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Hieronim Jakubowski - One of the best experts on this subject based on the ideXlab platform.
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quantification of Homocysteine Thiolactone in human saliva and urine by gas chromatography mass spectrometry
Journal of Chromatography B, 2020Co-Authors: Justyna Piechocka, Grazyna Chwatko, Hieronim Jakubowski, Monika Wronska, Rafal GlowackiAbstract:Abstract Homocysteine Thiolactone (HTL) is a chemically reactive thioester that has been implicated in cardiovascular disease. So far, its presence has been documented in human and mouse plasma and urine. Here, using a new method, we show that HTL is present in human saliva. The assay involves chloroform-methanol extraction of HTL, lyophilization, and derivatization with N-trimethylsilyl-N-methyl trifluoroacetamide (MSTFA) and trimethylchlorosilane (TMCS). The method is based on a gas chromatography coupled with mass spectrometry (GC–MS) and quantifies HTL in a linear range from 0.05 to 1 µmol L−1 saliva and urine. The limit of quantification (LOQ) was 0.05 µmol L−1. With respect to saliva specimen, the accuracy was 98.7–112.6%, and 90.2–100.5%, while the precision was 7.1–13.5% and 12.5–15.0% for the intra- and inter-day variation, respectively. In relation to urine samples, the accuracy was 91.9–110.9% and 91.2–103.3%, while the precision varied from 2.2% to 14.5% and 7.4% to 14.3% for intra- and inter-day measurements, respectively. Using this method, we show that in apparently healthy individuals (n = 18), HTL levels in saliva are not positively correlated with urinary HTL levels. Undoubtedly, larger population should be investigated to get more meaningful results.
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paraoxonase 1 q192r genotype and activity affect Homocysteine Thiolactone levels in humans
The FASEB Journal, 2018Co-Authors: Joanna Perlakajan, Rafal Glowacki, Kamila Borowczyk, Ottar Nygard, Hieronim JakubowskiAbstract:Genetic or nutritional deficiencies in 1 carbon and Homocysteine (Hcy) metabolism elevate Hcy-Thiolactone levels and are associated with cardiovascular and neurologic diseases. Hcy-Thiolactone causes protein damage, cellular toxicity, and proatherogenic changes in gene expression in human cells and tissues. A polymorphic cardio-protective enzyme, paraoxonase 1 (PON1), hydrolyzes Hcy-Thiolactone in vitro. However, whether Hcy-Thiolactone hydrolysis is a physiologic function of the PON1 protein and whether polymorphisms in the PON1 gene affect Hcy-Thiolactone levels in humans was unknown. Here we show that the PON1-192 genotype, which affects the enzymatic activity of the PON1 protein, also affected urinary Hcy-Thiolactone levels, normalized to creatinine. Carriers of the PON1-192R allele had significantly lower Hcy-Thiolactone/creatinine levels than individuals carrying the PON1-192Q allele. Individuals with low serum PON1 paraoxonase activity had significantly higher Hcy-Thiolactone/creatinine levels compared with individuals with high paraoxonase activity. In contrast, Hcy-Thiolactone/creatinine levels were unaffected by serum PON1 arylesterase activity or by PON1 protein levels. Taken together, these findings suggest that PON1 hydrolyzes Hcy-Thiolactone in humans and that the interindividual variations in PON1 genotype/activity can modulate the pathology of hyperHomocysteinemia.-Perla-Kajan, J., Borowczyk, K., Glowacki, R., Nygard, O., Jakubowski, H. Paraoxonase 1 Q192r genotype and activity affect Homocysteine Thiolactone levels in humans.
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effects of betaine on body composition performance and Homocysteine Thiolactone
Journal of The International Society of Sports Nutrition, 2013Co-Authors: Jason M Cholewa, Hieronim Jakubowski, Rafal Glowacki, Monika Wyszczelskarokiel, Tracey D Matthews, Richard J Wood, Stuart A S Craig, Vincent J PaoloneAbstract:Background This study investigated the effects of long term betaine supplementation on body composition, performance, and Homocysteine Thiolactone (HCTL) in experienced strength trained men.
