scholarly journals 2-(3-Hydroxy-5-phosphonooxymethyl-2-methyl-4-pyridyl)-1,3-thiazolidine-4-carboxylic Acid, Novel Metabolite of Pyridoxal 5′-Phosphate and Cysteine Is Present in Human Plasma—Chromatographic Investigations

2020 ◽  
Vol 21 (10) ◽  
pp. 3548 ◽  
Author(s):  
Justyna Piechocka ◽  
Monika Wrońska ◽  
Iwona E. Głowacka ◽  
Rafał Głowacki
Keyword(s):  
Carboxylic Acid ◽  
Human Plasma ◽  
Vitamin B6 ◽  
Active Form ◽  
Aqueous Environment ◽  
Breast Cancer Patients ◽  
Limit Of Quantification ◽  
Apparently Healthy

It is well-established that aminothiols, to which cysteine (Cys) belongs, are highly reactive towards aldehydes in an aqueous environment, forming substituted thiazolidine carboxylic acids. This report provides evidence that formation of the product containing a thiazolidine ring through non-enzymatic condensation of Cys and an active form of vitamin B6 pyridoxal 5′-phosphate (PLP) occurs in vivo in humans. To prove this point, a new method, based on a gas chromatography coupled with mass spectrometry (GC-MS), has been designed to identify and quantify Cys and PLP adduct, 2-(3-hydroxy-5-phosphonooxymethyl-2-methyl-4-pyridyl)-1,3-thiazolidine-4-carboxylic acid (HPPTCA) in human plasma. The GC-MS assay relies on sample deproteinization by ultrafiltration over cut-off membranes and preconcentration by drying under vacuum, followed by treatment of the residue with derivatization mixture containing anhydrous pyridine, N-trimethylsilyl-N-methyl trifluoroacetamide (MSTFA) and trimethylchlorosilane (TMCS). The method quantifies HPPTCA in a linear range from 1 to 20 µmol L−1, where the lowest standard on the calibration curve refers to the limit of quantification (LOQ). The validity of the method was demonstrated. Furthermore, the method was successfully applied to plasma samples donated by apparently healthy volunteers and breast cancer patients. The GC-MS assay provides a new tool that will hopefully facilitate studies on the role of HPPTCA in living systems.

scholarly journals Identification and Determination of 1,3-Thiazinane-4-carboxylic Acid in Human Urine—Chromatographic Studies

2022 ◽  
Vol 23 (2) ◽  
pp. 598
Author(s):  
Justyna Piechocka ◽  
Natalia Litwicka ◽  
Rafał Głowacki
Keyword(s):  
Carboxylic Acid ◽  
Human Urine ◽  
Clinical Analysis ◽  
Gas Chromatography Mass Spectrometry ◽  
Aqueous Environment ◽  
Limit Of Quantification ◽  
Isobutyl Chloroformate ◽  
United States Food ◽  
States Food

It is well established that homocysteine (Hcy) and its thiolactone (HTL) are reactive towards aldehydes in an aqueous environment, forming substituted thiazinane carboxylic acids. This report provides evidence that Hcy/HTL and formaldehyde (FA) adduct, namely 1,3-thiazinane-4-carboxylic acid (TCA) is formed in vivo in humans. In order to provide definitive proof, a gas chromatography–mass spectrometry (GC–MS) based method was elaborated to identify and quantify TCA in human urine. The GC–MS assay involves chemical derivatization with isobutyl chloroformate (IBCF) in the presence of pyridine as a catalyst, followed by an ethyl acetate extraction of the obtained isobutyl derivative of TCA (TCA-IBCF). The validity of the method has been demonstrated based upon United States Food and Drug Administration recommendations. The assay linearity was observed within a 1–50 µmol L−1 range for TCA in urine, while the lowest concentration on the calibration curve was recognized as the limit of quantification (LOQ). Importantly, the method was successfully applied to urine samples delivered by apparently healthy volunteers (n = 15). The GC–MS assay may provide a new analytical tool for routine clinical analysis of the role of TCA in living systems in the near future.

