CRON-WEB's Definitive Guide to Supplementation

Introduction

What is “life extension”?

Not accident avoidance, hygiene
Not correction for genetic disorders
Not correction for poor lifestyle
Extension of health (and therefore lifespan) beyond expectations for a basically healthy person living a basically healthy lifestyle.

CR [calorie restriction] “has been repeatedly shown to increase life span and delay the onset of age-associated pathologies in laboratory mice and rats.”[1] “For more than 60 years the only dietary manipulation known to retard aging was caloric restriction, in which a variety of species respond to a reduction in energy intake by demonstrating extended median and maximum life span.”[2]

No confirmed evidence for life-extending effects of any drug or supplement in normal, healthy mammals.

Animal Studies

‘Successes’ in short-lived strains, suboptimal environments (well-cared-for, normal mice, average LS ~900 days, max ~1200).
Failures in well-cared-for animals. Eg. “Mice were fed modified AIN76 diet or modified AIN76 supplemented with vitamin E, glutathione (GSH), vitamin E and GSH, melatonin, or strawberry extract starting at 18 months of age [~54 human years]. … Lesion burden and incidence of specific lesions observed amongst the various groups in this study did not differ. There were no differences observed for longevity of any of the study groups. The longevity observed in this study was similar to that previously reported for male C57BL/6 mice.”[3]
Eg. LEF LifeSpan Project: melatonin; CoQ10; lipoic acid (racemate); aminoguanidine; lipoic acid (racemate) + aminoguanidine + CoQ + Pregnenalone; ALCAR + lipoic acid (racemate) + lycopene + alpha-tocopherol; ALCAR + lipoic acid (racemate) + CoQ + NADH; melatonin + pregnenalone. No effect (see graphs)![4]
Deprenyl: data reviewed in ([5]). Only Knoll (inventor has reported increased lifespan for deprenyl; variations by strain, age, etc, in response; no reasonable basis for human dosing.

Human Studies

Epidemiology

Highly inconsistent; many negative findings; much weak design (case-control, external referent population etc).
Highly confounded. “[S]upplement use tended to increase with age, education, physical activity, fruit intake, and dietary fiber intake and to decrease with obesity, smoking, and dietary fat intake.”[6]
Hidden vulnerable subpopulations. Eg folate and colon cancer in Nurses' Health Study. Preliminary finding: “Higher … folate intake … was related to a lower risk for colon cancer (RR, 0.69 ... for intake > 400 microg/d compared with intake < or = 200 microg/d) … [Among] Women who used multivitamins containing folic acid … After 15 years of use … risk was markedly lower (RR, 0.25 [CI, 0.13 to 0.51]), … the benefit of long-term multivitamin use was present across all levels of dietary intakes.”[7]
- But: “Compared with women without a family history who consumed 200 µg or less of folate/day …, women without a family history who consumed >400 µg/day experienced a multivariate RR of 0.91 (95% CI, 0.69–1.19), … and women with a family history who consumed >400 µg/day experienced a RR of 1.30 (95% CI, 0.82–2.08). … Among women with a family history, the RR for those who consumed >400 µg/day was 0.48 (95% CI, 0.28–0.83) when compared with women with a family history who consumed 200 µg or less per day.”[8]

Most epidemiological results demonstrate danger of deficiency, not protective effect of megadosing (eg. folate (<200 vs >400 mg (DRI) and colorectal cancer, fish and CHD (no additional protective effect beyond 2 servings/week),[9] etc).

Randomized, Controlled Trials

Heart disease

Alpha-tocopherol
- CHAOS: 400-800 IU RRR-alpha-tocopherol/d vs placebo: “a significant reduction in the risk of non-fatal MI …; however, there was a non-significant excess of cardiovascular deaths”.[10]
- Several other large studies report no effect on any carciovascular outcome.[11],[12],[13],[14]
Antioxidant “Cocktails”
- WAVE: 400 IU alpha-tocopherol + 500 mg C ± HRT vs placebo. “Death, nonfatal MI, or stroke occurred in … 26 vitamin patients and 18 vitamin controls (HR, 1.5; 95% CI, 0.80-2.9).”[15]
- HATS: Simvastatin ± antioxidants (800 IU alpha-tocopherol, 1000 mg C, 25 mg natural beta-carotene, 1000 mcg Se) ± placebo. “The average stenosis progressed by 3.9 percent with placebos, 1.8 percent with antioxidants (P=0.16 … ), and 0.7 percent with simvastatin-niacin plus antioxidants (P=0.004) and regressed by 0.4 percent with simvastatin-niacin alone (P<0.001). The frequency of the clinical end point was 24 percent with placebos; 3 percent with simvastatin-niacin alone; 21 percent in the antioxidant-therapy group; and 14 percent in the simvastatin-niacin-plus-antioxidants group.”[16]
- Heart Protection Study: 600 mg alpha-tocopherol, 250 mg C, and 20 mg b-c daily) vs. placebo. “[N]o significant differences in all-cause mortality …, or in deaths due to vascular … or non-vascular … causes. Nor … in the numbers of … non-fatal myocardial infarction or coronary death …, non-fatal or fatal stroke …, or coronary or non-coronary revascularisation ... For the first occurrence of any of these “major vascular events”, there were no material differences … There were no significant effects on cancer incidence or on hospitalisation for any other non-vascular cause.”[17]

