Acoustic emission signals analysis to differentiate the damage mechanism in the drilling of Al-5%B4C metal matrix composite.

Tool wear leads to dimensional inaccuracy and low surface quality in the workpiece, and unexpected sudden tool failure. Detection of tool wear is essential to enhance the quality of manufacturing components and extend tool life. The present work is aimed to investigate the various damage mechanisms involved in the cutting tool and workpiece during drilling of Al-5%B4C composite using acoustic emission technique (AET). The dry drilling experiments were carried out at different spindle speeds and feed rates with high strength steel (HSS) tool. AE time-domain parameters such as count, energy, amplitude and root mean square (RMS) voltage were extracted from the signals and correlated with cutting parameters and tool damage. Fast Fourier transform (FFT) was applied to visualize the frequency components in the AE signals during the drilling process. The wavelet packet transform (WPT) approach was performed to the AE signals to identify and discriminate the various damage mechanism involved in the drilling. The differentiated damage mechanism and their corresponding wavelet energy content were studied. The wavelet energy ratio for decomposed components at different speeds was discussed. The vision measuring microscope was employed to measure the tool wear. The AE features, i.e., AERMS and wavelet coefficient increases with increasing tool wear. A scanning electron microscope was also utilized to characterize the microstructural damage present in the cutting tool and workpiece.

Development of in-bore magnetostrictive transducer for ultrasonic guided wave based-inspection of steam generator tubes of PFBR.

An in-bore magnetostrictive transducer is designed for the steam generator tubes of Prototype fast breeder reactor for the generation of L(0,2) modes of frequencies in the range of 250-350 kHz. Towards this, axi-symmetric finite element models are developed to optimize the coil parameters. The optimized length of the transmitter and the receiver coils turns out to be 10 mm (~half the wavelength) for the frequency of 300 kHz. The optimized width of the coils turns out to be 0.46 mm. FE models also show the generation, propagation and reception of L(0,2) modes in the frequency range of 250-350 kHz. The role of skin effect in the magnetostrictive based-generation of L(0,2) modes with frequency is also discussed. A transducer is designed based on the FE results. The transducer is tested for the generation of L(0,2) mode in the frequency range of 250-350 kHz in a 1 m long steam generator tube segment. A good agreement is observed between FE and experimental normalized amplitudes and the times of flight for different frequencies. L(0,2) modes are found to generate and propagate and received, as predicted by the finite element simulations. An excellent agreement is observed between the experimentally measured group velocities with those obtained from the dispersion curves in this frequency range. Experiments show the signal to noise ratio to be better than 15 dB. To ascertain the utility of the transducer in steam generator tubes for the long range testing, L(0,2) mode at 300 kHz frequency is propagated in a 1.5 m long tube. The resulted multiple end reflections amount to the propagation of 51 m distance. To check the capability of detection of defects, a short tube with a full circumferential defect of depth 0.46 mm (20%WT) and a short tube with a pin hole of 1.5 mm diameter are considered. Further, FE results for the case of the axi-symmetric circumferential defect are validated experimentally. For the case of the pinhole (non-axi-symmetric), the experimental signal to noise ratio turns out to be 6 dB, which is only 6 dB lower as compared to that obtained using a piezo based ultrasonic transducer of frequency 300 kHz coupled to the end of the tube.

Development of ultrasonic guided wave inspection methodology for steam generator tubes of prototype fast breeder reactor.

An ultrasonic guided wave based methodology is developed for inspection of steam generator tubes of the prototype fast breeder reactor. To this aim, axisymmetric longitudinal mode (L(0,2)) at the frequency of 250 kHz is optimized using 3D-finite element simulation and experiments. The group velocity of mode L(0,2) at 250 kHz is found to be 5387 m/s. First, the long range propagation of the L(0,2) mode at 250 kHz is examined and the mode is found to propagate over a distance of 45.6 m with a sufficiently good SNR. Secondly, the detection of multiple defects such as circumferential, axial, partial-pinholes and tapered defects lying in the same line of sight is investigated using 3D-finite element simulation and the results obtained are validated experimentally for the first three cases. The sensitivities achieved are 0.23 mm depth (10%WT) for circumferential, axial and tapered defects and for partial-pinholes: 1 mm diameter and 1.38 mm depth (60%WT). Thirdly, 3D-FE simulations with ID and OD pinhole defects are performed which show that the ID and OD defects are detected by L(0,2) with a fairly similar sensitivity. Finally, study on the thermal expansion bend (with three successive bends) shows that the bend does not have much influence on the mode and the multiple circumferential defects considered in the bend are detected with good sensitivity.