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effects of betaine on body composition performance and Homocysteine Thiolactone
Journal of The International Society of Sports Nutrition, 2013Co-Authors: Jason M Cholewa, Hieronim Jakubowski, Rafal Glowacki, Monika Wyszczelskarokiel, Tracey D Matthews, Richard J Wood, Stuart A S Craig, Vincent J PaoloneAbstract:Background: This study investigated the effects of long term betaine supplementation on body composition, performance, and Homocysteine Thiolactone (HCTL) in experienced strength trained men. Methods: Twenty-three subjects were matched for training experience (4.8 ± 2.3 years) and body fat percentage (BF%: 16.9 ± 8.0%), randomly assigned to either a placebo (PL; n= 12) or betaine group (BET; n= 11; 2.5 g/day), and completed a 6 week periodized training program consisting of 3 two-week micro-cycles. Bench press and back squat training volumes were recorded and changes in training volume were assessed at each micro-cycle. Fasting urine was collected at baseline (BL), weeks 2, 4 and 6, and assayed for HCTL. Subjects were tested prior to and following 6 weeks of treatment. Arm and thigh cross sectional area (CSA) was estimated via girth and skin fold measurements. Body density was estimated via skin fold calipers and used to estimate BF%, fat mass (FM), and lean body mass (LBM). Performance was assessed via vertical jump (VJ), bench press 1 RM (BP), and back squat 1 RM (BS). Results: Arm CSA increased significantly (p< .05) in BET but not PL. No differences existed between group and time for changes in thigh CSA. Back squat training volume increased significantly (p< .05) for both groups throughout training. Bench press training volume was significantly (p < .05) improved for BET compared to PL at microcycles one and three. Body composition (BF%, FM, LBM) improved significantly (p< .05) in BET but not PL. No differences were found in performance variables (BP, BS, VJ) between groups, except there was a trend (p = .07) for increased VJ power in BET versus PL. A significant interaction (p< .05) existed for HCTL, with increases from BL to week 2 in PL, but not BET. Additionally, HCTL remained elevated at week 4 in PL, but not BET. Conclusion: Six-weeks of betaine supplementation improved body composition, arm size, bench press work capacity, attenuated the rise in urinary HCTL, and tended to improve power (p = .07) but not strength.
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mechanism of the condensation of Homocysteine Thiolactone with aldehydes
Chemistry: A European Journal, 2006Co-Authors: Hieronim JakubowskiAbstract:: Chemical reactivity of Homocysteine Thiolactone (HTL) has been implicated in cardiovascular disease. Owing to its aminoacyl-thioester character, HTL undergoes facile electrophilic and nucleophilic reactions at its amino and activated-carboxyl group, respectively. To gain insight into the mechanism of the reactions involving its amino group, the kinetics of the condensation of Homocysteine Thiolactone with formaldehyde, acetaldehyde, and pyridoxal phosphate, were analyzed in the pH range from 5 to 10. The reactions were first order with respect to HTL, aldehyde, and hydroxide ion concentrations. Of the two ionic species of HTL (pKa=6.67+/-0.05), the acid form HTL+ was approximately 100-fold more reactive than the base form HTL(0). The reactions of HTL with aldehydes involve intermediate adducts. The conversion of the intermediate carbinolamine to a product, 1,3-tetrahydrothiazine-4-carboxylic acid or its 2-substituted analogue, occurs in a two-step reaction. The first step involves hydrolysis of the thioester bond in the intermediate, facilitated by anchimeric assistance by the oxygen of the carbinolamine group of the intermediate. The second step involves an attack of the liberated thiolate on the aldehyde-derived carbon of the intermediate, affording 1,3-tetrahydrothiazine-4-carboxylic acid or its 2-substituted analogue. An unusual feature of these reactions is that the formation of the carbinolamine group increases the reactivity of the thioester bond of HTL approximately 10(4)-fold. The facile formation of tetrahydrothiazines may contribute to HTL elimination from the human body.
Victor Sanchezmargalet - One of the best experts on this subject based on the ideXlab platform.
