A novel and sensitive assay for heme oxygenase activity

2015 ◽  
Vol 309 (7) ◽  
pp. F667-F671 ◽  
Author(s):  
Saki Iwamori ◽  
Emiko Sato ◽  
Daisuke Saigusa ◽  
Kouichi Yoshinari ◽  
Sadayoshi Ito ◽  
...  
Keyword(s):  
Heme Oxygenase ◽  
Internal Standard ◽  
Biliverdin Reductase ◽  
Protein Levels ◽  
Oxygenase Activity ◽  
Liquid Chromatography Tandem Mass ◽  
Cobalt Protoporphyrin ◽  
Two Temperatures ◽  
The Difference

Heme oxygenase (HO) is a renoprotective protein in the microsome that degrades heme and produces biliverdin. Biliverdin is then reduced to a potent antioxidant bilirubin by biliverdin reductase in the cytosol. Because HO activity does not necessarily correlate with HO mRNA or protein levels, a reliable assay is needed to determine HO activity. Spectrophotometric measurement is tedious and requires a relatively large amount of kidney samples. Moreover, bilirubin is unstable and spontaneously oxidized to biliverdin in vitro. We developed a novel and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to quantify biliverdin to measure HO activity in mice. Biliverdin and its internal standard, a deuterated biliverdin-d4, have MS/MS fragments with m/z transitions of 583 to 297 and 587 to 299, respectively. We prepared lysates of mouse kidneys, and added excess hemin, NADPH, and bilirubin oxidase to convert all bilirubin produced to biliverdin. After 30-min incubation at 37 or 4°C, the samples were analyzed by LC-MS/MS. The difference in the amount of biliverdin between the two temperatures is HO activity. Treating mice with cobalt protoporphyrin, which induces the expression of HO, increased HO activity as determined by biliverdin production. Measuring the production of biliverdin using LC-MS/MS is a more sensitive and specific way to determine HO activity than the spectrophotometric method and allows the detection of subtle changes in renal or other HO activity.

The protective properties of synthetic porphyrin tin complexes in toxic hyperbilirubinemia

2000 ◽  
Vol 04 (03) ◽  
pp. 243-247 ◽  
Author(s):  
T. O. PHILIPPOVA ◽  
B. N. GALKIN ◽  
N. YA. GOLOVENKO ◽  
Z. I. ZHILINA ◽  
S. V. VODZINSKII
Keyword(s):  
Heme Oxygenase ◽  
Serum Bilirubin ◽  
Protoporphyrin Ix ◽  
Serum Bilirubin Level ◽  
Oxygenase Activity ◽  
Tetraphenyl Porphyrin ◽  
Tin Complexes ◽  
Cytochrome P 450

Tin complexes of meso-substituted synthetic porphyrins, namely Sn 4+-meso-tetraphenyl- porphyrin ( Sn - TPP ) and Sn 4+-meso-tetrakis(N-methyl-3-pyridyl)porphyrin tetratosylate ( Sn - TMe -3- PyP ), efficiently decrease the serum bilirubin level when injected subcutaneously at a dose of 100 μM kg−1 body weight into mice. These compounds are active during hyperbilirubinemia, induced by phenylhydrazine, hemin and tetrachloromethane, and also during autoimmune hemolytic anemia. In the latter case a decrease in serum bilirubin content was observed, as well as a decrease in the amount of blood reticulocytes which reflects a milder course of the disease. The Sn complexes under study induce, in vivo, cytochrome P-450, inhibit microsomal heme oxygenase and decrease the intensity of lipid peroxidation. At the same time, in vitro the hepatic and splenic heme oxygenase activity is blocked only when a 0.1 μM concentration of Sn - TMe -3- PyP or Sn -protoporphyrin IX is added to the incubation mixture. Sn - TPP does not affect the activity of this enzyme in vitro.