Hepatitis B vaccination with or without hepatitis B immunoglobulin at birth to babies born of HBsAg-positive mothers prevents overt HBV transmission but may not prevent occult HBV infection in babies: a randomized controlled trial.

Vertical transmission of Hepatitis B virus HBV can result in a state of chronic HBV infection and its complications. HBV vaccination with or without hepatitis B immunoglobulin (HBIG) prevents transmission of overt infection to the babies. However, whether it also prevents occult HBV infection in babies is not known. Consecutive pregnant women of any gestation found to be HBsAg positive were followed till delivery, and their babies were included in the study. Immediately after delivery, babies were randomized to receive either HBIG or placebo in addition to recombinant HBV vaccine (at 0, 6, 10 and 14 weeks). The primary end-point of the study, assessed at 18 weeks of age, was remaining free of any HBV infection (either overt or occult) plus the development of adequate immune response to vaccine. The babies were further followed up for a median of 2 years of age to determine their eventual outcome. Risk factors for HBV transmission and for poor immune response in babies were studied. Of the 283 eligible babies, 259 were included in the trial and randomized to receive either HBIG (n=128) or placebo (n=131) in addition to recombinant HBV vaccine. Of the 222 of 259 (86%) babies who completed 18 weeks of follow-up, only 62/222 (28%) reached primary end-point. Of the remaining, 6/222 (3%) developed overt HBV infection, 142/222 (64%) developed occult HBV infection, and 12/222 (5%) had no HBV infection but had poor immune response. All 6 overt infections occurred in the placebo group (P=0.030), while occult HBV infections were more common in the HBIG group (76/106 [72%] vs. 66/116 [57%]; P=0.025). This may be due to the immune pressure of HBIG. There was no significant difference between the two groups in frequency of babies developing poor immune response or those achieving primary end-point. The final outcome of these babies at 24 months of age was as follows: overt HBV infection 4%, occult HBV infection 42%, no HBV infection but poor immune response 8% and no HBV infection with good immune response 28%. Women who were anti-HBe positive were a low-risk group, and their babies were most likely to remain free of HBV infection (occult or overt) and had good immune response to the vaccine. Maternal HBeAg-positive status and negativity for anti-HBe predicted not only overt but also any infection (both overt and occult) in babies. In addition, high maternal HBV DNA and treatment with vaccine alone were significant factors for overt HBV infection in babies. The current practice of administration of vaccine with HBIG at birth to babies born of HBsAg-positive mothers is not effective in preventing occult HBV infection in babies, which may be up to 40%. Because the most important risk factors for mother-to-baby transmission of HBV infection are the replicative status and high HBV DNA level in mothers; it will be worthwhile investigating the role of antivirals and HBIG administration during pregnancy to prevent mother-to-child transmission of HBV infection.

Ceruloplasmin and cardiovascular disease.

Transition metal ion-mediated oxidation is a commonly used model system for studies of the chemical, structural, and functional modifications of low-density lipoprotein (LDL). The physiological relevance of studies using free metal ions is unclear and has led to an exploration of free metal ion-independent mechanisms of oxidation. We and others have investigated the role of human ceruloplasmin (Cp) in oxidative processes because it the principal copper-containing protein in serum. There is an abundance of epidemiological data that suggests that serum Cp may be an important risk factor predicting myocardial infarction and cardiovascular disease. Biochemical studies have shown that Cp is a potent catalyst of LDL oxidation in vitro. The pro-oxidant activity of Cp requires an intact structure, and a single copper atom at the surface of the protein, near His(426), is required for LDL oxidation. Under conditions where inhibitory protein (such as albumin) is present, LDL oxidation by Cp is optimal in the presence of superoxide, which reduces the surface copper atom of Cp. Cultured vascular endothelial and smooth muscle cells also oxidize LDL in the presence of Cp. Superoxide release by these cells is a critical factor regulating the rate of oxidation. Cultured monocytic cells, when activated by zymosan, can oxidize LDL, but these cells are unique in their secretion of Cp. Inhibitor studies using Cp-specific antibodies and antisense oligonucleotides show that Cp is a major contributor to LDL oxidation by these cells. The role of Cp in lipoprotein oxidation and atherosclerotic lesion progression in vivo has not been directly assessed and is an important area for future studies.

