Zinc Affects Cholesterol Oxidation Products and Fatty Acids Composition in Rats' Serum.

The purpose of this work was to evaluate the effect of the nanosized or microsized zinc (Zn) particles on fatty acid profile, enzyme activity and the level of cholesterol, squalene and oxysterols in rats with breast cancer. Rats (female, n = 24) were divided into the following groups: control, and two test groups, whose diets were enriched with either Zn microparticles (342 nm) or Zn nanoparticles (99 nm). All rats were treated twice with the carcinogenic agent; 7,12-dimethylbenz[a]anthracene. In rats whose diet was enriched with zinc (especially in the form of nanoparticles), the number and sizes of tumors were lower. Diet supplementation also significantly reduced the cholesterol (p = 0.027) and COPs (cholesterol oxidation products) levels (p = 0.011) in rats serum. Enriching the diet with Zn microparticles decreased the Δ6-desaturase activity (p < 0.001). Zn influences fatty acids' profile in rats' serum as well as inhibiting desaturating enzymes. A reduced amount of pro-inflammatory arachidonic acid derivatives may be the expected effect.

Modificação de proteínas por produtos de oxidação do colesterol: mecanismos e implicações biológicas / Modification of proteins by oxidation products of cholesterol: mechanisms and biological implications

O colesterol é um importante componente das membranas celulares em eucariotos superiores, desempenhando papéis estruturais e funcionais. O colesterol possui uma insaturação em sua estrutura sendo, portanto, alvo de oxidação mediada por espécies reativas de oxigênio e/ou nitrogênio. A oxidação não enzimática do colesterol gera, como produtos primários, os hidroperóxidos de colesterol. Tais moléculas, por sua vez, são altamente reativas e podem reagir com metais livres e/ou metaloproteínas, trazendo consequências à celula. Neste sentido, o primeiro capítulo deste trabalho tem como objetivo estudar a reação dos hidroperóxidos de colesterol (ChOOH) com o citocromo c (citc), uma heme proteína envolvida no transporte de elétrons na mitocôndria. Análises de espectroscopia no UV-Vis mostraram que o ChOOH promove o bleaching da banda Soret do citc de uma maneira dose-dependente. Mais ainda, esta reação leva à formação de radicais centrados em carbono tanto na proteína como no lipídeo, sugerindo uma redução homolítica do ChOOH. Como consequências, pode-se observar a oligomerização do citc, um processo que pode influenciar no transporte de elétrons bem como na sinalização para a apoptose. A partir da reação do citc com ChOOH podem surgir, direta ou indiretamente, outras espécies reativas, como aldeídos, cetonas e epóxidos. Dentre estas, destacam-se os aldeídos de colesterol, em particular o colesterol secoaldeído (CSec) e o carboxialdeído (ChAld), uma vez que foram encontrados elevados em placas ateroscleróticas e em tecidos cerebrais de pacientes com doenças neurodegenerativas. Tais espécies podem reagir com resíduos de aminoácidos provocando alterações estruturais e funcionais em proteínas. Neste sentido, o segundo capítulo deste trabalho tem como objetivo estudar a reação do ChAld com citc. Usando modelos mimétivos de membrana e espectrometria de massas, foi mostrado que o ChAld modifica covalentemente o citc por um mecanismo consistente com a formação de bases de Schiff. Tal modificação ocorre preferencialmente em resíduos de lisina que interagem com a membrana. Estas modificações influenciam na afinidade do citc pela membrana, aumentando sua aderência, o que pode ter influência no transporte de elétrons e sinalização para a apoptose. No terceiro e último capítulo deste trabalho nós buscamos uma ferramente analítica que permitisse analisar modificação de proteínas promovidas por produtos de oxidação de colesterol e outros esteróis. Em um estudo realizado em colaboração com o grupo do professor Porter na Universidade de Vanderbilt, utilizamos ensaios baseados em click chemistry para buscar proteínas modificadas. Para isso, foram sintetizados derivados de colesterol e 7-deidrocolesterol (7-DHC, precursor imediato do colesterol) contendo um grupo alquinil na sua cadeia lateral. Este grupo pode ser ligado a um grupo azida por meio de uma reação de cicloadição, em um processo conhecido como click chemistry. Após a síntese e caracterização dos derivados lipídicos contendo o grupo alquinil na cadeia lateral, células Neuro2a foram tratadas com o alquinil-7-DHC e o alquinil-colesterol para averiguar seu metabolismo. Análises por HPLC-MS/MS mostraram que ambos derivados contendo o grupo alquinil foram metabolisados e convertdos nos respectivos ésteres. Usando um modelo celular para a doença conhecida como Sindrome de Smith-Lemli-Opitz (SLOS), doença caracterizada pela deficiência na enzima 7-deidrocolesterol redutase, foi mostrado que o acúmulo característico de 7-DHC nos pacientes pode levar a uma maior modificação de proteínas promovidas por seus derivados, o que pode contribuir para o desenvolvimento da doença

