An Anti-Aging Drug Cocktail


Drug combinations work to synergistically extend the life- and healthspan in worms.

The most successful drug combination tested almost doubled lifespan, a feat never reported before. Furthermore, more than half of the worms receiving the drug combination were still in optimal health after all control animals had died! Finally, the authors show that these drugs also extend the lifespan of fruit flies and in fact the same combination that almost doubled worm lifespan was also the most successful one at extending the lifespan of fruit flies.

In a new paper published on the preprint server bioRxiv Jan Gruber and colleagues test various combinations of life extending drugs in worms.

Preprint servers allow scientists to publish their article to the wide world for anyone to criticize before submitting it to a classical journal for publication.

Furthermore they allow for rapid dissemination of research results as the publication in a peer-reviewed journal can take months to years between submission and publication.

While around a thousand compounds have been found to extend lifespan in at least one study in a model organism few studies have been conducted using combinations of two or more compounds.

A rare example includes the combination of rapamycin with metformin which was shown to outperform either drug alone in mice.

The authors started by identifying well known mechanisms for lifespan extension based on a literature review. Then they looked for drugs that influence these mechanisms and had previously been found to extend lifespan leading to the selection of 12 compounds for testing in this study.

Next the researchers tested these compounds in roundworms, a common model organism in aging research. When tested in isolation five drugs significantly extended mean and maximal lifespan: Psora-4, rifampicin, rapamycin, metformin, and allantoin.

Next the researchers tested all pairwise combinations of these five compounds. The combination of metformin and rapamycin, both at optimal doses, did not lead to a further increase in lifespan.

However if the combination was tested at suboptimal doses than lifespan was further increased.

Given that the optimal dose of metformin and rapamycin in mice is unknown the beneficial effect of the combination observed in the mice study mentioned before could possibly be the result of suboptimal concentrations of both drugs.

Two combinations, rifampicin + rapamycin and rifampicin + Psora-4 all at optimal concentrations did result in synergistic increases in lifespan.

Next the researchers tested triple combinations of the various compounds. As testing all combinations would be impractical (220 different lifespan tests would be necessary) the researchers decided to first try the combination of the 3 most successful compounds so far (rapamycin + rifampicin + Psora-4).

However this combination resulted in a shorter lifespan than the two successful pairwise combinations. Next, the researchers decided to test the two pairwise combinations from before with allantoin added as the third drug.

They chose this combination because allantoin shares no mechanistic overlap with the other drugs. Both of these triple combinations resulted in a significant extension of mean and maximal lifespan with the most successful one (rapamycin + rifampicin + allantoin) resulting in a doubling of mean lifespan! The authors remark that this is the largest lifespan extension ever observed by a drug intervention initiated in adult worms.

Abbreviations: Rif = rifampicin; Rap = rapamycin, Allan = allantoin. Image credit: Sven Bulterijs Certain interventions that result in lifespan extension reduce fertility but neither of the triple drug combinations reduced total fertility and actually slightly extended the fertile period of life.

Furthermore, both drug combinations extended the period of life spend in good health (= the healthspan).

In fact, more than half of the worms receiving the triple drug combination were still in optimal health after all control animals had died! Old animals on the triple drug combinations were indistinguishable from young control worms when judged by spontaneous movement.

Worms really show a significant decrease in spontaneous movement with age. Finally, the two successful triple drug combinations also significantly increased resistance to oxidative and heat stress.

Total mortality is made up of two distinguishable parts: age-dependent and age-independent mortality. The age-dependent mortality is a measure for the rate of aging.

So the authors tested which mortality rate was reduced by the drug combinations. Interestingly, the rifampicin + Psora-4 + allantoin combination significantly reduced the rate of age-dependent and age-independent mortality showing that this drug slows down aging in addition to making them more robust at young ages.

Abbreviations: Rif = rifampicin; Rap = rapamycin, Allan = allantoin. Image credit: Sven Bulterijs

The evolutionary distance between fruit flies and worms is larger than between fruit flies and humans. Hence the fact that the drug combinations worked in both fruit flies and worms offers hope that their lifespan extending effect may be conserved in humans.