Role of hypoxia-inducible factor-1 in transcriptional activation of ceruloplasmin by iron deficiency.

A role of the copper protein ceruloplasmin (Cp) in iron metabolism is suggested by its ferroxidase activity and by the tissue iron overload in hereditary Cp deficiency patients. In addition, plasma Cp increases markedly in several conditions of anemia, e.g. iron deficiency, hemorrhage, renal failure, sickle cell disease, pregnancy, and inflammation. However, little is known about the cellular and molecular mechanism(s) involved. We have reported that iron chelators increase Cp mRNA expression and protein synthesis in human hepatocarcinoma HepG2 cells. Furthermore, we have shown that the increase in Cp mRNA is due to increased rate of transcription. We here report the results of new studies designed to elucidate the molecular mechanism underlying transcriptional activation of Cp by iron deficiency. The 5'-flanking region of the Cp gene was cloned from a human genomic library. A 4774-base pair segment of the Cp promoter/enhancer driving a luciferase reporter was transfected into HepG2 or Hep3B cells. Iron deficiency or hypoxia increased luciferase activity by 5-10-fold compared with untreated cells. Examination of the sequence showed three pairs of consensus hypoxia-responsive elements (HREs). Deletion and mutation analysis showed that a single HRE was necessary and sufficient for gene activation. The involvement of hypoxia-inducible factor-1 (HIF-1) was shown by gel-shift and supershift experiments that showed HIF-1alpha and HIF-1beta binding to a radiolabeled oligonucleotide containing the Cp promoter HRE. Furthermore, iron deficiency (and hypoxia) did not activate Cp gene expression in Hepa c4 hepatoma cells deficient in HIF-1beta, as shown functionally by the inactivity of a transfected Cp promoter-luciferase construct and by the failure of HIF-1 to bind the Cp HRE in nuclear extracts from these cells. These results are consistent with in vivo findings that iron deficiency increases plasma Cp and provides a molecular mechanism that may help to understand these observations.

Ceruloplasmin ferroxidase activity stimulates cellular iron uptake by a trivalent cation-specific transport mechanism.

The balance required to maintain appropriate cellular and tissue iron levels has led to the evolution of multiple mechanisms to precisely regulate iron uptake from transferrin and low molecular weight iron chelates. A role for ceruloplasmin (Cp) in vertebrate iron metabolism is suggested by its potent ferroxidase activity catalyzing conversion of Fe2+ to Fe3+, by identification of yeast copper oxidases homologous to Cp that facilitate high affinity iron uptake, and by studies of "aceruloplasminemic" patients who have extensive iron deposits in multiple tissues. We have recently shown that Cp increases iron uptake by cultured HepG2 cells. In this report, we investigated the mechanism by which Cp stimulates cellular iron uptake. Cp stimulated the rate of non-transferrin 55Fe uptake by iron-deficient K562 cells by 2-3-fold, using a transferrin receptor-independent pathway. Induction of Cp-stimulated iron uptake by iron deficiency was blocked by actinomycin D and cycloheximide, consistent with a transcriptionally induced or regulated transporter. Cp-stimulated iron uptake was completely blocked by unlabeled Fe3+ and by other trivalent cations including Al3+, Ga3+, and Cr3+, but not by divalent cations. These results indicate that Cp utilizes a trivalent cation-specific transporter. Cp ferroxidase activity was required for iron uptake as shown by the ineffectiveness of two ferroxidase-deficient Cp preparations, copper-deficient Cp and thiomolybdate-treated Cp. We propose a model in which iron reduction and subsequent re-oxidation by Cp are essential for an iron uptake pathway with high ion specificity.

