Paul McGlothin

Welcome, Nashua! I have been thinking about your post and how best to answer it. I think the research suggests that in most cases a well thought out calorie restriction regimen provides protection against Alzheimer's disease. I am sure you have seen encouraging studies like this: Calorie restriction attenuates Alzheimer's disease type brain amyloidosis in Squirrel monkeys (Saimiri sciureus). J Alzheimers Dis. 2006 Dec;10(4):417-22. Qin W, Chachich M, Lane M, Roth G, Bryant M, de Cabo R, Ottinger MA, Mattison J, Ingram D, Gandy S, Pasinetti GM. Recent studies from our laboratories and others suggest that calorie restriction (CR) may benefit Alzheimer's disease (AD) by preventing amyloid-beta (Abeta) neuropathology in the mouse models of AD. Moreover, we found that promotion of the NAD+-dependent SIRT1 mediated deacetylase activity, a key regulator in CR extension of life span, may be a mechanism by which CR influences AD-type neuropathology. In this study we continued to explore the role of CR in AD-type brain amyloidosis in Squirrel monkeys (Saimiri sciureus). Monkeys were maintained on the normal and CR diets throughout the entire lifespan until they died of natural causes. We found that 30% CR resulted in reduced contents of Abeta1-40 and Abeta1-42 peptides in the temporal cortex of Squirrel monkeys, relative to control (CON) fed monkeys. The decreased contents of cortical Abeta peptide inversely correlated with SIRT1 protein concentrations in the same brain region; no detectable change in total full-length amyloid-beta protein precursor (AbetaPP) level was found. Most interestingly, we found that 30% CR resulted in a select elevation of alpha- but not beta- or gamma- secretase activity which coincided with decreased ROCK1 protein content in the same brain region, relative to CON group. Collectively, the study suggests that investigation of the role of CR in non-human primates may provide a valuable approach for further clarifying the role of CR in AD. PMID:17183154 It would be possible to fill a page with studies that show CR protects against Alzheimer's disease. The problem is that studies are lacking for homozygous E4. That will change. As I write this, I am working on the launch of cognitive workshops that, besides improving cognition overall, will raise funds for a long-term study to look at ways to reduce Alzheimer's risk. The study will be for human participants. It would be disingenuous for me to promise that any diet/meal plan will definitely protect you from Alzheimer's. On the other hand, following a regimen that can produce measurable improvement in cognitive performance, as well as extraordinary results at your annual physical, is a step in the right direction. I speak of the Daily Intermittent fasting plan that follow and write about here: Daily Intermittent Fasting. Let me hasten to add that this is only one approach. There are many ways to practice CR. Your job is to find the way that's right for you. You mention that you are ready to make some drastic changes. Hold off approaches that are drastic, extreme, or severe. Take it slow and easy. CR is not about losing as much weight as you can stand. It's about activating longevity signaling. That is a joyous, energy-giving approach that should make every aspect of your life better. Have fun with it and I'll bet that soon you will look forward to every CR day and meal and you'll wonder how you could have ever lived any other way. Regarding Alzheimer's Disease, take heart! Help is on the way. My guess is that within five years, definite ways to measure diet and lifestyle changes and how they protect against Alzheimer's will be in place. You may not have to worry about Alzheimer's and by adopting CR lifestyle now that you can naturally follow, you will likely have a longer disease-free life to enjoy. Wishing you great success and much happiness with it, Paul

Thank you, Michael! I appreciate your response on multiple levels. Any good discussion on oxalates needs to include "basics" of oxalate management and you have provided some excellent information that everyone should keep in mind. I also appreciate your response here, in this forum. As you know, I believe that posts here have communication advantages, not the least of which is that the subject stays visible, so people can respond to it when time permits. It also allows us to add to the posts over time, as new information becomes available. Some CR folk consume almost 2000 milligrams of oxalate on certain days, enough if done consistently, to be of serious concern. When I was at that level, I noticed oxalate accumulation in my kidneys, which would have been a disaster if I had not monitored my kidney health with my superb physicians Drs. Rosen and Bromberg who caught it and pointed me in the direction I needed to correct the problem. Many people will not be so lucky. Especially at risk are those who have a lot of internal oxalate production because of pathogenic bacteria and/or an inability to degrade oxalate because of impaired gut microflora. I hope to post more about this and not leave this response without references, but the latest and exciting developments in CR research are calling, so I must go for now. Paul

Ann thoughtfully provided this study and questioned the dangers of oxalate consumption in the diet. This is the study she provided: Effects of 5 different diets on urinary risk factors for calcium oxalate kidney stone formation: evidence of different renal handling mechanisms in different race groups. J Urol. 2002 Sep;168(3):931-6. Rodgers AL, Lewandowski S. Source Department of Chemistry, University of Cape Town, South Africa. Abstract PURPOSE: Since the incidence of renal calculi in the South African black population is extremely rare while in white subjects it occurs at the same rate as elsewhere in the western world, we investigated the possibility that different renal handling mechanisms in response to different dietary challenges might occur in the 2 race groups. MATERIALS AND METHODS: We administered 5 different dietary protocols, including low calcium, high oxalate, vitamin C, high salt and lacto-vegetarian, to 10 healthy male subjects