for the development of new antimalarial drugs. In this respect, the vitamin B6 biosynthesis and the shikimate pathway are of particular interest, since these synthesis pathways are only present in the malarial parasites and not in their human host. Given their essentiality for the parasite, they would represent ideal targets for antimalarial drug development. Previous studies revealed that Plasmodium falciparum is able to produce PLP de novo through a bifunctional enzyme complex composed of the proteins Pdx1 and Pdx2, of which Pdx1 is the actual synthase and Pdx2 the glutaminase partner providing the nitrogen for the ring system. In addition, the parasites possess a salvage pathway for PLP, of which pyridoxal kinase, PdxK, is a key player. Knockout studies of the pdx1 in the rodent malaria system P. berghei showed, that pbpdx1 is required for the optimal development of parasite blood stages but is not essential for parasite survival. Here, I investigated the effect of a pbpdxK(-) knockout in the same system. A monoclonal knockout strain was obtained, indicating that PbPdxK is not essential for the survival of the parasite. Blood stages were not affected by the knockout. However, in the mosquito stages pbpdxK(-) showed a tremendous reduction of sporozoites numbers in the midgut and in the salivary glands, indicating that PbPdxK is essential for the survival of sporozoites. It was then also tried to knockout pfpdx1, pfpdx2 and pfpdxK in the P. falciparum 3D7 strain by using the single cross over strategy. However, no integration of the constructs in the corresponding gene locus could be detected by a PCR approach. Also an approach to complement the loss of endogenous gene function by generating a functional gene copy upon recombination failed. Thus, it remains unclear if pfpdx1, pfpdx2 and pfpdxK can be knocked out or are inaccessible for gene targeting in P. falciparum. Cultivation of P. falciparum 3D7 parasites in medium deficient of vitamin B6 showed no effect on the growth rate of the parasites. Analysis of protein extracts of these parasites revealed an upregulation of PfPdxK expression, whereas the level of PfPdx1 remained stable. Thus it seems that the parasite is fully able to compensate vitamin B6 malnutrition by the increased usage of the salvage pathway. Previous studies on the activity of the PLP synthase complex indicated that the C-terminal end of Pdx1 is involved in PLP formation. Therefore several C-terminal deletion mutants of PfPdx1 were constructed, removing up to 30 amino acids. These analyses revealed that the C-terminus has four distinct functionalities: assembly of the Pdx1 monomers, binding of the pentose substrate (ribose 5-phosphate), formation of the reaction intermediate I320, and finally PLP synthesis. Deletions of distinct C-terminal regions distinguish between these individual functions. All variants were able to activate the glutaminase PfPdx2, indicating that the dodecameric structure is not a prerequisite for Pdx2 activation. Due to the low PLP synthase activity in vitro it was assumed that the PfPdx1/PfPdx2 complex maybe activated by an additional protein. Hence a yeast 2-hybrid assay was performed, using PfPdx1 as prey and a PCR-amplified cDNA-library of P. falciparum 3D7 as bait. Several clones were detected on high stringency plates, containing all a region of Mal13P1.540, a putative Hsp70 protein. Trials to confirm protein-protein interaction with recombinantly produced proteins failed as well as protein expression of full length Mal13P1.540. It was also not possible to determine the localisation of Mal13P1.540 in vivo. Thus, an interaction of PfPdx1 with Mal13P1.540 could so far not be verified. Besides the vitamin B6 biosynthesis, some genes of the shikimate pathway were identified in Plasmodium. In P. berghei, the C-terminal part of the dehydroquinatesynthase (2) as well as the shikimate kinase (5) and 5-enoylpyruvylshikimate 3-phosphatesynthase (6) were found in a single open reading frame having the same organisation as the arom-complex of yeast. To proof the functionality of these genes a complementation assay with S. cerevisiae BY4741Δaro1 with the Pb2_6_5_ABC construct, comprising the above mentioned genes, was performed. However, transformded yeast strains were not able to grow on minimal media without aromatic amino acids, indicating that they were not able to produce chorismate. Recombinant expression of this constructs in the baculovirussystem did not yield any detectable protein. Expression of parts of this protein for immunisation was also not successful. Hence, the functionality of this protein remains to be established.

Graduation date: 1992

Graduation date: 1994