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Homocysteine Thiolactone inhibits insulin stimulated dna and protein synthesis possible role of mitogen activated protein kinase mapk glycogen synthase kinase 3 gsk 3 and p70 s6k phosphorylation
Journal of Molecular Endocrinology, 2005Co-Authors: Souad Najib, Victor SanchezmargaletAbstract:HyperHomocysteinemia and insulin resistance are independent factors for cardiovascular disease. Most of the angiotoxic effects of Homocysteine are related to the formation of Homocysteine Thiolactone and the consequent increase in oxidative stress. We have recently found that Homocysteine Thiolactone inhibits insulin receptor tyrosine kinase activity, which results in decreased phosphatidylinositol 3-kinase (PI3K) activity and inhibition of glycogen synthesis. Oxidative stress seemed to be the mechanism underlying these effects, since glutathione was able to restore the insulin signaling as well as the insulin-mediated glycogen synthesis. In the present work we have further investigated insulin receptor signaling studying mitogen-activated protein kinase (MAPK), glycogen synthase kinase-3 (GSK-3) and p70 S6K phosphorylation. Again, Homocysteine Thiolactone (50 µM) prevented insulin-mediated MAPK, GSK-3 and p70 S6K phosphorylation and these effects were blocked by glutathione (250 µM). Since MAPK and PI3K pathways, including GSK3 and S6K, seem to mediate insulin-mediated growth and proliferation, we measured DNA and protein synthesis. We have found that Homocysteine Thiolactone (50 µM) inhibits insulin-mediated growth and proliferation, as previously shown for glycogen synthesis. Again, these effects seem to be mediated by oxidative stress, since 250 µM glutathione completely abolished the effects of Homocysteine Thiolactone on insulin-stimulated DNA and protein synthesis. In conclusion, these data suggest that Homocysteine Thiolactone impairs insulin signaling by a mechanism involving oxidative stress, leading to a defect in the action of insulin on growth and proliferation.
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Homocysteine Thiolactone inhibits insulin signaling and glutathione has a protective effect
Journal of Molecular Endocrinology, 2001Co-Authors: Souad Najib, Victor SanchezmargaletAbstract:: HyperHomocysteinemia and insulin resistance are independent factors for cardiovascular disease. Most of the angiotoxic effects of Homocysteine are related to the formation of Homocysteine Thiolactone and the consequent increase in oxidative stress. The oxidative stress has also been shown to impair insulin action, therefore leading to insulin resistance. In order to study a putative direct effect of Homocysteine on insulin signaling, we have characterized the molecular counter-regulation of the early events in the signal transduction of the insulin receptor, and the metabolic end-point of glycogen synthesis. We employed HTC rat hepatoma cells transfected with the human insulin receptor. A 10 min exposure to Homocysteine Thiolactone (50 microM) resulted in a significant inhibition of insulin-stimulated tyrosine phosphorylation of the insulin receptor beta-subunit and its substrates IRS-1 and p60-70, as well as their association with the p85 regulatory subunit of phosphatidylinositol 3-kinase. These effects led to impairment of the insulin-stimulated phosphatidylinositol 3-kinase activity, which plays a central role in regulating insulin action. Thus, insulin-stimulated glycogen synthesis was also inhibited by Homocysteine Thiolactone. To investigate whether oxidative stress was mediating the counter-regulatory effect of Homocysteine Thiolactone on insulin signaling, we preincubated the cells (5 min) with 250 microM glutathione prior to the incubation with Homocysteine (10 min) and subsequent insulin challenge. Glutathione completely abolished the effects of Homocysteine Thiolactone on insulin-receptor signaling and restored the insulin-stimulated glycogen synthesis. In conclusion, these data suggest that Homocysteine Thiolactone impairs insulin signaling by a mechanism involving oxidative stress, leading to a defect in insulin action.
Rafal Glowacki - One of the best experts on this subject based on the ideXlab platform.
