The primary goal of my studies was to elucidate the mechanisms for the well-recognized interaction between two nutrients, vitamins E and K. The outcomes from my studies assess mechanisms for adverse effects of vitamin E and provide novel information on mechanisms for vitamin K homeostasis. These findings will provide information relevant for assessing optimal intakes of vitamins E and K. This dissertation presents studies aimed at evaluating three different mechanisms by which vitamin K status could be decreased by increases in whole body vitamin E concentrations in rats supplemented with vitamin E by subcutaneous injections (100 mg α-tocopherol (α-T)/ kg body weight per day), the model system developed in the Traber lab. The tested mechanisms by which vitamin E leads to reduced vitamin K status were: 1) increasing vitamin K metabolism, 2) decreasing menaquinone-4 (MK-4) synthesis from dietary phylloquinone (PK) and 3) potentiating vitamin K excretion through xenobiotic pathways. Two approaches were undertaken to evaluate the hypothesis that vitamin E increases vitamin K metabolism. In Aim 1.1, the in vitro omega-hydroxylation of vitamin K by human cytochrome P450 CYP4F2 (expressed in insect microsomes) was tested because CYP4F2 is considered the limiting step in the catabolism of both vitamins. Chapter 2 shows that CYP4F2 more rapidly hydroxylated vitamin K compared with vitamin E. Moreover, vitamin E did not stimulate vitamin K metabolism in vitro. Thus, it is unlikely vitamin E stimulates vitamin K metabolism in vivo by direct interaction with the CYP4F2 enzyme-substrate complex. In Aim 1.2, the in vivo urinary and biliary excretion of vitamin K metabolites was investigated. Chapter 3 shows that α-T-injected rats significantly increased urinary excretion of vitamin E catabolites, but no increases in urinary vitamin K catabolites were found. Chapter 4 shows that α-T-injected rats increased biliary excretion of 5C-aglycone, a major vitamin K catabolite shared by MK-4 and PK. However, the overall in vivo excretion of vitamin K catabolites was not changed when urinary excretion was also taken into account. Aim 2 evaluated the hypothesis that α-T interferes with the conversion of PK to MK-4 because α-T and PK have similar side-chains. In Aim 2.1, conversion of PK or MN to MK-4 was tested in vivo. Rats were fed semi-purified diets containing equimolar concentrations of either PK or MN for 10 days, then α-T injections were undertaken. Chapter 3 shows that extra-hepatic tissues from α-T injected rats contained significantly lower MK-4 concentrations irrespective of whether the rats were fed PK or MN. These findings show that if vitamin E is interfering with the metabolic mechanism of MK-4 synthesis, then it is not specific to the cleavage of PK's side chain. In Aim 2.2, conversion of deuterium-labeled PK (d₄-PK) to d₄-MK-4 was used to evaluate the extra-hepatic tissue uptake of d₄-PK in α-T-injected rats. Rats were fed semi-purified diets containing equimolar concentrations of d₄-PK similar to my previous study for 10 days then α-T injections were undertaken for 7 days. Chapter 5 shows that total (labeled and unlabeled) vitamin K concentrations decreased in extra-hepatic tissues from α-T injected rats fed d₄-PK. Both d₄-MK-4 and d₄-PK concentrations decreased, suggesting that MK-4 concentrations were dependent upon those of d₄-PK. These findings suggest that PK, and not MN, is the primary substrate for MK-4 synthesis in extra-hepatic tissues. Moreover, both d₄-MK-4 and d₄-PK decreased in α-T-injected rats demonstrating that vitamin E's untoward effect on vitamin K status is likely a mechanism that is shared by both vitamin K forms and not specific to MK-4 synthesis. Recycling of vitamin K from the epoxide was not examined in this study and interference with the recycling mechanism for either PK or MK-4 in α-T injected rats has not been examined. Vitamin E metabolism is greatly increased in α-T-injected rats by increasing various xenobiotic pathways. Thus, vitamin K status was hypothesized to decrease in α-T-injected rats as a result of the up-regulation of these pathways. As shown in Aim 1, urinary vitamin K metabolite excretion was not increased in α-T-injected rats. In Aim 3.1, the biliary excretion of vitamins E and K were examined to evaluate whether the increased expression in biliary transporters, such as MDR1, led to increased vitamin K and E excretion via the bile. Chapter 4 shows that α-T increased in bile over the week of vitamin E injections and α-CEHC was the major vitamin E form excreted in bile. Although biliary PK secretion was unchanged and biliary MK-4 was undetectable, increased excretion of a major catabolite of both PK and MK-4, 5C-aglycone, was observed. In Aim, 3.2, the gene expression of enzymes and transporters in liver and extra-hepatic tissues as mechanisms involved in regulating their concentrations in these tissues was assessed. In Chapters 3 and 5, increased expression of biliary transporters were observed, one of which is known to bind the vitamin K intermediate MN as its substrate. It is possible other vitamin K catabolites, in addition to 5C-and 7C-aglycone, may have been excreted that were unaccounted for, e.g. MN or vitamin K epoxide metabolites. In summary, my studies have shown vitamin K status is decreased in α-T-injected rats because PK and MK-4 concentrations are decreased in many extra-hepatic tissues. Although metabolism of vitamin K was not stimulated in response to α-T injections, increased excretion of a vitamin K catabolite was measured in the bile; however it may not account for all of the vitamin K loss observed in tissues. Alternatively, transport of PK and MN to extra-hepatic tissues or MK-4 recycling may have been inhibited in response to vitamin E. Further studies are needed to distinguish between these mechanisms. / Graduation date: 2013

