Formation and polymerisation of malonaldehyde during irradiation of aqueous solutions of d-glucose and lactose with ultrasound

Irradiation with ultrasound, vigorous stirring, or vibration of aqueous solutions of d-glucose and lactose can induce chemical reactions. Malonaldehyde has been identified as one product, and the influence of pH and gas atmosphere on its formation by irradiation with ultrasound has been investigated. Only traces were present in neutral solutions, but it was a major product in alkaline solutions. Malonaldehyde is unstable, especially in neutral and acidic solutions, and gives 1,3,5-benzentricarbaldehyde by 1,2-addition and 1 by 1,4-addition. There is evidence that cyclic structures also exist, the ring closure being brought about by an additional hemiacetal bond. To some extent also, 1,3,5-benzenetricarbaldehyde is incorporated into the oligomers.     

Lipid Peroxidation in Rat Brain Cortical Slices as Measured by the Thiobarbituric Acid Test

Abstract: Malonaldehyde formation by cortical brain slices from rat brain was determined as a function of incubation time and of oxygen pressure. This substance, a byproduct of lipid peroxidation, was detected by the thiobarbituric acid test. Significant amounts of malonaldehyde were formed by brain slices during incubation in the 0.2 (air) to 10 atm oxygen range, and a portion of it was released into the medium. The rate of malonaldehyde formation was the highest during the first 10 min. Elevation of oxygen pressure above 1 atm caused further increments in malonaldehyde production with kinetic properties similar to that seen at 1 atm pressure, but the increments per additional oxygen pressure were diminishing. The formation of a given amount of malonaldehyde can be expressed as a function of atm oxygen × min. This function has the shape of a saturation curve approaching a maximum at around 300 atm × min. The results indicate extensive lipid peroxidation in brain slices under standard incubation conditions.     

Enzymatic synthesis of malonaldehyde.

Malonaldehyde was prepared from 1,3-propanediol by alcohol dehydrogenase. The K_m for 1,3-propanediol was about 1.7 mM. The reaction proceeded best at low ionic strength and at pH 9. The reaction was unaffected by pyrophosphate, phosphate, bicarbonate, or N-ethylmorpholine buffers, or by Mg⁺², Ca⁺², EDTA, or citrate. However, the reaction was inhibited 50% by 1.5 mM borate, 1 mM cyanide, and 5 mM azide. Thiols, such as dithioerythritol, inhibited the reaction 50% at 50–100 μM, while others, such as mercaptoacetate, inhibited 50% at concentrations over 1 mM. Malonaldehyde was removed from the reaction mixture by evaporation at pH 3 and condensation at ?78°C. No other products associated with lipid peroxidation were produced. The method was useful for preparation of radiolabeled malonaldehyde.     

Determination of malonaldehyde in oxidized lipids by the Hantzsch fluorometric method

Malonaldehyde is a secondary product formed during lipid oxidation. We developed a sensitive and reliable Hantzsch fluorometric method for determination of malonaldehyde in oxidized lipids. The principle of the method is based on the formation of highly fluorescent 1,4-dimethyl-1,4-dihydropyridine-3,5-dicarbaldehyde MI by reaction of malonaldehyde, methylamine, and acetaldehyde under neutral conditions. Compound MI formed could be estimated by high-performance liquid chromatography. Free malonaldehyde, that liberated under neutral conditions (labile forms) and that liberated by acid pretreatment (acid labile forms), could be determined by use of the calibration curves of MI versus malonaldehyde sodium salt. Oxidized methyl linoleate with a peroxide value of 1600 neq/mg contained 0.95 (free and labile) and 1.3 nmol (acid labile) malonaldehyde/mg, oxidized sardine oil with a peroxide value of 640 neq/mg contained 1.1 (free and labile) and 3.0 nmol (acid labile) malonaldehyde/mg, and the lipid fraction of oxidized rat liver microsomes contained less than 0.2 (free and labile) and 0.8 nmol (acid labile) malonaldehyde/mg. The malonaldehyde contents were much lower than those obtained by traditional 2-thiobarbituric acid test. It appears likely that the malonaldehyde contents, both free and labile, and acid labile forms, in oxidized lipids are too low to be taken into account.     

