Recovery of malondialdehyde in urine as a 2,4-dinitrophenylhydrazine derivative analyzed with high-performance liquid chromatography

Malondialdehyde (MDA) in urine was measured as a 2,4-dinitrophenylhydrazine (DNPH) derivative using high-performance liquid chromatography (HPLC) for the analysis. MDA standard coeluted with a peak obtained from rat urine after i.p. administration of MDA standard. This peak was also the only peak containing 14C after injection of a [14C]MDA standard, and was shown by mass spectrometry to contain 1-(2,4-dinitrophenyl)pyrazole, the derivative formed when MDA is treated with DNPH. Depending on the amount given (0.3-5.5 mumol), the recovery (after 24 h sampling period) in urine was 0.7-2.6%. This apparent non-linear kinetics may relate to several factors, such as dose-dependent metabolism. However, the peak urinary concentration approached the expected plasma concentration and reproducible recovery data were obtained, suggesting that MDA was passively excreted in a reasonably stable form. These data indicate that monitoring MDA excretion in urine can give useful information about lipid peroxidation in vivo.     

Methyl malondialdehyde is not suitable as an internal standard for malondialdehyde detection in urine after derivatisation with 2,4-dinitrophenylhydrazine

A previously described method of measurement of malondialdehyde (MDA) in human urine after derivatisation with 2,4-dinitrophenylhydrazine (DNPH) was tested for a possibility of using methyl malondialdehyde (MeMDA) as an internal standard. Despite structural similarity, those compounds were found to produce different yields of derivatisation under the same conditions depending on urine matrix. We conclude, that MeMDA is not suitable as an internal standard for the measurement of MDA in urine under previously reported conditions when DNPH is used as a deriviatising agent.     

Measurement of free and bound malondialdehyde in plasma by high-performance liquid chromatography as the 2,4-dinitrophenylhydrazine derivative

We established a method for the detection of free and total (free and bound) malondialdehyde (MDA) in human plasma samples after derivatisation with 2,4-dinitrophenylhydrazine (DNPH). Free MDA was prepared by perchloric acid deproteinisation whereas an alkaline hydrolysation step for 30 min at 60°C was introduced prior to protein precipitation for the determination of total MDA. Derivatisation was accomplished in 10 min at room temperature subsequently chromatographed by HPLC on a reversed-phase 3 μm C₁₈ column with UV detection (310 nm). The detection limit was 25 pmol/ml for free and 0.3 nmol/ml for total MDA. The recovery of MDA added to different human plasma samples was 93.6% (n=11; RSD 7.1%) for the hydrolysation procedure. In samples from 12 healthy volunteers who underwent a hypoxic treatment (13% O₂ for 6 h) we estimated a baseline value of total MDA of 2.16 nmol/ml (SD 0.29) (ambient air) with a significant increase to 2.92 (nmol/ml, SD 0.57; P=0.01) after the end of this physiological oxidative stress challenge. Plasma values of free MDA in these samples were close to our detection limit. The presented technique can easily performed with an isocratic HPLC apparatus and provides highly specific results for MDA as do sophisticated GC–MS methods.     

Determination of malondialdehyde by liquid chromatography as the 2,4-dinitrophenylhydrazone derivative: a marker for oxidative stress in cell cultures of human hepatoma HepG2

The use of a rapid and sensitive assay for N-acetylaspartate (NAA) in urine or eluates from dried urine on filter paper to make a chemical diagnosis of Canavan disease (CD) is described. It involves a simplified urease pretreatment for sample preparation and gas chromatography-mass spectrometry (EI, scanning mode) with or without stable isotope dilution. Significant improvements in the recovery of NAA and the GC-MS data-handling device made the assay without stable isotope dilution sensitive and quantitative enough to diagnose CD: Its coefficient of variation (CV) was below 12%. The CV obtained with stable isotope dilution was below 9%. One patient with CD had an abnormal NAA level that was more than 6 S.D. above the mean of the age-matched controls. This diagnostic procedure is accurate for screening and for the chemical diagnosis of CD, with a good cost:benefit ratio. The urinary NAA levels of the healthy controls decreased significantly with age. This change should be considered in making a chemical diagnosis of this disease.     