from each race group. We collected 24-hour urine at baseline and after 4 days on the prescribed diet which were analyzed for biochemical and physicochemical risk factors. Dietary intake was controlled throughout the experimental period. A 24-hour dietary recall questionnaire was recorded at baseline and analyzed using food composition tables. Statistical analysis of variance was performed on all the data. RESULTS: The low calcium diet caused statistically significant changes only in black subjects, which consisted of urinary oxalate increase (0.17 to 0.23 mmol./24 hours, p = 0.01), relative supersaturation of calcium oxalate decrease (1.88 to 0.97, p = 0.03) and relative supersaturation of brushite increase (0.85 to 1.69, p = 0.03). The high oxalate diet caused statistically significant changes in both race groups but these changes were different in the 2 groups. In white subjects urinary pH increased (6.24 to 6.62, p = 0.01), potassium excretion increased (40.01 to 73.49, p = 0.01) and relative supersaturation of brushite increased (1.34 to 2.12, p = 0.05). In black subjects urinary citrate increased (1.94 to 2.99 mmol./24 hours, p = 0.01). Clinically unimportant changes occurred in both race groups after the other 3 diets. CONCLUSIONS: Renal handling of dietary calcium and oxalate in South African black and white subjects is different and may explain the different stone incidence in the 2 race groups Thank you for posting this interesting study, Ann. It compares different responses of blacks and whites to a high oxalate diet. The conclusion is that the high oxalate diet produced statistically significant changes in both groups. Do you have access to the full paper? I would like to see what the researchers considered to be a high oxalate diet. It is too bad they used 24-hour dietary recall for their study. This method is fraught with inaccuracy. Even if they had used more accurate methods of weighing dietary intake and entering into software at the time of eating, oxalate intake would still be a guessing game. AFAIK, at the time the study was published, no accurate oxalate database was available. Further the study was conducted over four days. It may take months or years for stones to form in the kidneys. Ann asked: Also, do you know if this particular patient was someone with underlying kidney issues, or some other condition? It was a dietary condition: He is not unlike scores of patients my doctors see, who begin to follow diets that are high in oxalates and then develop stones. We certainly don’t know all the reasons why some people develop oxalate deposits and others don’t. Many factors may singly or in combination contribute to oxalate deposits in tissues. I am glad I now have an accurate means of measuring what some of those dietary risk factors are. Paul

In response to my encouragement of caution when consuming high oxalate foods, Ann, one of the Society's facebook members, asked "What do Oxalates do?" I wish someone somewhere could definitively answer that question. Some who follow low oxalate diets would say that oxalates are a “junk” molecule, contained in many foods: plants, nut, beans, tea, chocolate, etc. – very deleterious to health. In fact, one group I belong to advocates a very low oxalate diet, but which is full of questionable high GI, high fat, and high protein foods – not a solution for CR folk. Another view is that oxalates play an important role in binding with calcium and removing it from the body. It would be great if that were an efficient, well understood process. However, calcium oxalate can accumulate in kidneys to form stones as well as in other tissues. The reasons why are yet to be fully understood. Recently a kidney expert on a livingthecrway teleconference told us about a patient who decided to eat handfuls of high oxalate nuts all day long. It caused oxalate deposits throughout his kidneys, as well as on the outside of the kidneys and on other organs— a death sentence! Further, kidney stones are an emerging problem -- rapidly increasing in many parts of the world, especially among young women. A problem among calorie restrictors is that many of our food selections are high in oxalates – sweet potatoes, walnuts , etc. Not only might that make oxalate deposits in tissues more likely, it might make CR folk more susceptible to pathogens.Take a look at this related study, for example: Oxalate toxicity in renal cells. > > Jonassen JA, Kohjimoto Y, Scheid CR, Schmidt M. > > Source > > Department of Physiology, University of Massachusetts Medical School, 55 Lake Avenue North, Worcester, MA 01655, USA. Julie.Jonassen@ Urol Res. 2005 Nov;33(5):329-39. Epub 2005 Nov 13. > > > > Abstract > > Exposure to oxalate, a constituent of the most common form of kidney stones, generates toxic responses in renal epithelial cells, including altered membrane surface properties and cellular lipids, changes in gene expression, disruption of mitochondrial function, formation of reactive oxygen species and decreased cell viability. Oxalate exposure activates phospholipase A2 (PLA2), which increases two lipid signaling molecules, arachidonic acid and lysophosphatidylcholine (Lyso-PC). PLA2 inhibition blocks, whereas exogenous Lyso-PC or arachidonic acid reproduce many of the effects of oxalate on mitochondrial function, gene expression and cell viability, suggesting that PLA2 activation plays a role in mediating oxalate toxicity. Oxalate exposure also elicits potentially adaptive or protective changes that increase expression of proteins that may prevent crystal formation or attachment. Additional adaptive responses may facilitate removal and replacement of dead or damaged cells. The presence of different inflammatory cells and molecules in the kidneys of rats with hyperoxaluria and in stone patients suggests that inflammatory responses play roles in stone disease. Renal epithelial cells can synthesize a variety of cytokines, chemoattractants and other molecules with the potential to interface with inflammatory cells; moreover, oxalate exposure increases the synthesis of these molecules. The present studies demonstrate that oxalate exposure upregulates cyclooxygenase-2, which