The antioxidative effect of de novo generated vitamin B6 in Plasmodium falciparum validated by protein interference

2012 ◽  
Vol 443 (2) ◽  
pp. 397-405 ◽  
Author(s):  
Julia Knöckel ◽  
Ingrid B. Müller ◽  
Sabine Butzloff ◽  
Bärbel Bergmann ◽  
Rolf D. Walter ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Vitamin B6 ◽  
De Novo ◽  
Active Form ◽  
Cellular Level ◽  
Dominant Negative ◽  
Dominant Negative Effect ◽  
Transcription Analysis

The malaria parasite Plasmodium falciparum is able to synthesize de novo PLP (pyridoxal 5′-phosphate), the active form of vitamin B6. In the present study, we have shown that the de novo synthesized PLP is used by the parasite to detoxify 1O2 (singlet molecular oxygen), a highly destructive reactive oxygen species arising from haemoglobin digestion. The formation of 1O2 and the response of the parasite were monitored by live-cell fluorescence microscopy, by transcription analysis and by determination of PLP levels in the parasite. Pull-down experiments of transgenic parasites overexpressing the vitamin B6-biosynthetic enzymes PfPdx1 and PfPdx2 clearly demonstrated an interaction of the two proteins in vivo which results in an elevated PLP level from 12.5 μM in wild-type parasites to 36.6 μM in the PfPdx1/PfPdx2-overexpressing cells and thus to a higher tolerance towards 1O2. In contrast, by applying the dominant-negative effect on the cellular level using inactive mutants of PfPdx1 and PfPdx2, P. falciparum becomes susceptible to 1O2. Our results demonstrate clearly the crucial role of vitamin B6 biosynthesis in the detoxification of 1O2 in P. falciparum. Besides the known role of PLP as a cofactor of many essential enzymes, this second important task of the vitamin B6de novo synthesis as antioxidant emphasizes the high potential of this pathway as a target of new anti-malarial drugs.

DETECTION OF INACTIVATED α2-MACROGLOBULIN (α2M) IN HUMAN PLASMA USING MONOCLONAL ANTIBODIES

1987 ◽  
Author(s):  
J Abbink ◽  
J Nuijens ◽  
C Huijbregts ◽  
E Hack
Keyword(s):  
Monoclonal Antibodies ◽  
Longitudinal Studies ◽  
Human Plasma ◽  
Dextran Sulfate ◽  
Optimal Conditions ◽  
Α2 Macroglobulin ◽  
In Vitro Activation

Monoclonal antibodies (mAbs) were raised against human a2M. Five mAbs that bound to α2M in ELISA were further analyzed by a radioimmunoassay (RIA) for their reaction with three types of α2M: native α2M, chemically inactivated α2M (iα2M) (methylamine treated), and proteolytically iα2M. One mAb reacted with all forms of α2M, while four mAbs bound both forms of ia2M but not native α2M. One of these latter mAbs (Ml) was used to develop a RIA (the Ml-assay) for the detection of iα2M in plasma: Ml coupled to Sepharose is incubated with the plasma to be tested, and bound iα2M is detected by a subsequent incubation with polyclonal 125I-anti-α2M antibodies. As little as 5 ng of iα2M can be detected with this assay in the presence of an excess of native α2M. This assay was then applied to measure inactivation of α2M in vitro and in vivo. In vitro activation of the contact system in plasma by dextran sulfate results in the inactivation of ca 10% of α2M. When blood from normal donors was collected under optimal conditions, about 0.5% of the total α2M content appeared to be iα2M. Longitudinal studies in patients (a.o. with septicaemie, during cardiopulmunary bypass) revealed that increased levels of iα2M occurred sporadically. The Ml-assay appears to be useful to monitor the role of α2M in human diseases.

scholarly journals Anti-Inflammatory and Antinociceptive Activities of Anthraquinone-2-Carboxylic Acid

2016 ◽  
Vol 2016 ◽  
pp. 1-12 ◽  
Author(s):  
Jae Gwang Park ◽  
Seung Cheol Kim ◽  
Yun Hwan Kim ◽  
Woo Seok Yang ◽  
Yong Kim ◽  
...  
Keyword(s):  
Carboxylic Acid ◽  
Molecular Mechanisms ◽  
Interleukin 1 ◽  
Nuclear Factor ◽  
Experimental Conditions ◽  
Gene Assay ◽  
Anti Inflammatory ◽  
Gastric Irritation

Anthraquinone compounds are one of the abundant polyphenols found in fruits, vegetables, and herbs. However, thein vivoanti-inflammatory activity and molecular mechanisms of anthraquinones have not been fully elucidated. We investigated the activity of anthraquinones using acute inflammatory and nociceptive experimental conditions. Anthraquinone-2-carboxylic acid (9,10-dihydro-9,10-dioxo-2-anthracenecarboxylic acid, AQCA), one of the major anthraquinones identified from Brazilian taheebo, ameliorated various inflammatory and algesic symptoms in EtOH/HCl- and acetylsalicylic acid- (ASA-) induced gastritis, arachidonic acid-induced edema, and acetic acid-induced abdominal writhing without displaying toxic profiles in body and organ weight, gastric irritation, or serum parameters. In addition, AQCA suppressed the expression of inflammatory genes such as cyclooxygenase- (COX-) 2 in stomach tissues and lipopolysaccharide- (LPS-) treated RAW264.7 cells. According to reporter gene assay and immunoblotting analyses, AQCA inhibited activation of the nuclear factor- (NF-)κB and activator protein- (AP-) 1 pathways by suppression of upstream signaling involving interleukin-1 receptor-associated kinase 4 (IRAK1), p38, Src, and spleen tyrosine kinase (Syk). Our data strongly suggest that anthraquinones such as AQCA act as potent anti-inflammatory and antinociceptive componentsin vivo, thus contributing to the immune regulatory role of fruits and herbs.