Further studies on lipid-peroxide formation in isolated hepatocytes.

Lipid peroxide formation was initiated by the addition of either ADP-complexed Fe3+ or cumene hydroperoxide to a suspension of isolated hepatocytes. The reaction was monitored by malonaldehyde measurements. Upon the addition of iron, malonaldehyde production in the cells started immediately but ceased within 30-60 min, and the response was dose-related with iron concentrations ranging from 19 to 187 muM. Malonaldehyde formation was associated with increased oxygen uptake and conjugated diene production. The addition in vitro of N,N,N',N'-tetramethyl-p-phenylenediamine, menadione or p-benzoquinone inhibited the iron-induced malonaldehyde production. It was also possible to demonstrate an apparent disappearance of malonaldehyde from fresh cells by addition of adequate amounts of N,N,N',N'-tetramethyl-p-phenylenediamine (100 muM). The attenuation of the iron-induced malonaldehyde production was found to be correlated with an increased binding of iron to an intracellular ferritin fraction. Further, malonaldehyde formation was also associated with a conversion of reduced glutathione to the oxidized form which, in turn, revealed a faster permeation out of the cells into the surrounding medium of the oxidized than of the reduced thiol. So, concomitant with the redox alterations, there was also an overall loss of glutathione from the cells. Cumene hydroperoxide-induced malonaldehyde production could be initiated by the addition of this peroxide in concentrations ranging from 150 muM to the liver cell incubate. With concentrations below 150 muM, a lag phase was present which seemed to be glutathione-dependent. It is concluded that iron enters the cell, then is probably reduced inside the cell by NADPH via the NADPH-cytochrome P-450 reductase, and in the reduced state initiates lipid peroxidation. The reaction is inhibited by intracellular mechanisms, the glutathione redox system being of principal importance, and possibly terminated by the iron-apoferritin complex formation.     

Altered DNA repair, oxidative stress and antioxidant status in coronary artery disease

Coronary artery disease (CAD) is a multifactorial disease caused by the interplay of environmental risk factors with multiple predisposing genes. The present study was undertaken to evaluate the role of DNA repair efficiency and oxidative stress and antioxidant status in CAD patients. Malonaldehyde (MDA), which is an indicator of oxidative stress, and mean break per cell (b/c) values, which is an indicator of decreased DNA repair efficiency, were found to be significantly increased in patients compared to normal controls (P?<?0.05) whereas ascorbic acid and GSH were found to be lower among patients than the control group. It has been found that elevated oxidative stress decreased antioxidant level and decreased DNA repair efficiency can contribute to the development of CAD. This study also showed that high MDA, low ascorbic acid and GSH were significantly associated with high b/c value.     

Effect of battery-manufacturing effluent on endogenous antioxidant in freshwater fish

Abstract

This study monitors and assesses the effect of battery-manufacturing effluent containing metals Pb, Zn and Cd on endogenous antioxidants. Malonaldehyde (MDA), reduced glutathione sulfyhydryl (GSH) and catalase (CAT) which are known biomarkers of effluent were exposed to 0%, 25%, 50%, 75% and 90% amendments for 74h on the gills, liver and kidneys of C. punctata. There was more metal Zn accumulation in the gills and GSH contents increased significantly in the gills (P<0.01), liver accumulation of Pb was found to be more (P<0.05), whereas lowest accumulation of Pb was found in kidneys and the highest accumulation of Cd (P<0.05). Over all amendments of the effluents, MDA contents were increased in the gills, liver and kidneys (P<0.01). GSH levels were decreased among the liver and kidneys compared to the gills (P<0.01) at 90% amendment. Effluent exposure caused a significant decrease in the activities of CAT in the gills, liver and kidneys (P<0.01, 0.05 and 0.05) of fish. Increased MDA activity was indicative of the formation of free radicals in the fish with exposure to amendments of battery manufacturing effluent, while increased levels of GSH pointed to the occurrence of a scavenging mechanism of free radicals.     