Cancer

ATBC: 20 mg synthetic b-c &/or 50 mg alpha-tocopherol vs. placebo in smokers. “No overall effect was observed for lung cancer from alpha-tocopherol supplementation … beta-carotene supplementation was associated with increased lung cancer risk (RR = 1.16 …). The beta-carotene effect appeared stronger, … in participants who smoked at least 20 cigarettes daily (RR = 1.25; …) compared with those who smoked five to 19 cigarettes daily (RR = 0.97; 95% CI = 0.76-1.23) and in those with a higher alcohol intake (> or = 11 g of ethanol/day [just under one drink per day]; RR = 1.35; 95% CI = 1.01-1.81) compared with those with a lower intake (RR = 1.03; 95% CI = 0.85-1.24).”[18] Later reports: no effect on gastric,[19] urinary tract,[20] colorectal,[21] cataract,[22] or maculopathy;[23] no clear effect on pancreatic[24] or colorectal[25] cancers; 50 mg alpha-tocopherol decreased, but synthetic b-c may have increased, clinical prostate cancer incidence and mortality.[26]
Physicians' Health Study: 50 mg synthetic b-c EOD vs. placebo. “Virtually no early or late differences in the overall incidence of malignant neoplasms or cardiovascular disease, or in overall mortality ... Among current and former smokers, there were also no significant early or late differences in any of these end points.”[27]
CARET: 30 mg synthetic b-c and 25 000 IU of retinol in smokers, former smokers, and workers exposed to asbestos vs. placebo. RR of lung cancer of 1.28; RR of death from any cause 1.17; of death from lung cancer, 1.46; and of death from cardiovascular disease, 1.26 (95 percent confidence interval, 0.99 to 1.61).[28]
- Supplementers lost benefit of fruits and veggies![29]
Antioxidant Polyp Prevention Study: Actually potentially positive (!): recurrence of colon adenoma. 25 mg synthetic b-c and/or vitamins C and ‘E’ in vs. placebos. “Among subjects who neither smoked cigarettes nor drank alcohol, beta-carotene was associated with a marked decrease in the risk of one or more recurrent adenomas (RR = 0.56, 95% CI = 0.35 to 0.89), but … a modest increase in the risk of recurrence among those who smoked (RR = 1.36, 95% CI = 0.70 to 2.62) or drank (RR = 1.13, 95% CI = 0.89 to 1.43). For participants who smoked cigarettes and also drank more than one alcoholic drink per day, beta-carotene doubled the risk of adenoma recurrence (RR = 2.07 … P for difference from nonsmoker/nondrinker RR <.001).”[30] Ambiguous evidence from CARET and ATBC on role of alcohol – more distal sites?
Clarke et al JAMA selenium trial: 200 micg Se/day vs. placebo in persons with a history of skin cancer. No effect on primary end points (incidences of basal and squamous cell skin cancer); post-hoc endpoints, “nonsignificant reduction in all-cause mortality … [RR; 0.83; 95% CI, 0.63-1.08] and significant reductions in total cancer mortality … [RR, 0.50], total cancer incidence … [RR, 0.63], and incidences of lung, colorectal, and prostate cancers.”[31]
- However…
- “The patient population in this trial was recruited from the eastern coastal plain of the [US], a region characterized by relatively low [Se] levels in soil and crops [refs], and by high rates of [squamous cell carcinoma] and [basal cell carcinoma] of the skin and cancer mortality.[ref]” 31
- “participants with baseline plasma selenium concentrations in the lowest two tertiles (<121.6 ng/ml) experienced reductions in total cancer incidence, whereas those in the highest tertile showed an elevated incidence (HR = 1.20, 95% CI = 0.77-1.86).”[32]
Linxian Trial: Also successful; also in an intentionally-selected deficient population.[33]

“Well, it Can’t Hurt, and it Might Help” Mistake

Vitamin A (Retinol/Retinyl Esters)

Higher Intake [34],[35] or Serum Levels[36] Increase Fracture risk
- ”For every 1 mg [3330 IU] increase in daily intake of retinol, risk for hip fracture increased by 68% ... For intake > 1.5 mg [5000 IU] /d compared with < 0.5 mg [1665 IU]/d, … risk for hip fracture was doubled”. 34
- Nurses’ Health Study: “women in the highest quintile of total vitamin A intake … had a significantly elevated relative risk (RR) of hip fracture compared with women in the lowest quintile of intake … attributable primarily to retinol (RR, 1.89 … comparing >/=2000 mcg [6660 IU]/d vs <500 mcg [1665 IU]/d). 35
Negative association of retinol intake with BMD; 34,[37],[38],[39] no association for serum levels in NHANES III.[40]
In animal studies,[41],[42] vitamin D does not fully counteract; most of the excess retinol in the first study 34 was from cod liver oil supplements taken for vitamin D in Nordic countries!
No risk from dietary carotenoids. 34, 35

Excess Zn or Zn:Cu

Competition for ligands (for absorption and utilization)
Frank Zn-induced Cu deficiency demonstrated in humans:
- Zn:Cu of 23.5 (24 subjects) led to low CuZnSOD, high TC and LDL, low HDL, reduction in enkephalins, cardiac abnormalities (heart block, tachycardia, MI) (reviewed in ([43])).
- Zn:Cu of 16 (one subject) led to decreases in ceruloplasmin, increased TC and LDL, arrhythmia. (reviewed in (43))
- 50 mg Zn/day depresses CuZnSOD 20%.[44]
Other Cu-deficiency sequelae likely: impaired bone metabolism, poor glucose control, increased AGE, etc.

Excess Mn

Welders, other industrial inhalation exposure associated with manganism (Parkinsonian syndrome); reproduced in high-dose animal feeding studies; ecological study showed increased frequency of neurological symptoms in community with ~3.6-4.6 mg Mn/day from water as vs. 0.0072-0.03 and 0.16-0.5 mg/day.[45]
New case-control study links highest quartile [DEFINE THIS] Mn intake with 1.7-fold increase in PD risk.[46]
Vegetables, tea rich sources; vegetarian diets may contain 13-20 mg.
LOAEL from diet 15 mg (increases in serum Mn (‘should’ be tightly regulated)); UL 11 mg from all sources.[47]

Unbalanced alpha-tocopherol?

Alpha-tocopherol depletes gamma-tocopherol levels below baseline levels at 100 IU [48] (& perhaps 31 IU![49]). After 1 y alpha-tocopherol supplementation (1200 IU), tissue gamma-tocopherol levels take 2y to recover. [50] 30% depletion by 371 mg alpha-, even when balanced by >400 mg “other” tocopherols. [51] Alpha-tocopherol blocks tocotrienol action on HMG-CoA reductase if >30% of supplement.[52],[53], [54]
Little evidence of adverse outcomes (no increased risk of MI etc among supplement users; NS trend to dose-dependent increased risk of prostate cancer progressing 100 IU-400 IU[55]).

Synthetic beta-carotene (below)?

Should we supplement? What, and how much?