Universiẗat, Diss., 2006--Giessen.

Zugl.: Giessen, Univ., Diss., 2006

Thesis (Ph. D.)--University of Wisconsin--Madison, 1953. / Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references (leaves 53-54).

This thesis describes a series of pyridine-N-oxide derivatives of naphthoquinone that were prepared by the author. These compounds will be tested for medicinal activity by Parke-Davis and Company.

LD2668 .T4 1942 E3 / Master of Science

Im Jahre 2004 wurden in unserem Labor zwei Gene einer neuen Proteinfamilie kloniert, deren Charakterisierung seither im Gange ist. Das eine Protein, VKORC1, konnte durch Mutationsanalysen und biochemische Untersuchungen als eine zentrale Komponente des so genannten Vitamin K-Zyklus identifiziert werden. Vitamin K wird für die γ-Carboxylierung der Vitamin K-abhängigen Proteine wie z.B. der Gerinnungsfaktoren II, VII, IX und X als Cofaktor der γ-Glutamyl-Carboxylase benötigt. Da Vitamin K essentiell ist, wird seine Epoxidform vom Organismus wieder in eine physiologisch aktive Hydrochinon-Form überführt. Für diese Reaktion wird die Vitamin K-Epoxid-Reduktase (VKORC1) benötigt. Mutationen in VKORC1 führen einerseits zu einem erblich bedingten Mangel an Vitamin K-abhängigen Gerinnungsfaktoren vom Typ 2 (VKCFD2) mit starken Blutungen durch eine nicht oder nur unvollständig ablaufende Blutgerinnung. Das VKORC1 ist andererseits auch das Ziel von Medikamenten der Coumaringruppe, der sog. Vitamin K-Antagonisten, die zur Verhinderung einer unzeitigen Gerinnung eingesetzt werden. In höheren Dosen wird diese Substanzklasse als Rattenbekämpfungsmittel eingesetzt. Bei manchen Rattenpopulationen, aber auch bei einigen Patienten, ist die Wirksamkeit der Coumarine durch bestimmte Mutationen im VKORC1-Protein, welche eine Warfarinresistenz hervorrufen, erheblich eingeschränkt. Zu diesem VKORC1 existiert ein paraloges Protein, das Vitamin K-Epoxid-Reduktase Komplex 1-like 1 Protein (VKORC1L1), dessen Funktion bislang unbekannt ist und welches Gegenstand der vorliegenden Arbeit war. Es wurden unterschiedliche Methoden angewandt, um das VKORC1L1-Protein zu charakterisieren und seine mögliche Funktion(en) aufzuklären. Zum einen sollte die Herstellung einer Knockout-Maus dazu dienen, durch den erhaltenen Phänotyp Hinweise auf die mögliche physiologische Aufgabe zu erhalten. Allerdings gelangten die Versuche nur bis zur Generierung der Chimären, so dass dieses Teilprojekt nicht zum Abschluss gebracht werden konnte. Die biochemische Charakterisierung des Proteins zeigte eine Expression des VKORC1L1-Gens in allen untersuchten Geweben, wobei keine starke Expression für ein bestimmtes Gewebe ermittelt werden konnte. Es konnte gezeigt werden, dass das Protein Vitamin K-Epoxid auf gleiche Weise wie das VKORC1 recyceln kann und durch Warfarin gehemmt wird. Einige der in eingeführten VKORC1L1 Mutationen vermitteln darüber hinaus eine Warfarinresistenz. Des Weiteren wurden Enzymkinetiken für die Spezies Maus und Ratte sowie für die Stachelmaus erstellt. Die erhaltenen Werte für die Michaelis-Menten-Konstante und die Maximalgeschwindigkeit sind untereinander sehr ähnlich und sprechen für eine Oxido-Reduktase-Aktivität des VKORC1L1-Proteins. Bioinformatische Analysen konzentrierten sich auf die Aufklärung von konservierten Aminosäureresten im VKORC1L1. Dadurch konnten funktionell wichtige Positionen des Proteins ermittelt werden. Ein evolutiver Stammbaum konnte weiterhin zeigen, dass die paralogen Proteine VKORC1 und VKORC1L1 sehr wahrscheinlich aus einem gemeinsamen Vorläuferprotein bei der Entwicklung der Wirbeltiere aus einer Duplikation entstanden sind und nach der Entstehung der Landwirbeltiere ihre spezifischen Funktionen ausgebildet haben. Ein Homologievergleich zwischen