Ceruloplasmin copper induces oxidant damage by a redox process utilizing cell-derived superoxide as reductant.

Oxidative damage by transition metals bound to proteins may be an important pathogenic mechanism. Ceruloplasmin (Cp) is a Cu-containing plasma protein thought to be involved in oxidative modification of lipoproteins. We have previously shown that Cp increased cell-mediated low-density lipoprotein (LDL) oxidation by a process requiring cell-derived superoxide, but the underlying chemical mechanism(s) is (are) unknown. We now show that superoxide reduction of Cp Cu is a critical reaction in cellular LDL oxidation. By bathocuproine disulfonate (BCS) binding and by superoxide utilization, we showed that exogenous superoxide reduces a single Cp Cu atom, the same Cu required for LDL oxidation. The Cu atom remained bound to Cp during the redox cycle. Three avenues of evidence showed that vascular cells reduce Cp Cu by a superoxide-dependent process. The 2-fold higher rate of Cp Cu reduction by smooth muscle cells (SMC) compared to endothelial cells (EC) was consistent with their relative rates of superoxide release. Furthermore, Cp Cu reduction by cells was blocked by Cu,Zn superoxide dismutase (SOD1). Finally, the level of superoxide produced by EC and SMC was sufficient to cause the amount of Cu reduction observed. An important role of Cp Cu reduction in LDL oxidation was suggested by results showing that SOD1 inhibited Cp Cu reduction and LDL oxidation by SMC with equal potency, while tumor necrosis factor-alpha stimulated both processes. In summary, these results show that superoxide is a critical cellular reductant of divalent transition metals involved in oxidation, and that protein-bound Cu is a substrate for this reaction. The role of these mechanisms in oxidative processes in vivo has yet to be defined.

Role of ceruloplasmin in cellular iron uptake.

Individuals with hereditary ceruloplasmin (Cp) deficiency have profound iron accumulation in most tissues, which suggests that Cp is important for normal release of cellular iron. Here, in contrast to expectations, Cp was shown to increase iron uptake by HepG2 cells, increasing the apparent affinity for the substrate by three times. Consistent with its role in iron uptake, Cp synthesis was regulated by iron supply and was increased four- to fivefold after iron depletion. Unlike other iron controllers that are posttranscriptionally regulated, Cp synthesis was transcriptionally regulated. Thus, iron-deficient cells could increase Cp synthesis to maintain intracellular iron homeostasis, so that defects would lead to global accumulation of iron in tissues.

Identification of the prooxidant site of human ceruloplasmin: a model for oxidative damage by copper bound to protein surfaces.

Free transition metal ions oxidize lipids and lipoproteins in vitro; however, recent evidence suggests that free metal ion-independent mechanisms are more likely in vivo. We have shown previously that human ceruloplasmin (Cp), a serum protein containing seven Cu atoms, induces low density lipoprotein oxidation in vitro and that the activity depends on the presence of a single, chelatable Cu atom. We here use biochemical and molecular approaches to determine the site responsible for Cp prooxidant activity. Experiments with the His-specific reagent diethylpyrocarbonate (DEPC) showed that one or more His residues was specifically required. Quantitative [14C]DEPC binding studies indicated the importance of a single His residue because only one was exposed upon removal of the prooxidant Cu. Plasmin digestion of [14C]DEPC-treated Cp (and N-terminal sequence analysis of the fragments) showed that the critical His was in a 17-kDa region containing four His residues in the second major sequence homology domain of Cp. A full length human Cp cDNA was modified by site-directed mutagenesis to give His-to-Ala substitutions at each of the four positions and was transfected into COS-7 cells, and low density lipoprotein oxidation was measured. The prooxidant site was localized to a region containing His426 because CpH426A almost completely lacked prooxidant activity whereas the other mutants expressed normal activity. These observations support the hypothesis that Cu bound at specific sites on protein surfaces can cause oxidative damage to macromolecules in their environment. Cp may serve as a model protein for understanding mechanisms of oxidant damage by copper-containing (or -binding) proteins such as Cu, Zn superoxide dismutase, and amyloid precursor protein.