catalyzes the rate-limiting step in the synthesis of prostanoids, compounds derived from arachidonic acid that can modify crystal binding and may also influence inflammation. In addition, renal cell oxalate exposure promotes rapid degradation of IkappaBalpha, an endogenous inhibitor of the NF-kappaB transcription factor. A similar response is observed following renal cell exposure to lipopolysaccharide (LPS), a bacterial cell wall component that activates toll-like receptor 4 (TLR4). While TLRs are primarily associated with immune cells, they are also found on many other cell types, including renal epithelial cells, suggesting that TLR signaling could directly impact renal function. Prior exposure of renal epithelial cells to oxalate in vitro produces endotoxin tolerance, i.e. a loss of responsiveness to LPS and conversely, prior exposure to LPS elicits a similar heterologous desensitization to oxalate. Renal cell desensitization to oxalate stimulation may have profound effects on the outcome of renal stone disease by impairing protective responses. PMID: 16284883 I So enough was enough. We consulted with several experts in the field and devised a new CR Way approach that eliminates excess oxalate and some related risk factors for oxalate accumulation. I have written about it here: http://calorierestri...e-your-kidneys/ http://blog.livingth...organ-function/ http://www.crsociety...ended-software/ There is much more to say about oxalates. I am working on the oxalate issue with professionals in the kidney field and will post more about it over the next year. Wishing you a healthful and not overly high in oxalate CR diet. Paul

You are welcome, Golden. Be careful about CR that is "Severe." To most people that means extreme -- a lot of weight loss. I approach it very differently. I try to activate known Longevity pathways that are responsible for CR benefits. That means keeping insulin/IGF-1 on the low side , for example. Resveratrol may be helpful, but I have never been able to detect measurable benefits from it. Paul

Wonderful, tantalizing , and provocative response, Brian. We need to do a human CR study that looks at a whole lot of us and that begins by analyzing our heritable genome. I am going to to explore this. Paul

Thanks to you and Brian for this extremely important discussion. When I was at Harvard Medical School a few years ago, I heard an impressive presentation by Lenny Guarente on CR, SIRT1 and Alzheimer's Disease. The abstract is now freely available: SIRT1 suppresses B-amyloid production. This tells me that generally CR in most people will likely be protective against Alzheimer's disease. Here's a link to 2 pages of studies that came up when I searched on calorie restriction and Alzheimer;s Disease: http://www.ncbi.nlm.nih.gov/pubmed?term=calorie%20restriction%20alzheimer%27s As you know the brain is one of the most energy sensitive organs. Finding out more about the brain's beneficial and neuroprotective adaptations in a low energy environment, motivated me to adopt a low glucose regimen in addition to CR. Here is a Society archive link that talks about it, when I first started such a regimen: http://arc.crsociety.org/read.php?2,166951,166951#msg-166951 It would seem to me that adopting a low glucose regimen would be beneficial. You have said that certain interventions like exercise are not protective if one has two copies of ApoE4 . Can you explain any more about how that in addition to increased risk for Alzheimer's that ApoE4 and two copies of ApoE4 are manifested clinically. What can you test at your doctor's office that indicates that two copies of ApoE are at work? I began to search for answers and found studies like this: http://www.molecularneurodegeneration.com/content/7/S1/L10. Although it provides hints, it is hardly an adequate answer to my question. I would think that establishing markers that can be easily tested would be a great help to you. I am delighted that you and Brian started this discussion here because the forums lend themselves to ongoing dialogue and this subject certainly merits it. Hoping to help, Paul

On a recent CR Way teleconference, Dr. Stephen Spindler discussed his milestone paper: Review of the literature and suggestions for the design of rodent survival studies for the identification of compounds that increase health and life span. Doctor Spindler suggests exacting standards for how longevity studies should conducted in mice. Hopefully diligent researchers who seek outcomes that are truly accurate will integrate Dr. Spindler’s suggestions into their studies. AGE: The Official Journal of the American Aging Association © The Author(s) 2011 Stephen Richard Spindler1 Department of Biochemistry, University of California, Riverside, CA 92521, USA Received: 2 September 2010 Accepted: 21 February 2011 Published online: 22 March 2011 Abstract Much of the literature describing the search for agents that increase the life span of rodents was found to suffer from confounds. One-hundred-six studies, absent 20 contradictory melatonin studies, of compounds or combinations of compounds were reviewed. Only six studies reported both life span extension and food consumption data, thereby excluding the potential effects of caloric restriction. Six other studies reported life span extension without a change in body weight. However, weight can be an unreliable surrogate measure of caloric consumption. Twenty studies reported that food consumption or weight was unchanged, but it was unclear whether these data were anecdotal or systematic. Twenty-nine reported extended life span likely due to induced caloric restriction. Thirty-six studies reported no effect on life span, and three a decrease. The remaining studies suffer from more serious confounds. Though still widely cited, studies showing life span extension using short-lived or “enfeebled” rodents have not been shown to predict longevity effects in long-lived animals. We suggest improvements in experimental design that will enhance the reliability of the rodent life span literature. First, animals should receive measured quantities of food and its consumption monitored, preferably daily, and