Cholesterol is an important component of eukaryotic cellular membranes, where it has an influence in the fluidity and stability. Due to the presence of a double bond in its structure, cholesterol can be oxidized by reactive oxygen and nitrogen species. This non-enzymatic oxidation generates, as primary products, cholesterol hydroperoxides. Such molecules, in turn, are highly reactive and can react with free metal ions and/or metalloproteins, affecting cell metabolism. Therefore, the first chapter of the present study aims to investigate the reaction of cholesterol hydroperoxides (ChOOH) with cytochrome c (cytc), a heme protein involved in the mitochondrial electron transport. Spectroscopic analyses in the UV-Vis region showed that ChOOH induces a dose-dependent bleaching of cytc's Soret band. In addition, this reaction leads to the formation of carbon-centered radicals on both protein and lipid, suggesting a homolytic reduction of ChOOH. As consequences, cytc undergoes oligomerization, a process that can influence electron transport and apoptosis signaling. The reaction of cytc and ChOOH can produce, directly or indirectly, reactive species such as epoxides, aldehydes and ketones. Among them, cholesterol aldehydes, such as cholesterol secoaldehyde (CSec) and cholesterol carboxyaldehyde (ChAld), are of particular interest, since they were previously found elevated in atherosclerotic plaques and brain tissue of patients bearing neurodegenerative diseases. These species can also react with amino acid residues leading to protein denaturation and malfunction. With that in mind, the second chapter of this study aims to investigate the reaction of ChAld and cytc. Using mimetic membrane models and mass spectrometry analyses, we showed that ChAld covalently modifies cytc through a mechanism consistent with the formation of Schiff base adducts. Such modification occurs mostly at lysine residues that are known to interact with the membrane. The modifications have an influence in the affinity of cytc to the membrane, where they increase its binding to the membrane, a process that could affect the electron transport and apoptosis signaling. In the last and third chapter of this study we wanted an analytical tool that allowed the investigation of protein adduction promoted by cholesterol and other sterols-derived oxidation products. In a study performed in collaboration with the Porter group from Vanderbilt University, we used analyses based on click chemistry to search for protein adduction. To address that, we first synthesized derivatives of cholesterol and 7-dehydrocholesterol (7-DHC, the immediate precursor of cholesterol) containing an alkynyl group in the side chain. The alkynyl group can be ligated to an azide group through a cycloaddition reaction, in a process known as click chemistry. After the synthesis and characterization of alkynyl derivatives, Neuro2a cells were treated with alkynyl-7-DHC and alkynyl-cholesterol to check their metabolism. HPLC-MS/MS analyses showed that both alkynyl derivatives are metabolized and converted into their respective esters. In addition, using a cell model for Smith-Lemli-Optiz Syndrome (SLOS), a disease characterized by the deficiency in the dehydrocholesterol reductase 7, we showed that the characteristic accumulation of 7-DHC in SLOS patients might be associated with protein adduction promoted by its oxidation products, which might contribute to the development of the disease

An amperometric cholesterol biosensor based on epoxy resin membrane bound cholesterol oxidase.

BACKGROUND & OBJECTIVES: The use of epoxy resin membrane as a support for immobilization of enzyme has resulted into improved sensitivity and stability of biosensors for uric acid, ascorbic acid and polyphenols. The present work was aimed to prepare an improved amperometric biosensor for determination of serum cholesterol required in the diagnostics and management of certain pathological conditions. METHODS: Epoxy resin membrane with immobilized cholesterol oxidase was mounted on the cleaned platinum (Pt) electrode with a parafilm to construct a working electrode. This working electrode along with Ag/AgCl as reference and Ag wire as an auxiliary electrode were connected through a three terminal electrometer to construct a cholesterol biosensor. RESULTS: The sensor showed optimum response within 25 sec at pH 7.0 and 45°C. The linear working range of biosensor was 1.0 to 8.0 mM cholesterol. K m and I max for cholesterol were 5.0 mM and 9.09 µA, respectively. The biosensor measured serum cholesterol. The minimum detection limit of the sensor was 1.0 mM. The mean analytical recoveries of added cholesterol in serum (2.84 and 4.13 mM) were 91.4 ± 2.8 and 92.3 ± 3.1 per cent (n=6), respectively. Within and between assay coefficient of variation (CV) were <2 and <4 per cent, respectively. Biosensor had a storage life of 6 months at 4 o C. INTERPRETATION & CONCLUSIONS: The use of epoxy resin membrane as a support for immobilization of cholesterol oxidase has resulted into an improved amperometric cholesterol biosensor. The present biosensor had an advantage over the existing biosensors as it worked at comparatively lower potential.