A chemiluminescence method to detect malondialdehyde in plasma and urine

A highly sensitive and convenient method to detect malondialdehyde (MDA) in specimens of plasma and urine was developed using high-performance liquid chromatography combining chemiluminescence detection. MDA was separated through a reverse-phase C18 column at a flow rate of 0.8 ml min⁻¹. It was then detected by a chemiluminescence detector. Variables that affected chemiluminescence reaction, including mobile phase, flow rate, chemiluminescence reagent, parameters of the photomultiplier tube, and temperature, were studied. This assay was linear from 0.50 to 50 μmol L⁻¹, with limits of detection and quantification of 0.08 and 0.30 μmol L⁻¹, respectively. The recoveries were in the range of 92.2 to 98.5%, and the intra- and interday reproducibilities were obtained with relative standard deviations of less than 4% and less than 6%, respectively. MDA in plasma and urine was stable for 12 h at 4 °C. MDA levels were 2.74, 3.19, and 3.20 μmol L⁻¹ for plasma and 1.25, 2.04, and 1.58 μmol L⁻¹ for urine, respectively. The proposed method is simple, rapid, and sensitive with low cost. Taken together, this method can be suitable for detecting MDA in tissue and feed specimens.     

Simplified 2,4-dinitrophenylhydrazine spectrophotometric assay for quantification of carbonyls in oxidized proteins

This work proposes a modification of the 2,4-dinitrophenylhydrazine (DNPH) spectrophotometric assay commonly used to evaluate the concentration of carbonyl groups in oxidized proteins. In this approach NaOH is added to the protein solution after the addition of DNPH, shifting the maximum absorbance wavelength of the derivatized protein from 370 to 450 nm. This reduces the interference of DNPH and allows the direct quantification in the sample solution without the need for the precipitation, washing, and resuspension steps that are carried out in the traditional DNPH method. The two methods were compared under various conditions and are statistically equivalent.     

Goldfish can recover after short-term exposure to 2,4-dichlorophenoxyacetate: Use of blood parameters as vital biomarkers

This study investigated the effects of 2,4-dichlorophenoxyacetic acid (2,4-D), a widely used herbicide, on the metabolism of goldfish, Carassius auratus, using only vital (non-lethal) approaches. After 96 h exposure to 1, 10 or 100 mg/L of 2,4-D selected hematological (total hemoglobin and hematocrit) and biochemical (glucose content, aspartate transaminase and acetylcholinesterase activities) parameters were unchanged in blood of exposed fish. At 100 mg/L of 2,4-D lymphocyte numbers decreased by 8%, whereas promyelocyte and metamyelocyte numbers increased by 7- and 2-fold, respectively. Exposure to 100 mg/L of 2,4-D also elevated carbonyl protein levels (by 2-fold), triglyceride content (by 43%) and alanine transaminase activity (by 46%) in goldfish plasma. All of these hematological and biochemical parameters reverted to control values after a 96 h recovery period. These data indicate that 2,4-D has toxicological effects on goldfish that can be monitored with multiple diagnostic tests using non-lethal blood testing.     

Hydrazone formation of 2,4-dinitrophenylhydrazine with pyrroloquinoline quinone in porcine kidney diamine oxidase

Homogeneous diamine oxidase (EC 1.4.3.6) from porcine kidney was treated with the inhibitor 2,4-dinitrophenylhydrazine (DNPH). The coloured compounds formed were detached with pronase and purified to homogeneity. When the reaction with DNPH was conducted under an O2 atmosphere, the product (obtained in a yield of 55%) was the C(5)-hydrazone of pyrroloquinoline quinone (PQQ) and DNPH, as revealed by its chromatographic behaviour, absorption spectrum and 1H-NMR spectrum. Only 6% of this hydrazone was formed under air, the main product isolated being an unidentified reaction product of DNPH with the enzyme. Porcine kidney diamine oxidase is the second mammalian enzyme shown to have PQQ as its prosthetic group. In view of the requirements for hydrazone formation with DNPH, it is incorrect to assume that inhibition of this type of enzymes with common hydrazines is simply due to blocking of the carbonyl group of its cofactor.     

Recovery of growth of Hyphochytrium catenoides after exposure to environmental stress

ABSTRACT. The survival of an isolate of Hyphochytrium catenoides collected from soil in the Blue Mountains in eastern New South Wales, Australia, was tested under extreme conditions in the laboratory. This isolate recovered growth after being subjected to drying on filter paper, to heat while desiccated, to hypersalinity, to strict anaerobic conditions, to freezing temperatures, and to a short period in solutions at pH 2.8–11.2. The capacity to survive under these conditions in the laboratory suggests adaptation to fluctuating conditions in the soil. The partial DNA sequence of the 28S ribosomal RNA gene in the isolate from New South Wales was 98% similar to that in an isolate from Arizona with a similar morphology.     

