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© 2016, Springer-Verlag Berlin Heidelberg. Introduction: Chemotherapy-induced peripheral neuropathy (CIPN) is a debilitating side effect resulting from neurotoxic chemotherapeutic agents. This study aimed to assess the efficacy and safety of an oral B group vitamin compared to placebo, in preventing the incidence of CIPN in cancer patients undergoing neurotoxic chemotherapy. Methods: A pilot, randomised, placebo-controlled trial was conducted. Newly diagnosed cancer patients prescribed with taxanes, oxaliplatin or vincristine were invited to participate. A total of 71 participants (female 68 %, male 32 %) were enrolled into the study and randomised to the B group vitamin (n = 38) arm or placebo (n = 33). The data from 47 participants were eligible for analysis (B group vitamins n = 27, placebo n = 22). The primary outcome measure was the total neuropathy score assessed by an independent neurologist. Secondary outcome measures included serum vitamin B levels, quality of life, pain inventory and the patient neurotoxicity questionnaires. Outcome measures were conducted at baseline, 12, 24 and 36 weeks. Results: The total neuropathy score (TNS) demonstrated that a B group vitamin did not significantly reduce the incidence of CIPN compared to placebo (p = 0.73). Statistical significance was achieved for patient perceived sensory peripheral neuropathy (12 weeks p = 0.03; 24 weeks p = 0.005; 36 weeks p = 0.021). The risk estimate for the Patient Neurotoxicity Questionnaire (PNQ) was also statistically significant (OR = 5.78, 95 % CI = 1.63–20.5). The European Organisation of Research and Treatment of Cancer (EORTC) quality of life, total pain score and pain interference showed no significance (p = 0.46, p = 0.9, p = 0.37 respectively). A trend was observed indicating that vitamin B12 may reduce the onset and severity of CIPN. Conclusion: An oral B group vitamin as an adjunct to neurotoxic chemotherapy regimens was not superior to placebo (p > 0.05) for the prevention of CIPN. Patients taking the B group vitamin perceived a reduction in sensory peripheral neuropathy in the PNQ. Moreover, a robust clinical study is warranted given that vitamin B12 may show potential in reducing the onset and severity of CIPN. Trial number: ACTRN12611000078954 Protocol number: UH2010000749

The formations of reactive oxygen species (ROS) and reactive nitrogen species (RNS) have long been considered as major contributors to the dysregulation of the inflammatory response. Reactive oxygen species and RNS productions often are reported to be associated with the development of chronic diseases and acceleration of the aging process. Mechanistically, this association has linked the phenomena of oxidative stress with the occurrence of random deleterious modifications of macromolecules with progressive development of pro-inflammatory conditions promoting age-associated systemic diseases. On the contrary the so-called random modification of macromolecules is incorrect rather ROS and RNS are molecular regulators (second messengers) and not universal toxins whose overproduction should be annulled by antioxidants. We have previously reviewed the physiological role of superoxide anion (and hydrogen peroxide) and nitric oxide (and peroxynitrite) and concluded that these reactive molecular species behave as pro-oxidant second messengers. Reactive oxygen species and RNS are produced at specific cellular locations and are essential for both the normal physiological function of the metabolome and the regulated inflammatory response. This brings into question the whole concept of the orally administering of antioxidant molecular species to down-regulate or abrogate an overproduction of free radical activity. There are no human clinical trials that demonstrate that small molecules, the so-called antioxidants (e.g., vitamins C, vitamin E and beta-carotene), confer a favorable clinical outcome of long-lasting control of inflammation.

Elderly patients undergoing cardiac surgery are more likely to suffer postoperative heart failure than younger patients. This phenomenon is mirrored by an age-related loss of mitochondrial function and by an in vitro loss of myocardial contractile force following a stress. To examine the possibility that loss of mtDNA integrity may be responsible, we quantified representative age-associated mtDNA mutations (mtDNA(4977) and mtDNA(A3243G)) and mtDNA copy number using quantitative polymerase chain reaction in atrial samples obtained during cardiac surgery. The myocardium underwent organ bath contractility testing before and after either an ischaemic or hypoxic stress. We found that with age, recovery of developed force after either stressor significantly declined (p < 0.0001). The abundance of mtDNA(4977) correlated weakly with loss of contractility (R(2) = 0.09, p = 0.047). However, the abundance level was low (average 0.0075% of total mtDNA) and the correlation disappeared when age was included in a multivariate analysis. Neither the abundance of mtDNA(A3243G) nor mtDNA copy number correlated with reduced recovery of developed force after stress. We conclude that, although mtDNA mutations (as exemplified by mtDNA(4977)) accumulate in the ageing heart, they are unlikely to make a major contribution to loss of contractile function. (C) 2009 Elsevier Ireland Ltd. All rights reserved.

The overarching role of coenzyme Q10 in gene regulation, bioenergy formation, cellular redox poise regulation, and hydrogen peroxide formation is presented. Coenzyme Q10 has a central role acting as a prooxidant in the generation of H2O2. Contrary to the dogma that superoxide and H2O2 formation are highly deleterious to cell survival this premise is rejected. Data are discussed that continuous superoxide and hydrogen peroxide formation are essential for normal cell function and that they play a major role in subcellular redox state modulation. It is the prooxidant activity of the so-called antioxidants that may be responsible for previously claimed benefits for high doses of oxido-reduction nutritional supplements such as alpha lipoic acid and coenzyme Q10. Oxygen-free radical formation is essential for the biological function and is not a direct causation of the mammalian aging process; aging is a multisystem stochastic process. © 2006 New York Academy of Sciences

In this communication, the concept is developed that coenzyme Q10 has a toti-potent role in the regulation of cellular metabolism. The redox function of coenzyme Q10 leads to a number of outcomes with major impacts on sub-cellular metabolism and gene regulation. Coenzyme Q10's regulatory activities are achieved in part, through the agency of its localization in the various sub-cellular membrane compartments. Its fluctuating redox poise within these membranes reflects the cell's metabolic micro-environments. As an integral part of this process, H2O2 is generated as a product of the normal electron transport systems to function as a mitogenic second messenger informing the nuclear and mitochondrial (chloroplast) genomes on a real-time basis of the status of the sub-cellular metabolic micro-environments and the needs of that cell. Coenzyme Q10 plays a major role both in energy conservation, and energy dissipation as a component of the uncoupler protein family. Coenzyme Q10 is both an anti-oxidant and a pro-oxidant and of the two the latter is proposed as its more important cellular function. Coenzyme Q10 has been reported, to be of therapeutic benefit in the treatment of a wide range of age related degenerative systemic diseases and mitochondrial disease. Our over-arching hypotheses on the central role played by coenzyme Q10 in redox poise changes, the generation of H2O2, consequent gene regulation and metabolic flux control may account for the wide ranging therapeutic benefits attributed to coenzyme Q10.