255 THE ESTROGENIC EVALUATION OF PARABENS USING RAT UTERINE CALBINDIN-D9k

2010 ◽  
Vol 22 (1) ◽  
pp. 285
Author(s):  
T. T. B. Vo ◽  
E. B. Jeung
Keyword(s):  
Gene Expression ◽  
Adverse Effects ◽  
Treated Group ◽  
Environmental Chemicals ◽  
Female Rats ◽  
Protein Levels ◽  
Calbindin D9k ◽  
The Difference

In the current study, calbindin-D9k (CaBP-9k), a potent biomarker for screening estrogen-like environmental chemicals in vivo and in vitro, was adopted to examine the potential estrogen-like property of the following parabens: propyl-, isopropyl-, butyl-, and isobutyl-paraben. Immature female rats were administered for 3 days from postnatal day 14 to 16 with 17?-ethinylestradiol (EE, 1 mg/kg of body weight (BW) per day) or parabens (62.5, 250, and 1000 mg/kg of BW per day). In uterotrophic assays, significantly increased uterus weights were detected in the EE-treated group and in the groups treated with the greatest dose of isopropyl-, butyl- and isobutyl-paraben. In addition, these parabens induced uterine CaBP-9k mRNA and protein levels, whereas co-treatment of parabens and fulvestrant (Faslodex, formerly known as ICI 182, 780), a pure estrogen receptor (ER) antagonist, completely reversed the paraben-induced gene expression and increased uterine weights. To investigate the ER-mediated mechanism(s) by which parabens exert their effects, the expression level of ERα and progesterone receptor (PR) was analyzed. Exposure to EE or parabens caused a dramatic decrease in expression of both ER? mRNA and protein levels, whereas co-treatment with fulvestrant reversed these effects. These data showed the difference of CaBP-9k and ER? expression, suggesting that CaBP-9k might not express via ER? pathway. In the effect of parabens on CaBP-9k expression through PR mediation, a significantly increased expression of uterine PR gene, a well-known ER regulating gene, at both transcriptional and translational levels was indicated in the greatest dose of isopropyl- and butyl-paraben. These parabens induced PR gene expression that was completely blocked by fulvestrant. This result indicates that CaBP-9k expression might involve PR mediates in the estrogenic effect of paraben in immature rat uteri. Taken together, parabens exhibited an estrogen-like property in vivo, which might be mediated by a PR and/or ER? signaling pathway. In addition, our results expanded the current understanding of the potential adverse effects of parabens associated with their estrogen-like activities. Further investigation is needed to elucidate in greater detail the adverse effects of parabens in humans and wildlife.

Modified method of erythropoietin (ESF) bioassay in vitro using mouse fetal liver cells. II. measurement of ESF in human serum

Blood ◽  
1978 ◽  
Vol 52 (3) ◽  
pp. 569-577 ◽  
Author(s):  
G de Klerk ◽  
AA Hart ◽  
C Kruiswijk ◽  
R Goudsmit
Keyword(s):  
Human Serum ◽  
Fetal Liver ◽  
Internal Standard ◽  
Immune Serum ◽  
Quantitative Detection ◽  
Modified Method ◽  
The Difference ◽  
Normal Males ◽  
Test Serum

Abstract A modification of the mouse fetal liver cell bioassay for erythropoietin (ESF) that allows quantitative detection of ESF in human serum is described. The modification consists of (1) correction for the effect of serum iron on 59Fe incorporation into heme and (2) the use of an “internal standard,” i.e., a standard ESF preparation dissolved in the assayed test serum. As a result of this modification the statistical method of bioassay analysis had to be changed fundamentally. The mean serum concentration of ESF measured in 20 normal males was 48 microunit/ml, as compared to 29 microunit/ml in 18 females. The difference was significant at p = 0.017. The stimulatory activity of a human serum on 59Fe incorporation into heme could be neutralized by an anti-human ESF immune serum.

scholarly journals The Heme Oxygenase(s)-Phytochrome System ofPseudomonas aeruginosa

2004 ◽  
Vol 279 (44) ◽  
pp. 45791-45802 ◽  
Author(s):  
Rosalina Wegele ◽  
Ronja Tasler ◽  
Yuhong Zeng ◽  
Mario Rivera ◽  
Nicole Frankenberg-Dinkel
Keyword(s):  
Heme Oxygenase ◽  
Pathogenic Bacteria ◽  
Nmr Studies ◽  
Biliverdin Reductase ◽  
Heme Oxygenases ◽  
Rate Limiting Step ◽  
Phytochrome System ◽  
Rate Limiting ◽  
C Nmr