reported. Weights should be measured regularly and reported. Second, a genetically heterogeneous, long-lived rodent should be utilized. Third, chemically defined diets should be used. Fourth, a positive control (e.g., a calorically restricted group) is highly desirable. Fifth, drug dosages should be chosen based on surrogate endpoints or accepted cross-species scaling factors. These procedures should improve the reliability of the scientific literature and accelerate the identification of longevity and health span-enhancing agents. Keywords Longevity therapeutics – CR mimetics – Geroprotectors – Health span – Life span – Longevity – Drug discovery – Pharmaceutical testing Introduction There are presently no authentic longevity therapeutics. Such compounds would intervene in the process of aging to extend mean and/or maximum life span, maintain physiological function, and mitigate the onset and severity of a broad spectrum of age-related diseases in mammals. Such drugs might engage the pathways used by caloric, methionine, and phenylalanine restriction, and the longevity-enhancing mutations (reviewed in Spindler 2009). The terms “CR mimetics” and “geroprotectors” have been used to describe such compounds (Weindruch et al. 2001; Roth et al. 2001; Cao et al. 2001; Anisimov 1982; Lippman 1981). In this report, we will use the general term “longevity therapeutics.” While a full understanding of the mechanisms of aging will greatly facilitate the development and deployment of longevity therapeutics, drug discovery and development have a long history of using surrogate assays for identifying therapeutics, often with little knowledge or understanding of the etiology of the diseases for which the therapeutics were intended (discussed in Spindler 2006). Indeed, most of the medications currently in our armamentarium were discovered using surrogate assays. Thus, the development and refinement of surrogate assays for longevity therapeutics should speed their identification. There have been multiple methods used in the attempt to identify such compounds. For example, we and others have utilized genome-wide microarray studies of treated mice to identify potential therapeutics (Barger et al. 2008; Spindler and Dhahbi 2007; Spindler and Mote 2007; Spindler 2006; Dhahbi et al. 2005; Corton et al. 2004). Another approach, which will be discussed here, is the direct assays of compounds for their effects on the life span of rodents. Longevity assays using genetically normal, healthy rodents In mice, a number of natural mutations, gene knockouts, and overexpressed transgenes are known to extend longevity and increase health span (Selman et al. 2008; Taguchi et al. 2007; Kurosu et al. 2005; Holzenberger et al. 2003; Flurkey et al. 2001; Coschigano et al. 2000; Zhou et al. 1997; Brown-Borg et al. 1996). Thus, potential therapeutic targets for life span extension exist in mammals. However, no robustly effective, safe, and widely recognized longevity therapeutics exist at present. The likely reason that such drugs have not been identified is that we have not mounted an effective search for them. Life span studies in rodents have been used in this search (Table 1 and Electronic supplementary material (ESM) Table 1). More recently, this literature benefits from the improved levels of hygiene used in animal husbandry (e.g., see Sebesteny 1991). For example, several older studies in Table 1 appear to report data consistent with the presence of infectious agents in the rodent colony (Ferder et al. 1993; LaBella and Vivian 1978; Sperling et al. 1978). Despite these improvements, the design and implementation of rodent life span studies could be improved further. Table 1 Summary appraisal of the published life span studies using healthy rodents 106 separate life span studies where compounds were administered to normala rodents (less 20 contradictory melatonin studies)b 6 studies found life span extension and showed food consumption was not responsible by measuring it: Deprenyl administered orally to female hamsters Deprenyl and Dinh lang root extract administered to mice Dinitrophenol administered to a short-lived, normal mouse strain l-dopa administered orally to male mice Marine collagen peptides extended the mean life span of Sprague–Dawley rats Reduced advanced glycation end products present in standard rodent diet 6 studies found life span extension and reported no change in weight, with data shown or details given (this list excludes studies which showed no change in food consumption listed above): Coenzyme Q10 administered orally to male Wistar rats a diet high in polyunsaturated fatty acids Ginkgo biloba administered orally to F344 rats Green tea polyphenols administered orally to mice 2-Mercaptoethanol administered orally to mice PBN administered orally to mice Piperoxane administered by injection to rats 20 studies report LS extension but potential CR effects cannot be excluded Body weight and/or food consumption called “unchanged”, but no data given or data given but not analyzed statistically (e.g., it remains unclear whether the data are anecdotal or systematic, when and how many times during the study measurements were taken, the means and standard deviations of the measurements, and what statistical methods were used to analyze the data?) 29 studies report results that are likely due to induced “voluntary” CR Body weights or food consumption were less than those of controls or neither was reported 36 studies report no effect on life span 3 studies report reduced LS 9 studies would be difficult to repeat or have methodological or reporting confounds that render their data of uncertain significance Only English language publications were reviewed b Normal in this context means the animals had no known genetic defect leading to an artificially decreased life span and were not given a physical or chemical treatment to stress the animals and shorten their life span bIf a publication reports the testing of a compound or compounds using more than one group of animals, each test was listed and counted separately. If a compound was tested in more than one publication, these studies are counted separately. If a compound