Recovery of yeast cells after exposure to ionizing radiation and hyperthermia

Quantitative regularities of recovery of wild-type diploid yeast cells irradiated with gamma-rays (60Co) simultaneously with exposure to high temperatures were studied. It was shown that in conditions of such a combined action the constant of recovery did not depend on the temperature at which the irradiation was carried out. However, with an increase of acting temperature an augmentation in the portion of irreversible component was registered. The analysis of cell inactivation revealed that the augmentation of the irreversible component was accompanied by a continuous increase of cell killing without any postirradiation division after which cells are incapable of recovery. The reproductive death was mainly exerted after ionizing radiation applied alone while in conditions of simultaneous thermoradiation action the interphase killing (cell death without division) predominated. It is concluded on this base that the mechanism of synergistic interaction of ionizing radiation and hyperthermia may be related with cardinal change in mechanisms of cell killing.     

The electrical response of Phaseolus vulgaris roots to abrupt exposure to hydroquinone

Previous reports have suggested the primary mode of action of the allelochemical hydroquinone involves disruption of root cell membrane transport. Here we report the effects of hydroquinone on common bean (Phaseolus vulgaris) plants. Growth of leaves, roots and stems were all inhibited by 14 day exposure to 0.01 mM or 0.25 mM hydroquinone. Chlorophyll fluorescence (Fv/Fm) was inhibited by 0.25 mM hydroquinone. The membrane potential of P. vulgaris root cortex cells briefly hyperpolarized and subsequently slowly transiently depolarized upon abrupt exposure to a range of hydroquinone concentrations. Both the hyperpolarization and depolarization were concentration dependent but appeared saturable. Root cells exposed to 0.03 mM hydroquinone hyperpolarized 3.4 mV (+/− 0.6 s.e.) 3 minutes after the start of exposure then depolarized 36.7 mV (+/− 3.9) with no effect evident after 24 hours. Individual recordings showed a response to as little as 0.001 mM hydroquinone. Exposure of P. vulgaris root cells to arbutin, a nontoxic monoglucoside of hydroquinone, produced a similar but much smaller (approximately 25%) electrical response. Exposure of root cells of Antennaria microphylla, a known allelopathic source (donor plant) of hydroquinone, also produced a much smaller hyperpolarization and depolarization response. It is concluded that the electrical response to hydroquinone by P. vulgaris root cells and the changes in membrane transport they represent are not sufficiently large or long lasting enough to disrupt mineral and water uptake leading to plant injury. The possibility, however, that these events are related to initiation of signal transduction events leading to cell death is discussed.Key words: allelopathy, hydroquinone, membrane potential, depolarization, hyperpolarization, Phaseolus vulgaris, Antennaria microphylla     

Porphyrin profiles in blood and urine as a biomarker for exposure to various arsenic species.

A sensitive method using HPLC with fluorescence detection has been established for the measurement of porphyrins in biological materials. The assay recoveries were 88.0+/-1.8% for protoporphyrin IX in the blood, and ranged from 98.3+/-2.7% to 111.1+/-7.4% for various porphyrins in the urine. This method was employed to investigate the altered porphyrin profiles in rats after a single dose of various arsenicals including soluble sodium arsenate and sodium arsenite, and the relatively insoluble calcium arsenite, calcium arsenate and arsenic-contaminated soils at dose rates of 5 mg/kg or 0.5 mg/kg body weight. Porphyrin concentrations increased within 2448 hr after the arsenic treatment in blood and urine. Protoporphyrin IX is the predominant porphyrin in the blood. In rats administered 5 mg As(III)/kg body weight, protoporphyrin IX concentration elevated to 123% of the control values in rats, 24 hr after the treatment. Higher increases were recorded in the urinary protoporphyrin IX (253% at 24 hr; 397% on day 2), uroporphyrin (121% at 24 hr; 208% on day 2) and coproporphyrin III (391% at 24 hr; 304% on day 2), while there was no significant increase (109% on day 3) observed in the urinary coproporphyrin I excretion. In rats administered 5 mg As(V)/kg, urinary excretion of protoporphyrin LX, uroporphyrin, coproporphyrin III and coproporphyrin I elevated to the maximum levels by 48 hr with the corresponding percentage values compared to the control being 177%, 158%, 224% and 143%, respectively. In rats dosed with 5 mg As(III)/kg, the increases (expressed as % of the control values) of protoporphyrin IX in the blood were in the order: sodium arsenite (144%) > sodium arsenate (125%) > calcium arsenite (123%) > calcium arsenate. In contrast, there was no significant increase of protoporphyrin IX, when the six arsenic-contaminated cattle dip soils and nine copper chrome arsenate (CCA-contaminated) soils were administered to the rats. Probable explanations are discussed.     