Immunofluorescence study of the formation of evolutionary stable lens proteins in chick embryos]

In the lens of fishes (carp, spiny dogfish) beta-crystallins were identified which were characteristic also of reptiles, amphibians, birds and mammals (evolutionary stable beta-crystallins). The dynamics of the formation of such beta-crystallins in 5--14 days old chick embryos was studied by the indirect immunofluorescence method with antisera to fish lens. These proteins are reliably indentified first at the lens sections from 7--8days old chick embryos. At all stages under study these beta-crystallins are localized mainly in the epithelial cells and practically not found in the lens fibers. They were, however, found in the fibrous (central) part of developing lens as well by the method of immunoelectrophoresis.     

Lipolysis-induced iron release from diferric transferrin: Possible role of lipoprotein lipase in LDL oxidation.

Conditions leading to oxidation of LDL in vivo are still unknown. While the occurrence of oxidized lipoproteins and catalytic free iron in advanced atherosclerotic lesions has been demonstrated, the origin of both is unclear. In vivo, iron metabolism is tightly regulated by iron-binding proteins that ensure that virtually no free iron exists. We examined whether physiological events such as lipolysis might reduce pH, facilitate iron release from transferrin (Tf), and promote low density lipoprotein (LDL) oxidation. Lipolysis is brought about by lipoprotein lipase (LpL), a triglyceride hydrolase present on endothelial cell surfaces and in atherosclerotic lesions. LpL hydrolysis of Intralipid lowered pH from 7.40 to 7.00 in 10% human serum and from 7.40 to 6.88 in phosphate-buffered saline. Similar decreases in pH were also observed when very low density lipoproteins were hydrolyzed by LpL. Lipolysis was accompanied by a 2-fold increase in the release of 59Fe from Tf. Tf binding to subendothelial matrix (SEM), a site of key events in atherosclerosis, increased 2-fold as the pH decreased from 7.40 to 6.00. More free iron also bound to SEM as the pH decreased below 7.40. We next tested whether a reduction in pH promotes LDL oxidation. More oxidation products were found in LDL incubated at low pH for 24 h in 10% human serum. Malonaldehyde contents (nmol/mg protein), measured as TBARS, were 7.11 +/- 0.34 at pH 7.40, 7.65 +/- 0.49 at pH 7.00, 9.00 +/- 1.18 at pH 6.50, and 11. 54 +/- 0.63 at pH 6.00.Based on these results, we hypothesize that lipolysis-induced acidic conditions enhance iron release from Tf and increase formation of oxidized LDL.     

5-methoxytryptophol preserves hepatic microsomal membrane fluidity during oxidative stress

Lipid peroxidation is a degenerative chain reaction in biological membranes that may be initiated by exposure to free radicals. This process is associated with changes in the membrane fluidity and loss of several cell membrane-dependent functions. 5-methoxytryptophol (ML) is an indole isolated from the mammalian pineal gland. The purpose of this study was to investigate the effects of ML (0. 01mM-10mM) on membrane fluidity modulated by lipid peroxidation. Hepatic microsomes obtained from rats were incubated with or without ML (0.01-10 mM). Then lipid peroxidation was induced by FeCl(3), ADP, and NADPH. Membrane fluidity was determined using fluorescence spectroscopy. Malonaldehyde (MDA) +4-hydroxyalkenals (4-HDA) concentrations were estimated as an indicator of the degree of lipid peroxidation. With oxidative stress, membrane fluidity decreased and MDA+4-HDA levels increased. ML (0.01-3 mM) reduced membrane rigidity and the rise in MDA+4-HDA formation in a concentration-dependent manner. 10 mM ML protected against lipid peroxidation but failed to prevent the membrane rigidity. In the absence of oxidative reagents, ML (0.3-10 mM) decreased membrane fluidity whereas MDA+4-HDA levels remained unchanged. This indicates that ML may interact with membrane lipids. The results presented here suggest that ML may be another pineal indoleamine (in addition to melatonin) that resists membrane rigidity due to lipid peroxidation.     