Activation of membrane cholesterol by 63 amphipaths.

A few membrane-intercalating amphipaths have been observed to stimulate the interaction of cholesterol with cholesterol oxidase, saponin and cyclodextrin, presumably by displacing cholesterol laterally from its phospholipid complexes. We now report that this effect, referred to as cholesterol activation, occurs with dozens of other amphipaths, including alkanols, saturated and cis- and trans-unsaturated fatty acids, fatty acid methyl esters, sphingosine derivatives, terpenes, alkyl ethers, ketones, aromatics and cyclic alkyl derivatives. The apparent potency of the agents tested ranged from 3 microM to 7 mM and generally paralleled their octanol/water partition coefficients, except that relative potency declined for compounds with >10 carbons. Some small amphipaths activated cholesterol at a membrane concentration of approximately 3 mol per 100 mol of bilayer lipids, about equimolar with the cholesterol they displaced. Lysophosphatidylserine countered the effects of all these agents, consistent with its ability to reduce the pool of active membrane cholesterol. Various amphipaths stabilized red cells against the hemolysis elicited by cholesterol depletion, presumably by substituting for the extracted sterol. The number and location of cis and trans fatty acid unsaturations and the absolute stereochemistry of enantiomer pairs had only small effects on amphipath potency. Nevertheless, potency varied approximately 7-fold within a group of diverse agents with similar partition coefficients. We infer that a wide variety of amphipaths can displace membrane cholesterol by competing stoichiometrically but with only limited specificity for weak association with phospholipids. Any number of other drugs and experimental agents might do the same.

[Concentration of the oxygenated derivates of cholesterol in pregnant women suffering from diabetes type I]. / Stezenie utlenowanych pochodnych cholesterolu u kobiet ciezarnych z cukrzyca typu 1.

Free-radical peroxidation of cholesterol results in, among others, another products of its oxidation, called oxysterols. Scientists are still more and more interested in oxysterols, because, like the products of polyunsaturated fatty acids, show a strong biological activity, which effects physiology, pathology and pharmacology. The aim of the work was to investigate whether pre-pregnancy diabetes effects cholesterol oxidation process studied on the basis of the concentration of the chosen oxysterols. The chosen oxysterols were determined in 45 patients suffering from diabetes for various time period before pregnancy (diabetes type according to White from B to R) and in a control group (n = 27). Oxysterols (7-ketocholesterol, 7 alpha and 7 alpha-hydroxycholesterols and the sum of 5 alpha, 6 alpha and 5 beta, 6 beta-epoxycholesterols) were determined by thin-layer chromatography with densitometric detection according to the methodology which had been developed in the Chemical Department, the Silesian Medical Academy. The analysis scheme included plasma sample hydrolysis and lipid extraction, oxysterol fraction isolation by solid phase extraction (SPE), separation and identification of individual sterols by TLC technique, and densitometric quantitative analysis of the cholesterol oxidized derivatives mentioned above. We found statistically considerable differences between the concentrations of epoxycholesterol sum and 7-ketocholesterol in both groups, and the concentration was higher in the control group. While analysing the concentrations of the investigated parameters in diabetic pregnant women in II and III trimester we found a statistically considerable increase in oxysterol concentration in III trimester, compared to II trimester. The authors suggest that during complicated diabetic pregnancy the cholesterol oxidation process becomes more intensive, particularly in III trimester, compared to II trimester, both in normal pregnant women and in type I diabetic pregnant women as well.