The two drug combinations (Rap + Rif and Rap + Rif + Allan) significantly extended mean and maximum lifespan in fruit flies.

How the war on Cholesterol caused our diabetes epidemic


Cholesterol is a molecule required by every cell of the body in fairly large amounts. It can be easily synthesised by these cells, or taken up by them from LDL and other ApoB lipoproteins, but cannot be broken down. Cholesterol is not soluble in water, and thus must be carried through the blood on lipoprotein particles. When the cholesterol produced or taken up by the cells of the body becomes surplus to requirements it is extracted by HDL (ApoA1 lipoproteins) and carried back to the liver for disposal as bile salts and acids (most of this cholesterol is reabsorbed and recycled, but there is also a variable amount lost in faeces). Reverse cholesterol transport (RCT) is the term used for this extraction of unneeded cholesterol. Here we describe a simplified version of reverse cholesterol transport, how this has been modified by new research into HDL, and we explain the effect of raising or lowering insulin and insulin sensitivity on RCT.

This video gives a good overview of the systems we’ll be describing. (The brain has its own, largely separate cholesterol system which we’ll ignore for now).

Cholesterol and insulin

We have about 30g of cholesterol in our bodies, and synthesise well over a gram a day. Only 10% of this is synthesised in the liver, and even less if we eat cholesterol or have a reduced requirement. Our requirement goes up when we are growing (cells are expanding and new cells being made) and down when we are fasting or losing weight (when fat cells and glycogen cells are shrinking, and autophagic processes are clearing unwanted cells). Thus it makes sense, and helps to keep cholesterol in balance with requirements, that cholesterol synthesis is stimulated by insulin (the fed state hormone) and inhibited by glucagon (the fasting state hormone).[1] An additional check on cholesterol synthesis in the fasting state is the activation of AMPK by the ketone body B-hydroxybutyrate.[2] No surprises then that cholesterol synthesis is found to be increased in type 2 diabetes.[3]

If scientists want to create the early signs of heart disease in animals, they need to feed them doses of cholesterol much larger than the total capacity of the body to make cholesterol.[4] However, Jerry Stamler, one of the founding fathers of the diet heart hypothesis, found in the early 1960’s that animals treated in this way got better when the cholesterol feeding stopped – unless they were given extra insulin.[5] This vital clue was missed in the later rush to change our diets – Jerry Stamler advised the population to avoid egg yolk and replace fat with refined carbs, yet human diets never supplied the amount of cholesterol he fed his animals – unfortunately, the new, modified human diet would start to increase insulin to the high levels seen in those chickens once the diabesity epidemic got underway.


Reverse Cholesterol Transport

Fortunately our gut and liver cells make a protein called ApoA1, which the liver turns into something called a nascent HDL particle. Unlike VLDL and the other ApoB particles, which are released from the liver as large, fat and cholesterol laden spheres, HDL is produced in an embryonic state, just a few proteins with little if anything in the way of lipids (lipid-poor ApoA1), and only becomes what we call HDL by performing its cholesterol-gathering role out in the body.


If we focus on the cells believed to play the major role in atherosclerosis, macrophages (large immune cells) which can turn into foam cells if they become overloaded with cholesterol, we can see HDL at work. Macrophages clear the blood of infectious agents and damaged particles, and have a particular affinity for oxidised LDL particles (oxLDL).[6] LDL becomes oxidised if it stays in the blood too long (more likely with higher levels or small, dense particles) and is exposed to excessive glucose and fructose levels after meals, or to smoking and other oxidative stressors.[7,8,9] Brown and Goldstein, who won the Nobel Prize for discovering the LDL receptor, estimated that 30-60% of LDL is cleared from circulation by macrophages. (Macrophages exposed to excess insulin increase their uptake of oxLDL by 80%).[10] The oxLDL is then broken down and the cholesterol stored – remember it can’t be broken down. As in other cells, any excess is sent to the surface of the cell, to transporters and other structures that make it available for HDL to pick up, as free cholesterol (cholesterol efflux). If this doesn’t happen for some reason, over a long period, there’s a risk of foam cell formation and atherosclerosis. (Macrophages exposed to excess insulin decrease their efflux of cholesterol to HDL by 25%).[10]