Induction of ceruloplasmin synthesis by IFN-gamma in human monocytic cells.

Ceruloplasmin is a 132-kDa glycoprotein abundant in human plasma. It has multiple in vitro activities, including copper transport, lipid pro- and antioxidant activity, and oxidation of ferrous ion and aromatic amines; however, its physiologic role is uncertain. Although ceruloplasmin is synthesized primarily by the liver in adult humans, production by cells of monocytic origin has been reported. We here show that IFN-gamma is a potent inducer of ceruloplasmin synthesis by monocytic cells. Activation of human monoblastic leukemia U937 cells with IFN-gamma increased the production of ceruloplasmin by at least 20-fold. The identity of the protein was confirmed by plasmin fingerprinting. IFN-gamma also increased ceruloplasmin mRNA. Induction followed a 2- to 4-h lag and was partially blocked by cycloheximide, indicating a requirement for newly synthesized factors. Ceruloplasmin induction in monocytic cells was agonist specific, as IL-1, IL-4, IL-6, IFN-alpha, IFN-beta, TNF-alpha, and LPS were completely ineffective. The induction was also cell type specific, as IFN-gamma did not induce ceruloplasmin synthesis in endothelial or smooth muscle cells. In contrast, IFN-gamma was stimulatory in other monocytic cells, including THP-1 cells and human peripheral blood monocytes, and also in HepG2 cells. Ceruloplasmin secreted by IFN-gamma-stimulated U937 cells had ferroxidase activity and was, in fact, the only secreted protein with this activity. Monocytic cell-derived ceruloplasmin may contribute to defense responses via its ferroxidase activity, which may drive iron homeostasis in a direction unfavorable to invasive organisms.

Evolutionary significance of vitamin C biosynthesis in terrestrial vertebrates.

Evolution of vertebrates from aquatic medium to the terrestrial atmosphere containing high concentration of environmental oxygen was accompanied by tissue-specific expression of the gene for L-gulonolactone oxidase (LGO). LGO is the terminal enzyme in the pathway of biosynthesis of ascorbic acid in animals. In this paper we present data to indicate that emergence of LGO is apparently to provide the terrestrial vertebrates with adequate amount of ascorbic acid and thereby protect their tissues against oxygen toxicity. Superoxide dismutase (SOD) was not induced in the early tetrapods. However, SOD activity has increased in the mammals which is accompanied by a decrease in the LGO activity. In fact, there has been an inverse relationship between LGO and SOD in the progress of evolution. SOD activity is markedly high in the guinea pig, flying mammal, monkey and man, the species those lack LGO. The inverse relationship between LGO and SOD is also observed in rats during postnatal development, that is when the new born rats are exposed to high concentration of atmospheric oxygen. Recent results from our laboratory indicate that ascorbic acid is specifically needed for protection of microsomal membranes against cytochrome P450-mediated lipid peroxidation and protein oxidation, where SOD is ineffective. Data presented in this paper also indicate an apparent tissue-specific correlation among LGO activity, P450 level and O2.- production during phylogenetic evolution.

Ceruloplasmin enhances smooth muscle cell- and endothelial cell-mediated low density lipoprotein oxidation by a superoxide-dependent mechanism.

Cultured vascular smooth muscle cells (SMC) and endothelial cells (EC) stimulate low density lipoprotein (LDL) oxidation by free radical-mediated, transition metal-dependent mechanisms. The physiological source(s) of metal ions is not known; however, purified ceruloplasmin, a plasma protein containing 7 coppers, oxidizes LDL in vitro. We now show that ceruloplasmin also increases LDL oxidation by vascular cells. In metal ion-free medium, human ceruloplasmin increased bovine aortic SMC- and EC-mediated LDL oxidation by up to 30- and 15-fold, respectively. The maximal response was at 100-300 microg ceruloplasmin/ml, a level at or below the unevoked physiological plasma concentration. Oxidant activity was dependent on protein structure as a specific proteolytic cleavage or removal of one of the seven ceruloplasmin copper atoms inhibited activity. Three lines of evidence indicated a critical role for cellular superoxide (O2.) in ceruloplasmin-stimulated oxidation. First, the rate of production of O2. by cells correlated with their rates of LDL oxidation. Second, superoxide dismutase effectively blocked ceruloplasmin-stimulated oxidation by both cell types. Finally, O2. production by SMC quantitatively accounted for the observed rate of LDL oxidation. To show this, the course of O2. production by SMC was simulated by repeated addition of xanthine and xanthine oxidase to culture medium under cell-free conditions. Neither ceruloplasmin nor O2. alone increased LDL oxidation, but together they completely reconstituted the oxidation rate of ceruloplasmin-stimulated SMC. These results are the first to show that ceruloplasmin stimulates EC- and SMC-mediated oxidation of LDL and that cell-derived O2. accounts quantitatively for metal-dependent, free radical-initiated oxidation of LDL by these cells.