Recovery after exposure to near-ultraviolet light of cells containing 5-bromodeoxyuridine.

The survival of synchronized V79 Chinese hamster cells irradiated with near-ultraviolet light after a 1-h labeling with 5-bromodeoxyuridine (BrdUrd) is highly dependent upon the cell's position in the cell cycle at the time of irradiation (Hagan, M., and M. M. Elkind. Biophys. J. 1979. 27:75-86). In this report, we show that cells irradiated in the same S phase after BrdUrd incorporation demonstrate an ability to repair sublethal damage, in contrast to the lack of an increase in survival with dose fractionation in template-labeled cells (Ben-Hur, E., and M. M. Elkind. Mutat. Res. 1972. 14:236-245). In addition, we show that pulse-labeled cells in S phase can repair potentially lethal damage expressed by caffeine. The kinetics of these recovery processes and the absence of a caffeine effect on the repair of sublethal damage indicate that these two processes are to a large degree unrelated. We conclude that in template-labeled cells inadequate time to effect prereplicational repair precludes effective contributions to cell survival from other kinds of DNa repair processes.     

Effect of alpha-tocopherol on the production of malondialdehyde in rat tissue homogenates after hypobaric exposure

Alpha-tocopherol content and production of malondialdehyde (MDA) was measured in liver, kidney, heart, lung, brain and skeletal muscle homogenates of control and hypoxic rats (following a 2-h-exposure to 200 mm Hg): the samples were incubated at 37 degrees C in air for 1 h. MDA production showed no relation with the content of alpha-tocopherol in control and hypoxic rats. In control animals, the lowest MDA level was found in lungs: it was several fold lower than in other tissues. After hypobaric exposure, a marked increase in MDA level could be observed in lungs only. No marked changes in alpha-tocopherol concentration could be observed in any of the tissues tested. A single i.p. injection of 25 and 50 mg/alpha-tocopherol acetate/kg body mass, 2 hours prior to the exposure produced organ-specific accumulation of alpha-tocopherol. Both doses of alpha-tocopherol resulted in a reduced (by about 40%) production of MDA in lung homogenates. The addition of alpha-tocopherol (750 nmol/g wet tissue mass) to homogenates from control and hypoxic rats prior to the incubation resulted in a marked inhibition of MDA production in all tissues (49-70%).     

Methods for determination of carbonyl compounds by 2,4-dinitrophenylhydrazine and their application to the assay of aldehyde dehydrogenase

Two 2,4-dinitrophenylhydrazine methods are presented for the determination of small amounts of carbonyl compounds when present in biological materials as a single carbonyl compound. When the hydrazones of the compounds are soluble in ethanolic alkaline solution, a direct method is carried out omitting an extraction procedure with organic solvent. On the other hand, the extraction procedure is used when the hydrazones of the compounds are scarcely soluble in ethanolic alkaline solution.     

Recovery from a near-lethal exposure to ultraviolet-C radiation in a scleractinian coral

Hermatypic (reef building) corals live in an environment characterized by high ambient levels of photosynthetically active radiation (PAR) and ultraviolet radiation (UVR). Photoadaptive mechanisms have evolved to protect the sensitive cell structures of the host coral and their photosynthetic, endosymbiotic zooxanthellae. Environmental stressors may destabilize the coral-zooxanthellae system resulting in the expulsion of zooxanthellae and/or loss of photosynthetic pigment within zooxanthellae, causing a condition known as bleaching. It is estimated that 1% of the world’s coral population is lost yearly, partly due to bleaching. Despite intensive research efforts, a single unified mechanism cannot explain this phenomenon. Although UVA and UVB cellular damage is well documented, UVC damage is rarely reported due to its almost complete absorption in the stratosphere. A small scale coral propagation system at the University of Maine was accidentally exposed to 15.5 h of UVC radiation (253.7 nm) from a G15T8 germicidal lamp, resulting in a cumulative surface irradiance of 8.39 × 10⁴ J m⁻². An experiment was designed to monitor the progression of UVC induced damage. Branch sections from affected scleractinian corals, Acropora yongei and Acropora formosa were submitted to histopathology to provide an historical record of tissue response. The death of gastrodermal cells and necrosis resulted in the release of intracellular zooxanthellae into the gastrovascular canals. Zooxanthellae were also injured as evidenced by pale coloration, increased vacuolization and loss of membrane integrity. The recovery of damaged coral tissue likely proceeds by re-epithelialization and zooxanthellae repopulation of gastrodermal cells by adjacent healthy tissue.