LCAT and esterification

After HDL picks up free cholesterol, this is esterified by an enzyme called lecithin cholesterol acyltransferase (LCAT), making the HDL particle larger. Cholesteryl ester (CE) is cholesterol joined to a fatty acid, usually an unsaturated fatty acid, which is supplied from the phospholipids also picked up from cells by HDL. The more effectively HDL can esterify cholesterol, the sooner it can return to pick up more from the macrophage (or other cell) – this is called HDL efflux capacity – and the phospholipids found in egg yolk have been shown to increase HDL efflux capacity.[11] Phospholipids, found in all whole foods, especially fatty ones like eggs, nuts, seeds, liver, shellfish, and soya beans, are good things to have in your diet; you won’t get them from eating flour, sugar, and oil.


Cholesteryl oleate – a cholesteryl ester


CETP – swapping cholesteryl esters for triglycerides

HDL renews itself in the bloodstream by moving cholesteryl esters onto VLDL and other ApoB particles, in more-or-less equal exchange for triglycerides (TGs), through a banana-shaped protein tunnel called Cholesteryl Ester Transport Protein (CETP) which docks between ApoA1 and ApoB particles. HDL can then shed the TGs picked up to help feed cells along its path (as ApoB particles also do), turning them into free fatty acids and glycerol by the action of lipase enzymes. However, the CETP exchange is another place where things can go wrong. If there are too many TGs on VLDL, and too many TG-rich VLDL particles, and fats are not being burned by the body (yes, we’re talking about insulin resistance again), then the piling of TGs onto HDL via CETP will result in its recall to the liver after limited efflux.[12] Carrying lots of TGs back to the liver that made them is not a good use of HDL’s time. And the cholesterol esters being transferred to former TG-rich VLDL is what makes the “Pattern B” lipoproteins, small dense LDL, which are more likely to oxidise and more easily taken up by macrophages. LDL really, once it’s done its job of delivering fat, cholesterol, antioxidants and proteins to cells that need them, ought to be helping in reverse cholesterol transport by ferrying the extra cholesterol esters it received from HDL back to the liver. Large, cholesterol-dense LDL particles – “Pattern A” – are better at this. Small, dense LDL particles aren’t taken up as avidly by the liver, so tend to stay in circulation and oxidize. Hence the TG/HDL ratio is a critical predictor of cardiovascular risk, whether or not we factor in LDL.

The exchange via CETP action is thought responsible for the inverse relationship between levels of TG and HDL-C. Specifically, the larger the VLDL pool (higher TG level), the greater the CETP-mediated transfer of CE from HDL to VLDL in exchange for TG, resulting in TG-rich small, dense HDL which are catabolized more rapidly, leading to low levels of HDL-C. These small, dense HDL also have reduced antioxidant and anti-inflammatory properties. Thus, the greater the increase in hepatic VLDL-TG synthesis and secretion that characterizes insulin-resistant/hyperinsulinemic individuals, the lower will be the HDL-C concentration.[12]


Insulin LDL

Insulin resistance in this population (n=103,000) was stratified by tertiles of TG and HDL, with the insulin sensitive tertile having a mean TG/HDL ratio of 1.1 [13]

Fasting, weight loss, and LDL

People who are naturally lean and active and have good insulin sensitivity are at very low risk of cardiovascular disease; they tend to burn fat and have low TG/HDL ratios on any diet. Paradoxically, LDL rises sharply when such people fast for long periods.[14] Despite this, no-one as far as we know has ever suggested that not eating enough causes atherosclerosis. Of course TGs and insulin also fall, while HDL stays the same. But strangely, this rise in LDL does not happen in obese people or people with atherosclerosis.[15]