Ascorbic acid prevents lipid peroxidation and oxidative damage of proteins in guinea pig extrahepatic tissue microsomes.

It has recently been indicated that in the absence of free iron, NADPH initiates oxidative damage of proteins in guinea pig liver microsomes and also lipid peroxidation and protein damage in cardiac microsomes and that ascorbic acid specifically inhibits both the lipid peroxidation and protein damage [Mukhopadhyay CK, Chatterjee IB: J Biol Chem 269: 13390-13397, 1994; Mukhopadhyay M et al.: Mol Cell Biochem 126: 69-75, 1993]. In this paper we demonstrate that Fe(III)-independent NADPH-initiated lipid peroxidation and oxidative damage of proteins occur in the microsomes of all the extrahepatic tissues including lung, kidney, adrenal gland and brain and that both the lipid peroxidation and protein damage are specifically prevented by ascorbic acid. We further demonstrate that when NADPH is replaced by O2 as the electron donor, the O2-initiated lipid peroxidation and protein damage are also inhibited by ascorbic acid.

Free metal ion-independent oxidative damage of collagen. Protection by ascorbic acid.

In this paper we demonstrate that in the absence of free metal ions, active oxygen species, generated by activated macrophages or xanthine/xanthine oxidase (XOD), carry out oxidative degradation of collagen fibrils type I in conjunction with proteases. The collagen degradation is completely prevented by ascorbate (AH2) but not by catalase. The free metal ion-independent collagen degradation is a two-step process: (i) oxidation of collagen and (ii) subsequent proteolytic cleavage of the oxidatively modified collagen. AH2 completely prevents collagen oxidation and thereby protects the collagen from subsequent proteolytic degradation. This is in contrast to free metal ion-catalyzed spontaneous fragmentation of collagen, which is accelerated by AH2 and inhibited by catalase (Kato, Y., Uchida, K., and Kawakishi, S. (1992) J. Biol. Chem. 267, 23646-23651). Studies using xanthine/XOD and model polypeptides, namely, poly-L-Pro, poly-L-hydroxyproline, poly-L-Lys, and poly(Pro-Gly-Pro) indicate that although O2-. is needed along with XOD, oxidation of model polypeptides appears to be a direct function of XOD iron, which is also stimulated by cytochrome P450.

NADPH-initiated cytochrome P450-mediated free metal ion-independent oxidative damage of microsomal proteins. Exclusive prevention by ascorbic acid.

In this paper we demonstrate that NADPH-initiated oxidative damage of microsomal proteins occurs in the absence of free metal ions and that this protein oxidation is mediated by cytochrome P450 (cyt P450). Oxidized proteins are rapidly degraded by proteases. Ascorbate (AH2) specifically inhibits free metal ion-independent cyt P450-mediated protein oxidation and thereby prevents subsequent proteolytic degradation. Other scavengers of reactive oxygen species including superoxide dismutase, catalase, and glutathione are ineffective. This is in variance with free metal ion-catalyzed protein oxidation, which is accelerated by AH2 and inhibited by catalase. Oxidative damage of proteins has been assessed by the production of carbonyl groups, bityrosine formation, and tryptophan loss. The mechanism of protein oxidation has been studied using a reconstituted system comprised of purified NADPH-cyt P450 reductase, cyt P450, and isolated microsomal proteins as well as model polypeptides, e.g. poly-L-proline and poly-L-lysine. Cyt P450 Fe3+ is reduced by NADPH-cyt P450 reductase to cyt P450 Fe2+, which consumes oxygen in a stoichiometric proportion to produce cyt P450 Fe2+ O2, the resonance form of which is a perferryl moiety, cyt P450 Fe3+.O2-.. It is proposed that cyt P450 Fe3+.O2-. abstracts hydrogen from amino acid side chains leading to the production of carbonyl derivatives. Tentatively, AH2 prevents protein oxidation by interacting with cyt P450 Fe3+.O2-..