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quantification of Homocysteine Thiolactone in human saliva and urine by gas chromatography mass spectrometry
Journal of Chromatography B, 2020Co-Authors: Justyna Piechocka, Grazyna Chwatko, Hieronim Jakubowski, Monika Wronska, Rafal GlowackiAbstract:Abstract Homocysteine Thiolactone (HTL) is a chemically reactive thioester that has been implicated in cardiovascular disease. So far, its presence has been documented in human and mouse plasma and urine. Here, using a new method, we show that HTL is present in human saliva. The assay involves chloroform-methanol extraction of HTL, lyophilization, and derivatization with N-trimethylsilyl-N-methyl trifluoroacetamide (MSTFA) and trimethylchlorosilane (TMCS). The method is based on a gas chromatography coupled with mass spectrometry (GC–MS) and quantifies HTL in a linear range from 0.05 to 1 µmol L−1 saliva and urine. The limit of quantification (LOQ) was 0.05 µmol L−1. With respect to saliva specimen, the accuracy was 98.7–112.6%, and 90.2–100.5%, while the precision was 7.1–13.5% and 12.5–15.0% for the intra- and inter-day variation, respectively. In relation to urine samples, the accuracy was 91.9–110.9% and 91.2–103.3%, while the precision varied from 2.2% to 14.5% and 7.4% to 14.3% for intra- and inter-day measurements, respectively. Using this method, we show that in apparently healthy individuals (n = 18), HTL levels in saliva are not positively correlated with urinary HTL levels. Undoubtedly, larger population should be investigated to get more meaningful results.
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determination of Homocysteine Thiolactone in human urine by capillary zone electrophoresis and single drop microextraction
Analytical Biochemistry, 2020Co-Authors: Krystian Purgat, Rafal Glowacki, Patrycja Olejarz, Izabella Kośka, Pawel KubalczykAbstract:Abstract A simple, fast, sensitive and reproducible capillary zone electrophoresis (CZE) method with single drop microextraction (SDME) for determination of Homocysteine Thiolactone (HTL) in human urine has been developed and validated. The method is characterized by good precision, high accuracy, short analysis time and low consumption of reagents. The procedure consists only of few steps: urine sample centrifugation, dilution with phosphate buffer and methanol, chloroform addition onto the top of donor phase, on-line SDME in CE system, sample separation by CZE and ultraviolet detection of HTL at 240 nm. The background electrolyte was 0.1 M pH 4.75 phosphate buffer. Effective separation was achieved within 6.04 min under the separation voltage of 24 kV (~110 μA). The LOQ and LOD for HTL were 50 and 25 nM urine, respectively. The calibration curve in urine showed linearity in the range of 50–200 nM, with R2 0.9995. The intra- and inter-day precision and recovery were 4.0–14.5% (average 8.7% and 9.3%) and 92.7–115.5% (average 103.6% and 104.8%), respectively. The procedure was successfully applied to analysis of urine samples.
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higher levels of low molecular weight sulfur compounds and Homocysteine Thiolactone in the urine of autistic children
Molecules, 2020Co-Authors: Paulina Gątarek, Rafal Glowacki, Kamila Borowczyk, Angelina Rosiak, Joanna KaluznaczaplinskaAbstract:In this study, the levels of concentration of Homocysteine Thiolactone (HTL), cysteine (Cys), and cysteinylglycine (CysGly) in the urine of autistic and non-autistic children were investigated and compared. HTL has never been analyzed in autistic children. The levels of low molecular weight sulfur compounds in the urine of both groups were determined by validated methods based on high-performance liquid chromatography with spectrofluorometric and diode-array detectors. The statistical data show a significant difference between the examined groups. Children with autism were characterized by a significantly higher level of HTL (p = 5.86 × 10−8), Cys (p = 1.49 × 10−10) and CysGly (p = 1.06 × 10−8) in urine compared with the control group. A difference in the p-value of <0.05 is statistically significant. Higher levels of HTL, Cys, and CysGly in the urine of 41 children with autism, aged 3 to 17, were observed. The obtained results may indicate disturbances in the metabolism of methionine, Cys, and glutathione in some autistic patients. These preliminary results suggest that further research with more rigorous designs and a large number of subjects is needed.