scholarly journals Mutations in efflux pump Rv1258c (Tap) cause resistance to pyrazinamide and other drugs inM. tuberculosis

2018 ◽  
Author(s):  
Jiayun Liu ◽  
Wanliang Shi ◽  
Shuo Zhang ◽  
Gail Cassell ◽  
Dmitry A. Maslov ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Drug Targets ◽  
Drug Exposure ◽  
Efflux Pump ◽  
Active Form ◽  
Point Mutations ◽  
Drug Susceptibility ◽  
Clinical Isolates

AbstractAlthough drug resistance inM. tuberculosisis mainly caused by mutations in drug activating enzymes or drug targets, there is increasing interest in possible role of efflux in causing drug resistance. Previously, efflux genes are shown upregulated upon drug exposure or implicated in drug resistance in overexpression studies, but the role of mutations in efflux pumps identified in clinical isolates in causing drug resistance is unknown. Here we investigated the role of mutations in efflux pump Rv1258c (Tap) from clinical isolates in causing drug resistance inM. tuberculosisby constructing point mutations V219A, S292L in Rv1258c in the chromosome ofM. tuberculosisand assessed drug susceptibility of the constructed mutants. Interestingly, V219A, S292L point mutations caused clinically relevant drug resistance to pyrazinamide (PZA), isoniazid (INH), and streptomycin (SM), but not to other drugs inM. tuberculosis. While V219A point mutation conferred a low level resistance, the S292L mutation caused a higher level of resistance. Efflux inhibitor piperine inhibited INH and PZA resistance in the S292L mutant but not in the V219A mutant. S292L mutant had higher efflux activity for pyrazinoic acid (the active form of PZA) than the parent strain. We conclude that point mutations in the efflux pump Rv1258c in clinical isolates can confer clinically relevant drug resistance including PZA and could explain some previously unaccounted drug resistance in clinical strains. Future studies need to take efflux mutations into consideration for improved detection of drug resistance inM. tuberculosisand address their role in affecting treatment outcome in vivo.

scholarly journals Role of reversible phosphorylation in mediating changes in the activity of hepatic 3-hydroxy-3-methylglutaryl-CoA reductase during pregnancy and lactation in the rat

1987 ◽  
Vol 248 (3) ◽  
pp. 993-996 ◽  
Author(s):  
R A Easom ◽  
V A Zammit
Keyword(s):  
Reductase Activity ◽  
Post Partum ◽  
Active Form ◽  
Total Activity ◽  
Female Rats ◽  
Hmg Coa Reductase ◽  
Pregnancy And Lactation ◽  
Coa Reductase

1. The expressed and total (completely dephosphorylated) activities of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase were measured in microsomal fractions isolated from cold-clamped liver samples from female rats in various stages of the reproductive cycle. 2. There was little change in total HMG-CoA reductase activity during pregnancy and early lactation, but after 2 days post partum there was a marked increase in total activity. 3. The expressed/total activity ratio of HMG-CoA reductase showed a profound decrease during the last 2 days of pregnancy. The fraction of the enzyme in the active form increased progressively during the first 2 days of lactation. 4. The combined effect of these changes was that the expressed activity of HMG-CoA reductase changed in parallel with the known changes in the hepatic rate of cholesterogenesis during pregnancy and lactation in vivo.

Isolation of a New Enzyme from Human Plasma Fractions and its Effects on Coagulation

1979 ◽  
Author(s):  
Michael H. Coan ◽  
Duane D. Schroeder ◽  
Milton M. Mozen
Keyword(s):  
Human Plasma ◽  
Human Factor ◽  
Active Form ◽  
Factor V ◽  
Diisopropyl Fluorophosphate ◽  
Human Factor Viii ◽  
Plasma Fractions ◽  
Related Proteins