Tier I: Supplementation Necessities

Correct deficiencies
- New IOM Dietary Reference Intakes (DRIs) grounded in solid science.
- Exception: Vitamin D: Plenty of evidence for safety, clinical efficacy (osteoporosis), and biochemical optimization (plateau of PTH) at 1000 IU.[56],[57],[58] Even summer outdoor workers (agriculture, landscaping, etc) do not store up enough D to lat through the winter.[59] Canadians, Northern US, sunscreen users are not getting enough; attempts to do so increase skin cancer risk. Use a pill.
- Subclinical deficiencies and cancer: See Clarke Se trial; 31 long-standing, Ames thesis[60] (misappropriated by supplement propagandists by implying “deficient” = “sub-megadose”).
- CR Folks (CRONies)!
  - Common deficiencies in low-Calorie diets: Ca, Fe, Mg, Zn, B1, B2, B6; B12 (semi-vegetarian); w3, fat-soluble vitamins (low-fat).[61]
  - CR-induced deficiencies: Evidence of increased need for protein, Fe,[62] Cu,[63] Ca[64] during weight loss in ‘CR’ diet – others??
    - OTOH, CR protects against effects of some deficiencies: B1,[65] Mg,[66] Cu.[67]
- Biochemical individuality: extra folate for MTHFR polymorphism; riboflavin for NQO; other, speculative cases;[68] ‘common’ variance in requirements[69]? RDAs meet 95th percentile of population; average person will have an unusual need for 1 in 20 nutrients.
- Strategy: get a balanced multiple (1.5-2 fold) of RDA for most nutrients; use verified testing (eg ferritin) when warranted and verified.
Selected ‘pharmacologic’ supplementation for high-risk/unwell individuals:
- CR-related disease susceptibility: Loss of bone mass. Especially critical to ensure standard osteo support nutrients (Ca, Mg, vit D, B, Zn, Cu, Si, Mn); ‘pharmacologic’ Sr[70],[71],[72],[73],[74] and menatetrenone (MK-4).[75],[76],[77],[78]
- I3C for cervical dysplasia (carcinoma in situ).[79] (Animal studies report good or bad effects on cancer risk, depending on site [80]).
- Lipoic acid,[81] benfotiamine[82] for neuropathy.
- Pantethine, megadose niacin vs. hyperlipidemia
- Epidemiology, animal studies, and biochemistry for some nutrients vs. bad genes/family history of diseases:
  - Folate vs. colon cancer. 8
  - D-glucarate vs. hormonal cancers (animal studies)?[83]

This is what we know. Everything beyond this is increasingly speculative.

“You can’t win if you don’t play” lottery logic is very bad thinking for the life extensionist. Killing yourself with supplements is just too damned stupid for words. If you’re going to take a supplement, insist on meaningful evidence that it will help you. Human RCTs in healthy people with clinical endpoints > epidemiology with clinical endpoints > studies in vivo in healthy people and rodents showing health benefits/favorable surrogate endpoints. Make-the-case mechanistic arguments, inbred genetic fuckup rodents, or animals exposed to massive doses of hideous toxins may mean nothing at all.

Bad-Evidence Supplementation Schemes:

Alpha-tocopherol succinate: “Better cancer fighter!”
- Extensive literature (eg. ([84],[85])), but all in vitro.
- Physiologically irrelevant. All esters removed by GI esterases; appear in plasma as free phenol.[86]
- Kline: “vitamin E succinate … loses its anticancer properties when the succinate moiety is removed by cellular etherases”; she’s accordingly working with a novel tocopherol analog instead.[87]
Many Flavonoids: Test-tube and rodent studies use pure compound – not good evidence, because humans biotransform flavonoids much more heavily than rodents; can’t extrapolate human results without human evidence.
- Curcumin requires multi-gram doses to elevate blood levels in humans [88],[89],[90],[91],[92],[93] unless combined with piperine. See relative jump in curcumin bioavailability with piperine (inhibits glucuronidation), humans 2000% vs. rodents 154%. 90 (Yes, piperine is scary!).
- “Consumption of [500 mL] cranberry juice [total phenols 893 units/L; 1.53 mM vitamin C] resulted in a significant increase in ... measures of antioxidant capacity ... [which] corresponded to a 30% increase in vitamin C and a small but significant increase in total phenols in plasma. Consumption of [500 mL] blueberry juice [2589 phenol units/L; ~0 C] had no such effects [my emphasis].”[94] Cf. rodent blueberry studies.
“It’s an antioxidant!!”
- Many in vitro ‘antioxidants’ are not so in vivo;[95] and so what if it is? “Antioxidant” is not the same as “beneficial.” Cyanide is a great antioxidant, etc.

Acute, massive-dose carcinogens do not reflect real etiology of disease in humans: low-level, chronic, endogenous and exogenous factors. Want evidence it’s effective in spontaneous carcinogenesis in healthy organisms.
No record for long-term use of herbals, or use of herbals by the healthy. “Natural,” but xenobiotic. IMO life extensionists can’t afford the risk of silymarin, ginseng, Ginkgo, St. John’s Wort etc. if healthy and other remedies available: orthomolecules preferred, but incl. often drugs, which are better-tested.
Clinical endpoints (morbidity/mortality), not putative surrogates.

Tier II: Disease Risk Reduction in the Healthy?

The Supplement Paradox: massive epidemiological support for fruit and vegetable intake – and intake of many of the nutrients they contain – and lower cancer &CVD risk. Yet supplements keep failing! Why??

[Tautology]: Supplements fail to reflect the key aspects of healthy diets.

[Inference]: Supplement programs which better reflect healthy diets are more likely to yield risk reduction.

How to identify ‘healthy diets’? What are their key (supplementable) components? How can supplements better reflect them?

Best current long-term, healthy human evidence is epidemiology, testing independent variables and clinical endpoints.

Independent variables (nutrients and foods), not dependent variables (eg serum levels):
- RAGE Polymorphism: “HbA(1c), Amadori, and AGE did not reveal any significant association with any of the polymorphisms analyzed. However, significant differences between … “wild-type majority” … and subjects with “mutated” genotypes were found for total carotenoids (P =.001), alpha-carotene (P =.046), beta-carotene (P =.028), lutein (P =.001), lycopene (P =.006), and alpha-tocopherol (P =.047). … The extent of diabetic vascular disease is related to the plasma levels of antioxidants.”[96]
- Inflammation: “Serum vitamin C was strongly and inversely related to systemic inflammation [ie, CRP].”[97]
- High-GI Diet: “The age-adjusted mean concentrations (micmol/L) for increasing quintiles of dietary GI were 29.3, 27.0, 25.5, 24.6, and 23.6 (p<0.0001 for trend) for serum alpha-tocopherol and 46.2, 43.2, 39.7, 37.9, and 34.8 for vitamin C (P<0.001 for trend). … [Persisted after] adjustment for sex, ethnicity, education, smoking status, BMI, alcohol intake, physical activity, percent calories from carbohydrates and fat, and total energy intake. [After] adjusting for the same covariates, higher … GI was also associated with lower … alpha-carotene, beta-carotene, cryptoxanthin, and lutein/zeaxanthin.”[98]

How do Supplements and Good Diets Differ?