Rápida identificación del 7-dehidrocolesterol en pacientes con Síndrome de Smith-Lemli-Opitz / Rapid identification of 7-dehidrocholesterol in patients with Smith-Lemli-Optiz syndrome

El síndrome de Smith-Lemli-Opitz es un trastorno autosómico recesivo caracterizado por unos bajos niveles de colesterol sérico y un acúmulo de su precursor, el 7-dehidrocolesterol. En los pacientes con este síndrome, el método enzimático normalmente utilizado para la medida del colesterol (basados en la colesterol oxidasa) da unos valores falsamente altos. El diagnóstico del síndrome siempre se tiene que confirmar poniendo de manifiesto valores elevados de 7-dehidrocolesterol. Hemos usado un método rápido y simple mediante espectrofotometría ultravioleta para la detección de dicho compuesto. El 7-dehidrocolesterol mostró un espectro ultravioleta característico, con unas absorciones máximas a 271, 282 y 293 nm. Este test parece ser suficientemente sensible para detectar niveles elevados de 7-dehidrocolesterol. Concluimos que la determinación por espectrofotometría ultravioleta del 7-dehidrocolesterol sérico es un método simple y barato para detectar rápidamente el síndrome de Smith-Lemli-Opitz en la mayoría de los laboratorios cínicos (AU)

Tiroiditis autoinmune en población mayor de 50 años: Aspectos clínicos, evolutivos y epidemiológicos / Autoimmune thyroiditis in a population over 50 years of age: clinical evolutive, and epidemiological aspects

Para conocer la frecuencia de la tiroiditis autoinmune y sus consecuencias clínicas en población mayor de 50 años de área urbana de Ciudad de La Habana, se seleccionó, en 1989, mediante muestreo aleatorio estratificado, al 8 por ciento de la población mayor de 50 años (555 sujetos) del área de salud del policlínico "Rampa" de Ciudad de La Habana, a cada uno se le realizó examen clínico y se le determinaron niveles de anticuerpos antimicrosomales, antitiroglobulina, de TSH, T4t, T3t, CT3. Luego se evaluó la respuesta de TSH por TRH en sujetos con anticuerpos positivos y en grupos sin anticuerpos y, más tarde, mediante la determinación del tiempo de intervalo sistólico, de los niveles de colesterol sérico y de turbiedad, se precisó la repercusión periférica de una respuesta alterada de TSH por TRH. Se encontró: 1. Bocio en el 9,5 por ciento de los sujetos; 2. En el 12 y 23 por ciento anticuerpos antiTg y antiMC positivos, respectivamente, que constituyen un factor de riesgo para desarrollar hipofunción tiroidea; 3. Prevalencia de hipotiroidismo primario del 3,9 por ciento y de hipertiroidismo del 0,3 por ciento y 4. Que no siempre una respuesta alterada de TSH pos TRH se acompaña de cambios periféricos que sugieran disfunción tiroidea subclínica. En conclusión, en población mayor de 50 años es preciso buscar tiroiditis autoinmune que constituye un factor de riesgo para el hipotiroidismo primario y que, de existir disfunción tiroidea subclínica, no instituir tratamiento hasta demostrar similar trastorno en otros órganos(AU)

Tiroiditis autoinmune en población mayor de 50 años: Aspectos clínicos, evolutivos y epidemiológicos / Autoimmune thyroiditis in a population over 50 years of age: clinical evolutive, and epidemiological aspects

Para conocer la frecuencia de la tiroiditis autoinmune y sus consecuencias clínicas en población mayor de 50 años de área urbana de Ciudad de La Habana, se seleccionó, en 1989, mediante muestreo aleatorio estratificado, al 8 por ciento de la población mayor de 50 años (555 sujetos) del área de salud del policlínico "Rampa" de Ciudad de La Habana, a cada uno se le realizó examen clínico y se le determinaron niveles de anticuerpos antimicrosomales, antitiroglobulina, de TSH, T4t, T3t, CT3. Luego se evaluó la respuesta de TSH por TRH en sujetos con anticuerpos positivos y en grupos sin anticuerpos y, más tarde, mediante la determinación del tiempo de intervalo sistólico, de los niveles de colesterol sérico y de turbiedad, se precisó la repercusión periférica de una respuesta alterada de TSH por TRH. Se encontró: 1. Bocio en el 9,5 por ciento de los sujetos; 2. En el 12 y 23 por ciento anticuerpos antiTg y antiMC positivos, respectivamente, que constituyen un factor de riesgo para desarrollar hipofunción tiroidea; 3. Prevalencia de hipotiroidismo primario del 3,9 por ciento y de hipertiroidismo del 0,3 por ciento y 4. Que no siempre una respuesta alterada de TSH pos TRH se acompaña de cambios periféricos que sugieran disfunción tiroidea subclínica. En conclusión, en población mayor de 50 años es preciso buscar tiroiditis autoinmune que constituye un factor de riesgo para el hipotiroidismo primario y que, de existir disfunción tiroidea subclínica, no instituir tratamiento hasta demostrar similar trastorno en otros órganos

Clinical efficacy of the direct assay method using polymers for serum high density lipoprotein cholesterol.

