Pamela Starke-Reed

Protein oxidation and proteolysis during aging and oxidative stress.

Previous studies in this laboratory have shown that glutamine synthetase (GS) and other key metabolic enzymes are inactivated by metal-catalyzed oxidation reactions in vitro. Oxidative inactivation renders these proteins highly susceptible to proteolysis, especially to a class of newly identified alkaline proteases which exhibit little or no activity against the native enzymes. These studies have suggested that oxidative inactivation may be an important marking step for intracellular protein degradation. Because many of the enzymes which have been shown to accumulate as inactive or less active forms during aging are readily inactivated by metal-catalyzed oxidation reactions in vitro, we have investigated the possible relationship between protein oxidation and proteolysis during aging and oxidative stress in vivo. Oxidized proteins accumulate in hepatocytes of rats exposed to 100% oxygen during the first 48 h of oxygen treatment. In the interval between 48 and 54 h the levels of oxidized proteins decline sharply. The specific activities of at least two liver enzymes, glutamine synthetase and glucose-6-phosphate dehydrogenase (G-6-PDH), decrease during the 54-h experiment. GS and G-6-PDH specific immunological cross-reactivity remains high during the first 48 h of oxygen treatment and then declines in the interval between 48 and 54 h. During this same interval the levels of alkaline proteases which degrade oxidized proteins increase, indicating that these activities are induced or activated in response to oxidative stress and subsequently degrade the proteins which have become oxidized during the initial phase of oxygen treatment. Oxidized proteins accumulate progressively during aging in hepatocytes from rats 3 to 26 months old, with the largest incremental increase between 20 and 26 months. The increase in protein oxidation is correlated with a loss of specific activity of GS and G-6-PDH without a concomitant loss of immunological cross-reactivity. The levels of alkaline proteases which degrade oxidized proteins in hepatocytes from 26-month-old rats is only 20% that of 3-month-old rats, suggesting that oxidized proteins accumulate in hepatocytes from old rats, in part, because the proteases which degrade them are deficient or defective. moreover, when old rats are subjected to treatment with 100% oxygen, the levels of oxidized proteins continue to increase and the alkaline protease activity remains low, indicating that these protease activities are not increased in response to oxidative stress in old rats.

Protein modification in aging.

The age-related accumulation of abnormal forms of enzymes is attributable to posttranslational modification of protein structure and to a progressive loss with age of proteases that preferentially degrade the modified forms. The protein modifications include, but are not limited to: the oxidation of amino acid side chains (especially, side chains of prolyl, arginyl, lysyl and histidinyl residues) by mixed-function oxidation systems; the deamidation of asparaginyl and glutaminyl residues; the racemization and isomerization of aspartyl and asparaginyl residues; the isomerization of prolyl residues; the oxidation of cysteine sulfhydryl groups; and spontaneous changes in protein conformation that are apparently unlinked to changes in amino acid composition. Evidence supporting the roles of these protein modifications and of the proteases that degrade abnormal enzymes during aging is discussed, as well as a consideration of some technical limitations of the methods used in their study.

Dietary Reference Intakes for vitamin D: justification for a review of the 1997 values

Recent Institute of Medicine (IOM) reviews of the process for deriving Dietary Reference Intakes (DRIs) suggest that determining the need for a new nutrient review should be evaluated against criteria set a priori. After selecting the criterion of significant new and relevant research, a working group of US and Canadian government scientists used results from a systematic review and 2 conferences on vitamin D and health to evaluate whether significant new and relevant scientific evidence had become available since the 1997 IOM publication of the DRIs for vitamin D. This working group concluded that there appears to be new research meeting the criteria for 4 key DRI questions. The new research is of larger quantity and quality for the elderly than for other groups, but overall 1) adds to the bone-related and status evidence available to the 1997 DRI Committee for several of the life-stage groups, 2) identifies new outcomes with respect to risk of falls and performance measures in the elderly and potential adverse effects, and 3) provides additional information on dose-response relations between intakes and circulating 25-hydroxyvitamin D concentrations and between 25-hydroxyvitamin D concentrations and several health outcomes (ie, bone-related outcomes for all ages and risk of falls and performance measures in older adults). Members of the working group concluded that significant new and relevant research was available for reviewing the existing DRIs for vitamin D while leaving the decision of whether the new research will result in changes to the current DRIs to a future IOM-convened DRI committee.