Different molecules: Food forms vs. supplement forms
- Vitamin E: Numerous studies find dietary – and not supplement – “vitamin E” protects against CVD,[99] ,[100],[101] and AD.[102],[103],[104]
  - Alpha-tocopherol is the sole E vitamer in supplements in trials, epidemiology to date, but is minority of “vitamin E” in diet (gamma-tocopherol is plurality).[105],[106],[107],[108],[109]
  - High plasma gamma- – and not alpha- – tocopherol associated with reduced risk of CVD,[110],[111],[112],[113] MI,[114] prostate cancer.[115],[116]
  - Selective gamma-tocopherol depletion and nitration in AD.[117],[118]
  - Gamma-, not alpha-tocopherol reduces PGE2, LTB4, signs of inflammatory damage in vivo[119] and COX-2 activity in macrophages and epithelial cells ;[120] superior RNS-quenching capacity in vitro; [121],[122],[123] preferential uptake by macrophages;[124] more favorable effect on LDL metabolism in vitro.[125]
- Beta-carotene: Extensive review of epidemiology finds b-c from food protective against lung and other cancers. [126]
  - Nearly all trials used synthetic (all-trans) b-c; food b-c is mixture (9,cis- and all-trans-).
  - Synthetic b-c has lower antioxidant activity in vitro[127] and in vivo.[128],[129]
  - Synthetic b-c genotoxic in vitro (!), unlike natural.[130]
- Selenium: Extensive epidemiology for dietary inverse risk with cancer.
  - Much Se in diet is from grains, meat as selenomethionine – not foods associated with low cancer risk. Se in cruciferous and Allium vegetables (strong inverse association with cancer) contains Se-methylselenocysteine.
  - Animal studies show SeMC a superior anticancer form.[131]
- Vitamin K: Phylloquinone must be metabolized into menatetrenone (MK-4).
  - ”The tissue specific localization of MK-4 and a metabolic pathway for its production from phylloquinone strongly suggest that there is a yet-to-be-discovered unique role for this form of vitamin K that is independent of the currently recognized coenzyme function.”[132]
  - MK-4 has unique effects on bone tissue and cells in vitro, despite similar gamma-carboxylation coenzymatic activity.[133]
  - Dietary MK-4, not phylloquinone, associated with reduced risk of aortic atherosclerosis.[134]
Different dose: Just how much are “the group eating the most X” getting in the epidemiology?
- Too much beta-carotene: Extensive review of epidemiology finds b-c from food protective against lung and other cancers. 126
  - Highest quintile intakes of 8950 mcg (2484 IU)/day,[135] 5902 mcg/day (older, incomplete database),[136] 11399 mcg/day;[137] trials and most supplements 25 mg (6937 IU).
  - Oxidative metabolites of b-c, if not detoxified by compensatory C and E, may increase carcinogenesis in smokers;[138] more b-c = more imbalance.
  - In ferrets, pharmacologic dose of synthetic b-c results in keratinized squamous metaplasia, with or without exposure to cigarette smoke; not observed with physiologic dose.[139]
- Too little lycopene: Lots of good epidemiology for tomatoes, tomato paste, dietary and plasma lycopene vs. various cancers.[140]
  - Highest quintile intakes of 18, 135 9 (older, incomplete database), 136 13 137 mg/day; typical multis contain 0.3-6 mg (vs. lowest quintile intake of 6 mg! 135).
  - Very strong surrogate results in RCTs in prostate cancer patients with lycopene (30 mg/day led to lower DNA damage and incidence of high-grade carcinoma in situ, and PSA)[141] or tomato paste (22 mg lycopene from target 30 mg (3/4 C tomato paste) led to lower DNA damage &PSA).[142]
  - Earlier epidemiology underestimated lycopene intake due to incomplete database for tomato products. 29,[143]
- Too little Se? Plenty of good epidemiology for Se intake (not tissue levels (eg toenail)) vs. cancer.[144]
  - Much, again, supports simple correction of deficiency, but world intakes often much higher (800-1600 mcg in China).
  - With bulk of dietary Se (SeMet, some inorganic), methylselenol (anticancer metabolite) only formed at extreme end and full benefits only seen at supranutritional levels – at or near toxic range.
  - SeMC forms methylselenol at much lower doses (above).
- Excellent evidence for optimal vitamin C 100-200 mg.
  - LPI review of epidemiology: CVD, possibly cance4r benefits of ~90-100 vs ~<45 mg/day and no further.[145] Similar data, but ‘no conclusions can be drawn,’ from IOM; new DRI, 90 mg/day.[146]
  - Plasma levels do not increase at intakes >100 mg/day when tested 1 x 30 - 4 x 45 mg, but decreasing-returns increases from 4 x 250 and 4 x 500 mg;[147] plasma levels essentially plateau at 200 mg w/small increases 500-2500 mg, but tissue saturation (selective active transport into leukocytes) complete at ~100 mg/day and excretion of unmetabolized C jumps at >60 mg/day.[148]
    -No evidence for reduced DNA damage from 60-600 mg/day; lipid damage almost entirely bogus measures (ex vivo LDL oxidizability unphysiologic; TBARS, MDA reflect all kinds of other stuff); F2 isoprostanes inconsistent;145, 146 continues in later studies. 51,[149],[150],[151],[152]
  - Pauling et al ‘making the case’ from animals who synthesize their own C, etc – very weak ‘evidence.’
  - No data on humans consuming 10 g C/day! 95th percentile intake 1200 mg. 146
  - ‘Hypoascorbemic’ guinea pigs have shorter mean, max, minimum LS when given 10-20 ‘RDAs.’[153]
  - Uric acid and other factors may have replaced C in humans.[154]
- B-Vitamin Doses Whacked: Many times greater than food intakes or RDAs with no good evidence of benefit for most.
  - Can’t absorb >~8-12 mg thiamin dose. [155],[156],[157],[158],[159],[160]
  - PABA of no nutritional value to humans: needed by bacteria to synthesize folate, but PABA-depleting sulfa drugs harmless to mammalian cells; ‘it’s an antioxidant!!’ – so??
  - Why 50 mg of everything?? Strange ‘coincidence:’ purely arbitrary.
Missing molecules:
- C and ‘E’ for detoxing oxidized b-c products. 138
- b-c partly a ‘marker’ for other carotenoids.
- Unknown dietary constituents: eg. new vitamin (?), pyrroloquinoline quinone.[161],[162],[163],[164]
- Phytochemicals. A plethora! Which are important?
- CVD and Stroke: less clear, beyond folate, minerals, antioxidant vitamins; likely much of inverse association due to low GI and GL of vegetables, displacement of saturated fat.
  - Strongest evidence for flavonoids: notably tea, onions (quercetin); berries?[186] – but ubiquitous and so hard to deconfound. 166

Tier III: Slowing Aging?

