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Characterisation of rat and human cytosolic cysteine conjugate #beta#-lyaseHarries, Helen M. January 1997 (has links)
No description available.
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Donor Substrate Specificity of Bovine Kidney Gamma-GlutamyltransferaseAgblor, Anita 14 December 2012 (has links)
Mammalian γ-glutamyltransferase (GGT) is a glycoprotein consisting of two subunits - a light chain and a heavy chain. The light chain contains the catalytic activity; the heavy chain anchors the protein to the membrane. GGT catalyzes the hydrolysis of the γ-glutamyl isopeptide bond of glutathione conjugates, releasing glutamic acid, or the transfer of the γ-glutamyl group to an acceptor substrate. The specificity of the enzyme for xenobiotic donor substrates has not been fully characterized. The transpeptidation activity of bovine kidney GGT was measured with glycylglycine as acceptor substrate and several glutathione conjugate donor substrates, representative of detoxication products of polycyclic aromatic xenobiotics. HPLC separation with UV detection was used for quantitation. The commonly-used chromogenic donor substrate γ-glutamyl-p-nitroanilide was also tested. Michaelis constants (Km) were obtained for γ-glutamyl-p-nitroanilide (0.74 mM), 4-nitrobenzyl glutathione (0.075 mM), 2,4-dinitrophenyl glutathione (0.30 mM), 4-methylbiphenylyl glutathione (0.12 mM), 1-menaphthyl glutathione (0.23 mM), and 9-methylanthracenyl glutathione (0.22 mM), indicating that enzyme activity is affected, but not strongly, by the nature of the S-substituent attached to glutathione, and there is a slight trend of higher Km values with bulkier aromatic S-substituents.
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Hydrolysis of S-aryl-cysteinylglycine conjugates catalyzed by porcine kidney cortex membrane dipeptidasePoon, James 31 August 2012 (has links)
Following conjugation with glutathione, xenobiotics are converted into cysteinylglycine conjugates, cysteine conjugates, and, finally, mercapturic acids. The structural factors determining the activities of dipeptidases for the metabolism of toxicologically-relevant cysteinylglycine conjugates are not well understood. I purified porcine kidney cortex membrane dipeptidase (MDP) to homogeneity, via phosphatidylinositol-specific phospholipase C-mediated cleavage of the protein’s membrane anchor and cilastatin affinity chromatography. The homodimeric structure of the MDP protein was confirmed by mass spectrometry. To test the enzyme activity of purified MDP, the cysteinylglycine conjugates of 1-(chloromethyl)naphthalene, 4-nitrobenzyl chloride, and 1-chloro-2,4-dinitrobenzene were synthesized and HPLC separation methods for their quantitation were developed. MDP catalyzed the hydrolysis of all three conjugates, but the rate of this activity was strongly dependent on the nature of the substituent on the cysteine sulfur atom.
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Mercaptursäure und Nukleosidaddukt im Harn als Biomarker in 1-Hydroxymethylpyren-exponierten RattenMa, Lan January 2002 (has links)
1-Methylpyren (MP) ist hepatokanzerogen in neugeborenen männlichen Mäusen. Durch Hydroxylierung an der benzylischen Stelle und anschließende Sulfonierung wird MP zu DNA-reaktivem 1-Sulfooxymethylpyren (SMP) aktiviert. In der Ratte führt die Exposition des benzylischen Alkohols, 1-Hydroxymethylpyren (HMP), zur DNA-Adduktbildung in verschiedenen Geweben. Eventuelle Konsequenz der Toxifizierung ist die Ausscheidung entsprechender Mercaptursäure und Nukleosidaddukt im Harn, welche aufgrund ihrer Herkunft als Biomarker eignen könnten. In dieser Arbeit wird die Ausscheidung der Mercaptursäure und des N2-Desoxyguanosinadduktes in HMP-exponierten Ratten untersucht. <br />