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application of gc ms technique for the determination of Homocysteine Thiolactone in human urine
Journal of Chromatography B, 2018Co-Authors: Monika Wronska, Grazyna Chwatko, Pawel Kubalczyk, Kamila Borowczyk, Justyna Piechocka, Rafal GlowackiAbstract:Abstract It is well established that Homocysteine Thiolactone (HTL) is associated with some health disorders, including cardiovascular diseases. HTL is a by-product of sulfur metabolic cycle. So far, its presence has been confirmed in human plasma and urine. It has been also shown that a vast majority of HTL is removed from human body through kidney. Thus, the aim of the current investigations has been the identification, separation and quantification of HTL in urine samples. For the first time a cheap, reliable and robust GC–MS method was developed for the determination of HTL in human urine in the form of its volatile isobutyl chloroformate derivative. Separation of the analyte and internal standard (homoserine lactone (HSL)) was achieved in 15 min followed by mass spectrometry detection (MS). Isocratic elution was accomplished with helium at a flow rate of 1 mL min−1 and a gradient of the column temperature was concomitant with the analysis. The mass spectrometer was set to the electron impact mode at 70 eV. The ion source, quadrupole and MS interface temperatures were set to 230 °C, 150 °C and 250 °C, respectively. Elaborated analytical procedure allows quantification of analyte in a linear range of 0.01–0.20 nmol mL−1 urine. The LOQ and LOD values were 0.01 and 0.005 nmol mL−1, respectively. The method accuracy ranged from 98.0% to 103.2%, while precision varied from 6.4% to 9.5% and from 10.7% to 16.9% for intra- and inter-day measurements, respectively. Finally, the method has been successfully implemented in the analysis of 12 urine samples donated by apparently healthy volunteers. Concentration of HTL ranged from
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paraoxonase 1 q192r genotype and activity affect Homocysteine Thiolactone levels in humans
The FASEB Journal, 2018Co-Authors: Joanna Perlakajan, Rafal Glowacki, Kamila Borowczyk, Ottar Nygard, Hieronim JakubowskiAbstract:Genetic or nutritional deficiencies in 1 carbon and Homocysteine (Hcy) metabolism elevate Hcy-Thiolactone levels and are associated with cardiovascular and neurologic diseases. Hcy-Thiolactone causes protein damage, cellular toxicity, and proatherogenic changes in gene expression in human cells and tissues. A polymorphic cardio-protective enzyme, paraoxonase 1 (PON1), hydrolyzes Hcy-Thiolactone in vitro. However, whether Hcy-Thiolactone hydrolysis is a physiologic function of the PON1 protein and whether polymorphisms in the PON1 gene affect Hcy-Thiolactone levels in humans was unknown. Here we show that the PON1-192 genotype, which affects the enzymatic activity of the PON1 protein, also affected urinary Hcy-Thiolactone levels, normalized to creatinine. Carriers of the PON1-192R allele had significantly lower Hcy-Thiolactone/creatinine levels than individuals carrying the PON1-192Q allele. Individuals with low serum PON1 paraoxonase activity had significantly higher Hcy-Thiolactone/creatinine levels compared with individuals with high paraoxonase activity. In contrast, Hcy-Thiolactone/creatinine levels were unaffected by serum PON1 arylesterase activity or by PON1 protein levels. Taken together, these findings suggest that PON1 hydrolyzes Hcy-Thiolactone in humans and that the interindividual variations in PON1 genotype/activity can modulate the pathology of hyperHomocysteinemia.-Perla-Kajan, J., Borowczyk, K., Glowacki, R., Nygard, O., Jakubowski, H. Paraoxonase 1 Q192r genotype and activity affect Homocysteine Thiolactone levels in humans.
Souad Najib - One of the best experts on this subject based on the ideXlab platform.
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Homocysteine Thiolactone inhibits insulinstimulated DNA and protein synthesis: possible role of mitogen-activated protein kinase (MAPK), glycogen synthase kinase-3
2015Co-Authors: Souad Najib, V Sánchez-margaletAbstract:HyperHomocysteinemia and insulin resistance are independent factors for cardiovascular disease. Most of the angiotoxic effects of Homocysteine are related to the formation of Homocysteine Thiolactone and the consequent increase in oxidative stress. The oxidative stress has also been shown to impair insulin action, therefore leading to insulin resist-ance. In order to study a putative direct effect of Homocysteine on insulin signaling, we have charac-terized the molecular counter-regulation of the early events in the signal transduction of the insulin receptor, and the metabolic end-point of glycogen synthesis. We employed HTC rat hepatoma cells transfected with the human insulin receptor.