A hypothesis as to the nature of the ageing process is presented. Our studies lead us to emphasise that ageing is a stochastic process; it is individualistic, it is species specific, it is different for individuals within a species, it is organ and tissue specific as well as varying among individual cells of a tissue. Human ageing is a slow process which takes place over decades; it is a cellular process in a dynamic equilibrium of continuing damage and repair. Tissues are damage mosaics. We hypothesise that the characteristic of ageing post-mitotic tissues is the progressive, slow loss of cells; those cells which no longer adequately function are removed by an apoptotic or non-inflammatory necrotic process. A case is made for an encompassing role for coenzyme Q10 in the regulation of systemic disease, cellular metabolism and ageing.

younger muscle. 4)Oral CoQ10 therapy before cardiac surgery improves efficiency of mitochondrial energy production, improves post-operative heart function, reduces intra-operative myocardial damage and shortens hospital stay.

In this paper, we report results obtained from a continuing clinical trial on the effect of coenzyme Q10 (CoQ10) administration on human vastus lateralis (quadriceps) skeletal muscle. Muscle samples, obtained from aged individuals receiving placebo or CoQ10 supplementation (300mg per day for four weeks prior to hip replacement surgery) were analysed for changes in gene and protein expression and in muscle fibre type composition. Microarray analysis (Affymetrix U95A human oligonucleotide array) using a change in gene expression of 1.8-fold or greater as a cutoff point, demonstrated that a total of 115 genes were differentially expressed in six subject comparisons. In the CoQ10-treated subjects, 47 genes were up-regulated and 68 down-regulated in comparison with placebo-treated subjects. Restriction fragment differential display analysis showed that over 600 fragments were differentially expressed using a 2.0-fold or greater change in expression as a cutoff point. Proteome analysis revealed that, of the high abundance muscle proteins detected (2,086 +/- 115), the expression of 174 proteins was induced by CoQ10 while 77 proteins were repressed by CoQ10 supplementation. Muscle fibre types were also affected by CoQ10 treatment; CoQ10-treated individuals showed a lower proportion of type I (slow twitch) fibres and a higher proportion of type IIb (fast twitch) fibres, compared to age-matched placebo-treated subjects. The data suggests that CoQ10 treatment can act to influence the fibre type composition towards the fibre type profile generally found in younger individuals. Our results led us to the conclusion that coenzyme Q10 is a gene regulator and consequently has wide-ranging effects on over-all tissue metabolism. We develop a comprehensive hypothesis that CoQ10 plays a major role in the determination of membrane potential of many, if not all, sub-cellular membrane systems and that H2O2 arising from the activities of CoQ10 acts as a second messenger for the modulation of gene expression and cellular metabolism.

Proteomics provides an extremely powerful tool for the study of variations in protein expression between individuals, different disease states and different conditions. One of the major challenges facing the medical profession in the forthcoming decades is to understand the changes that occur in individuals as they become older and to attempt to develop means to improve the quality of life for the otherwise healthy ageing population. The present study describes the first phase of such an investigation in which the protein composition of human skeletal muscle samples from young and aged subjects are compared. These results provide the beginning of a Human Aged Skeletal Muscle Profile reference map which is an essential first step to further investigations.

Extra-long PCR (XL-PCR) was used to assess the relative concentration of functional full-length mitochondrial DNA (mtDNA) in single type I human vastus lateralis muscle fibres of defined cytochrome c oxidase (COX) activity. Type I muscle fibres rely more on mitochondrial oxidative phosphorylation for their energy demands, compared to the other common fibre types (IIa, IIab and IIb) that principally depend on glycolysis for their energy requirements. A total of 60 single type I fibres were analyzed from 15 individuals (8 males and 7 females) of various ages. COX positive muscle fibres were shown to contain amplifiable full-length mtDNA together with a small number of mtDNA rearrangements. By contrast, COX negative fibres did not contain detectable full-length mtDNA, but did contain a heterogeneous mixture of rearranged mtDNA species with the frequency and occurrence of each deletion varying considerably from fibre to fibre. These data lead us to the conclusion that the level of COX activity in type I muscle fibres is reflected by the amount of amplifiable full-length mtDNA. It is proposed that the amount of amplifiable full-length mtDNA constitutes the functional fraction of the total mtDNA. A comprehensive hypothesis that relates the dynamics of mtDNA turnover, mtDNA mutations, mtDNA damage and repair to the ageing process is discussed.

A phenomenon associated with the aging process is a general age-dependent decline in cellular bioenergetic capacity that varies from tissue to tissue and even from cell to cell within the same tissue. This variation eventually forms a tissue bioenergy mosaic. Recent evidence by our group suggests that the accumulation of mitochondrial DNA mutations, in conjunction with a concurrent decrease in full-length mtDNA in tissues such as skeletal and cardiac muscle, strongly correlates with decreased mitochondrial function and accounts for the bioenergy mosaic. Evidence is also presented suggesting that amelioration with coenzyme Q10 may restore some of the age-associated decline in bioenergy function, in effect providing the potential for a “redox therapy”. Coenzyme Q is a naturally occurring material that is present in the membranes of all animal cells. Its primary function is to act as an electron carrier in the mitochondrial electron transport chain enabling the energy from substrates such as fats and sugars (in the form of reducing equivalents) to be ultimately captured in the form of ATP, which in turn may be utilised as a source of cellular bioenergy. Coenzyme Q10 has no known toxic effects and has been used in a limited number of animal studies and human clinical trials; however, the mechanism of action of coenzyme Q10 remains unclear. A series of experiments by this group aimed at determining the efficacy of coenzyme Q10 treatment on ameliorating the bioenergy capacity at the organ and cellular level will also be reviewed.