had differential effects on the lifespan of mice of different strains in a single report, these effects were counted under multiple categories. Table 1 and ESM Table 1 summarize and evaluate all of the rodent life span studies we found using repeated key word searches of the online databases. In ESM Table 1, under the heading “Evaluation,” we present our evaluation of the study results. ESM Table 1 presents 106 life span studies performed with healthy rodents. We excluded from this table 20 rodent life span studies performed with melatonin, which are contradictory in their results and which have been reviewed elsewhere (Anisimov et al. 2006). Despite the fact that the effects of caloric restriction on life span were described 76 years ago (McCay et al. 1935), drug screening studies which regulate or measure food consumption are rare. We found only six studies which measured food consumption and also found life span extension (Liang et al. 2010; Caldeira da Silva et al. 2008; Cai et al. 2007; Stoll et al. 1997; Yen and Knoll 1992; Cotzias et al. 1977). These were deprenyl fed to Syrian hamsters (Stoll et al. 1997); deprenyl and Dinh lang root extract fed to mice (Yen and Knoll 1992); dinitrophenol fed to normal mice of a short-lived strain (Caldeira da Silva et al. 2008); l-dopa fed to male mice (Cotzias et al. 1977); marine collagen peptides fed to Sprague–Dawley rats (Liang et al. 2010); and reduced advanced glycation end product-containing standard mouse diet fed to mice (Cai et al. 2007). These are the only studies in the literature showing an increase in rodent longevity for which the potential effects of “voluntary” CR on life span can be confidently excluded. Four studies which controlled or measured caloric intake found no change in life span with various treatments (Smith et al. 2010; Spindler and Mote 2007; Lee et al. 2004; Pugh et al. 1999b). Six other studies found life span extension and reported the effects of the treatments on body weight as a surrogate measure of food consumption (Table 1 and ESM Table 1). However, there are demonstrated instances in which a discordance was found between body weight and food consumption, making body weight a potentially unreliable surrogate measure of caloric consumption (see below). These treatments are: coenzyme Q10 administered orally to male Wistar rats fed a diet high in polyunsaturated fatty acids (Quiles et al. 2004); Ginkgo biloba extract administered orally to male F344 rats (Winter 1998); green tea polyphenols administered in drinking water to male C57BL/6 (B6) mice (Kitani et al. 2007); 2-mercaptoethanol administered orally in food to male BC3F1 mice (Heidrick et al. 1984); PBN fed to B6 male mice (Saito et al. 1998); piperoxane administered by injection to F344 rats (Compton et al. 1995). Twenty studies found extended life span, but potential CR effects cannot be excluded based on the data available (Table 1 and ESM Table 1). Many of these reports include statements to the effect that no change in body weight (most common) or food intake (rarely) occurred, but no data or analysis are shown. No indication is given of whether the data were anecdotal or systematic, when and how many times during the study the measurements were taken, or what statistical methods were used to analyze the data. These uncertainties, coupled with the potential fallibility of weight as a biomarker for food consumption (see below), make these studies less persuasive. Twenty-nine other studies report life span extension by treatments, but the body weight and/or food consumption data presented in the publication suggest that induced voluntary CR was responsible for the longevity effects observed. Of the remaining studies, nine would be difficult to repeat because the composition, preparation, or mode of delivery of the treatment agents are published in difficult to obtain journals or are not reported. Food consumption should be measured Body weight is often used in longevity studies as a surrogate measure of caloric consumption (Table <a href="http://www.springerlink.com/content/g327u8582pm05q62/fulltext.html#Tab1">1 and ESM Table 1). The vast majority of the studies using body weight as a surrogate do not report the methods used or the results obtained (ESM Table 1). Thus, the reader cannot know whether the conclusions drawn used systematic or anecdotal measures. The number of animals weighed, the number of times they were weighed, and the statistics used are not reported. Such problems are evident in two reports from the NIA Interventions Testing Program (NIH-ITP; Harrison et al. 2009; Strong et al. 2008; Miller et al. 2007). While the studies are unusually robust in many aspects of experimental design, including large cohorts of genetically heterogeneous mice of both sexes tested at multiple sites, most of their reports give no details regarding body weight measurements (Harrison et al. 2009; Strong et al. 2008; Miller et al. 2007). Thus, NIH-ITP investigators have reported that the same concentration of rapamycin fed to HET3 mice produced either no effect on body weight (methods and data unspecified; Harrison et al. 2009) or a 6% or 10% decrease in body weight (for females and males, respectively; Miller et al. 2011). Thus, it is possible that the mice in the first study experienced an undetected reduction in body weight. It also is unclear whether the reductions in body weight found in the second study were due to reduced caloric intake. Thus, “voluntary CR” may have played a role in the longevity effects observed. While one may seek further information from these investigators at this time, our publications are likely to outlive us. Body weight is an unreliable surrogate measure of caloric intake. Both dietary l-dopa and dietary dinitrophenol reduce body weight without changing food consumption (Caldeira da Silva et al. 2008; Cotzias et al. 1977). A drug-induced discordance between body weight and food intake may not be uncommon. We found five agents or combination of agents that significantly decreased body weight and four agents or combination of agents which significantly increased the body weight of mice fed isocalorically (unpublished results). For