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Homocysteine Thiolactone inhibits insulin stimulated dna and protein synthesis possible role of mitogen activated protein kinase mapk glycogen synthase kinase 3 gsk 3 and p70 s6k phosphorylation
Journal of Molecular Endocrinology, 2005Co-Authors: Souad Najib, Victor SanchezmargaletAbstract:HyperHomocysteinemia and insulin resistance are independent factors for cardiovascular disease. Most of the angiotoxic effects of Homocysteine are related to the formation of Homocysteine Thiolactone and the consequent increase in oxidative stress. We have recently found that Homocysteine Thiolactone inhibits insulin receptor tyrosine kinase activity, which results in decreased phosphatidylinositol 3-kinase (PI3K) activity and inhibition of glycogen synthesis. Oxidative stress seemed to be the mechanism underlying these effects, since glutathione was able to restore the insulin signaling as well as the insulin-mediated glycogen synthesis. In the present work we have further investigated insulin receptor signaling studying mitogen-activated protein kinase (MAPK), glycogen synthase kinase-3 (GSK-3) and p70 S6K phosphorylation. Again, Homocysteine Thiolactone (50 µM) prevented insulin-mediated MAPK, GSK-3 and p70 S6K phosphorylation and these effects were blocked by glutathione (250 µM). Since MAPK and PI3K pathways, including GSK3 and S6K, seem to mediate insulin-mediated growth and proliferation, we measured DNA and protein synthesis. We have found that Homocysteine Thiolactone (50 µM) inhibits insulin-mediated growth and proliferation, as previously shown for glycogen synthesis. Again, these effects seem to be mediated by oxidative stress, since 250 µM glutathione completely abolished the effects of Homocysteine Thiolactone on insulin-stimulated DNA and protein synthesis. In conclusion, these data suggest that Homocysteine Thiolactone impairs insulin signaling by a mechanism involving oxidative stress, leading to a defect in the action of insulin on growth and proliferation.
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Homocysteine Thiolactone inhibits insulin signaling and glutathione has a protective effect
Journal of Molecular Endocrinology, 2001Co-Authors: Souad Najib, Victor SanchezmargaletAbstract:: HyperHomocysteinemia and insulin resistance are independent factors for cardiovascular disease. Most of the angiotoxic effects of Homocysteine are related to the formation of Homocysteine Thiolactone and the consequent increase in oxidative stress. The oxidative stress has also been shown to impair insulin action, therefore leading to insulin resistance. In order to study a putative direct effect of Homocysteine on insulin signaling, we have characterized the molecular counter-regulation of the early events in the signal transduction of the insulin receptor, and the metabolic end-point of glycogen synthesis. We employed HTC rat hepatoma cells transfected with the human insulin receptor. A 10 min exposure to Homocysteine Thiolactone (50 microM) resulted in a significant inhibition of insulin-stimulated tyrosine phosphorylation of the insulin receptor beta-subunit and its substrates IRS-1 and p60-70, as well as their association with the p85 regulatory subunit of phosphatidylinositol 3-kinase. These effects led to impairment of the insulin-stimulated phosphatidylinositol 3-kinase activity, which plays a central role in regulating insulin action. Thus, insulin-stimulated glycogen synthesis was also inhibited by Homocysteine Thiolactone. To investigate whether oxidative stress was mediating the counter-regulatory effect of Homocysteine Thiolactone on insulin signaling, we preincubated the cells (5 min) with 250 microM glutathione prior to the incubation with Homocysteine (10 min) and subsequent insulin challenge. Glutathione completely abolished the effects of Homocysteine Thiolactone on insulin-receptor signaling and restored the insulin-stimulated glycogen synthesis. In conclusion, these data suggest that Homocysteine Thiolactone impairs insulin signaling by a mechanism involving oxidative stress, leading to a defect in insulin action.
Isiah M Warner - One of the best experts on this subject based on the ideXlab platform.