Nach der Applikation von HMP bzw. MP wurden weniger als 1 % der Dosis als MPMA über Urin und Faeces ausgeschieden (0 - 48 h). Die Ausscheidung erfolgt hauptsächlich in den ersten 24 h nach der Applikation. MPdG konnte weder in Urin noch in Faeces der HMP-behandelten Tieren identifiziert werden. Nach direkter SMP-Applikation wurde MPdG nur in sehr geringe Menge (weniger als 0,9 ppm in 12 h) im Urin gefunden. <br />
Aufgrund der geringen Menge eignet sich MPdG nicht als Biomarker. MPMA dagegen, lässt sich analytisch gut erfassen. Es sollte daher untersucht werden, ob MPMA die Toxifizierung des HMP wiederspiegelt. Die Voraussetzung dafür ist die Kenntnisse über das Metabolismusmuster von HMP. <br />
Es wurde daher umfassende Untersuchungen zum Metabolismus des HMP durchgeführt. Die Ergebnisse zeigten, dass mehr als 80 % der Metaboiten in ihrer oxidierten Form (PCS, deren Glucuronsäure-Konjugate sowie phenolische Sulfatester der PCS) ausgeschieden wurden. Demnach spielt die Oxidation des HMP zu PCS eine sehr wichtige Rolle bei der Detoxifizierung und Ausscheidung von HMP. Ferne konnte nachgewiesen werden, dass die Enzyme Alkohol- und Aldehyd-Dehydrogenase an der Oxidation von HMP beteiligt waren. Die Inhibitoren Disulfiram und Ethanol der o. g. Enzyme wurde daher zur Modulation der Detoxifizierung in vivo eingesetzt. Die Veränderungen in der Toxifizierung von HMP zu SMP wurden durch die SMP-Konzentration im Plasma, die DNA-Addukthäufigkeit und die MPMA-Ausscheidung erfasst. Die Vorbehandlung von Disulfiram und Ethanol führte zu tendentielle Erhöhung der SMP-Konzentration im Plasma, DNA-Addukthäufigkeit in der Leber und die MPMA-Ausscheidung. Bemerkenswert ist jedoch, dass bereits eine Dosis von 0,2 g Ethanol/kg Körpermasse bereits zu statistisch signifikanten Erhöhungen der MPMA-Ausscheidung bei weiblichen Ratten. / 1-Methylpyrene is hepatocarcinogenic in rodents. It is metabolized primarily to 1-hydroxymethylpyrene (HMP) by various cDNA-expressed rat and human cytochromes P450. HMP is activated to a highly reactive sulfuric acid ester, 1-sulfooxymethylpyrene (SMP), by sulphotransferases. In the rat, this activation pathway leads to the formation of DNA adducts in various tissues. Possible consequences of the toxification could be the excretion of the corresponding mercapturic acid and nucleosidadduct in urine and feces. Because of their origin, these substances should reflex the toxification process may be used as biomarkers. We investigate the excretion of 1-methylpyrenyl-mercapturic acid (MPMA) and the excretion of N2-(1-methylpyrenyl)-desoxyguanosin (MPdG) in urine and feces of HMP-treated rats. These studies showed that only a minor portion (< 1 %) of the administered dose of 1-HMP was excreted as mercapturic acid. MPdG could not be identified in urine and feces of HMP-treated rats. Treating rats with the active spieces sulfooxymethylpyrene, 0.9 ppm of the dose was found excreted within 12 h. I now investigated the alternative metabolic pathways of HMP. More than 50 % of the dose (administered intraperitoneally) was excreted as free 1-pyrenyl carboxylic acid and its glucuronic acid conjugate primarily in the urine. Other major urinary metabolites were phenolic sulpho conjugates of ring-oxidized 1-pyrenyl carboxylic acid (> 30 %). Minor metabolites were phenolic sulpho conjugates of HMP (< 5 %). The glucuronic acid conjugate of HMP was found in very small amounts. In total, > 80 % of the metabolites excreted were oxidized at the exocyclic carbon. This side-chain oxidation, probably catalyzed by alcohol and aldehyde dehydrogenases, appears to represent a detoxification pathway. Indeed, administration of ethanol shortly before the administration of HMP to rats increased the levels of SMP detected in blood, of DNA adducts formed in tissues and of mercapturic acid excreted. These effects were observed even at very low dose levels of ethanol (0.2 g per kg body weight). Similar effects were shown after administration of Disulfiram, an inhibitor of aldehyde dehydrogenase.