Changes in lipid peroxidation during pregnancy and after delivery in rats: effect of pinealectomy

Pregnancy is a physiological state accompanied by a high energy demand of many bodily functions and an increased oxygen requirement. Because of the increased intake and utilization of oxygen, increased levels of oxidative stress would be expected. In the present study, the degree of lipid peroxidation was examined in different tissues from non-pregnant and pregnant rats after the delivery of their young. Melatonin and other indole metabolites are known to be direct free radical scavengers and indirect antioxidants. Thus the effect of pinealectomy at 1 month before pregnancy on the accumulation of lipid damage was investigated in non-pregnant and pregnant rats after the delivery of their young. Malonaldehyde and 4-hydroxyalkenal concentrations were measured in the lung, uterus, liver, brain, kidney, thymus and spleen from intact and pinealectomized pregnant rats soon after birth of their young and at 14 and 21 days after delivery. The same parameters were also evaluated in intact and pinealectomized non-pregnant rats. Shortly after delivery, lipid oxidative damage was increased in lung, uterus, brain, kidney and thymus of the mothers. No differences were detected in liver and spleen. Pinealectomy enhanced this effect in the uterus and lung. It is concluded that during pregnancy high levels of oxidative stress induce an increase in oxidative damage to lipids, which in some cases is inhibited by the antioxidative actions of pineal indoles.     

Effects of catechin and epicatechin on superoxide dismutase and glutathione peroxidase activity,in vivo

Abstract

Objectives

The objective of this study was to investigate the effects of catechin and epicatechin on the activity of the endogenous antioxidant enzymes superoxide dismutase (SOD) and glutathione peroxidase (GPx) (as well as the total antioxidant capacity (TAC)) of rats after intra-peritoneal (i.p.) administration.

Methods

Twenty-four Wistar rats were randomly divided into two groups: the experimental group which was administered daily with a 1:1 mixture of epicatechin and catechin at a concentration of 23 mg/kg body weight for 10 days and the control group which was injected daily with an equal amount of saline. Blood and urine samples were collected before and after the administration period, as well as 10 days after (follow-up).

Results

Intra-peritoneal administration of catechins led to a potent decrease in GPx levels and a significant increase in SOD levels. TAC was significantly increased in plasma and urine. Malonaldehyde levels in urine remained stable. In the animals treated with catechins, SOD activity showed a moderate negative correlation with GPx activity.

Discussion

Boosting the activity of the antioxidant enzymes could be a potential adjuvant approach for the treatment of the oxidative stress-related diseases.     

空心莲子草具有较强抗盐能力的原因初探

为了探讨空心莲子草(Alternantheraphiloxeroides(Mart.)Griseb)的抗盐机理,测定了盐胁迫条件下的空心莲子草和水稻中保护酶活性和渗透调节物质的变化。结果表明,在盐胁迫下,空心莲子草中的脯氨酸、甜菜碱以及SOD、POD等含量增加速度比水稻快,膜脂过氧化产物丙二醛的含量在两种植物中虽亦增加,但在空心莲子草叶中的增加幅度小于水稻。证明盐胁迫下保护酶活性和渗透调节物质含量的迅速增加是空心莲子草具有较强抗盐能力的重要生理学基础。     

Appearance of acid phosphatase activity in the developing anterior pituitary of the rat