The inferior recovery of cardiac function after interventional cardiac procedures in elderly patients compared to younger patients suggests that the aged myocardium is more sensitive to stress. We report two studies that demonstrate an age-related deficit in myocardial performance after aerobic and ischemic stress and the capacity of CoQ10 treatment to correct age-specific diminished recovery of function. In Study 1 the functional recovery of young (4 mo) and senescent (35 mo) isolated working rat hearts after aerobic stress produced by rapid electrical pacing was examined. After pacing, the senescent hearts, compared to young, showed reduced recovery of pre-stress work performance. CoQ10 pretreatment (daily intraperitoneal injections of 4 mg/kg CoQ10 for 6 weeks) in senescent hearts improved their recovery to match that of young hearts. Study 2 tested whether the capacity of human atrial trabeculae (obtained during surgery) to recover contractile function, following ischemic stress in vitro (60 min), is decreased with age and whether this decrease can be reversed by CoQ10. Trabeculae from older individuals (or = 70 yr) showed reduced recovery of developed force after simulated ischemia compared to younger counterparts (70 yr). Notably, this age-associated effect was prevented in trabeculae pretreated in vitro (30 min at 24 degrees C) with CoQ10 (400 MicroM). We measured significantly lower CoQ10 content in trabeculae fromor = 70 yr patients. In vitro pretreatment raised trabecular CoQ10 content to similar levels in all groups. We conclude that, compared to younger counterparts, the senescent myocardium of rats and humans has a reduced capacity to tolerate ischemic or aerobic stress and recover pre-stress contractile performance, however, this reduction is attenuated by CoQ10 pretreatment.

Post-mitotic tissues such as skeletal muscle develop a tissue bioenergy mosaic during the process of normal aging that eventually culminates into a bioenergetically diverse tissue containing cells ranging in their oxidative phosphorylation capacity from normal to grossly defective. The mosaic is postulated to develop continuously from birth with the relative proportions of cytochrome c oxidase (COX) proficient (positive) and COX deficient (negative) muscle fibers differing dramatically as a function of age. Generally, young individuals only display the rare fiber deficient in COX activity while aged individuals show a significantly higher proportion of negative fibers. There appears to be a random element governing which cells will be affected. Consequently, adjacent cells within a given tissue may exhibit vastly differing COX activities. Multiple mitochondrial DNA (mtDNA) deletions also appear to accumulate in skeletal muscle, similarly displaying a dramatic disparity as a function of age. Our previous findings have indicated that the accumulation of multiple mtDNA deletions, along with a concurrent decrease in wild-type mtDNA, strongly correlates with the age-associated decrease in COX activity observed in skeletal muscle. Although no definitive associations were established at the cellular level, an important prediction arose from this study. Cells that accumulate large numbers of mitochondrial mutations and have reduced levels of full-length mtDNA would be expected to be severely affected and show reduced COX activity as a consequence. Cells that accumulate fewer mutations or retain adequate amounts of wild-type mtDNA would be predicted to be less affected or even retain normal oxidative metabolism. In order to establish a link associating COX activity to the status of mtDNA within individual fibers, we developed single cell extra-long PCR (XL-PCR). The procedure was used to assess the relative concentration of full-length mtDNA with respect to any mtDNA deletions detected in individual human skeletal muscle fibers of `pre-established' COX activity. Single cell XL-PCR analysis of COX positive fibers dissected from a 5-year old and 90-year old individual showed that 80% or more of the fibers contained full length mtDNA and few, if any, mtDNA rearrangements. COX deficient or COX intermediate fibers taken from the same individuals, by contrast, depicted a heterogeneous population of rearranged mtDNA species with no detectable full-length mtDNA. The data presented here indicates that COX deficient muscle fibers extracted from individuals, regardless of age, were accompanied by extensive mtDNA rearrangements and reduced levels of full-length mtDNA. This provides compelling evidence linking mtDNA mutations to COX activity decline in skeletal muscle and has important implications when considering the molecular basis of the aging process.

Seven mtDNA mutations (five base substitutions and two deletions) were studied in skeletal muscle samples of 18 human subjects aged 1 hr to 90 years. Quantitative PCR procedures were applied to determine the incidence (frequency of occurrence) and abundance (percentage of mutant mtDNA out of total mtDNA). The base substitutions, in general, showed a very early onset, three such mutations being detectable in the muscles of infants aged 1 hr and 5 weeks. Of two disease-associated point mutations studied, 3243 A-->G showed significant accumulation with age (P G showed no significant age accumulation (P > 0.1). Moreover, three arbitrarily chosen mutations (not disease-associated) showed no age-associated accumulation: two (7029 C-->T and 7920 A-->G) showed little change over the years (P > 0.1), while the other (13167 A-->G) showed a significant decrease (P < 0.05). both the 4,977-bp and 7,436-bp deletions showed a significant age-associated occurrence (P < 0.01 and P < 0.05, respectively). The age of onset of detectable deletions is about 20-40 years; thereafter, the incidence and abundance of deletions tend to increase as a function of advancing age. The seven specific mutations were found to occur independent of each other, indicating the random nature of mtDNA mutations in skeletal muscle. Moreover, the age-associated accumulation of multiple deletions was observed in the same set of muscle tissues, each extract displaying a unique set of multiple PCR products. Thus, mutations in mtDNA occur differentially in human skeletal muscle during aging.

Extra long PCR analysis of mitochondrial DNA (mtDNA) isolated from skeletal muscle of humans of different ages revealed three phenomena: (i) the amount of normal length mtDNA (16.5 kb) was progressively reduced with age, such that the cells of old age individuals (about 90 years) contained little, or undetectable amounts of normal length mtDNA; (ii) the total amount of mtDNA did not appear to be greatly decreased rather the extent of mtDNA deletions greatly increased; (iii) in old age subjects, considerable amounts of over-sized mtDNA (more than 16.5 kb) was observed. Enzyme histochemical analysis of cytochrome-c oxidase (COX) activity in the muscle tissue of all subjects evidenced a cellular bioenergy mosaic with cells ranging from high to zero detectable enzyme activity in the muscle samples. The frequency of COX deficient muscle fibres was highly dependent on the age of the subject. We have found that the extent of the mtDNA mutational changes strongly correlate with the observed progressive decrease in COX activity. Therefore, it was suggested that the total extent of mtDNA mutation is very large in old age subjects and is sufficient to account for the decline in cellular COX activity with age and for a progressive decrease of overall mitochondrial bioenergetic capacity.

We have developed an improved allele-specific polymerase chain reaction (AS-PCR) procedure that can selectively amplify mutant DNA sequences (which differ from the normal sequences by a single base pair) in the presence of large excess of normal sequences. We applied this procedure to quantification of mutant molecules of human mitochondrial DNA (mtDNA). Conditions for AS-PCR have been systematically varied, encompassing DNA template input, annealing temperature, and PCR cycle number. Adjustment of these three reaction parameters to optimal conditions, using plasmids containing cloned segments of mutant and normal mtDNA, enabled the reliable detection of as little as 0.01% of mutant mtDNA. By standardising the DNA input for AS-PCR, the percentage of mutant molecules can be accurately quantified. This improved procedure was used here to detect and quantify the base substitution at nucleotide position 3243 (AG) in mtDNA from total cellular DNA isolated from various tissues of both infants and adults. We observed a 5- to 10-fold higher mutant mtDNA (3243 AG) frequency in adult tissues than in infant tissues. The results are consistent with the hypothesis that the accumulation of mtDNA mutations is an important feature of the human aging process. The quantitative and sensitive allele-specific amplification system described here is applicable to the quantification of low levels of somatic mutations in oncogenes and tumour suppressor genes in the context of human mutation, and could be extended to any biological situation in which only a small proportion of a DNA molecular population is subjected to a particular base substitution. Hum Mutat 9: 265–271, 1997. © 1997 Wiley-Liss, Inc.