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Metabolismus von alkylierten polyzyklischen aromatischen Kohlenwasserstoffen : Einfluss der Struktur auf benzylische Hydroxylierung und Sulfonierung in vitro und Modulation des Metabolismus in vivo / Metabolism of alcylated polycyclic aromatic hydrocarbons : influence of the structure on benzylic hydroxylation and sulfonation in vitro and modulation of the metabolism in vivoBatke, Monika January 2008 (has links)
Die Toxizität und Kanzerogenität von rein aromatischen polyzyklischen aromatischen Kohlenwasserstoffen (PAK) ist seit Jahrzehnten bekannt und umfassend erforscht. Die alkylierten PAK (alkPAK) besitzen jedoch aufgrund ihrer Alkylgruppe eine weitere Möglichkeit zur Bioaktivierung und müssen daher gesondert betrachtet werden. Die Alkylgruppe wird zunächst hydroxyliert, anschließend zur Säure oxidiert oder direkt konjugiert. Entstehen hierbei instabile benzylische Sulfokonjugate, so können diese DNA-Addukte bilden und zu Mutationen führen.
In Hinblick auf die Bioaktivierung von alkPAK galt es daher zu klären welchen
Einfluss die Struktur auf die benzylische Hydroxylierung hat und welche humanen Formen der löslichen Sulfotransferasen besonders an der Umsetzung der alkPAK-Derivate beteiligt sind. Die Untersuchung der Albuminbindung von Schwefelsäureestern sowie ihre Aufnahme in Nierenzellen sollten Aufschluss hinsichtlich möglicher Transportvorgänge geben. Für die in-vivo-Situation wurde weiterhin die Modulation des Metabolismus ausgewählter benzylischer Alkohole durch verschiedene Nahrungsmittelbestandteile, Arzneimittel und Fremdstoffe an Ratten untersucht. Als Biomarker wurden benzylische Carbonsäuren im Urin und die entsprechenden Mercaptursäuren in Urin und Fäzes betrachtet.
Zunächst wurde anhand von Inkubationen mit Rattenlebermikrosomen festgestellt,
dass insbesondere größere Ringsystemen wie etwa alkylierte Benzo[a]pyrene im Gegensatz zu Methylpyrenen in wesentlich geringerem Umfang zum benzylischen Alkohol umgesetzt werden. Dies wurde auch in Untersuchungen mit humanen Lebermikrosomen bestätigt. Untersuchungen an einzelnen humanen Cytochromen P450 zeigten, dass insbesondere die durch PAK induzierbaren Formen hCYP1A1 und 1B1 hohe Umsatzraten aufwiesen. Die hepatisch exprimierten Formen hCYP1A2 und 3A4 waren jedoch auch zur Bildung der benzylischen Alkohole in der Lage.
Für die anschließende Sulfonierung der benzylischen Alkohole wurden besonders
hohe Aktivitäten mit den humanen Sulfotransferasen hSULT1A1, 1A2, 1C2 und 1E1
festgestellt. Aufgrund der Enzymexpression und der guten Durchblutung, die eine gute Substratversorgung ermöglicht, ist die Leber als Hauptort der benzylischen Hydroxylierung und Sulfonierung anzusehen.
Ergebnisse unserer Arbeitsgruppe zeigen jedoch, dass nach 1-Hydroxymethylpyren-Applikation bei Ratten die Niere die höchste Zahl an DNA-Addukten aufweist. Wegen der Fokussierung der Sulfonierung auf die Leber ist die systemische Verteilung der Schwefelsäureester die einzig plausible Erklärung. So wurde im Rahmen dieser Arbeit eine hochaffine Bindungsstelle für 2-Sulfoxymethylpyren an Albumin beschrieben und die Aufnahme von benzylischen Sulfaten durch die humanen organischen Anionentransporter hOAT1, 3 und 4 in Nierenzellen in vitro gezeigt.