The ultrastructure of the anterior pituitary gland in developing rats was investigated according to Gomori's method for acid phosphatase. During the earlier period of development (day 14 to 16 of gestation), enzyme activity could not be found, although nonspecific deposits of lead were observed within the nuclear envelope, ER, and Golgi cisternae. This facilitated observation of the topographical relationship of the intracellular membrane system and suggestive evidence was obtained that the nuclear envelope in the pituitary anlage is involved in formation of the Golgi apparatus.During days 17 and 18 of gestation, when granule formation begins, little acid phosphatase activity was detectable in the Golgi apparatus and in the secretory granules. A polarized distribution of acid phosphatase was first detected in the Golgi apparatus on day 20 of gestation, with a concomitant increase of lysosomes.From these findings it seems that acid phosphatase begins to contribute to the secretory process a few days after granule formation has started.     

Tracking the changes in unloaded bone: Morphology and gene expression

Bone formation and growth are controlled by genetic, hormonal and biomechanical factors. In this study, an established rat disuse osteoporosis model, hindlimb-suspension (HLS), was used to relate morphological change and gene expression to altered mechanical load in the underloaded femora and the ostensibly normally loaded humeri of the suspended rats (39 days old at onset; 1, 3, 7 and 14 days suspension). Morphological change was measured by labelling new bone formation with fluorescent agents during the experimental period and subsequent histological analysis of bone sections post-sacrifice. Hindlimb suspension reduced both the total amount of bone present, assessed as cross-sectional area, and the bone formation rate at the mid-diaphysis of the unloaded femora while no significant effect was found in the loaded humeri. In addition, the femora of the suspended animals were found to have a markedly increased circularity as a result of unloading. A sensitive semi-quantitative method of gene expression analysis, involving the creation of SMART cDNA arrays, was successfully implemented. This technique amplified all populations of mRNA to levels where they could be assessed using standard molecular biology protocols. Gene expression patterns of two candidate genes, c-fos and osteocalcin were assessed in periosteal tissue. Altered gene expression patterns were identified and tracked over the suspension period. The altered levels of both candidate genes were found to be consistent with the changes observed in the histological analysis.     

Collagen-IV supported embryoid bodies formation and differentiation from buffalo (Bubalus bubalis) embryonic stem cells

Embryoid bodies (EBs) are used as in vitro model to study early extraembryonic tissue formation and differentiation. In this study, a novel method using three dimensional extracellular matrices for in vitro generation of EBs from buffalo embryonic stem (ES) cells and its differentiation potential by teratoma formation was successfully established. In vitro derived inner cell masses (ICMs) of hatched buffalo blastocyst were cultured on buffalo fetal fibroblast feeder layer for primary cell colony formation. For generation of EBs, pluripotent ES cells were seeded onto four different types of extracellular matrices viz; collagen-IV, laminin, fibronectin and matrigel using undifferentiating ES cell culture medium. After 5days of culture, ESCs gradually grew into aggregates and formed simple EBs having circular structures. Twenty-six days later, they formed cystic EBs over collagen matrix with higher EBs formation and greater proliferation rate as compared to other extracellular matrices. Studies involving histological observations, fluorescence microscopy and RT-PCR analysis of the in vivo developed teratoma revealed that presence of all the three germ layer derivatives viz. ectoderm (NCAM), mesoderm (Flk-1) and endoderm (AFP). In conclusion, the method described here demonstrates a simple and cost-effective way of generating EBs from buffalo ES cells. Collagen-IV matrix was found cytocompatible as it supported buffalo EBs formation, their subsequent differentiation could prove to be useful as promising candidate for ES cells based therapeutic applications.     