As part of the ongoing studies aimed at elucidating the mechanism of the energy conserving function of mitochondrial complex I, NADH: ubiquinone (Q) reductase, we have investigated how short-chain Q analogs activate the proton pumping function of this complex. Using a pH-sensitive fluorescent dye we have monitored both the extent and initial velocity of proton pumping of complex I in submitochondrial particles. The results are consistent with two sites of interaction of Q analogs with complex I, each having different proton pumping capacity. One is the physiological site which leads to a rapid proton pumping and a stoichiometric consumption of NADH associated with the reduction of the most hydrophobic Q analogs. Of these, heptyl-Q appears to be the most efficient substrate in the assay of proton pumping. Q analogs with a short-chain of less than six carbons interact with a second site which drives a slow proton pumping activity associated with NADH oxidation that is overstoichiometric to the reduced quinone acceptor. This activity is also nonphysiological, since hydrophilic Q analogs show little or no respiratory control ratio of their NADH:Q reductase activity, contrary to hydrophobic Q analogs.

The prevalence in tissues of mtDNA deletions was compared by PCR between humans and rats of similar "biological ages". Pairs of species-specific primers were used which spanned similar portions of the human and rat mtDNA genomes. There were much fewer PCR products amplified from rat mtDNA than from human mtDNA in each of the three tissues initially analysed: heart, liver and skeletal muscle. By contrast, many more PCR products were amplified from rat kidney than from human kidney. Therefore, while there were far more deletions in heart, liver and skeletal muscle of humans than in corresponding rat tissues, the prevalence of mtDNA deletions was markedly less in human kidney than in rat kidney. The data also indicate that human kidney contains less mtDNA deletions than heart, liver and skeletal muscle in humans; whereas in rat kidney there are more mtDNA deletions than in those three tissues of rat. It is further suggested that, when utilising rodents as experimental models for human ageing, the appropriate tissues should be considered, since not all tissues of rats accumulate mtDNA mutations in the same manner as those of humans.

A genetic and molecular analysis of the oli2 region in mitochondrial DNA of Saccharomyces cerevisiae has been carried out. The oli2 gene codes for subunit 6 of the mitochondrial ATPase complex. We have isolated a series of 41 mit (-), temperature sensitive and oligomycin resistant mutants containing mutations located in the oli2 region. In addition the sequence in wild type mtDNA of 2820 nucleotides covering the oli2 gene and its flanking regions has been determined. A library of petite mutants which have various deletion end points in the oli2 region has been used to generate marker rescue data that allow the assignment of each mit (-), temperature sensitivity and oligomycin resistance mutation to one of seven uniquely ordered genetic groups. The following physical reference points allow the limits of the physical map positions of a number of the genetic groups to be established: DNA sequence data have been obtained for three of the petites so as to determine unambiguously their relevant deletion end points and the base substitution constituting the mtDNA sequence change in one oligomycin resistant mutant [oli2-23r] has been determined to lie at nucleotide +523 of the oli2 coding region. One group of mit (-) mutations (group N) are known to map within the aap1 gene that is found some 700 bases upstream of the oli2 gene, and which codes for subunit 8 of the mitochondrial ATPase complex. Mutations in the remaining six groups (A-F) map within (or very close to) the coding region of the oli2 gene. Various features of the DNA sequence in and around the oli2 gene are considered, including protein coding regions, sequence divergence in the A,T-rich spacer regions, properties of G,C-rich clusters, and potential ribosome binding sites. Consideration of the transcriptional map of this region of mtDNA suggests that an abundant 4,500 nucleotide transcript may represent the first defined example of a dicistronic messenger RNA in yeast mitochondria carrying distinct coding regions (aap1 and oli2) for two different proteins.

We have studied the interaction of idebenone (2,3-dimethoxy-5-methy-6-(10-hydroxy)decyl-1,4-benzoquinone) with the energy-conserving complexes of the respiratory chain in beef heart mitochondria and compared its energetic efficiency with that of other analogs of coenzyme Q. Idebenone is a very effective substrate for succinate:Q reductase and ubiquinol:cytochrome c reductase, but it is clearly a poor substrate for NADH:Q reductase (complex I). Indeed, idebenone is a strong inhibitor of both the redox and proton pumping activity of complex I, showing effects in part similar to those of coenzyme Q-2. However, the mechanism of idebenone interaction with complex I may be different from that of Q-2 because of its different sensitivity to inhibitors. The possible relevance of the present findings to the therapeutic use of idebenone is discussed.

The tumour-associated epitope recognised by monoclonal antibody (MAb) 4D3 is expressed on a high m.w. mucin glycoprotein preparation known as small intestinal mucin antigen (SIMA). This epitope is detected in tissue from a high proportion of patients with colorectal cancer, and elevated levels occur in serum from a significant number of such patients, highlighting the potential clinical utility of MAb 4D3. In the present study, insight into the composition and structure of the carbohydrate epitope recognised by MAb 4D3 was gained following characterisation of 2 glycopeptides that co-purified with SIMA. Sequence analysis of 1 of these glycopeptides revealed that it was identical to the glycoprotein α-1-anti-chymotrypsin. This glycoprotein was subsequently deglycosylated to yield 5 forms corresponding to α-1-anti-chymotrypsin substituted with 4, 3, 2, 1 or no branched glycans. MAb 4D3 was reactive with each of the glycosylated forms, including the form carrying only 1 branched glycan, but did not react with fully deglycosylated α-1-anti-chymotrypsin. MAb 4D3 also reacted to different extents with ovine, bovine or porcine submaxillary mucins, each of which has a different amount of the O-linked sialylated disaccharide known as sialosyl Tn. Of these mucins, MAb 4D3 was most reactive with ovine submaxillary mucin, in which almost all of the carbohydrate chains are sialosyl Tn. Reactivity of MAb 4D3 towards isolated glycans, sialosyl Tn and related structures led to the conclusion that the preferred MAb 4D3 epitope involves the sialylated N-acetyl galactosamine disaccharide as well as an additional monosaccharide present on a neighbouring carbohydrate chain. Although the preferred epitope recognised by MAb 4D3 involves this sialylated disaccharide, the specificity of MAb 4D3 was different from that of other MAbs with a reported specificity for sialosyl Tn. © 1996 Wiley-Liss, Inc.