example, mice fed food supplemented with four doses of nordihydroguaiaretic acid (NDGA) experienced an approximately dose-responsive decrease in body weight without a corresponding decrease in food consumption (Fig. 1). Food was packed in 1-g pellets and fed daily. Food intake for each of these groups was carefully monitored and recorded. Any uneaten food, even when masticated and dropped into the bedding, was readily identifiable by shape, color, and texture. Quantitatively, the group fed the highest dose of NDGA weighed the same or less than a 20% calorie-restricted (20% CR) group at most times during the study (Fig. 1). Others have reported, without showing data, that mice consuming NDGA-supplemented diets ad libitum have no change in body weight relative to controls (Strong et al. 2008). Thus, it is possible that the mice in this published study maintained their body weight by increasing food consumption. Feeding measured quantities of food and monitoring of its consumption ensures that life span data are not confounded by changes in caloric consumption. This reduces the likelihood of CR-related changes in life span (Merry 2002; Compton et al. 1995). Monitoring of both food consumption and body weight will identify instances in which a compound produces a discordance between them. Drug-induced changes in activity, metabolic rate, or intestinal absorption of calories might lead to such a discordance, which would not be detected by monitoring of only body weight. Once detected, a discordance can be investigated further using measurements of spontaneous activity, metabolic rate, and absorption of calories (e.g., Westbrook et al. 2009; Adams et al. 2006). Thus, measured feeding coupled with body weight monitoring is a much more robust approach to life span studies than body weight monitoring alone. Methods for isocaloric feeding In the author’s experience, measuring food consumption is less difficult and expensive than it is sometimes assumed to be. In an ongoing longevity study involving 2,400 mice, measured feeding is ~9% of total costs. To deliver a known amount of food to each cage conveniently, we use the method described by Weindruch and colleagues (Pugh et al. 1999a). The food (AIN-93M) and any additional components are cold-packed into 1-g pellets by Bio-Serv (Frenchtown, NJ). These round pellets are conveniently scooped into a 1.6-cm inner diameter Plexiglas tube fitted with a commercially available plastic cap. Tubes cut to different lengths are used to deliver different numbers of pellets to the cages. If a supplemented diet is under-consumed, flavoring can be added, the supplement can be changed to an agent with similar actions, the supplement concentration in the food can be reduced, or, if desired, the amount of food given to a control group can be decreased to that of the test agent. We slightly underfeed all the mice in our studies to insure that all food is eaten. Healthy, long-lived rodents, such as an F1 hybrid or a more genetically heterogeneous mouse should be used for compound screening During our survey of the literature, we found many reports of life span-based compound screening performed with short-lived or enfeebled rodents (data not shown). By “enfeebled,” we mean natural or sel-ected rodent lines that have genetic (or possibly epigenetic) changes that reduce longevity and health relative to their unaltered parental or control strains. For example, many studies utilized senescence-accelerated prone mouse strains (SAMP1 through SAMP9) to rapidly screen for longevity therapeutics (Rodriguez et al. 2008; Li et al. 2007; Umezawa et al. 2000; Boldyrev et al. 1999; Kumari et al. 1997; Edamatsu et al. 1995; Zhang et al. 1994). SAMP mice suffer from the early onset of a spectrum of age-related pathologies which abbreviate their life span. We found only one study in which the effectiveness of an agent was tested in both an SAMP mouse (SAMP8) and in one of its associated control mouse strains (SAMPR1; Zhang et al. 1994). In this study, a botanical which extend the life span of SAMP8 mice did not extend the life span of the control strain. Similarly, resveratrol was reported to extend the life span of high fat-fed, obese, and diabetic mice (Baur et al. 2006). While this article has been cited by many as evidence that resveratrol can extend mammalian life span, the results did not translate to healthy mice (Pearson et al. 2008). Thus, screening agents in enfeebled rodents has not yet been shown to facilitate the identification of compounds which extend the life span of healthy animals. For these reasons, studies designed to identify longevity therapeutics should utilize long-lived mice, such as an F1 hybrid or more genetically heterogeneous mouse. F1 hybrid mice, which are widely available, are genetically heterozygous at all loci for which their parents are heteroallelic. They are more disease- and stress-resistant and have larger litters and longer life spans than their inbred parental lines (Flurkey et al. 2009). HET3 mice, which are produced using a four-way crossbreeding scheme, are more genetically heterogeneous than F1 mice and are used by the NIH-ITP. However, they are more difficult to produce and have shorter life spans than some F1 mice. For example, B6C3F1 mice have a mean life span of about 915 days (Spindler and Mote 2007; Pugh et al. 1999b; Smith and Walford 1977), while HET3 mice have a mean life span of about 800 days (Strong et al. 2008). Longer life spans are usually regarded as signs of greater vigor. Outbred mice, which are even more genetically heterogeneous than HET3 mice, are more vigorous and less expensive than inbred mice (Flurkey et al. 2009). However, they have the disadvantage of being genetically undefined. Because each mouse is genetically unique, study results can be more varied and thus more difficult to reproduce. Chemically defined diets should be used for gerontological research There are three general categories of rodent diets: cereal-based (non-purified), purified, and chemically defined (Kozul et al. 2008; Reeves et al. 1993; American Institute of Nutrition ad hoc Committee on Standards for Nutritional Studies 1977). The