den humanen Chromosomen 7 und 16 und den jeweiligen Chromosomen verschiedener Spezies zeigte, dass sich nach der Duplikation die Gene für das VKORC1 und das VKORC1L1 bei fast allen betrachteten Spezies unabhängig voneinander auf verschiedenen Chromosomen evolutiv entwickelt haben. Dies ist ein weiteres Indiz dafür, dass die Duplikation schon sehr lange zurück liegt. / In 2004 two genes of a new protein family were cloned in our institute. Since then the characterization of this protein family is in progress. Through mutation and biochemical analyses one protein, VKORC1, could be identified as a central component of the so-called vitamin K cycle. Vitamin K is required as a cofactor of the γ-glutamyl carboxylase for the γ-carboxylation of the vitamin K-dependent proteins such as the coagulation factors II, VII, IX and X. As vitamin K is essential, the epoxide form is again transferred by the organism into a physiologically active hydrochinone form. For this reaction vitamin K epoxide reductase (VKORC1) is necessary. On the one hand mutations in VKORC1 lead to a hereditary combined deficiency of vitamin K-dependent clotting factors of type 2 (VKCFD2) with heavy bleedings because of missing or insufficient blood coagulation. On the other hand VKORC1 is also the target of medical treatment with the coumarin derivative group, the so-called vitamin K antagonists, which are used for preventing untimely coagulation. In higher doses this class of substances is used as rodenticides. In some rat populations as well as in case of some human patients the efficiency of the coumarins is considerably reduced through specific mutations in the VKORC1 protein leading to a warfarin resistance. There is a paralogue protein to VKORC1, the vitamin K epoxide reductase complex 1-like 1 protein (VKORC1L1), the function of which is not known so far and which is the subject of this study. Different methods have been applied in order to characterize the VKORC1L1-protein and to explain its potential functions. One the one hand, the creation of a knockout mouse was to give information on potential physiological tasks through its specific phenotype. The experiments, however, were only successful as far as the creation of chimeras was concerned. Thus, this part of the project could not be completed totally. The biochemical characterization showed an expression of the VKORC1L1-gene in all tissues examined. It was, however, not possible to find a strong expression for a specific tissue. We were able to show that the protein can recycle vitamin K epoxide in the same way as VKORC1 and that it is inhibited by warfarin. Some of the mutations within the VKORC1L1 lead to a warfarin resistance. Moreover, enzyme kinetics were applied for the mouse, rat and acomys species. The calculated values of the Michaelis-Menten constant and of the maximal speed Vmax are very similar to each other and support an oxido-reductase activity for the VKORC1L1-protein. Bioinformatic analyses were focused on the explanation of the conserved amino acids within VKORC1L1. So, functionally important positions could be determined. Moreover, an evolutionary life tree showed that the paralogue proteins VKORC1 and VKORC1L1 have probably been arisen from a common precursor protein by duplication when vertebrates developed and formed its specific functions after the tetrapod vertebrates came into being. A homological comparison between the human chromosomes 7 and 16 and the corresponding chromosomes of different species showed that the duplication of the genes for VKORC1 and VKORC1L1 evolved independently of each other on different chromosomes within all species examined. This is a further indication that the duplication took place a long time ago.