Fasting LDL Apo B

In healthy lean individuals, LDL and ApoB rise as Insulin-like growth factor falls during a fast.[14]

Fasting Chol athero

In obesity or T2DM, or in this case a patient with arteriosclerosis, cholesterol and LDL do not rise during a fast.[15]

Phinney and colleagues found that LDL first fell, then rose significantly, during major weight loss. They calculated that this was due to the delayed removal of around 100g extra cholesterol from the adipose of obese people. LDL became normal when a weight maintenance diet replaced the (low fat, reduced calorie) weight loss diet.[16]
Think about this – all of this extra 100g of cholesterol, 3x the usual whole body content, was eventually removed by reverse cholesterol transport. Some of it ended up on LDL, increasing the LDL count to the level where statins would be indicated according to guidelines. This did not prevent its removal. There is no “LDL gradient” that forces cholesterol back into the body. The LDL level doesn’t tell you whether cholesterol is coming or going – the TG/HDL ratio is the best guide to that.[17]


Hepatic lipase – burning fat.

ApoA1 and HDL production increases the release of hepatic lipase, so in a sense ApoA1 is a fat-burning protein, which helps to explain why eating fat increases ApoA1 output.[18, 19] More lipase means lower TGs all round. So, making more HDL can lower TGs, just as making too many TGs can lower HDL – but only the latter is likely to be harmful.

Of course, a low fat, high carbohydrate diet decreases ApoA1, but this doesn’t mean it’s bad if you’re insulin sensitive and have low TGs (and low LDL) eating such a diet, as many people do; the lower lipid circulation all round probably just means that less ApoA1 will be required for equilibrium. However, the old assumption that the lower fat higher carb diet is the “Prudent” diet hasn’t aged well.

We have previously reported that apoA-I and HDL directly affect HL-mediated triacylglycerol hydrolysis, and showed that the rate of triacylglycerol hydrolysis is regulated by the amount of HDL in plasma.

The antioxidant and antiinflammatory benefits of HDL.

Reverse cholesterol transport is the core business of HDL, but it isn’t the only business; HDL is like a busy doctor with a useful bag of healing tricks trundling up and down your bloodstream. For example, HDL carries an antioxidant protein, PON1. When a fatty hamburger meal rich in lipid peroxides was fed to 71 subjects, those with higher HDL experienced a much smaller rise in oxLDL.[20]

The pre-meal HDL level was associated with the extent of the postprandial rise in oxidized LDL lipids. From baseline to 6 h after the meal, the concentration of ox-LDL increased by 48, 31, 24, and 16 % in the HDL subgroup 1, 2, 3, and 4, respectively, and the increase was higher in subgroup 1 compared to subgroup 3 (p = 0.028) and subgroup 4 (p = 0.0081), respectively. The pre-meal HDL correlated with both the amount and the rate of increase of oxidized LDL lipids. Results of the present study show that HDL is associated with the postprandial appearance of lipid peroxides in LDL. It is therefore likely that the sequestration and transport of atherogenic lipid peroxides is another significant mechanism contributing to cardioprotection by HDL.

Tregs or T regulatory cells are a type of immune cell that switches off inflammatory responses. They are also a type of cell that takes up HDL, and HDL selectively promotes their survival. This is a good thing.[21]

Can LDL help in reverse cholesterol transport?


The answer is yes – if it’s large LDL particles (Pattern A), not so much small dense ones. Triglycerides and VLDL, on the other hand, are no help at all.