majority of the studies summarized in ESM Table 1 appeared to have used non-purified or purified cereal-based diets. However, cereal-based diets are often variable in composition (American Institute of Nutrition ad hoc Committee on Standards for Nutritional Studies 1977), and this variability, and the presence of trace contaminants, can strongly influence experimental results (Kozul et al. 2008; Jensen and Ritskes-Hoitinga 2007; Allred et al. 2004; Thigpen et al. 2004, 2003). For example, Prolab-RMH 1000 rodent chow contains appreciable quantities of polychlorinated dibenzo-p-dioxins and dibenzofurans, probably from pesticide residues (Schecter et al. 1996). Purina Laboratory Rodent diet 5001 (LRD-5001) contains high concentrations of methylmercury and a mixture of inorganic and organic arsenic compounds at a concentration 36 times the EPA-recommended level for drinking water (Kozul et al. 2008; Weiss et al. 2005). The specifications for diets such as NIH-31 allow manufacturers to use any of a number of sources of protein, including fish meal, a possible source of arsenic and other contaminants, or soy, a possible source of pesticide residue. Thus, purified, defined diets are preferable. Use of a positive control is highly desirable Many life span studies are published without the benefit of a positive control group, such as a 40% CR group. If none of the compounds tested in a study extend life span, the possibility cannot be excluded that the rodents would not respond to a longevity treatment under the study conditions. Few reviewers would endorse the publication of negative biochemical data without the inclusion of a positive control to show that the assay was working. This should be similarly important for rodent life span studies. Dosages of agents tested in rodents The dosages at which potential therapeutics are tested in rodents must balance a number of competing theoretical and practical issues. Ideally, one would like to know that a therapeutic level of the agent is maintained throughout a life span study. Of course, the ideal therapeutic level of an agent is not known for most life span studies. Furthermore, food intake, body volume, intestinal absorption, and metabolism may change with age. Monitoring the blood levels of an agent throughout a life span study would be difficult and expensive. Group sizes which would make rodents available for testing throughout the study are often impractical. Several approaches can mitigate these limitations. Published studies with well-defined treatment endpoints can be used to estimate dosages. In this way, one can be reasonably certain that a therapeutic level of the agent is achieved. Initial signs that a dosage is too high, such as reduced food intake or inattention to grooming, can be used to adjust dosages “on the fly.” Where rodent studies cannot be found, equivalent rodent dosages can be calculated from human dosages using default cross-species scaling factors (Reagan-Shaw et al. 2008; US EPA 2005; Rhomberg and Lewandowski 2004; Dourson et al. 1992, 1996; Dourson and Stara 1983). These scaling factors are often used to set and access drug dosages in human and animal studies (e.g., Chalastanis et al. 2010). Empirically, small animals have been found to require larger dosages per gram body weight than larger animals. These differences are due to pharmacokinetic differences (e.g., rates of uptake, metabolism, and clearance of compounds) and to pharmacodynamic differences (e.g., rates of damage to macromolecules, cellular repair and regeneration, signaling cascades, and proliferative responses) between small and large animals. One widely used scaling formula increases the human dosage in milligrams per kilogram body weight/day by tenfold to obtain the equivalent mouse dosage. Another scaling factor also in use is based on the 3/4 power of body weight [i.e., (milligrams/kilogram body weight)3/4/day], which leads to equivalent mouse dosages that are about sevenfold higher than the equivalent human dosages. While these calculations were initially developed for chemotherapeutics, they are also used as starting points when human dosages must be extrapolated from preclinical rodent data (Chalastanis et al. 2010). Summary: the preferred design for testing potential longevity therapeutics using mouse life span studies Based on the information reviewed above, we recommend a number of design parameters essential or highly desirable for rodent life span assays: (1) The diets should be fed in measured amounts and consumption monitored. Body weight should be monitored regularly. These measurements and their statistical analysis should be reported. (2) A long-lived, healthy rodent strain should be used, preferably an F1 or further outcrossed strain. (3) Chemically defined diets should be used. They ensure the greatest degree of reproducibility and avoid the confounds introduced by contaminants or compositional variability. (4) Use of a positive control is highly desirable. Without a positive control, negative results are of questionable significance. We use a 40% CR control, which also allows us to calibrate the effects of a treatment (e.g., Fig. 1). (5) Dosages can be chosen using treatment endpoints gleaned from the literature or, where necessary, from human dosages using accepted cross-species scaling factors. Use of these methods will produce a more reliable literature on which to base further studies. Acknowledgments: The author would like to thank Mehgan Hassanzadah and Patricia Mote for their help in the preparation of this manuscript. Open Access This article is distributed under the terms of the Creative Commons Attribution Noncommercial License which permits any noncommercial use, distribution, and reproduction Electronic supplementary material Here is the link to the electronic version of the supplementary material. Supplementary Table 1 Summary of all published compound testing studies using healthy, long-lived rodents using life span as an endpointa (DOC 566 kb) The full paper is available here I suggest that longevity studies on any species, especially humans must be done in the same precise way. 