A new hydrolase enzyme has been isolated from human plasma fractions. This enzyme has been purified to homogeneity by adsorption to and elution from DEAE-5ephadex, Benzamidine- EACA-Sepharose, and Heparin-5epharose. The molecular weight, as judged by SDSpolyacrylamide gel electrophoresis, is 70,000 daltons; and there are 2 peptide chains (45,000 and 25,000) after reduction. This enzyme, isolated in an active form, hydrolyzes 5-2238 (H-D-Phe-Pip-Arg-PNA), a chromogenic substrate (AB Kabi) , at a rapid rate, S-225l to a slishtly lesser extent, and S-2222, S-2302, and 5-2160 about equally and much more slowly. CaCl2 greatly enhances its activity. The enzyme is inhibited by antithrombin III especially in the presence of heparin, but poorly by soybean trypsin inhibitor or by diisopropyl fluorophosphate. The enzyme binds to and can be eluted from insoluble barium salts. In vitro, the protein will activate and degrade human Factor VIII, will activate human Factor V, and will inhibit epinephrine-induced platelet aggregation; however, the in vivo function is unknown . Comparison with known properties of protein C and other coagulation-related proteins indicates. that this enzyme has not been previously described.

scholarly journals What is molt-inhibiting hormone? The role of an ecdysteroidogenesis inhibitor in the crustacean molting cycle

1989 ◽  
Vol 86 (17) ◽  
pp. 6826-6829 ◽  
Author(s):  
Yoko Naya ◽  
Mayumi Ohnishi ◽  
Midori Ikeda ◽  
Wataru Miki ◽  
Koji Nakanishi
Keyword(s):  
Procambarus Clarkii ◽  
Active Form ◽  
Xanthurenic Acid ◽  
Sinus Gland ◽  
Inhibitory Effects ◽  
Molting Cycle ◽  
Biosynthesis Inhibitors

The in vivo molt-inhibitory effects of the ecdysone biosynthesis inhibitors 3-hydroxy-L-kynurenine and xanthurenic acid were investigated. These ecdysone biosynthesis inhibitors, isolated from the eyestalks of blue crabs (Callinectes sapidus), were injected into eyestalk-ablated crayfish (Procambarus clarkii). The active factor was found to be species-nonspecific within crabs and crayfish. The seasonal profiles of the xanthurenic acid and ecdysone titers exhibited a staggered relationship. Moreover, the activity of a 3-hydroxy-L-kynurenine aminotransferase varied during the molting cycle. The data suggested that 3-hydroxy-L-kynurenine, which is secreted from the X-organ-sinus gland complex of crustaceans, is released into the hemolymph, and after accumulating at the surface of the Y-organ, is converted into the active form, xanthurenic acid. Xanthurenic acid was found to profoundly repress ecdysteroidogenesis in vitro.

Systemic activation of glutamate dehydrogenase increases renal ammoniagenesis: implications for the hyperinsulinism/hyperammonemia syndrome

2010 ◽  
Vol 298 (6) ◽  
pp. E1219-E1225 ◽  
Author(s):  
Jason R. Treberg ◽  
Kathy A. Clow ◽  
Katie A. Greene ◽  
Margaret E. Brosnan ◽  
John T. Brosnan
Keyword(s):  
Skeletal Muscle ◽  
Carboxylic Acid ◽  
Glutamate Dehydrogenase ◽  
Glutamate Oxaloacetate Transaminase ◽  
Gain Of Function ◽  
Ammonia Metabolism ◽  
Allosteric Activator

The hyperinsulism/hyperammonemia (HI/HA) syndrome is caused by glutamate dehydrogenase (GDH) gain-of-function mutations that reduce the inhibition by GTP, consequently increasing the activity of GDH in vivo. The source of the hyperammonemia in the HI/HA syndrome remains unclear. We examined the effect of systemic activation of GDH on ammonia metabolism in the rat. 2-Aminobicyclo[2,2,1]heptane-2-carboxylic acid (BCH) is a nonmetabolizable analog of the natural GDH allosteric activator leucine. A dose of 100 μmol BCH/100 g rat resulted in a mild systemic hyperammonemia. Using arterial-venous (A-V) differences, we exclude the liver, intestine, and skeletal muscle as major contributors to this BCH-induced hyperammonemia. However, renal ammonia output increased, as demonstrated by an increase in A-V difference for ammonia across the kidney in BCH-treated animals. Isolated renal cortical tubules incubated with BCH increased the rate of ammoniagenesis from glutamine by 40%. The flux through GDH increased more than twofold when BCH was added to renal mitochondria respiring on glutamine. The flux through glutaminase was not affected by BCH, whereas glutamate-oxaloacetate transaminase flux decreased when normalized to glutaminase flux. These data show that increased renal ammoniagenesis due to activation of GDH can explain the BCH-induced hyperammonemia. These results are discussed in relation to the organ source of the ammonia in the HI/HA syndrome as well as the role of GDH in regulating renal ammoniagenesis.

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