Für die in-vivo-Situation wurde der Einfluss von Ethanol, 4-Methylpyrazol, Pentachlorphenol, Quercetin und Disulfiram untersucht. Neben der durch die Detoxifizierung mittels Alkoholdehydrogenase und Aldehyddehydrogenase entstandenen benzylischen Carbonsäure kann als Biomarker die entsprechende Mercaptursäure herangezogen werden. Sie ist ein indirekter Nachweis für die reaktiven und toxischen benzylischen Sulfate der alkPAK. Für die beiden im Tierversuch eingesetzten benzylischen Alkohole (1-Hydroxymethylpyren und 1-Hydroxymethyl-8-methylpyren) konnte sie in Urin und Fäzes nachgewiesen werden. Es wurde jedoch ein deutlicher Unterschied in der gebildeten Menge sowie der Verteilung zwischen Urin und Fäzes für die beiden Mercaptursäuren festgestellt. Hierfür sind wahrscheinlich Unterschiede im Transport der benzylischen Schwefelsäureester sowie der Spezifität der an der Mercaptursäurebildung beteiligten Enzyme verantwortlich. In diesem Zusammenhang konnte gezeigt werden, dass der humane organische Anionentransporter hOAT1 1,8-Dimethylpyrenmercaptursäure nicht und der hOAT3 nur mit niedrigen Umsatzraten transportiert.
Bei den Modulatoren zeigte die Gabe der kompetitiven Alkoholdehydrogenase-Hemmstoffe Ethanol und 4-Methylpyrazol die Bedeutung der Alkoholdehydrogenasen für die Entgiftung der benzylischen Alkohole: Die Oxidation zur entsprechenden Carbonsäure war reduziert und die Bildung der Mercaptursäure erhöht. Eine Hemmung der Toxifizierung vermittelt durch Sulfotransferase-Inhibitoren konnte nur für Pentachlorphenol beim Metabolismus des 1-Hydroxymethylpyrens beobachtet werden. Gleichzeitig erwies sich Pentachlorphenol als kompetitiver Alkoholdehydrogenase-Inhibitor, da eine signifikant geminderte Carbonsäureausscheidung zu beobachten war. Bei 1-Hydroxymethyl-8-methylpyren traten diese Effekte nicht auf. Die unterschiedlichen bzw. unterschiedlich starken Effekte der Modulatoren beim Metabolismus der verschiedenen benzylischen Alkohole bestätigen die Beobachtungen aus den in-vitro-Untersuchungen, dass unterschiedliche Enzym- und Transporteraffinitäten und –aktivitäten vorliegen. / The toxicity and carcinogenicity of purely aromatic polycyclic aromatic hydrocarbons (PAH) is known since decades and has been thoroughly investigated. Compared to the purely aromatic PAH the alcylated PAH (alcPAH) can additionally be biologically activated because of their alcyl group. The alcyl group is hydroxylated and subsequently oxidised to the corresponding acid or conjugated. If unstable benzylic sulfoconjugates arrise from this bioactivation DNA adducts may be formed and could induce mutations.
Concering the bioactivation of alcPAH this work should help to get to know which influence the structure has on the benzylic hydroxylation and it should be clarified which forms of human soluble sulfotransferases catalyse the sulfonation of benzylic alcohols. Furthermore the albumin binding of sulfuric acid esters and the uptake into kidney cells by human organic anion transporters in vitro have been analysed to get inside into transport processes. For the in vivo situation the modulation of enzyme
activities by food compounds, pharmaceuticals and xenobiotics is of
interest. As biomarkers the respective benzylic carboxylic acid and
mercapturic acid were measured in urine only and feces.
By the use of incubations with rat liver microsomes it turned out that larger
ring systems were benzylically hydroxylated to a remarkable less extent then alcyl pyrenes. This observation was also made for human liver microsomes. In vitro experiments addressing the activity of single human cytochromes P450 revealed that PAH inducable forms hCYP1A1 and 1B1 had highest hydroxylation rates, but also the hepatically expressed forms hCYP3A4 and CYP1A2 catalysed the benzylic hydroxylation. The subsequently following sulfonation of the benzylic alcohols was found to be catalysed with high formation rates by human sulfotransferase hSULT1A1, 1A2, 1C2 and 1E1. Due to the enzyme expression and the high blood circluation ensuring the substrate supply it can be assumed that liver is the main organ for benzylic hydroxylation and sulfonation.
Nevertheless results from our group showed that after 1-hydroxy methyl pyrene exposure, rats had higher levels of DNA adducts in kidneys than in liver. Thus, it has to be assumed that the sulfuric acid esters are systemically distributed. In the course of this work a high affinity albumin binding site for 2-sulfoxy methyl pyrene
was identified and the uptake of sulfuric acid esters mediated by
human organic anion tranporter 1, 3 and 4 to kidneys cells in vitro was shown.