For many pathogenic bacteria likePseudomonas aeruginosaheme is an essential source of iron. After uptake, the heme molecule is degraded by heme oxygenases to yield iron, carbon monoxide, and biliverdin. The heme oxygenase PigA is only induced under iron-limiting conditions and produces the unusual biliverdin isomers IXβ and IXδ. The gene for a second putative heme oxygenase inP. aeruginosa,bphO, occurs in an operon with the genebphPencoding a bacterial phytochrome. Here we provide biochemical evidence thatbphOencodes for a second heme oxygenase inP. aeruginosa. HPLC,1H, and13C NMR studies indicate that BphO is a “classic” heme oxygenase in that it produces biliverdin IXα. The data also suggest that the overall fold of BphO is likely to be the same as that reported for other α-hydroxylating heme oxygenases. Recombinant BphO was shown to prefer ferredoxins or ascorbate as a source of reducing equivalentsin vitroand the rate-limiting step for the oxidation of heme to biliverdin is the release of product. In eukaryotes, the release of biliverdin is driven by biliverdin reductase, the subsequent enzyme in heme catabolism. BecauseP. aeruginosalacks a biliverdin reductase homologue, data are presented indicating an involvement of the bacterial phytochrome BphP in biliverdin release from BphO and possibly from PigA.

scholarly journals Stability of Extemporaneously Prepared Lansoprazole Suspension at Two Temperatures

2013 ◽  
Vol 18 (2) ◽  
pp. 122-127 ◽  
Author(s):  
Jordan T. Morrison ◽  
Ralph A. Lugo ◽  
Jim C. Thigpen ◽  
Stacy D. Brown
Keyword(s):  
Sodium Bicarbonate ◽  
Room Temperature ◽  
Internal Standard ◽  
Storage Condition ◽  
Significant Difference ◽  
Expiration Time ◽  
Liquid Chromatography Tandem Mass ◽  
Two Temperatures ◽  
The Stability ◽  
Oral Sodium

OBJECTIVE The purpose of this study was to examine the stability of a generic lansoprazole product in a 3 mg/mL sodium bicarbonate suspension under room temperature and refrigerated conditions. METHODS Lansoprazole suspensions (3 mg/mL) were prepared in triplicate using an 8.4% sodium bicarbonate vehicle for each storage condition (room temperature and refrigerated). During 1 month, samples from each replicate were periodically removed and analyzed for lansoprazole concentration by liquid chromatography–tandem mass spectrometry (LC-MS/MS). Each sample was spiked with 10 mg/L omeprazole to serve as the internal standard. A positive electrospray LC-MS/MS method was validated over the calibration range of 5 to 25 mg/L using Food and Drug Administration Guidance. The identities of the analyte and internal standard in the samples were verified by monitoring the MS/MS transitions of m/z 370 to m/z 252 and m/z 346 to m/z 198 for lansoprazole and omeprazole, respectively. Additionally, the pH of the suspensions was monitored throughout the study. RESULTS The stability of lansoprazole in the oral sodium bicarbonate suspension under refrigeration is compromised prior to what has been previously reported in the literature. Samples kept at room temperature lost >10% of the lansoprazole after 48 hours compared with the refrigerated samples, which maintained integrity up to 7 days. No statistically significant difference was found between the pH of the room temperature and refrigerated suspension samples, indicating that this factor is not the cause for the differences in stability at these two conditions. CONCLUSIONS This study suggests that the extemporaneously compounded lansoprazole oral suspension prepared in 8.4% sodium bicarbonate should not be stored in plastic oral syringes longer than 48 hours at room temperature and no longer than 7 days when refrigerated. These data indicate an expiration time earlier than that previously reported for the refrigerated product (14 days).

scholarly journals In Vitro and in Vivo Immunomodulatory Effects of RDP1258, a Novel Synthetic Peptide

1999 ◽  
Vol 10 (9) ◽  
pp. 1997-2005
Author(s):  
COLM C. MAGEE ◽  
HARUHITO AZUMA ◽  
ANDREAS KNOFLACH ◽  
MARK D. DENTON ◽  
ANIL CHANDRAKER ◽  
...  
Keyword(s):  
Heat Shock ◽  
Heme Oxygenase ◽  
Computer Assisted ◽  
Dependent Manner ◽  
Immunomodulatory Effects ◽  
Heme Oxygenase Activity ◽  
Oxygenase Activity ◽  
Dose Dependent