Ascorbate protects guinea pig tissues against lipid peroxidation.

In recent years we and others have shown that ascorbic acid (AH2) is a potential scavenger of superoxide (O2.-) and peroxyl (LOO.) radicals, the species involved in lipid peroxidation (LPO) in animal tissues. In this paper we have demonstrated that AH2 protects guinea pig tissues from LPO both in vivo and in vitro. The extent of LPO has been determined by estimating malonaldehyde using the thiobarbituric acid test and HPLC and also by measuring the accumulation of fluorescent pigment and occurrence of protein changes in the microsomal membranes. In AH2-deficiency, LPO occurs progressively in guinea pig tissues, despite the presence of adequate levels of antioxidants like alpha-tocopherol, GSH, protein thiols, and scavenging enzymes, namely, superoxide dismutase, catalase, and glutathione peroxidase. In a model in vitro system, microsomal LPO initiated by O2.- is completely prevented by AH2 but not by alpha-tocopherol, GSH, uric acid, and catalase. AH2 is also the most effective antioxidant in preventing microsomal LPO mediated by tert-butylhydroperoxide or the chain propagating species LOO., generated from 2,2'-azobis (2-amidinopropane) hydrochloride. The results obtained with guinea pigs may be applicable to humans, because humans are also dependent on dietary AH2. Our data suggest that an adequate vitamin C nutrition may prevent common cellular degenerative diseases associated with LPO.

Protective effect of ascorbic acid against lipid peroxidation and oxidative damage in cardiac microsomes.

One of the current theories of cardiovascular disease is that it may begin with oxygen radical-induced damages. Extensive studies have been made in different laboratories to elucidate the mechanism of oxidative damages in the presence of added iron salts. However, those in vitro studies are unlikely to be relevant to the in vivo situation, where in the normal physiological condition most of the iron remains bound with proteins. In the present study we have demonstrated that an in vitro system containing desferrioxamine, a strong iron chelator, superoxide generated by the action of xanthine oxidase on acetaldehyde initiates lipid peroxidation and protein changes in the guinea pig cardiac microsomes. We have further demonstrated that superoxide-initiated lipid peroxidation and protein changes are completely prevented by ascorbic acid. SOD also prevents but catalase, alpha-tocopherol, glutathione, uric acid, thiourea, mannitol and histidine are without effect. When NADPH is used instead of generated superoxide, the lipid peroxidation and protein changes are exclusively inhibited by ascorbic acid. SOD, catalase and other antioxidants are ineffective. The results obtained with guinea pigs may be extrapolated to humans, because like guinea pigs humans are also incapable of synthesizing ascorbic acid.

Protective role of ascorbic acid against lipid peroxidation and myocardial injury.

Ascorbic acid (AH2) is a potential scavenger of superoxide radical and singlet oxygen. In the guinea pig, marginal AH2 deficiency results in intracellular oxidative damage in the cardiac tissue as evidenced by lipid peroxidation, formation of fluorescent pigment and loss of structural integrity of the microsomal membranes. The oxidative damage does not occur due to lack of enzymatic scavengers of reactive oxygen species such as superoxide dismutase, catalase and glutathione peroxidase. Also, glutathione transferase activity is not decreased in AH2 deficiency. Lipid peroxidation, fluorescent pigment formation and protein modification disappear after AH2 therapy. These results, if extra-polated to human beings, would indicate that chronic subclinical AH2 deficiency may result in progressive oxidative damage which in the long run may lead to permanent degenerative diseases in the heart.