For the further estimation of the in vivo bioactivation of alcPAH the
modulation of enzyme activities by ethanol, 4-methylpyrazole, quercetin, pentachlorophenol and disulfiram was explored. The carboxylic acids formed via alcohol dehydrogenase and aldehyde dehydrogenase were used as biomarkers as well as the respective mercapturic acids. The occurence of the mercapturic acids is an indirect proof for the reactive and toxic benzylic sulfo conjugates. In the urine and fecal samples of rats treated with either 1-hydroxymethyl pyrene or 1-hydroxymethyl 8-methyl pyrene the corresponding mercapturic acids of the sulfuric acid esters were
found. Even though the absolute amount excreted and the distribution in urine and fecal samples were quite different. This observation may be explained by differences in transport of the sulfuric acid esters as well as by different specificities of the enzymes responsable for mercapturic acid formation. Additionally it was shown that the human organic anion transporter 1 does not transport 1,8-dimethyl pyrenyl mercapturic acid and the human organic anion transporter 3 only with very little turnover. Whereas 1-methyl pyrenyl mercapturic acid was well transported by both of these proteins.
With regard to the modulation the concurrent application of ethanol or 4-methyl pyrazole to rats revealed the important role of alcohol dehydrogenase for the detoxification of benzylic alcohols: The oxidation leading to the corresponding carboxylic acid was remarkably reduced and the excretion of the mercapturic acid via urine and feces was enhanced. In order to observe an inhibition of sulfotransferases
pentachlorophenol and quercetine were concurrently applied to rats. An inhibitory effect by the means of an reduced excretion of mercapturic acid was only observed for pentachlorophenol in animals treated with 1-hydroxymethyl pyrene. In addition it turned out, that pentachlorophenol was a potent competitive alcohol dehydrogenase inhibitor as the renal excretion of the corresponding carboxylic acid was remarkably reduced. For 1-hydroxymethyl 8-methyl pyrene this modulation was not observed. These differences in effects and strenght of effects may be ascribed to different enzymatic and transport affinities and activities which have already been observed in in vitro experiments.
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Urinary 1,4–dihydroxynonene mercapturic acid (DHN–MA) and 8–hydroxy–2'–deoxyguanosine (8–OHdG) as markers of oxidative damage : the SABPA study / by Leandrie SteenkampSteenkamp, Leandrie January 2010 (has links)
The human body has evolved certain defence mechanisms to cope with the high occurrence of free radicals. These radicals are obtained endogenously from the mitochondria, peroxisomes, the cytochrome P450 (CYP 450) system and neutrophils, or exogenously from the environment. Lack of antioxidants and/or increased production of free radicals will result in oxidative stress, which has been implicated in certain human diseases such as hypertension, inflammation, ageing, autoimmunity, atherosclerosis, Parkinson?s disease, cancer and diabetes.
Although the initial aim was to standardise a single assay to quantify both 8–OHdG and DHN–MA, this could not be achieved in this study due to the vast difference in the chemical properties of these two metabolites. Following the decision to use two separate assays for the quantification of the mentioned biomarkers, the 8–OHdG assay was standardised and validated. The intrabatch variation of the assay was 4.18% and the interbatch variation was 17.37%. Unfortunately, the DHN–MA assay could not be standardised within the time frame of this study due to experimental difficulties. Therefore, only urinary 8–OHdG and serum ROS levels were quantified.
Urinary 8–OHdG levels were measured in 409 participants (209 Caucasians, 101 males and 108 females and 200 Africans, 100 males and 100 females) from the SABPA study. After removal of outliers from the data matrix, the effect of gender and ethnicity was investigated on the measured urinary 8–OHdG levels. No significant difference in the urinary 8–OHdG levels between Caucasian males (n=87) and females (n=96) were observed (p = 0.68). A similar observation was made for the African males (n=86) and females (n=84), where no significant difference in 8–OHdG levels was detected (p = 0.053). Thus, from the results obtained in this study, it seems that urinary 8–OHdG levels are not influenced by gender. However, 8–OHdG levels were dramatically influenced by ethnicity. Caucasian males (n=87) excreted 70% higher amounts of 8–OHdG compared to African males (n=86) (p < 0.001). Caucasian females (n=96) also excreted larger urinary 8–OHdG amounts (42%) compared to African females (n=84) (p < 0.001). Therefore, it seems that urinary 8–OHdG levels are dramatically influenced by ethnicity. Finally, urinary 8–OHdG levels were compared to serum ROS levels, but no significant correlation between the measured metabolites was observed (r = –0.045). Hence, urinary 8–OHdG and serum ROS levels are not related in these subjects.