Inhibition of postcardiac arrest brain protein oxidation by acetyl-l-carnitine

Free radical mediated, site-specific protein oxidation has been implicated in the pathophysiology of ischemia/reperfusion brain injury. The purpose of this study was to determine whether this form of molecular damage could be detected in a clinically relevant model employing 10-min cardiac arrest in dogs followed by restoration of spontaneous circulation for up to 24 h. The effects of postischemic acetyl-L-carnitine administration on protein oxidation were also tested due to its previously reported improvement of brain energy metabolism and neurological outcome in this model. Following the experimental period, soluble proteins were extracted from a sample of frontal cortex and reacted with dinitrophenylhydrazine for spectrophotometric measurement of protein carbonyl groups. The most important results of this study were that brain protein carbonyl groups were significantly elevated following 2 and 24 h of reperfusion compared to nonischemic controls, and that postischemic IV administration of acetyl-L-carnitine eliminated the increase in carbonyl groups observed at the 24-h period. These results indicate that brain protein oxidation does occur in a clinically relevant model of complete global cerebral ischemia and reperfusion, and that oxidation is inhibited under treatment conditions that improve neurological outcome.

Guidance from an NIH Workshop on Designing, Implementing, and Reporting Clinical Studies of Soy Interventions

The NIH sponsored a scientific workshop, “Soy Protein/Isoflavone Research: Challenges in Designing and Evaluating Intervention Studies,” July 28–29, 2009. The workshop goal was to provide guidance for the next generation of soy protein/isoflavone human research. Session topics included population exposure to soy; the variability of the human response to soy; product composition; methods, tools, and resources available to estimate exposure and protocol adherence; and analytical methods to assess soy in foods and supplements and analytes in biologic fluids and other tissues. The intent of the workshop was to address the quality of soy studies, not the efficacy or safety of soy. Prior NIH workshops and an evidence-based review questioned the quality of data from human soy studies. If clinical studies are pursued, investigators need to ensure that the experimental designs are optimal and the studies properly executed. The workshop participants identified methodological issues that may confound study results and interpretation. Scientifically sound and useful options for dealing with these issues were discussed. The resulting guidance is presented in this document with a brief rationale. The guidance is specific to soy clinical research and does not address nonsoy-related factors that should also be considered in designing and reporting clinical studies. This guidance may be used by investigators, journal editors, study sponsors, and protocol reviewers for a variety of purposes, including designing and implementing trials, reporting results, and interpreting published epidemiological and clinical studies.

Protein oxidation and myelinolysis occur in brain following rapid correction of hyponatremia.

Myelinolysis occurs following rapid correction of hyponatremia in both humans and experimental animals. Although the mechanism of this effect at present is unknown, we have examined the possibility that a rapid rise in serum sodium following hyponatremia potentiates an oxidative stress and results in the oxidation of cellular proteins. In these studies, rats treated with 1 M NaCl following 3 days of vasopressin-induced hyponatremia exhibited myelinolysis in the corpus striatum and thalamus as well as significant increases in soluble oxidized proteins in the brain. These changes did not occur in rats treated with 0.155 M (0.9%) NaCl following 3 days of hyponatremia.

Challenges and New Opportunities for Clinical Nutrition Interventions in the Aged

Nutritional status plays a critical role in the prevention and management of many chronic health conditions that are common in the elderly and are likely to become more prevalent as the population ages. This paper highlights several aspects of nutrition that require additional basic science and clinical application research to improve the health and well-being of older adults. Topics addressed are selected demographic and health indices, the uncertain benefits of energy restriction in aged humans compared with other species, the impact of food insecurity on health, the relationship between dietary protein and sarcopenia, the prevention and management of obesity while maintaining muscle mass and functional status, and controversy regarding high intakes of folic acid. Research needs regarding the safety, efficacy, and application of clinical interventions related to these topics also are discussed.