Even if we get full lifestyle benefits, so what? Average person's longevity gain from the eradication of cancer <=3.2 years of life. Ischemic heart disease? <=3.55 years. Both together <= 7.83 years. Both, plus all circulatory diseases and diabetes:15.3 years. (And, hint: we’re already living pretty healthily …).

Retarding aging to extend life span. Logically includes healthspan!

Only one proven intervention: CR [CRON] – and that in rodents! Hence, warning: maximum speculation follows!!

What factors are associated across mammalian species, and within mammalian spp AL [ad lib] vs CR, with slower aging and extended maximum lifespan?

Only 2 AFAIK [as far as I know]:

Mitochondrial ROS issues: mtROS production, mtDNA (not nuDNA) damage, mt membranes oxidative susceptibility inversely associated with max LS interspecies, and reduced by CR.[187],[188]
- Antioxidant enzymes inversely associated with max LS! 187
- Dietary AOs, and prob. AO enzymes, do not reduce these factors and do not affect aging.
- You must reduce production.
- mtROS production is the great majority of ROS in vivo.
  - Food e- moved to NAD+ (forming NADH) by glycolysis and then Krebs/Citric Acid/TCA cycle.
  - NADH transfers e- to Complex I, passed along ETS to pump protons, create electrochemical gradient for Complex V (ATP synthase): hydro dam system, Complex V (ATP synthase) a literal turbine.
  - “Fumbling” by CoQ in transferring e- from Complex I to Complex III allows e- to react with O2, forming superoxide.
- In CR and longevous spp, lower mtROS production occurs because of less reduction of Complex I by NADH. Fewer e- pass thru’ CoQ, fewer ‘fumblings.’ 188
- Interspp difference genetic by definition; perhaps Complex I structure?
- de Grey proposes CR redirects NADH to PMRS.[189] Explains lower ROS production, Complex I location, no reduction in metabolic rate.
  - Also (unbeknownst to him) lower peak sprint speed in CR (seen in rodents[190] and anecdotally in some humans (eg. me!)) despite much higher overall activity levels (“CR-induced hyperactivity,” longer running at any age, continuing running after AL are dead); consistent w/high NADH levels reported by Dr. Robert Lord (MetaMetrix) in Society subjects?[191]
- Intervention: R(+)-lipoic acid.
  - Hagen and Ames: R(+) reduces mtROS production, improves mt function, lowers mt damage in normal, healthy aging rodents (no significant effect in youngsters).[192],[193]
  - Reducing ROS generation or just mopping up afterward? Hagen (personal communication) and de Grey think the latter most likely. However:
    - R(+) specifically reduced to DHLA in mt PDH by NADH, leaving NAD;[194], [195],[196] less NADH means fewer e- transfered to Complex I: lower ROS?
    - In cell culture [197] and in diabetic rodents,[198] NAD:NADH is indeed increased. (Latter study used racemate; R(+) should mean higher ratio in mt, lower in cytosol.
    - Packer believes that increased NAD:NADH is responsible for improvements in diabetic neuropathy (corrects for diabetic hypoxia). 197
    - Diabetics overexpress PMRS;[199] consistent with high NADH:NAD and need for use of PMRS as exit valve. Consequent cell-surface reductive stress would explain high oxLDL in NIDDM (mechanism proposed in MiFRA for how so few cells drive oxidative stress thru’out the body.[200]).
    - Alternatively/additionally, DHLA can quench ubisemiquinone (CoQ radical which causes fumbling) in membranes in vitro.[201]
  - Even racemate LA reported to reduce cochlear mtDNA ‘common deletion” in normally-aging rodents.[202]
    - So why no LS bennies from racemate? Not enough effect, due to low R(+) content and interference of R(+) reduction by S(-) 194? Negative side-effects of S(-) (eg. thru’ reduced GSH recycling, NADPH?). Dose? (Seidman used 300 mg/kg; after metabolic scaling (300-400 g rats), human equivalent 17.659 g! LEF studies used human-equivalent 518 mg.[203] at standard energy density).
  - R(+), not S(-) or racemate, increased “max LS” in athymic mice (weak evidence: short-lived strain, increase only based on 2 outliers in small cohort).[204]
  - NIA-funded Hagen and Ames LS study with R(+)/ALCAR cocktail.[205]
- Dose: Human-equivalent 612.6 - 1225.2 mg.[206],[207],[208],[209],[210],[211] Voodoo: coincidental equivalence to RCT dosage for racemate in diabetes, AD. But Seidman et al 203 and LEF LifeSpan studies 4, 203 (using racemate, NB) might suggest higher dose.
- Intervention 2: Metformin + R(+)
  - Roth and Lane: metformin appears “to mimic some of the bioeffects of [2-deoxyglucose] without apparent toxicity. Preliminary experiments suggest that [metformin] can increase median and maximal survival of rats to the same extent as 30% CR.”[212] Spindler quoted as claiming ~20% increase in LS.[213],[214]
    - Most assuming works thru’ antidiabetic mechanisms (increased insulin sensitivity and glycolysis inhibition), and perhaps crypto-CR noted by Patrick[215]); this would likely be irrelevant in normal, healthy folk (basic weight-loss program (7% weight reduction thru' low-calorie, low-fat diet and moderate exercise (eg. brisk walking), for at least 150 min/week) more effective than M in preventing the conversion of prediabetics to full fleged NIDDM);[216] “Metformin does not improve insulin sensitivity nor insulin secretion in obese female patients with normal glucose tolerance.”[217] But:
      - Gene expression changes for wider range of effects that overlap CR. 213 “These findings suggest that metformin has more beneficial effects than the reduction of blood glucose and insulin, and that it may be an authentic anti-aging therapy.”
      - Much closer overlap than other diabetic drugs (incl. insulin-sensitizing ’glitazones).
    - Metformin inhibits Complex I in vitro[218] and ex vivo.[219]