Early quantitative method for measuring germination in non-green spores of Dryopteris paleacea using an epifluorescence-microscope technique

A method is described to determine germination by blue-light excited red fluorescence in the positively photoblastic spores of Dryopteris paleacea Sw. This fluorescence is due to chlorophyll as evidenced from 1) a fluorescence-emission spectrum in vivo, where a bright fluorescence around 675 nm is obtained only in red light (R)-irradiated spores and 2) in vitro measurements with acetone extracts prepared from homogenized spores. Significant amounts of chlorophyll can be found only in R-treated spores; this chlorophyll exhibits an emission band around 668 nm, when irradiated with 430 nm light at 21°C.
Compared to other criteria for germination, such as swelling of the cell, coat splitting, greening, and rhizoid formation, which require longer periods after induction for their expression, chlorophyll fluorescence can be used to quantify germination after two days. This result is confirmed by fluence-response curves for R-induced spore germination; the same relationship between applied R and germination is obtained by the evaluation with the epifluorescence method 2 days after the light treatment as compared with the evaluation with bright-field microscopy 5 days after the inducing R.
Using this technique we show for the first time that Ca²⁺ contributes to the signaltransduction chain in phytochrome-mediated chlorophyll synthesis in spores of Dryopteris paleacea .     

The Development of the Graft Union

In this paper a new method is described for the examinationof graft formation. The method depends in principle on the measurementof the tensile strength of the graft at intervals of 2 days.It has been applied to the analysis of the development of asimple graft, made by cutting across an internode of a tomatoseedling and rejoining the separated portions. The method requiresthat the treatment be applied to a group of a hundred seedlingsfrom which samples are taken at intervals of 2 days. With thisgeneral technique and with the system used it has been shownthat the development of the graft continues steadily after thesecond day of the experiment, that graft formation is most rapidwhen the different tissues of the stem coincide in the stockand the scion, and that the tensile strength of the graft isreduced markedly when the turgidity of the tissues is depressed.In the last section of the paper the significance of these severalobservations is discussed.     

Effects of lipid peroxidation on adrenal microsomal monooxygenases

Incubation of guinea pig adrenal microsomes with 10^?6 M ferrous (Fe²⁺) ion and adrenal cytosol initiated high levels of lipid peroxidation as measured by the production of malonaldehyde. Cytosol or Fe²⁺ alone had little effect on microsomal malonaldehyde formation. When microsomes were incubated in the presence of Fe²⁺ and cytosol, malonaldehyde levels continued to increase for at least 60 min. Accompanying the lipid peroxidation was a decline in adrenal microsomal monooxygenase activities. The rates of metabolism of xenobiotics (benzphetamine demethylase, benzo[α]pyrene hydroxylase) as well as steroids (21-hydroxylation) decreased as malonaldehyde levels increased. In addition, cytochrome P-450 levels, NADPH- and NADH-cytochrome c reductase activities, and substrate interactions with cytochrome(s) P-450 decreased as lipid peroxidation progressed. Inhibition of lipid peroxidation by increasing microsomal protein concentrations during the incubation period prevented the changes in microsomal metabolism. Malonaldehyde had no direct effects on adrenal microsomal enzyme activities. The results indicate that lipid peroxidation may have significant effects on adrenocortical function, diminishing the capacity for both xenobiotic and steroid metabolism.     

Postnatal differentiation of the Golgi apparatus and the dendrites of Purkinje cells of the rat cerebellum

Summary The morphology of postnatal differentiation of the Golgi apparatus, the nucleus, the perikaryon, and the dendrites was studied in Purkinje cells of the rat cerebellum for 30 days after birth using histochemical, histological, and electron microscopic methods.The Golgi apparatus during differentiation undergoes morphological and positional changes. From the 1st to 7th postnatal day, the Golgi apparatus is found in a supranuclear position, and is connected with the axes of differentiating primary dendrites by beam-like processes. From days 8 to 11 this connection disappears, and most of the Golgi apparatus assumes a lateronuclear and infranuclear position. After the 11th or 12th day, the Golgi apparatus is found in perinuclear and peripheral cytoplasmic positions. The formation of granular endoplasmic reticulum occurs in the vicinity of the perinuclear Golgi apparatus. The differentiation of cell and nuclear forms requires approximately 20 days. The morphological changes of differentiation are discussed in relation to the participation of the Golgi apparatus in the differentiation of dendrites and in the formation of the granular endoplasmic reticulum.