The clinical efficacy and accuracy of the homogeneous assay method for the serum high density lipoprotein (HDL)-cholesterol determination were evaluated. The principle is as follows: low density lipoproteins (LDL) and very low density lipoproteins (VLDL) were coated by polymers and polyanion to be blocked from cholesterol esterase and cholesterol oxidase. The reaction of these enzymes for HDL cholesterol was enhanced with a detergent, and HDL cholesterol was selectively measured. Both within-run (n = 3, 20 times) and between-run (n = 3, 7 days) CVs were < 2%. The repeated freezing and thawing (4 times) of three distinct sera resulted in no changes of HDL cholesterol values. Additions of lipid emulsion (Triglyceride = 100 mg/dl) and free bilirubin (20 mg/dl) gave no effect. Linearity was found up to 300 mg/dl. Increases in HDL cholesterol values by the addition of VLDL (total cholesterol (TC) = 300 mg/dl) or LDL (TC = 300 mg/dl) to the tested sera were < 0.5%. The correlation coefficient of the new method with a precipitation method was 0.995 (n = 64). HDL-C values for patients with hyperlipidemia (Type IIa, IIb, or III, IV, and V) by this method were comparable with those obtained by the precipitation method. From these results, we concluded that the new method meets the requirements for accuracy, precision, ease of handling massive samples, and was clinically useful.

[Induction of lipid peroxidation in the erythrocytes during cholesterol oxidation catalysed by cholesterol oxidase]. / Induktsiia perekisnogo okisleniia lipidov v éritrotsitakh v khode okisleniia kholesterina, kataliziruemogo kholesterinoksidazoi.

Using the chemiluminescence technique to assay the activity of cholesterol oxidase it has been shown that enzymic oxidation of cholesterol to cholest-4-en-3-one red cell membranes is accompanied by accumulation of lipid peroxidation products--malonyl dialdehyde (MDA). The amount of MDA formed was dependent on the amount of cholesterol oxidized. The free radical scavenger 4-methyl-2,6-ditretbutylphenol, the transition metal chelator EDTA and catalase inhibited lipid peroxidation in red blood cells. The participation of OH radicals in the initiation of lipid peroxidation in red cell membranes in the course of cholesterol oxidation is discussed.

Cholesterol oxidase susceptibility of the red cell membrane.

We have used the highly variable and conditional susceptibility of cholesterol oxidase to probe molecular rearrangements in the human red cell membrane. Cholesterol in the intact erythrocyte normally is not a substrate for this enzyme. Susceptibility was induced however, by these pretreatments: mild enrichment in membrane cholesterol, exposure to greater than or equal to 0.03% (3 mM) glutaraldehyde and warming in dilute salt solutions (mu approx. 0.001). Cholesterol reactivity in dilute salt solutions emerged only following a lag of 30 min or more. The lag time was shortened by raising the temperature, by reducing the salt concentration or by treating with glutaraldehyde. The induced sensitivity to the enzyme was inhibited by restoring physiologic ionic strength or by introducing 0.1 mol lysophosphatidylcholine per mol cholesterol into the membrane. (In striking contrast, lysophosphatidylethanolamine and lysophosphatidylserine did not inhibit oxidation). The various effectors of cholesterol oxidase sensitivity strongly influenced the impact of the others, suggesting that each shifted cholesterol toward or away from an enzyme-sensitive disposition. None of these effects was observed in pure cholesterol or red cell membrane lipids dissolved in detergent, which were uniformly highly reactive with the enzyme. We conclude that the observed variation in cholesterol oxidase sensitivity reflects changes in the organization of the bilayer, perhaps a lateral redistribution of lipids which creates cholesterol-rich phases or domains in which cholesterol is more or less accessible to the enzyme. If so, the time-dependent increase in cholesterol susceptibility during warming at low ionic strength might be a novel indicator of the kinetics of phase changes in the bilayer of the red cell.