Chemotherapy induced peripheral neuropathy [CIPN] is a common significant and debilitating side effect resulting from the administration of neurotoxic chemotherapeutic agents. These pharmaco-chemotherapeutics can include taxanes, vinca alkaloids and others. Moderate to severe CIPN significantly decreases the quality of life and physical abilities of cancer patients and current pharmacotherapy for CIPN e.g. Amifostine and antidepressants have had limited efficacy and may themselves induce adverse side effects. To determine the potential use of nutraceuticals i.e. vitamin E, acetyl-L-carnitine, glutamine, glutathione, vitamin B6, omega-3 fatty acids, magnesium, calcium, alpha lipoic acid and n-acetyl cysteine as adjuvants in cancer treatments a systematic literature review was conducted. Revised clinical studies comprised of randomized clinical trials that investigated the anti-CIPN effect of nutraceuticals as the adjuvant intervention in patients administered chemotherapy. Twenty-four studies were assessed on methodological quality and limitations identified. Studies were mixed in their recommendations for nutraceuticals. Currently no agent has shown solid beneficial evidence to be recommended for the treatment or prophylaxis of CIPN. The standard of care for CIPN includes dose reduction and/or discontinuation of chemotherapy treatment. The management of CIPN remains an important challenge and future studies are warranted before recommendations for the use of supplements can be made.

We have considered the regulatory interrelationship of the plasma membrane oxidoreductase (PMOR) system and the mitochondrial respiratory capacity of human Namalwa (lymphoblastoid) cells. To this end, we made use of mitchondrially respiratory competent (ϱ+) cells and ϱ0 cells, which lack mitochondrial DNA (mtDNA) and consequently mitochondrial respiratory activity. NADH-fer-ricyanide reductase activity of the PMOR system is increased 3-fold in ϱ0 Namalwa cells compared to ϱ+ cells. It is also shown for the first time that addition of coenzyme Q10 and coenzyme Q10-ana-logues, which can rescue ϱ0 Namalwa cells in the absence of pyravate, gives rise to a further 2–3-fold increase in plasma membrane NADH-ferricyanide reductase activity. These systems were examined to determine if there exists a correlation between the regulation of the PMOR system and extracellular Superoxide radical formation as measured with the fluorescence probe L-012. No correlation was found between NADH-ferricyanide reductase activity and extracellular Superoxide radical production. PMOR function in cellular proliferation appears therefore not to involve extracellular Superoxide radical production.

Genes in the 16.6 kb human mitochondrial DNA (mtDNA) are concerned exclusively with bioenergy production. Mutations in mtDNA can, therefore, lead to bioenergy decline and so contribute to various age-related degenerative diseases and even to 'natural' ageing (1-3). Large deletions in mtDNA occur in tissues of patients with mitochondrial myopathies and also occur in normal ageing, particularly in postmitotic tissues characterised by high energy demands and low rates of cell division, notably skeletal muscle, cardiac muscle and brain (4-11). The frequency in ageing of deletion-bearing mtDNA is much lower than that in overt mitochondrial diseases, but does regularly increase with advancing age. The age-associated deletions affecting mtDNA frequently include a 4977 bp deletion that is known as the 'common deletion' and is generated between a pair of 13 bp direct repeats, located at nucleotide (nt) positions 8470-8482 and nt 13447-13459, respectively (Table 1, Deletion 1). This deletion was detected by the polymerase chain reaction (PCR) in every postmitotic tissue examined, including skeletal muscle, heart and brain, from adult humans 40 years and over (4,7,13). In addition, a 7436 bp deletion, occurring between a pair of 12 bp direct repeats at nt 8637-8648 and nt 16073-16084 (Table 1, Deletion 2), has been observed in many adult postmitotic tissues, along with other deletions (2,6,9,14).

We have studied aspects of the regulatory interrelationship between the plasma membrane oxidoreductase (PMOR) system and the mitochondrial respiratory capacity of human Namalwa cells. Although the role of mitochondria in the maintenance of cellular redox and energetic states is well established, the PMOR system in comparison is a poorly characterized enzyme system whose functions, particularly in relation to cellular metabolism, have not been clearly elucidated. Therefore we compared the PMOR and mitochondrial respiratory activities of human Namalwa cells during the induction by ethidium bromide treatment of rho 0 cells, which lack a functional mitochondrial respiratory system. The plasma membrane NADH-ferricyanide reductase activity of the PMOR system was found to increase in a stepwise manner concomitant with a decline in cellular mitochondrial respiratory activity. Addition of p-chloromercuriphenylsulfonic acid to the culture medium, at a concentration totally inhibiting the plasma membrane NADH-ferricyanide reductase in vitro, leads to cell death of rho 0 but not of rho + cells. Thus, the up-regulation of a functional PMOR system is a necessary phenomenon in maintaining the viability of mammalian rho 0 cells.

In this paper the specific mitochondrial respiratory chain inhibitors rotenone and antimycin A and the highly specific mitochondrial ATP-synthase inhibitor oligomycin are shown to induce an apoptotic suicide response in cultured human lymphoblastoid and other mammalian cells within 12–18 h. The mitochondrial inhibitors do not induce apoptosis in cells depleted of mitochondrial DNA and thus lacking an intact mitochondrial respiratory chain. Apoptosis induced by respiratory chain inhibitors is not inhibited by the presence of Bcl-2. We discuss the possible role of mitochondrial induced apoptosis in the ageing process and age-associated diseases.

A variety of techniques have been applied to investigate the interrelationship between age-related atrophy of rat soleus muscle and other signs of muscle aging, such as changes in muscle fibre type composition, decrease in bioenergy capacity and accumulation of mitochondrial DNA (mtDNA) arrangements. Age-related atrophy of rat soleus muscle was shown to start at the age of about 28 months. It was accompanied by a decrease in the number of slow twitch muscle fibres (type I) and an increase in the proportion of muscle fibres co-expressing slow and fast myosins (type Ic and IIc fibres). Bioenergy capacity of the soleus muscle, assessed by the level of measurable cytochrome c oxidase (COX) activity, was found to be decreased both in the middle age and old rats compared to the young animals. Muscle atrophy was also accompanied by a decrease in the amount of full-length mitochondrial DNA (FL-mtDNA) amplifiable by the extra-long PCR (XL-PCR) and the increase in the number of mtDNA deletions. The results of the study show that the decline in the bioenergy capacity of the rat soleus occurs by the middle age. It is followed by the onset of the age-related muscle atrophy that is accompanied by both fibre type changes and functional mtDNA degradation.