      - Associated in vitro with inhibition of gluconeogenesis from L-lactate 218 – observed in vivo as dangerous lactic acidosis side-effect.
      - “Because it is established that [metformin] is not metabolized, these [ex vivo] results suggest the existence of a new cell-signaling pathway targeted to the respiratory chain complex I with a persistent effect after cessation of the signaling process.” 219
      - “We conclude that the drug's pharmacological effects are mediated, at least in part, through a time-dependent, self limiting inhibition of the respiratory chain that restrains hepatic gluconeogenesis while increasing glucose utilization in peripheral tissues.” 218
      - De Grey was intrigued, even before Roth’s announcement: “Interesting … I think that TOTALLY inhibiting the respiratory chain is unconditionally bad (at least if the affected cells accumulate), but partial inhibition may be very different” depending on “how the cells react to the inhibition” (per his CR model).[220]
      - More evidence for R(+)? In isolated perfused rat liver, “RLA reduces hepatic glucose release by inhibiting lactate-dependent glucose production in a concentration-dependent fashion.”[221]
      - Previously observed in vitro,[222] and “antigluconeogenic effects of lipoic acid in liver can be attributed largely, if not entirely, to sequestration of intramitochondrial coenzyme A” (??).
      - ”When transported into the mitochondrion, pyruvate [product of glycolysis used by TCA to reduce NADH] encounters two principal metabolizing enzymes: pyruvate carboxylase (a gluconeogenic enzyme) and pyruvate dehydrogenase (PDH), the first enzyme of the PDH complex [requires R(+) –MR]. With a high cell-energy charge [high NADH:NAD?-MR], coenzyme A (CoA) is highly acylated … and able allosterically to activate pyruvate carboxylase, directing pyruvate toward gluconeogenesis. When the energy charge is low CoA is not acylated, pyruvate carboxylase is inactive, and pyruvate is preferentially metabolized via the PDH complex and the enzymes of the TCA cycle to CO2 and H2O. Reduced NADH and FADH2 generated during the oxidative reactions can then be used to drive ATP synthesis via oxidative phosphorylation.”[223]
    - Net effect on safety of metformin/R(+) cocktail??
      - In vitro, “Parallel decreases (30%) in cellular NADH/NAD+ and in lactate/pyruvate ratios were observed in [R(+)-]alpha-lipoate-treated cells [former should be reflected in latter as re: glycolytic pathway].” 197
      - “Treatment of guinea pigs (250 g weight) with a-lipoic acid (0.5 mg) for 10 d substantially increased the level of lactic acid (30% increase with respect to control values), and decreased the level of citric acid (60% decrease compared to control). These data have been interpreted as lipoic acid stimulating the anaerobic conversion of pyruvic acid to lactic acid, a reaction that other authors have indicated to occur in both directions.”[224]
      - IDDM rodent study: “Gastrocnemius lactic acid was increased in diabetic rats … and was normal in LA-treated diabetic rats”.[225]
      - Suicide attempts using 10-40 g racemate have resulted in lactic acidosis. 224
      - Human RCT: “lactate and pyruvate before and after glucose loading were ~45% lower in lean and obese diabetic patients after LA treatment.”[226]
      - Potential synergism of inhibited Complex I (metformin) + reduced NADH:NAD (R(+))?
- Dose: PDR, for diabetics: 500 mg bid, increasing by 500 mg weekly, up to 2g; or 850 mg, increasing by 850 up to 2550.[227] Combine with R(+), 600 mg.
- Safety: Lactic acidosis serious and unpredictable.
- Steve Harris: “Metformin's an especially scary drug in this regard, causing liver necrosis in those susceptible without a lot of warning first, and at doses which are (or were thought to be) more or less therapeutic. … The LDH on your liver enzyme panel tells you exactly NOTHING about how well your liver's lactate processing system is working. Nor really the AST and ALT – they're simply markers of acute liver cell damage and leakage.” ALT and AST “won't tell you that you're close to … lactic acidosis … Don't think of metformin like Jack Daniels or statins, think of it like digitalis and coumadin. [Cf PDR: “Side effects cannot be anticipated.”] 227 The idea of skinny nondiabetic calorie restricted laypeople willy nilly using it as part of a life extension program in the absence of good data for the pro side, and probably without good lab support in many cases, scares me. Not a good idea.”
- Per LEF,[228] “According to the Physician's Desk Reference, clinically significant responses in Type II diabetics are not seen at doses below 1500 mg a day”. Not in online version 227 …
- IMO: Not ready for prime time. This is a potentially toxic xenobiotic drug, and we’re basing all of this on a single, unpublished rodent study!! Wait for repetition (Spindler, Roth).
- Negative Intervention: Avoid n3 HUFA (EPA/DHA).
  - As noted, mt inner membrane (MIM) ‘peroxidizability index’ (number of double bonds/FA in membrane PL) inversely associated with max LS; aging increases, and CR reduces, MIM index via lower HUFA, esp n3 HUFA and esp DHA.[229] Possibly related to membrane peroxidizability and physical attachment of MIM to mtDNA (ROS damage to MIM à mtDNA deletions).
  - Feeding rodents fish oil increases mt inner membrane DHA. See details at ([230]).
  - In parallel, unusually low desaturase activity consistently observed in human CR. 191
  - Cardio benefits seen equally or more so with increased ALA intake, per epidemiology and RCTs. 230,[231],[232]
  - No benefit to more than 1-2 fish servings/week in any case,[233],[234] so no justification for supplementation.
- Dose: Maximum avoidance. Ensure adequate intake of n3 with 2-8 g ALA.