Even though the initial aim of this study was to standardise an analytical method to quantify both urinary 8–OHdG and DHN–MA, this could not be achieved due to time constraints. vi
The human body has evolved certain defence mechanisms to cope with the high occurrence of free radicals. These radicals are obtained endogenously from the mitochondria, peroxisomes, the cytochrome P450 (CYP 450) system and neutrophils, or exogenously from the environment. Lack of antioxidants and/or increased production of free radicals will result in oxidative stress, which has been implicated in certain human diseases such as hypertension, inflammation, ageing, autoimmunity, atherosclerosis, Parkinson?s disease, cancer and diabetes.
Although the initial aim was to standardise a single assay to quantify both 8–OHdG and DHN–MA, this could not be achieved in this study due to the vast difference in the chemical properties of these two metabolites. Following the decision to use two separate assays for the quantification of the mentioned biomarkers, the 8–OHdG assay was standardised and validated. The intrabatch variation of the assay was 4.18% and the interbatch variation was 17.37%. Unfortunately, the DHN–MA assay could not be standardised within the time frame of this study due to experimental difficulties. Therefore, only urinary 8–OHdG and serum ROS levels were quantified.
Urinary 8–OHdG levels were measured in 409 participants (209 Caucasians, 101 males and 108 females and 200 Africans, 100 males and 100 females) from the SABPA study. After removal of outliers from the data matrix, the effect of gender and ethnicity was investigated on the measured urinary 8–OHdG levels. No significant difference in the urinary 8–OHdG levels between Caucasian males (n=87) and females (n=96) were observed (p = 0.68). A similar observation was made for the African males (n=86) and females (n=84), where no significant difference in 8–OHdG levels was detected (p = 0.053). Thus, from the results obtained in this study, it seems that urinary 8–OHdG levels are not influenced by gender. However, 8–OHdG levels were dramatically influenced by ethnicity. Caucasian males (n=87) excreted 70% higher amounts of 8–OHdG compared to African males (n=86) (p < 0.001). Caucasian females (n=96) also excreted larger urinary 8–OHdG amounts (42%) compared to African females (n=84) (p < 0.001). Therefore, it seems that urinary 8–OHdG levels are dramatically influenced by ethnicity. Finally, urinary 8–OHdG levels were compared to serum ROS levels, but no significant correlation between the measured metabolites was observed (r = –0.045). Hence, urinary 8–OHdG and serum ROS levels are not related in these subjects.
Even though the initial aim of this study was to standardise an analytical method to quantify both urinary 8–OHdG and DHN–MA, this could not be achieved due to time constraints.
However, an LC–MS/MS analytical assay was standardised and validated for the quantification of urinary 8–OHdG. The method proved reliable for the quantification of 8–OHdG from urine samples and can thus be used for further studies on oxidative DNA damage. / Thesis (M.Sc. (Biochemistry))--North-West University, Potchefstroom Campus, 2011.
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Urinary 1,4–dihydroxynonene mercapturic acid (DHN–MA) and 8–hydroxy–2'–deoxyguanosine (8–OHdG) as markers of oxidative damage : the SABPA study / by Leandrie SteenkampSteenkamp, Leandrie January 2010 (has links)
The human body has evolved certain defence mechanisms to cope with the high occurrence of free radicals. These radicals are obtained endogenously from the mitochondria, peroxisomes, the cytochrome P450 (CYP 450) system and neutrophils, or exogenously from the environment. Lack of antioxidants and/or increased production of free radicals will result in oxidative stress, which has been implicated in certain human diseases such as hypertension, inflammation, ageing, autoimmunity, atherosclerosis, Parkinson?s disease, cancer and diabetes.
Although the initial aim was to standardise a single assay to quantify both 8–OHdG and DHN–MA, this could not be achieved in this study due to the vast difference in the chemical properties of these two metabolites. Following the decision to use two separate assays for the quantification of the mentioned biomarkers, the 8–OHdG assay was standardised and validated. The intrabatch variation of the assay was 4.18% and the interbatch variation was 17.37%. Unfortunately, the DHN–MA assay could not be standardised within the time frame of this study due to experimental difficulties. Therefore, only urinary 8–OHdG and serum ROS levels were quantified.