We have developed a sensitive ELISA using MAb 4D3 for the detection of a novel epitope on Small Intestinal Mucin Antigen (SIMA) and report here that SIMA is present in the serum of patients with colorectal cancer. SIMA has been shown to occur in tissue from a high proportion of patients with colorectal cancer. SIMA derived from serum was similar to tissue-derived SIMA: both eluted in the void volume of a Superose 6 column indicating a molecular weight above 5,000 kDa and they exhibited similar buoyant densities on CsCI gradients. The ELISA was most reliable after pre-treatment of serum with 0.4 M perchloric acid to remove interfering substances. The upper limit for SIMA in normal serum was set as the mean plus 2 standard deviations determined from a group of 97 healthy control subjects. In a samsle of 113 patients with colorectal cancer, SIMA serum levels were elevated in 15% of patients with Dukes' Stage A, 38% with Stage B, 32% with Stage C and 75% with Stage D colorectal cancer. SIMA serum levels were compared with those of the widely used tumor marker, carcino-embryonic antigen (CEA). The SIMA assay detected a significant number of sera that were not detected by the test for CEA. We propose that SIMA will prove to be a valuable serological tumor marker, in combination with CEA and other tumor markers, for the detection of colorectal cancer.

We have devised a sensitive method based on the polymerase chain reaction (PCR) to detect expression of human interferon-alpha-encoding genes (IFN-A) in general, and specifically, expression of the IFN-A2 or IFN-A4 genes. The utility of the PCR approach was assessed by analysis of cloned IFN-A genes, as well as genomic DNA and mRNA isolated from peripheral blood mononuclear cells. We demonstrate the specific amplification of sequences encoding IFN subtypes IFN-alpha-2 and IFN-alpha-4 from as little as 0.1 pg of IFN-A mRNA. The PCR technique has potential clinical application for the detection of IFN-A expression and, thus, identification of the IFN-alpha subtypes produced, particularly in small biopsy samples or otherwise, where only low numbers of cells are available.

This study has identified the expression in normal and neoplastic gastrointestinal (GI) tract of epitopes on the colonic mucin LIMA (large intestinal mucin antigen), which are unique markers of normal colonic differentiation. Six anti-LIMA monoclonal antibodies (MAbs) (22D4, 9B5, 2C3, 23B2, 46A2, and 10B3) were studied immunohistochemically in normal GI tract, colorectal adenomas, and colorectal and gastric cancers. All MAbs showed specificities consistent with distinct epitopes, five of which were neuraminidase-resistant and four periodate-sensitive. Each reacted with mucin in 60-100 per cent normal colons--MAbs 10B3 and 23B2 also with small intestinal mucin--but none with gastric mucin. Five MAbs showed crypt and regional gradients in normal colon, MAbs, 22D4, 9B5, and 2C3 showing a hierarchy of reactivities in the crypt. Individual adenomas showed decreasing goblet cell (GC) LIMA expression with increasing size. However, 30 per cent of familial adenomatous polyposis (FAP) patients had generalized background losses of 9B5 and 2C3 GC reactivity, retaining 22D4, whilst 44 per cent of non-FAP patients lost 22D4 GC reactivity, regaining 9B5 and 2C3--evidence for polymorphism of mucin expression. All colorectal cancers expressed LIMA epitopes (frequently weaker than normal), and three MAbs (22D4, 9B5, and 2C3) showed deeper than normal staining in adjacent crypts. Eighty-five per cent of gastric cancers also expressed LIMA epitopes.

We have investigated changes in mucin antigenicity and morphology in the perineoplastic mucosa adjacent to rare, predominantly non-mucosal gastrointestinal (GI) tumours. Twenty-nine tumours of small and large intestine, including primary mesenchymal and ectodermal tumours, were examined immunohistochemically using 11 monoclonal antibodies (MAbs) raised against SIMA and LIMA (small and large intestinal mucin antigens). Non-epithelial GI tumours were essentially non-reactive, while adjacent mucosa showed altered mucin expression and morphology, in particular, features of transitional mucosa (TM). Combinations of different SIMA epitopes were detected adjacent to all colorectal tumours, and, similarly, LIMA epitopes adjacent to small intestinal tumours. Specific patterns adjacent to certain tumours may reflect influences of factors produced by individual tumours on mucin composition. Altered antigenicity and morphology in TM thus appear to be reactive changes in response to a wide range of GI tumours, presumably as a consequence of factors secreted by the tumour and/or a host response to the tumour.

The constitutive production of interferon-alpha (IFN-alpha) subtypes by the lymphoblastoid cell lines, Namalwa, Daudi and Raji, was investigated using sensitive and semi-quantitative flow cytometric techniques. Further, we sought to determine whether the previously described failure of these cell lines to produce IFN-alpha-4 was a result of the deletion of the IFN A4 gene. Cytoplasmic production of IFN-alpha-2 and IFN-alpha-4 was assessed using IFN-alpha subtype-specific antipeptide antibodies and FITC-labelled secondary antibodies in indirect immunofluorescence-flow cytometry studies. The constitutive production of IFN-alpha-2 was detected in all three cell lines. Significant increases in fluorescence representing increased production of IFN-alpha-2 and possibly other IFN-alpha subtypes were detected after induction by Sendai virus. Approximately 100 per cent of cells in the Namalwa, Daudi and Raji cell populations contained IFN-alpha-2 before and after induction. However, no cells from the same cell populations contained the IFN-alpha-4 subtype. Analysis of genomic DNA isolated from the lymphoblastoid cells using the Polymerase Chain Reaction (PCR) and oligonucleotide primers specific for IFN A2 or IFN A4 confirmed the presence of the genes encoding both IFN-alpha subtypes. Furthermore, using reverse transcriptase-PCR amplification, mRNAs for both IFN-alpha-2 and IFN-alpha-4 were detected. Therefore, in contrast to some leukaemias and derived cell lines where IFN A genes have been deleted, these cell lines of B cell lineage exhibit selective expression of IFN A genes, as a result of altered transcriptional/translational control of IFN-alpha expression.