Abstract. Peptides derived from certain regions of human class I MHC molecules are known to have immunomodulatory effects. In particular, amino acid residues 75-84 of the HLA-B7 and HLA-B2702 molecules have demonstrated allele nonspecific immunosuppression in several animal transplant models. There is evidence that these effects are mediated by binding to intracellular heat shock proteins, including heme oxygenase-1. A new derivative of these peptides, RDP1258, was developed using a novel computer-assisted rational design technique. In vitro, RDP1258 peptide inhibited rat heme oxygenase activity in a dose-dependent manner. Similar to observations made with other in vitro heme oxygenase inhibitors, in vivo administration of RDP1258 peptide to naïve rats resulted in upregulation of splenic heme oxygenase activity. The effects of the peptide on alloimmune responses were then tested. Addition of RDP1258 to rat and human mixed leukocyte reactions inhibited proliferation in a dose-dependent manner. In a rat renal transplantation model, peptide therapy combined with a sub-therapeutic dose of cyclosporin A significantly prolonged allograft survival. These data provide further evidence that modulation of the heat shock protein heme oxygenase by rationally designed peptides affects immune effector functions and may allow the development of novel immunomodulatory strategies in organ transplantation.

Testosterone treatment increases thromboxane function in rat cerebral arteries

2005 ◽  
Vol 289 (2) ◽  
pp. H578-H585 ◽  
Author(s):  
Rayna J. Gonzales ◽  
Amir A. Ghaffari ◽  
Sue P. Duckles ◽  
Diana N. Krause
Keyword(s):  
Cerebral Arteries ◽  
Underlying Mechanism ◽  
Cerebral Vessel ◽  
Testosterone Treatment ◽  
Protein Levels ◽  
Tp Receptors ◽  
The Difference ◽  
Synthase Inhibitor

We previously showed that testosterone, administered in vivo, increases the tone of cerebral arteries. A possible underlying mechanism is increased vasoconstriction through the thromboxane A2 (TxA2) pathway. Therefore, we investigated the effect of chronic testosterone treatment (4 wk) on TxA2 synthase levels and the contribution of TxA2 to vascular tone in rat middle cerebral arteries (MCAs). Using immunofluorescence and confocal microscopy, we demonstrated that TxA2 synthase is present in MCA segments in both smooth muscle and endothelial layers. Using Western blot analysis, we found that TxA2 synthase protein levels are higher in cerebral vessel homogenates from testosterone-treated orchiectomized (ORX+T) rats compared with orchiectomized (ORX) control animals. Functional consequences of changes in cerebrovascular TxA2 synthase were determined using cannulated, pressurized MCA segments in vitro. Constrictor responses to the TxA2 mimetic U-46619 were not different between the ORX+T and ORX groups. However, dilator responses to either the selective TxA2 synthase inhibitor furegrelate or the TxA2-endoperoxide receptor (TP) antagonist SQ-29548 were greater in the ORX+T compared with ORX group. In endothelium-denuded arteries, the dilation to furegrelate was attenuated in both the ORX and ORX+T groups, and the difference between the groups was abolished. These data suggest that chronic testosterone treatment enhances TxA2-mediated tone in rat cerebral arteries by increasing endothelial TxA2 synthesis without altering the TP receptors mediating constriction. The effect of in vivo testosterone on cerebrovascular TxA2 synthase, observed here after chronic hormone administration, may contribute to the risk of vasospasm and thrombosis related to cerebrovascular disease.

scholarly journals THE EFFECT OF ANTICOAGULANT TYPES ON THE IN VITRO ANALYSIS OF CLOPIDOGREL IN HUMAN PLASMA USING LIQUID CHROMATOGRAPHY TANDEM-MASS SPECTROMETRY

2018 ◽  
Vol 10 (1) ◽  
pp. 392
Author(s):  
Yahdiana Harahap ◽  
Anisa Maulidina ◽  
Delly Ramadon
Keyword(s):  
Liquid Chromatography ◽  
Internal Standard ◽  
Linear Calibration ◽  
Column Temperature ◽  
Heparin Plasma ◽  
Liquid Chromatography Tandem Mass ◽  
Edta Plasma ◽  
The Stability ◽  
Vitro Analysis