Oxidative Modification of Enzymes During Aging and Acute Oxidative Stress

Previous studies have demonstrated that many cellular enzymes accumulate as catalytically inactive or less active forms during normal aging.1,2 The mechanism(s) for these changes has (have) not been determined. However, because many of the enzymes which exhibit loss of enzymatic activity during aging are also inactivated by mixed-function oxidation (MFO) systems in vitro,3,4 we have considered the possibility that these changes may be due to oxidative inactivation by MFO systems in vivo. In the studies reported here we have examined the levels of oxidatively modified proteins in hepatocytes isolated from young and old rats and from young rats subjected to treatment with 100% oxygen.

Diet, Microbiome, and Epigenetics in the Era of Precision Medicine

Precision medicine is a revolutionary approach to disease prevention and treatment that takes into account individual differences in lifestyle, environment, and biology. The US National Institutes of Health has recently launched The All of Us Research Program (2016) to extend precision medicine to all diseases by building a national research cohort of one million or more US participants. This review is limited to how the human microbiome factors into precision medicine from the applied aspect of preventing and managing cancer. The Precision Medicine Initiative was established in an effort to address particular characteristics of each person with the aim to increase the effectiveness of medical interventions in terms of prevention and treatment of multiple diseases including cancer. Many factors contribute to the response to an intervention. The microbiome and microbially produced metabolites are capable of epigenetic modulation of gene activity, and can influence the response through these mechanisms. The fact that diet has an impact on microbiome implies that it will also affect the epigenetic mechanisms involving microbiota. In this chapter, we review some major epigenetic mechanisms, notably DNA methylation, chromatin remodeling and histone modification, and noncoding RNA, implicated in cancer prevention and treatment. Several examples of how microbially produced metabolites from food influence cancer risk and treatment response through epigenetic mechanisms will be discussed. Some challenges include the limited understanding of how diet shapes the microbiome and how to best evaluate those changes since both, diet and the microbiota, exhibit daily and seasonal variations. Ongoing research seeks to understand the relationship between the human microbiome and multiple diseases including cancer.

Optimization of ESI-Source Parameters for Lipidomics Reduces Misannotation of In-Source Fragments as Precursor Ions

Lipidomics requires the accurate annotation of lipids in complex samples to enable determination of their biological relevance. We demonstrate that unintentional in-source fragmentation (ISF, common in lipidomics) generates ions that have identical masses to other lipids. Lysophosphatidylcholines (LPC), for example, generate in-source fragments with the same mass as free fatty acids and lysophosphatidylethanolamines (LPE). The misannotation of in-source fragments as true lipids is particularly insidious in complex matrixes since most masses are initially unannotated and comprehensive lipid standards are unavailable. Indeed, we show such LPE/LPC misannotations are incorporated in the data submitted to the National Institute of Standards and Technology (NIST) interlaboratory comparison exercise. Computer simulations exhaustively identified potential misannotations. The selection of in-source fragments of highly abundant lipids as features, instead of the correct recognition of trace lipids, can potentially lead to (i) missing the biologically relevant lipids (i.e., a false negative) and/or (ii) incorrect assignation of a phenotype to an incorrect lipid (i.e., false positive). When ISF is not eliminated in the negative ion mode, ∼40% of the 100 most abundant masses corresponding to unique phospholipids measured in plasma were artifacts from ISF. We show that chromatographic separation and ion intensity considerations assist in distinguishing precursor ions from in-source fragments, suggesting ISF may be especially problematic when complex samples are analyzed via shotgun lipidomics. We also conduct a systematic evaluation of electrospray ionization (ESI) source parameters on an Exactive equipped with a heated electrospray ionization (HESI-II) source with the objective of obtaining uniformly appropriate source conditions for a wide range of lipids, while, at the same time, reducing in-source fragmentation.