Previous theories of aging based on somatic mutation neglected mtDNA, which has a high propensity for mutational error. Knowledge of yeast mtDNA mutations and their functional effects, and of human mtDNA mutations identified in the mitochondrial cytopathies, provides for a concept of aging based on the cumulative effect of mutations affecting human mtDNA. An essential feature of this concept is heteroplasmy, representing mixtures of normal and mutant mtDNA at the cellular and mitochondrial level, resulting in a "tissue mosaic" of focal bioenergetic deficits. Direct evidence for the concept is provided by (i) focal loss of staining for mitochondrially encoded enzymes, such as cytochrome c oxidase, in tissues of aged individuals (humans and rats) and (ii) an age-related increase in deletional mutations in mtDNA demonstrable by application of the polymerase chain reaction to DNA templates from individuals of different ages.

BACKGROUND: Human melanomas have shown only limited responsiveness to clinical therapy with interferon (IFN). PURPOSE: Our aim was to determine the most effective class of IFN for inhibiting growth of melanoma cells and to establish whether variation exists in response of various cell lines to different IFNs. METHODS: We compared the direct antiproliferative effects of the type I IFN alpha-2b, IFN alpha-4a, and IFN-beta and the type II IFN-gamma on eight melanoma cell lines grown in vitro. We did this comparison by determining the concentration of each IFN that resulted in 50% growth inhibition, using the MTT [3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium tetrazolium bromide] dye uptake method. We also tested IFN alpha-2a and IFN-beta for their ability to inhibit the growth of xenografts of the LiBr melanoma cell line in vivo in nude mice. Receptor binding was determined using [35S]methionine-labeled IFN alpha-4a, in competition with unlabeled IFN alpha-2b, IFN alpha-4a, and IFN-beta. RESULTS: The melanoma cell lines differed markedly in their sensitivity to the IFNs tested: Five were sensitive to low concentrations (less than 30 pM) of IFN-beta, only one was sensitive to similar concentrations of IFN alpha-2b, and none were sensitive to IFN alpha-4a at concentrations up to 920 pM. For all cell lines, the antiproliferative potency of the type I IFNs was IFN-beta greater than IFN alpha-2b greater than IFN alpha-4a. IFN-gamma was less active than IFN-beta on all except one of the cell lines. Similarly, IFN-beta was more potent than IFN alpha-2a in inhibiting the growth of the LiBr xenograft in nude mice. Labeled IFN alpha-4a bound with high specificity in all four melanoma lines tested, and competitive binding experiments showed that the order of binding affinity (IFN-beta greater than IFN alpha-2b greater than IFN alpha-4a) correlated with the order of antiproliferative potency. CONCLUSION: The finding that melanoma cell lines differ intrinsically in their sensitivity to IFNs may explain differences in clinical response. Our results suggest that IFN-beta may be the most effective IFN in the treatment of melanoma, although confirmation will require clinical trials involving large numbers of patients.

The highly conserved amino acid residues Leu-30 and Arg-33 of human interferon-alpha 4 (IFN-alpha 4) have previously been identified as important for biological activity. In this study, the sequence around Arg-33 was targeted to determine the importance of other residues in this region. A library of analogues containing amino acid substitutions spanning residues 26-37 was generated using site-directed and random mutagenesis. Analogues were expressed in vitro and assayed for antiviral and antiproliferative activity on human cells. No significant separation between the antiviral and antiproliferative activities was observed for any of the analogues tested. Substitutions at positions 26, 27, 31, 32, 34, 35, and 37, did not substantially affect biological activity. However, substitution of Phe-36 with arginine resulted in a greater than 100-fold decrease in biological activity. Thus, together with previous data, the residues in this region identified as most important for biological activity include Leu-30, Arg-33, and Phe-36. Recently published models for the three-dimensional structure of human IFN-alpha and the X-ray crystallographic structure of murine IFN-beta, suggest that the region investigated in this study forms a loop at the surface of the protein. Thus, residues Leu-30, Arg-33, and Phe-36, could be involved in binding to the Type-I IFN receptor, or in interactions with signal-transducing molecules.

We describe the production, immunochemical and immunohistochemical characterization of monoclonal antibodies (MAbs) raised against the oncofetal small intestinal mucin antigen (SIMA). Four MAbs, reacting with distinct neuraminidase-sensitive SIMA epitopes, were shown to define a novel differentiation-associated relationship of SIMA epitopes within the normal small intestinal villus. Using Swiss rolls of 15 entire colorectal cancer resections, inappropriate expression of SIMA epitopes was detected in all cancers, in adjacent transitional mucosa and remote morphologically normal mucosa, extending as far as resection margins (73%). SIMA expression, whether preexisting or reactive to the tumor, may predispose to malignant change and tumor recurrence.

A strategy for the production of human interferon-alpha (IFN-alpha) subtype-specific antibodies, based on immunizing rabbits with short unique synthetic peptides coupled to protein carriers, has been validated. These peptides correspond to amino acid residues 99-111 of IFN-alpha 1, 50-57 and 103-116 of IFN-alpha 2, and 37-50 of IFN-alpha 4. The antipeptide antibodies [anti-IFN alpha 1(99-111), anti-IFN alpha 2(50-57C), anti-IFN alpha 2(103-116) and anti-IFN alpha 4(C37-50)] were tested by ELISA and Western blotting for their reactivity with immunoaffinity-purified recombinant human IFN-alpha 1, -alpha 2b and -alpha 4a. The anti-IFN alpha 1(99-111) and anti-IFN alpha 2(50-57C) reacted with their corresponding IFN-alpha and did not crossreact with the other IFN subtypes. The anti-IFN alpha 2(103-116) reacted with IFN-alpha 2b and also crossreacted slightly with the other subtypes. The anti-IFN alpha 4(C37-50) reacted well with IFN-alpha 4a, crossreacted with significantly lower affinity with IFN-alpha 1 and did not bind IFN-alpha 2b. Residues 104-107 and 108-111 are the major components of the epitopes recognized by anti-IFN alpha 1(99-111) and anti-IFN alpha 2(103-116), respectively, as determined by ELISA against overlapping octapeptides.