Objective: The aim of this study was to optimize and validate a plasma clopidogrel analysis method using liquid chromatography tandem-massspectrometry.Methods: Plasma samples were analyzed using a BEH C18 column (1.7 μm; 100 mm×2.1 mm), the mobile phase was 0.1% formic acid in acetonitrile(30:70, v/v). The flow rate was 0.2 mL/min, with a column temperature set to 35°C, an injection volume of 5 μL, an analysis time of 4 min, andirbesartan as the internal standard. Aliquots were obtained by liquid-liquid extraction using ammonium acetate and diethyl ether. The stability andpeak area ratio of the respective plasma area responses were evaluated using ANOVA.Results: No significant differences (p>0.05) were observed between anticoagulants regarding analyte stability. However, the peak area ratioshowed significant differences (p<0.05) between the anticoagulants. The accuracy and precision of the analysis with citrate, heparin, andethylenediaminetetraacetic acid (EDTA) plasma met the quality requirements, and a linear calibration curve was created with concentrations rangingfrom 0.02 to 5.0 ng/mL.Conclusion: The results showed that improved analysis of clopidogrel was achieved using citrate or heparin plasma compared with EDTA plasma.

scholarly journals Cannabidiol Protects Dopaminergic Neurons in Mesencephalic Cultures against the Complex I Inhibitor Rotenone Via Modulation of Heme Oxygenase Activity and Bilirubin

Antioxidants ◽  
2020 ◽  
Vol 9 (2) ◽  
pp. 135 ◽  
Author(s):  
Johanna Catharina Duvigneau ◽  
Alice Trovato ◽  
Andrea Müllebner ◽  
Ingrid Miller ◽  
Christopher Krewenka ◽  
...  
Keyword(s):  
Stress Response ◽  
Heme Oxygenase ◽  
Dopaminergic Neurons ◽  
Complex I ◽  
Neuroblastoma Cells ◽  
Enzyme Reaction ◽  
Stress Markers ◽  
Potent Inhibition ◽  
Oxygenase Activity

Phytocannabinoids protect neurons against stressful conditions, possibly via the heme oxygenase (HO) system. In cultures of primary mesencephalic neurons and neuroblastoma cells, we determined the capability of cannabidiol (CBD) and tetrahydrocannabinol (THC) to counteract effects elicited by complex I-inhibitor rotenone by analyzing neuron viability, morphology, gene expression of IL6, CHOP, XBP1, HO-1 (stress response), and HO-2, and in vitro HO activity. Incubation with rotenone led to a moderate stress response but massive degeneration of dopaminergic neurons (DN) in primary mesencephalic cultures. Both phytocannabinoids inhibited in-vitro HO activity, with CBD being more potent. Inhibition of the enzyme reaction was not restricted to neuronal cells and occurred in a non-competitive manner. Although CBD itself decreased viability of the DNs (from 100 to 78%), in combination with rotenone, it moderately increased survival from 28.6 to 42.4%. When the heme degradation product bilirubin (BR) was added together with CBD, rotenone-mediated degeneration of DN was completely abolished, resulting in approximately the number of DN determined with CBD alone (77.5%). Using N18TG2 neuroblastoma cells, we explored the neuroprotective mechanism underlying the combined action of CBD and BR. CBD triggered the expression of HO-1 and other cell stress markers. Co-treatment with rotenone resulted in the super-induction of HO-1 and an increased in-vitro HO-activity. Co-application of BR completely mitigated the rotenone-induced stress response. Our findings indicate that CBD induces HO-1 and increases the cellular capacity to convert heme when stressful conditions are met. Our data further suggest that CBD via HO may confer full protection against (oxidative) stress when endogenous levels of BR are sufficiently high.

scholarly journals Modified method of erythropoietin (ESF) bioassay in vitro using mouse fetal liver cells. II. measurement of ESF in human serum

Blood ◽  
1978 ◽  
Vol 52 (3) ◽  
pp. 569-577
Author(s):  
G de Klerk ◽  
AA Hart ◽  
C Kruiswijk ◽  
R Goudsmit
Keyword(s):  
Human Serum ◽  
Fetal Liver ◽  
Internal Standard ◽  
Immune Serum ◽  
Quantitative Detection ◽  
Modified Method ◽  
The Difference ◽  
Normal Males ◽  
Test Serum

A modification of the mouse fetal liver cell bioassay for erythropoietin (ESF) that allows quantitative detection of ESF in human serum is described. The modification consists of (1) correction for the effect of serum iron on 59Fe incorporation into heme and (2) the use of an “internal standard,” i.e., a standard ESF preparation dissolved in the assayed test serum. As a result of this modification the statistical method of bioassay analysis had to be changed fundamentally. The mean serum concentration of ESF measured in 20 normal males was 48 microunit/ml, as compared to 29 microunit/ml in 18 females. The difference was significant at p = 0.017. The stimulatory activity of a human serum on 59Fe incorporation into heme could be neutralized by an anti-human ESF immune serum.

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