There are two pathways by which RCT can occur. In the first, the scavenger receptor class B type 1 (SRB-1) mediates hepatic uptake of CE from HDL particles without uptake of apoA-I or the whole HDL particle [74]. In the second pathway, cholesteryl ester transfer protein (CETP) catalyzes the transfer of CE from HDL to apoB-containing lipoproteins (VLDL and LDL) in exchange for TG from the apoB-containing lipoproteins (Fig. 1) [21, 75]. This exchange results in apoB-containing lipoproteins which are enriched with CEs and depleted of TGs, and HDL particles which are depleted of CEs and enriched with TGs. The TG-rich and CE-poor HDL particles are catabolized faster than large, CE-rich HDL (apoA-I FCR is increased as noted in Fig. 1), a finding resulting in lower levels of HDL-C in the setting of high TG levels [76]. The apoB-containing lipoproteins, now enriched in CE, can also be taken up by the liver receptors as previously described [75]. When TG levels are high, the apoB particles are TG-enriched and hepatic lipase then hydrolyzes the TGs within the TG-rich LDL to release FFAs, a process which remodels the LDL particles into smaller and denser LDL particles which can enter the arterial intima more easily than larger LDL particles, thus making them more atherogenic (Fig. 1). Small, dense LDL particles also bind less avidly to the LDL receptor, thus prolonging their half-life in the circulation and making these particles more susceptible to oxidative modification and to subsequent uptake by the macrophage scavenger receptors [12].

An unusual experiment (using a radioactive nanoemulsion mimetic of LDL) showed that LDL cholesterol is removed from circulation more rapidly in resistance-trained healthy men than in sedentary healthy men. oxLDL was 50% lower in the resistance-trained men – but total LDL levels were the same, probably as a result of increased beta-oxidation (fat burning).[22]

Why are doctors being confused about HDL and reverse cholesterol transport?

There’s a trend in mainstream medicine to be dismissive of HDL and treat reverse cholesterol transport as unimportant; LDL lowering is the thing. New evidence from genetics, epidemiology, and drug trials is increasingly misinterpreted in this way – probably because drugs that increase HDL have, with some exceptions, been failures. However, drugs that raise HDL, and lower LDL, by inhibiting CETP are not helping either particle do its job; so far, they have neither decreased nor increased the rate of heart attacks in people taking them. Drugs that raise HDL by increasing ApoA1 and nascent HDL output, like the fibrates (e.g. gemfibrozil), do decrease CHD – but only in people with lower HDL and higher TGs! Moderate alcohol use, which also increases ApoA1 output, seems to have a similar effect, though the first randomised controlled trial of this observational hypothesis is only beginning.[23, 24] Even statins help with RCT by decreasing the synthesis of cholesterol in peripheral tissues, thus leaving more room on HDL for efflux cholesterol – again, statins only seem to reduce CHD in the subgroup of people with lower HDL. Clearly reverse cholesterol transport is very important, and efficient reverse cholesterol transport can best explain why so many people with high LDL and high cholesterol do enjoy long lives free from cardiovascular disease. Some ApoA1 genes that especially promote RCT are associated with reduced CVD risk, notably ApoA1 milano – which is actually associated with low HDL, because HDL clearance is so rapid – a paradox which highlights the trickiness of measuring a dynamic process across all tissues only by what appears in the blood.[25] Efficient RCT is associated with lean genes, but it’s largely something you have to work for – eating right, exercising, and looking after yourself in various ways – including giving up smoking, or not starting – which may be why the drug industry has largely given up on it.[26]

We observed that normolipidemic smokers present higher total plasma and HDL phospholipids (PL) (P < .05), 30% lower postheparin hepatic lipase (HL) activity (P < .01), and 40% lower phospholipid transfer protein (PLTP) activity (P < .01), as compared with nonsmokers. The plasma cholesteryl ester transfer protein (CETP) mass was 17% higher in smokers as compared with controls (P < .05), but the endogenous CETP activity corrected for plasma triglycerides (TG) was in fact 57% lower in smokers than in controls (P < .01). Lipid transfer inhibitor protein activity was also similar in both groups. In conclusion, the habit of smoking induces a severe impairment of many steps of the RCT system even in the absence of overt dyslipidemia.

The latest study on very, very high HDL – why isn’t it good?