Rate of AGE Accumulation: Inversely correlated across species; not fully correlated with blood sugar levels (eg “hyperglycemic” hummingbirds,[235] but also seen within mammalian class[236]) and hence evidently genetically regulated. Miller et al zeroing in on such genes in mice.[237] CR lowers both blood sugar and AGE, and “markers of skin collagen glycation and glycoxidation rates can predict early deaths in AL and CR C57BL/6NNia mice.”
- Nearly all “anti-glycation” nutrients have only ever been tested in vitro, and work at Schiff base formation, not AGE formation per se: ineffective strategy in vivo (sheer stoichiometry and reversibility).[238] In vitro conditions extremely misleading in any case. 238
- Includes carnosine
  - No in vivo data.
  - In vitro, prevents Schiff base formation, but little effect on AGE.[239]
    - Recent LEF ad: “research has shown that metals, predominantly copper, are culprits that promote excess glycation … A new study[240] concludes that carnosine not only inhibits glycation earlier than aminoguanidine, but that it is 625 times more potent at chelating ... the harmful copper. This new study confirms that carnosine is the most effective anti-glycating agent.” Misleading – see cited study:
    - Looking for mechanisms and to undo confounding, not clinical utility.
    - In vitro, and not even measuring AGE formation!!
    - Physiological concentration already 5 times IC50 for chelation mechanism!
    - Chelation of unlikely mechanistic significance in vivo. 238
    - “Earlier” not “better” re: AGE.
  - OTOH, in vitro studies suggest alternative mechanism for in vivo effect via increased proteolysis;[241],[242] but physiological relevance still unknown.
- Aminoguanidine does not inhibit AGE formation in areas distal from the circulation (skin and tail collagen),[243] is quite toxic (antinuclear antibodies (sign of autoimmunity) and flulike symptoms) and of no clear benefit in diabetic humans at 300-600 mg/day,[244] and was of no benefit in the LEF LifeSpan studies. 4
- Intervention 1: Pyridoxamine
  - Post-Amadori AGE inhibitor. 238,[245]
  - Extensive animal evidence: lowers AGE (&ALE) accumulation, including in skin and tail collagen;[246],[247],[248],[249] prevents or treats nephropathy in insulin-resistant hyperlipidemia, 246 NIDDM[250] and IDDM, 249 with stronger effects than aminoguanidine;[251] reduces atherosclerosis in hyperlipidemia; 246 reduces retinopathy in IDDM. 247
  - Human Phase II Studies: Well-tolerated at doses which create plasma levels similar to those in rodent studies;[252] in 12 patients with diabetic nephropathy, “The average decrease from baseline in 24 hour urinary albumin excretion was 32% at day 45 ... Three patients receiving Pyridorin had also converted from macroalbuminuria [>300 mg albumin excretion/24h] to microalbuminuria [<300 mg/24h] by the end of the treatment period.”[253]
  - “Should” primarily work on extracellular proteins (connective tissue, some parts of glomeruli, retina), as phosphorylated upon (regulated) intracellular uptake.
  - Dose: 500 mg PM dihydrochloride (~300 mg “elemental” PM) used in Phase II study, yields similar plasma levels to 2 g/L used in rodent studies.
  - Possible neuropathic side-effects: observed with pyridoxine: cutoff dose controversial. LOAEL 500 mg pyridoxine; NOAEL 200 mg. No increase in pyridoxine seen with PM, 253, but in vitro, pyridoxine and pyridoxamine of equal neurotoxicity.[254]
    - Unusual B6 metabolism in CR rodents[255] and possibly humans. 191
- Intervention 2: Benfotiamine
  - Dose: 320-400 mg first month, 120-160 mg thereafter in diabetic neuropathy.
- Intervention 3: Interprandial arginine
  - Carbonyl scavenger. 238 Reduces AGE accumulation in diabetic rodent kidneys[275],[276],[277] and hearts;[278] results in humans inconsistent.[279],[280]
  - Dose: 2g/day, taken ~40 min before meals to address postprandial dicarbonyl surge.[281]