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gold nanoparticle sensor for Homocysteine Thiolactone induced protein modification
Langmuir, 2008Co-Authors: Arther T Gates, Sayo O Fakayode, Mark Lowry, Gabriela M Ganea, Abitha Murugeshu, J W Robinson, Robert M Strongin, Isiah M WarnerAbstract:Homocysteine Thiolactone-induced protein modification (HTPM) is a unique post-translational protein modification that is recognized as an emergent biomarker for cardiovascular disease. HTPM involves the site-specific acylation of proteins at lysine residues by Homocysteine Thiolactone (HTL) to produce protein homocystamide, which has been found at elevated levels in patients with coronary heart disease. Herein, we report the development of a novel gold nanoparticle (GNP) biochemical sensor for detection of protein homocystamide in an in vitro serum protein-based model system. Human serum albumin (HSA) and human sera were subjected to HTPM in vitro to produce HSA−homocystamide or serum protein homocystamide, respectively, which was subsequently treated with citrate-capped GNPs. This GNP sensor typically provided instantaneous visual confirmation of HTPM in the protein model systems. Transmission electron microscopy images of the GNPs in the presence of HSA−homocystamide suggest that modification-directed n...
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gold nanoparticle sensor for Homocysteine Thiolactone induced protein modification
Langmuir, 2008Co-Authors: Arther T Gates, Sayo O Fakayode, Mark Lowry, Gabriela M Ganea, Abitha Murugeshu, J W Robinson, Robert M Strongin, Isiah M WarnerAbstract:Homocysteine Thiolactone-induced protein modification (HTPM) is a unique post-translational protein modification that is recognized as an emergent biomarker for cardiovascular disease. HTPM involves the site-specific acylation of proteins at lysine residues by Homocysteine Thiolactone (HTL) to produce protein homocystamide, which has been found at elevated levels in patients with coronary heart disease. Herein, we report the development of a novel gold nanoparticle (GNP) biochemical sensor for detection of protein homocystamide in an in vitro serum protein-based model system. Human serum albumin (HSA) and human sera were subjected to HTPM in vitro to produce HSA-homocystamide or serum protein homocystamide, respectively, which was subsequently treated with citrate-capped GNPs. This GNP sensor typically provided instantaneous visual confirmation of HTPM in the protein model systems. Transmission electron microscopy images of the GNPs in the presence of HSA-homocystamide suggest that modification-directed nanoparticle assembly is the mechanism by which the biochemical sensor produces a colorimetric signal. The resultant nanoparticle-protein assembly exhibited excellent thermal and dilutional stability, which is expected for a system stabilized by chemisorption and intermolecular disulfide bonding. The sensor typically provided a linear response for modified human sera concentrations greater than approximately 5 mg/mL. The calculated limit of detection and calibration sensitivity for the method in human sera were 5.2 mg/mL and 13.6 AU . (microg/mL)-1, respectively.
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capillary electrophoretic screening for the inhibition of Homocysteine Thiolactone induced protein oligomerization
Analytical Chemistry, 2007Co-Authors: Arther T Gates, Mark Lowry, Abitha Murugeshu, Robert M Strongin, Kristin A Fletcher, Oleksandr Rusin, James W Robinson, Isiah M WarnerAbstract:We report the first demonstration of rapid electrophoretic monitoring of Homocysteine Thiolactone-induced protein oligomerization (HTPO), a unique type of post-translational protein modification that may have clinical significance as an indicator of cardiovascular and neurovascular diseases. HTPO of the model protein bovine cytochrome c was initiated in vitro. The relative monomer and aggregate levels of the resultant protein mixtures were determined following separation using capillaries coated with the cationic polymer, poly(diallyldimethylammonium chloride). UV detection provided adequate sensitivity for the monitoring of higher order species, which exist at relatively low concentrations in the protein reaction mixture as compared to the monomeric species. Separations performed under standard injection conditions were optimized on the basis of applied voltage and sample denaturation conditions. Separations performed using short-end injection allowed for more rapid analyses, typically in less than 70 s. Relative errors for run-to-run migration times were less than 0.5%. This novel oligomeric system provides a rapid and straightforward in vitro method to screen therapeutic agents for their ability to inhibit HTPO. Changes in peak area for monomer and aggregate species were used to assess HTPO inhibition as a function of pyridoxal 5-phosphate (PLP) concentration. PLP was shown to effectively inhibit HTPO in vitro. Rapid analysis times of approximately 1.5 min were achieved for inhibition screening.