Vitamin K ist ein essentieller Cofaktor für die posttranslationale Gamma-Carboxylierung von sog. Vitamin K-abhängigen Gerinnungsfaktoren, Knochenproteinen, Zellwachstum-regulierenden und weiteren Proteinen mit noch unbekannter Funktion. Defekte im Vitamin K-Stoffwechsel führen einerseits zu zwei verschiedenen Formen des familiären Mangels aller Vitamin K-abhängigen Gerinnungsfaktoren (VKCFD1 und 2) und andererseits zur Resistenz oder Hypersensitivität gegenüber Cumarinderivaten, wie Warfarin, die als Vitamin K-Antagonisten zur Antikoagulationstherapie bei thromboembolischen Erkrankungen, aber auch zur Bekämpfung von Ratten und Mäusen eingesetzt werden. Die Aufklärung und Charakterisierung der molekularen Ursachen dieser Erkrankungen wird in dieser Doktorarbeit anhand von Veröffentlichungen dokumentiert. Ausgehend von der Charakterisierung zweier Familien mit dem VKCFD2-Phänotyp, wird die Kartierung des VKCFD2-Locus auf dem kurzen Arm von Chromosom 16 beschrieben. Durch eine systematische Mutationssuche in der ca. 130 Gene umfassenden Kandidatenregion von Chromosom 16 konnte das für diese Erkrankung und die Warfarinresistenz ursächliche Gen ausfindig gemacht werden. Dabei handelt es sich um das Gen für die entscheidende Komponente der Vitamin K-Epoxid-Reduktase (VKORC1), die den Recycling-Prozess von Vitamin K im sog. Vitamin K-Zyklus katalysiert. Die Charakterisierung des VKORC1-Proteins umfasst dessen subzelluläre Lokalisation, den Vergleich orthologer Proteine in verschiedenen Species und die funktionelle Charakterisierung von rekombinant exprimiertem VKORC1. Durch positionsspezifische Mutagenesen und anschließende Expression in humanen Nierenzellen konnten mehrere für die Funktion der VKORC1 relevante Aminosäuren identifiziert werden. Die posttranslationale Modifikation der Vitamin K-abhängigen Proteine wird von der Gamma-Glutamyl-Carboxylase (GGCX) katalysiert. Defekte in diesem Enzym wurden von zwei verschiedenen Arbeitsgruppen als Ursache für die erste Form der VKCFD-Erkrankung nachgewiesen. In dieser Doktorarbeit werden drei weitere, von unserer Arbeitsgruppe identifizierte Mutationen im GGCX-Gen beschrieben, unter denen sich ein nachgewiesener Founder-Effekt an Position 485 des Proteins befindet. Die Arg485Pro-Variante wurde rekombinant in Insektenzellen exprimiert und konnte mittels kinetischer Studien als VKCFD1-verursachende Mutation verifiziert werden. / Vitamin K is an essential cofactor for the posttranslational gamma-carboxylation of the so-called vitamin K-dependent coagulation factors, bone proteins, cell growth regulating proteins and others of unknown function. Defects in vitamin K metabolism cause two different forms of combined deficiency of vitamin K-dependent coagulation factors (VKCFD type 1 and type 2) as well as resistance or hypersensitivity to coumarin derivates, such as warfarin, which act as vitamin K antagonists. Coumarins are used for anticoagulation therapy of thromboembolic diseases and in higher dosis also for rodent pest control. The aim of this thesis is to characterize the molecular basis of these diseases. This work has led to six publications. The VKCFD type 2 phenotype as described in two unrelated families was used to perform a homozygosity mapping of the VKCFD2 locus on the short arm of chromosome 16. A systematic mutation screening in the candidate region on chromosome 16 comprising approximately 130 putative genes resulted in the identification of the gene causative for VKCFD2 and the allelic phenotype warfarin resistance. This gene encodes the first identified component of the vitamin K epoxide reductase (VKORC1) which catalyzes the reduction of vitamin K epoxide as an important part of the so-called vitamin K cycle. Characterization of the VKORC1 protein includes its subcellular localization, comparison of orthologous proteins in different species and functional studies of the recombinant VKORC1. Amino acids which are relevant for protein structure or function were identified by site-directed mutagenesis experiments and subsequent expression in human embryonic kidney cells (HEK293). Posttranslational modification of the vitamin K-dependent proteins is catalysed by the gamma-glutamyl carboxylase (GGCX), an enzyme of the endoplasmic reticulum. Mutations in the gene encoding this enzyme were demonstrated to be causative for VKCFD type 1 by two different working groups. Our working group identified three additional mutations in the GGCX gene. Recurrent mutations at position 485 of the protein were shown to result from a founder effect. The Arg485Pro variant was recombinantly expressed in insect cells using the baculovirus system and could be verified as a causative mutation for the VKCFD1 phenotype by kinetic studies.

Thesis (Ph. D.)--University of Washington, 2001. / Vita. Includes bibliographical references (leaves 117-153).

This paper describes the preparation of pyridine derivatives of 2,3-dichloro-5(8?)-nitro-1,4-naphthoquinone. A method for the nitration of 2,3-dichloro-1,4-naphthoquinone is also described. Certain 4-n-alkyl, 3,4-dialkyl, and 4-cycloalkyl pyridine derivatives are caused to undergo condensation reactions with the nitrated naphthoquinone, giving rise to a series of compounds of the preceding type (X). All of the compounds prepared will be tested for anti-tubercular activity by Parke-Davis and Company.