A mucin preparation from a colonic adenocarcinoma was used to prepare monoclonal antibodies (MAbs) that reacted specifically either with normal adult small-intestine mucin antigen(s) (SIMA), or normal adult large-intestine mucin antigen(s) (LIMA). Both SIMA and LIMA show a unique oncofetal pattern of expression. Thus SIMA was expressed in early fetal stomach, large and small intestines but thereafter only in the normal small intestine. SIMA expression was detected immunohistochemically in cancers of the colorectum (82/112) and stomach (48/86). LIMA was detected in the stomach of the early fetus but thereafter only in the normal large intestine. LIMA expression was detected in 61/86 cancers of the stomach. Moreover, both SIMA and LIMA were expressed inappropriately in mucosa adjacent to tumors, indicative of the detection of possible pre-malignant epithelium. We used a sandwich ELISA and biochemical procedures to show that the SIMA and LIMA molecules were large extensively glycosylated multi-unit mucin glycoproteins that differed markedly from each other. SIMA, whether extracted from normal small-intestine or colonic cancers, had a molecular weight above 1.000 kDa, a mean buoyant density 1.33 g/ml and s value of 4.8. LIMA had a molecular weight above 10.000 kDa, a mean buoyant density 1.45 g/ml and an s value 9.5. The SIMA and LIMA epitopes were judged to be carbohydrate in nature by reason of their resistance to harsh physical chemical treatments or protease digestion, and sensitivity to periodate oxidation, neuraminidase or beta elimination. Only the SIMA epitope was sensitive to neuraminidase. In conclusion, MAbs to carbohydrate-dependent epitopes on SIMA and LIMA identify the oncofetal pattern of expression of these distinct intestinal mucin glycoproteins in colonic and gastric carcinoma. These MAbs will be useful in further studies of the significance of oncofetal mucin expression during carcinogenesis.

Small intestine mucin antigen (SIMA) is an oncofoetal antigen for the colon and is distinct from the normal large intestinal mucin antigen (LIMA). In the present study, a panel of anti-SIMA and anti-LIMA monoclonal antibodies (MAb) was used to charaterise altered mucin expression in colorectal adenocarcinomas, by immunohistochemistry and quantitative immunoassays of tissue extracts. These results are compared with CEA expression and correlated with various clinicopathological indices. All mucin MAb reacted with a high proportion of the 100 colon cancers of every stage, histological type (including non-mucinous cancers), differentiation, site, or size. Inappropriate SIMA production was detected by either anti-SIMA MAb 4D3 or 4A1, even in 85% of early stage cancers. MAb 4D3 reacted with a higher proportion of cancers of smaller size and better differentiation. At the subcellular level, both anti-SIMA MAb showed reactivity typical of normal mucin, i.e., goblet cell and extracellular mucin. The normal colonic antigen, LIMA, was also detectable in the majority of cases, but quantitatively overproduced in some cases and reduced in others. However, in contrast to SIMA, LIMA was detected in predominantly undifferentiated cancer cells but not in goblet cells. Heterogeneity of MAb reactivity between cases and complementarity within each cancer was frequently observed. Mucin reactive with at least one of the MAb was detected in all of the CEA-negative cancers. A high rate of inappropriate SIMA expression was also detected in the perineoplastic transitional mucosa (88%, c.f. CEA, 35%) and adjacent, morphologically normal mucosa (80% c.f. CEA, 24%), indicating biochemical changes similar to the cancer. This panel of anti-mucin MAb demonstrated altered mucin glycoprotein metabolism associated with the development and progression of most colorectal cancers, which emphasises their utility as indicators of neoplastic change in the colon, and their superiority to CEA. Images Figure 1 Figure 2 Figure 3 Figure 4

Coenzyme Q10 plays a central role in cellular bioenergy generation and its regulation. Closed membrane systems generate a proton motive force to create transient localized bio-capacitors; the captured energy is used for the synthesis of mitochondrial ATP but also for many other processes, such as metabolite translocations, nerve conduction and a host of other bioenergy requiring processes. Coenzyme Q10 plays a key role in many of these sub-cellular membrane energy generating systems. Integral to this phenomenon is the prooxidant role of coenzyme Q10 in generating the major superoxide anion/hydrogen peroxide second messenger system. This messenger system, largely but not exclusively, arises from coenzyme Q10 semiquinone function; it contributes to the regulation of sub-cellular redox potential levels; transcription/gene expression control; is essential for modulated protein turnover and activation; mediates hormone and growth factor extracellular signaling. The regulated prooxidant formation of the superoxide anion/H2O2 second messenger system is essential for the normal physiological function of the metabolome. The normally functioning metabolome is the expression of a finely tuned dynamic equilibrium comprised of thousands of anabolic and catabolic reactions and all cellular signaling systems must be finely regulated. There is still much to be learnt about the up/down regulation of the H2O2 messenger system. The concept that superoxide anion/H2O2 cause random macromolecular damage is rebutted. The administration of antioxidants to quench the inferred toxicity of these compounds as a therapy for age associated diseases is unsupported by extant mammalian clinical trials and should be subject to serious re-evaluation. The role of ascorbic acid as a beneficial hydrogen peroxide prodrug is discussed.

The production of reactive oxygen species (ROS) and reactive nitrogen species (RNS) has long been proposed as leading to random deleterious modification of macromolecules with an associated progressive development of age associated systemic disease. ROS and RNS formation has been posited as a major contributor to the aging process. On the contrary, this review presents evidence that superoxide anion (and hydrogen peroxide) and nitric oxide (and peroxynitrite) constitute regulated prooxidant second messenger systems, with specific sub-cellular locales of production and are essential for normal metabolome and physiological function. The role of these second messengers in the regulation of the metabolome is discussed in terms of radical formation as an essential contributor to the physiologically normal regulation of sub-cellular bioenergy systems; proteolysis regulation; transcription activation; enzyme activation; mitochondrial DNA changes; redox regulation of metabolism and cell differentiation; the concept that orally administered small molecule antioxidant therapy is a chimera. The formation of superoxide anion/hydrogen peroxide and nitric oxide do not conditionally lead to random macromolecular damage; under normal physiological conditions their production is actually regulated consistent with their second messenger roles.

Contrary to the dogma that superoxide anion and hydrogen peroxide formation are highly deleterious to cell function and healthy aging, we suggest this premise is flawed. Superoxide anion and hydrogen peroxide formation are essential to normal cellular function; they constitute a second messenger system absolutely required for the regulation of the metabolome. Embraced within this regulation is the modulation of cellular redox poise, bioenergy output, gene expression and cell differentiation. A key component in the overall process is coenzyme Q10 whose prooxidant function through the formation of superoxide anion and hydrogen peroxide is a major factor in the overall processes. The free radical gas, nitric oxide (similarly to superoxide anion), functions in the regulation of a wide range of cell systems. As part of the normal physiological process, superoxide anion and NO function separately and interactively as second messengers. Superoxide anion and nitric oxide play an intrinsic role in the regulated ordered turnover of proteins, rather than randomly cause protein damage and their inactivation. The proposition that metabolic free radical formation is unequivocally deleterious to cell function is rebutted; their toxicity as primary effectors in the aging process has been overemphasized. The concept that a dietary supplement of high concentrations of small-molecule antioxidants is a prophylactic/amelioration therapy for the aging process and age-associated diseases is questioned as to its clinical validity.