Alternate (even more speculative!) CR-Mimetic Make-the-Case Speculations:
- SIR2, PNC1 Gene Silencing [282] ,[283]
  - Free NAD+ or NAD:NADH ratio prevents inhibition of SIR2.
  - Yeast. Mammals do not form rDNA circles; “aging” in yeast entirely replicative (relevance to mammals questionable: postreplicative brain, heart, muscle tissues).[284] If Guarente correct on mechanism (CR in yeast causes reduced glycolytic flux, hence higher NAD:NADH), possibly irrelevant (mammals apparently show no reduced metabolism in response to CR). If Anderson correct (PNC1 converts nicotinamide to nicotinic acid), greater potential relevance.
- Intervention: R(+)-lipoic acid.
  - See above: lowers cytosolic and mt NADH:NAD.
- Insulin/IGF1 Signaling [285]
  - ”Reduced signaling of insulin-like peptides increases the [max] life-span of nematodes, flies, and rodents.” 285
  - CR lowers insulin and IGF-1
  - Anti-mitotic (anti-cancer; preserves immune reserve and prevents autoimmunity (CR)?)
  - Reduced mtROS seen in Ames dwarf mice.[286]
  - Reduced IGF-1 signalling in mammals does not fully reproduce CR effects.[287],[288],[289]
- Intervention: Benfotiamin?
  - Monnier: Activation of PPP by B might reduce glycolytic flux, reduce deleterious insulin-related signaling.[290]
  - I don’t buy it.
    - Benfotiamin effective during intracellular hyperglycemia in cells whose uptake of glucose (via GLUT1) is not insulin-mediated.
    - Likely little reduction in glycolysis per se, esp in normoglycemic folk.
    - Irrelevant to heart, skeletal muscle.
- Negative intervention: Avoid GH-boosting supplements, hGH injections, etc.
- PMRS Modulation
  - See mt discussion above. De Grey’s e- shunt through PMRS 189 would create mild REDOX stress. Lawen et al [291] suggest ‘rescue’ of bioenergetically deficient cells by taking e- at PMRS; could make PMRS e- less toxic, extend CR LS bennies per MiFRA.
- Intervention: CoQ10?
  - Tissue uptake negligible outside of liver and spleen,[292] so cardio effects not due to mt enrichment. Lawen et al 291 show that CoQ unloads e- from PMRS; proposed as mechanism.[293]
  - Several LS Studies Negative:
  - Favorable, but not definitive, result from Bliznakov:[294] injects 50 mcg/week/mouse, beginning at ~510 days (~51 human years); av’g LS increased to ~728 from ~650 days (12% increase); max LS either unrecorded or 1084 days. Promising, but no clear effect vs. optimally-cared-for rodents; pharmacokinetic issues.
  - Null effect from 10 mg/kg/day oral CoQ in rats (scales to 213 mg[295]).[296] Older (16 mo) dams of experimental populations supplemented during pregnancy; complications? (Not teratogenic; did not affect litter size; no difference in BW thru’out lifespan).
  - Another null effect from Lonnrot et al mice:[297] prob. Scales to ~100 mg/day.
  - Null effect, or even slightly unfavorable, LEF LifeSpan studies, alone or in combination; 4 dose 86 mg.
  - Steve Harris[298] unpublished LS study at human-equivalent 500-750 mg/day: robust 23% curve-squaring. Video suggests v. healthy.
  - Extended LS from CoQ-free diet in Clk-KO flatworms[299] irrelevant to mammals: mutant strain probably CoQ-deficient, so CoQ-free diet causes halt in respiratory chain.[300]
  - Most studies[301] (but not all[302]) report lower tissue CoQ with CR; not relevant to present mechanism or pharmacokinetics of CoQ itself, and could be a limiting factor rather than a mechanism.
  - Dose: 750 mg/day, per Harris study; 100 insufficient, per Lonnrot, and >200 mg normally required to achieve therapeutic levels in human heart failure (>2.0 mcg/L), w/ highly variable bioavailability.[303] ,[304] 1200 mg needed for clear-cut effect in Parkinson’s;[305] responders in prostate cancer case series all achieved > 3.0 mcg/L, nonresponders all < 2.0 mcg/L, at 600 mg/day.[306] Use softgels or dissolve in oil; take with fat-containing meal.
- Carnosine?
  - Reverses cellular senescence in vitro.[307] ,[308]
  - In vitro!!
  - -Role of cellular senescence in aging debatable; possible indirect effects thru’ secreted factors.[309]
  - Selective inhibition of cancer cell growth: no effect on normal[310] or embryonic stem cells.[311]
  - In vitro!!
  - In SAM-P mice, increases mean LS by 20%, reduces some ‘aging’ phenotypes (hair fullness and color, skin ulcers, periopthalmic lesions, spinal curvature) and normalizes binding of NMDA receptors, MAOb, Na/K-ATPase.[312]
  - SAM-P mice are fuckups; still much shorter LS than normal mice.
  - No effect on max LS.
  - SAM-P mice have ‘naturally’ low carnosine levels.[313]
  - Painfully speculative: zero data on normal, healthy organisms, no epidemiology, nada. But intriguing …
  - Dose: Good question!
    - 100 mg/kg used in SAM-P 312 and in studies on ischemic assaults.[314],[315],[316] Human-equivalent ~1000 mg.
    - In rodent studies, human-equivalent 500 mg/day does not elevate brain or muscle levels [317],[318],[319] and do not protect rodent vascular system from fructose-rich diets.[320] Human-equivalent doses of ~945 mg a day raise tissue levels 317, 320 and protect vascular system.[321]
    - However, carnosinase enzyme degrades carnosine to beta-alanine + histidine. Rodents only have a nonspecific dipeptidase with carnosinase activity by default. Humans have an additional, specific serum carnosinase;[322] ,[323] higher than proportional scaled doses will be required.
    - Strangely, oral supplementation leads to increased urinary C, yet no increase in plasma levels, in humans;[324],[325] “it seems that absorbed carnosine may be very rapidly cleared from the plasma and sequestered in some compartment before it is excreted by the kidneys.” 324 Alternately, hydrolyzed and resynthesized?
    - IAS claims lower doses reduce urinary MDA;[326] MDA is a bad marker of oxidative stress, and in any case also seen in rodent studies using TBARS, despite a failure to increase tissue levels (associated with conserved vitamin E in liver). 318 Uncontrolled, unpublished trial by unpublished ‘researcher.’
  - Dose Conclusion: Substantially > 1 g will be required; 1500 mg?
    - High-protein diet leads to higher C absorption;[327] Met-free diets reduce absorption.[328] Prob due to density or activity of dipeptide transporters.
    - Various dipeptides (eg glycyl-L-proline, alanine) compete with C for transporters, inhibit absorption:[329] take on an empty stomach.
- ALCAR issues. Human RCTs for Alzheimer’s disease;[330] also seems effective for spatial learning in healthy young subjects.[331]
  - Improves various aspects of mt structure and function in rodents;[332],[333],[334] supplementation in middle-aged (16 mo) rats had mixed effects on pathology but improves survival to 22 mo (38/45 vs. 29/45 survivors).[335] But Hagen and Ames reported increased mtROS production in old supplemented rodents.[336] Yikes!
  - Only observed in old rodents. 336
  - Observed at v. high dose: 1.5% in drinking water, scales to 12.9 g.[337] In dose-ranging studies, “lower concentrations of ALCAR (0.15% and especially 0.5% [scales to 1.29 to 4.3 g]) ameliorated the age-associated decline in ambulatory activity (TABLE 2) and mitochondrial cristae loss in the dentate gyrus of the hippocampus (FIG. 12) more effectively [MR's emphasis] than the 1.5% dose. The lower doses had no effect on protein oxidation, in contrast to the 1.5% dose, which caused an increase in protein carbonyls in the brain. Furthermore, lower doses (0.15%) also reduced the age-dependent increase in malondialdehyde … more effectively than the 1.5% dose”. 332 Likewise, “Lower doses of ALCAR, that is, below 1.0% (wt/vol) in the drinking water did not increase hepatocellular oxidative stress”. 333
  - Tissue-specific effects: In heart, even high dose (1.5% in drinking water, = 12.9 g) caused “no alterations in oxidant production in isolated cardiac myocytes ... Furthermore, myocardial levels of ascorbic acid, which decline significantly (P < 0.02) with age (FIG. 5) did not exhibit a further ALCAR-induced decline”. 333 (Cf. tissue-specific effects of aging and CR on mt function[338],[339]).
  - ALCAR reduces mtDNA deletions in cochlea per Seidman at massive dose (17.7 g, scaled); 202 again, tissue-specific (local bioavailability eg)?
  - mt structural improvements (retarded loss of cristae) 332 at lower doses argue for direct benefit, in addition to metal chelation, improved bioenergetics, and other antioxidant mechanisms.
  - R(+) corrects ALCAR-induced mtROS even at high dose; 334, 336 as argued above, likely a real, primary effect
  - Dose: 1.29 to 4.3 g – again, suggestive coincidence, as this is the standard therapeutic range for AD. 330 Conservatives might lean lower than lower end, but not clinically supported.