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The CR Society Intl and the CR Way are working together to support CR research. For every CR Society member who becomes a CR Way member, the CR Way will contribute 20% of the membership fee to the CR Society for research.You can learn more by visiting the LivingTheCRWay Store: Store.LivingTheCRWay.com. Since its beginning, The CR Way has strongly supported the Society, encouraging its growth and fostering its meetings. Now supporting the Society research has become urgent:Blood has already been drawn and the shelf life, even at sub-zero storage conditions, is limited. Whether we are successful in looking at the many aspects of cellular aging, envisioned by the Spindler-Dhahbi research team, depends on you. The situation we face now is parallel in many ways to that of the launch of human CR research at Washington University in St. Louis School of Medicine. At the time it was important to know if the research findings in calorie restricted animals would be similar to calorie restricted humans. Who could have known that the research there would indicate that CR in humans could have such extraordinary results on the cardiovascular system? Remember this: Long-term calorie restriction is highly effective in reducing the risk for atherosclerosis in humans. Fontana L, Meyer TE, Klein S, Holloszy JO. Proceedings of the National Academy of Sciences of the U. S. A. 2004 Apr 7; 101(17):6659-63. PMID: 15096581, NIH, NLM, PubMed access to MEDINE citations The group from the Calorie Restriction Society showed virtually no evidence of risk for atherosclerosis. Many evaluative measures – such as total cholesterol, LDL, triglycerides, insulin, and high sensitivity C-reactive protein were significantly lower in the CR group, while the cardio-protective HDL was higher. Carotid artery wall thickness, a diagnostic indicator for coronary artery disease, was 40% less than that of the controls, with no evidence of plaque accumulation. Long-term caloric restriction ameliorates the decline in diastolic function in humans. Meyer TE, Kovacs SJ, Ehsani AA, Klein S, Holloszy JO, Fontana L. Journal of the American College of Cardiology. 2006 Jan 17;47(2):398-402. PMID: 16412867, NIH, NLM, PubMed access to MEDINE citations Decline in the heart's diastolic function occurs with age. The results of this study showed that the diastolic function of our CR cohort resembled that found in people about 15 years younger. New Research! Now Drs. Spindler and Dhahbi have discovered that another biological system responds to certain longevity protocols that can produce life extension. The findings will likely be published sometime this year. To study this idea in humans fully requires that we raise more money for research. Further, the Spindler-Dhahbi research focus in microRNA and epigenetic aging markers will provide multiple paths for ways to slow cellular deterioration even more. If you are in a position to make a major contribution to this extraordinary opportunity to extend healthspan, contact me directly, and we will arrange for a private phone call with the scientists who will discuss the confidential details of the project. But even if you can’t make a major contribution, at least consider contributing to your own better health and the possibility of additional opportunities to live better longer by becoming a CR Way member and seeing 20% of your dues going to CR research immediately. You can choose from a number of memberships, depending upon your budget and goals. Paul McGlothin, Vice President for Research Research@CRSociety.org Phone: 914-762-8878 or (toll free) or 877-481-4841

Our recent visit to the Spindler Lab in California was extraordinary. Have you ever talked with someone whose words were so profound that they affected you for years or perhaps even your whole life? I felt that happening to me at a recent meeting with Dr. Joseph Dhahbi who explained his vision for analyzing the effects of calorie restriction on aging at the cellular level. Never have I heard so comprehensive a vision for thorough analysis of cell signaling as it relates to CR's health benefits. It was refreshing but challenging . We must act to take full advantage of Dr. Dhahbi’s vision, or we risk losing a golden opportunity to slow the aging process. Earlier in the day, Dr. Stephen Spindler talked with Meredith and me about his latest work on identifying substances that slow aging. The information that will come from this work will change your lives. Dr. Spindler has identified molecules that have surprising effects on aging and some that hitherto have been overlooked by longevists, searching for the best ways to extend their lives. The work of these two geniuses is so important that I’ve decided to create a blog that ties in to the CR Research forum so you can be informed of the latest developments. Meanwhile take advantage of this new forum by asking relevant questions of these researchers and other leading CR scientists. We are beginning a new era – one in which aging can be slowed. I hope you will save your life and join us. Paul P.S. Take a moment to enjoy our trip to the Spindler Lab: https://picasaweb.go...Lab?locked=true

Calorie restriction has changed! Eleven years ago, we were in the dark ages. People knew little about how Calorie restriction works and the CR world was dominated by demagogues who tried to shove piecemeal research down the throats of new CR members or anyone else gullible enough to listen. So we rolled up our sleeves and went to work. Money needed to be raised and CR research was desperately needed on humans.Now, 12 years later, science has prevailed. We know that human CR follows the same biological patterns of CR’ed animals. And though we will always be deluged with disinformation, we have many human CR studies that provide a road map for how to practice calorie restriction. Take a look at what we have accomplished and what is planned. Don’t think for a minute we are resting on our laurels. For many of us feel strongly that opportunities to slow aging and improve health span are greater than ever before. We must act on that now! Paul Source: CR Science has made progress, but must move forward rapidly!