Urinary 8–OHdG levels were measured in 409 participants (209 Caucasians, 101 males and 108 females and 200 Africans, 100 males and 100 females) from the SABPA study. After removal of outliers from the data matrix, the effect of gender and ethnicity was investigated on the measured urinary 8–OHdG levels. No significant difference in the urinary 8–OHdG levels between Caucasian males (n=87) and females (n=96) were observed (p = 0.68). A similar observation was made for the African males (n=86) and females (n=84), where no significant difference in 8–OHdG levels was detected (p = 0.053). Thus, from the results obtained in this study, it seems that urinary 8–OHdG levels are not influenced by gender. However, 8–OHdG levels were dramatically influenced by ethnicity. Caucasian males (n=87) excreted 70% higher amounts of 8–OHdG compared to African males (n=86) (p < 0.001). Caucasian females (n=96) also excreted larger urinary 8–OHdG amounts (42%) compared to African females (n=84) (p < 0.001). Therefore, it seems that urinary 8–OHdG levels are dramatically influenced by ethnicity. Finally, urinary 8–OHdG levels were compared to serum ROS levels, but no significant correlation between the measured metabolites was observed (r = –0.045). Hence, urinary 8–OHdG and serum ROS levels are not related in these subjects.
Even though the initial aim of this study was to standardise an analytical method to quantify both urinary 8–OHdG and DHN–MA, this could not be achieved due to time constraints. vi
The human body has evolved certain defence mechanisms to cope with the high occurrence of free radicals. These radicals are obtained endogenously from the mitochondria, peroxisomes, the cytochrome P450 (CYP 450) system and neutrophils, or exogenously from the environment. Lack of antioxidants and/or increased production of free radicals will result in oxidative stress, which has been implicated in certain human diseases such as hypertension, inflammation, ageing, autoimmunity, atherosclerosis, Parkinson?s disease, cancer and diabetes.
Although the initial aim was to standardise a single assay to quantify both 8–OHdG and DHN–MA, this could not be achieved in this study due to the vast difference in the chemical properties of these two metabolites. Following the decision to use two separate assays for the quantification of the mentioned biomarkers, the 8–OHdG assay was standardised and validated. The intrabatch variation of the assay was 4.18% and the interbatch variation was 17.37%. Unfortunately, the DHN–MA assay could not be standardised within the time frame of this study due to experimental difficulties. Therefore, only urinary 8–OHdG and serum ROS levels were quantified.
Urinary 8–OHdG levels were measured in 409 participants (209 Caucasians, 101 males and 108 females and 200 Africans, 100 males and 100 females) from the SABPA study. After removal of outliers from the data matrix, the effect of gender and ethnicity was investigated on the measured urinary 8–OHdG levels. No significant difference in the urinary 8–OHdG levels between Caucasian males (n=87) and females (n=96) were observed (p = 0.68). A similar observation was made for the African males (n=86) and females (n=84), where no significant difference in 8–OHdG levels was detected (p = 0.053). Thus, from the results obtained in this study, it seems that urinary 8–OHdG levels are not influenced by gender. However, 8–OHdG levels were dramatically influenced by ethnicity. Caucasian males (n=87) excreted 70% higher amounts of 8–OHdG compared to African males (n=86) (p < 0.001). Caucasian females (n=96) also excreted larger urinary 8–OHdG amounts (42%) compared to African females (n=84) (p < 0.001). Therefore, it seems that urinary 8–OHdG levels are dramatically influenced by ethnicity. Finally, urinary 8–OHdG levels were compared to serum ROS levels, but no significant correlation between the measured metabolites was observed (r = –0.045). Hence, urinary 8–OHdG and serum ROS levels are not related in these subjects.
Even though the initial aim of this study was to standardise an analytical method to quantify both urinary 8–OHdG and DHN–MA, this could not be achieved due to time constraints.
However, an LC–MS/MS analytical assay was standardised and validated for the quantification of urinary 8–OHdG. The method proved reliable for the quantification of 8–OHdG from urine samples and can thus be used for further studies on oxidative DNA damage. / Thesis (M.Sc. (Biochemistry))--North-West University, Potchefstroom Campus, 2011.
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