Last year we wrote about the CANHEART study, which seemed to show adverse health effects of higher HDL. We wrote then that this was probably showing an effect of alcoholism, hereditary CETP defects, and other factors, and not an increase in heart disease. A new study allows us to look at this problem in more detail.[27]

When compared with the groups with the lowest risk, the multifactorially adjusted hazard ratios for all-cause mortality were 1.36 (95% CI: 1.09–1.70) for men with HDL cholesterol of 2.5–2.99 mmol/L (97–115 mg/dL) and 2.06 (1.44–2.95) for men with HDL cholesterol ≥3.0 mmol/L (116 mg/dL). For women, corresponding hazard ratios were 1.10 (0.83–1.46) for HDL cholesterol of 3.0–3.49 mmol/L (116–134 mg/dL) and 1.68 (1.09–2.58) for HDL cholesterol ≥3.5 mmol/L (135 mg/dL).

Those are some very high HDL levels, and not surprisingly fewer than 4% of men and even fewer women had HDL levels so high that they were associated with any extra risk.
Compare that with 40% of both men and women having low HDL levels that were associated with an equally elevated risk!
Further, the risk associated with very high HDL, though it does include cardiovascular deaths, doesn’t seem to include much increased risk of heart attacks and strokes.


This is consistent with alcoholism (a confounder not measurable with accuracy, as we described in the CANHEART analysis) increasing deaths from heart failure, cancer, and other causes, and with no further benefit (but maybe not much harm overall) from CETP variants elevating HDL.[28] Furthermore, interactions between heavy alcohol consumption and genes associated with higher HDL have been noted in some populations.[29]
Note that the HDL level associated with lowest heart disease and stroke incidence in this study is well to the right of the bell curve of population HDL distribution. Most of these people could have done with more HDL.
Madsen et al discuss their results soberly; although they fail to discuss the potential role of alcohol, which would explain the exact pattern of increased mortality seen well, and don’t highlight the 10-fold larger impact associated with low HDL in their study, there is nothing biased about their analysis. The European Heart Journal’s editorial was also worth reading.[30]


However, as reported in medical media, the message changed a bit.

“It appears that we need to remove the focus from HDL as an important health indicator in research, at hospitals and at the general practitioner. These are the smallest lipoproteins in the blood, and perhaps we ought to examine some of the larger ones instead. For example, looking at blood levels of triglyceride and LDL, the ‘bad’ cholesterol, are probably better health indicators,” he notes.

Well yes, looking at everything is good, and TGs and the TG/HDL ratio as well as LDL will give you extra information about the likely reasons for low HDL and whether you need to worry about it. However, Denmark, where the extremely high HDL study was done, is a place where high LDL (the ‘bad’ cholesterol, remember) is associated with lower all-cause mortality in those over 50 free from diabetes or CVD at the start of the study.[31] Over 50 is when most CVD and type 2 diabetes is diagnosed, so LDL might not be all that informative unless you can look at the subclasses of oxLDL, sdLDL, particle number, and so on (of course part of the effect of LDL in Denmark will be due to that country’s higher dairy fat intake, which will also raise HDL and LDL particle size, maybe helping to explain why the association is so favourable in that population).

If we look at the PURE study, higher fat consumption is associated with both higher LDL and higher ApoA1 and HDL, with saturated fat (like all fat types) tending to improve the ApoB/ApoA1 ratio.[32] This is consistent with many other lines of evidence.

Intake of total fat and each type of fat was associated with higher concentrations of total cholesterol and LDL cholesterol, but also with higher HDL cholesterol and apolipoprotein A1 (ApoA1), and lower triglycerides, ratio of total cholesterol to HDL cholesterol, ratio of triglycerides to HDL cholesterol, and ratio of apolipoprotein B (ApoB) to ApoA1 (all ptrend<0·0001).

This is just what fat-burning does, and there’s maybe not a lot of reason to think it’s good or bad per se. What is good about it is, that fat burning lowers insulin. Insulin is what makes your cells hoard cholesterol, and it’s also one of the things that can mess with reverse cholesterol transport. If you’re making or using excess insulin, the switch to a fat burning metabolism allows the insulin to normalise and causes your cells, including the macrophages, to let go of cholesterol – and when they do, the lipoproteins are there ready to carry it away.


Reverse cholesterol transport manages cholesterol flux through all cells and helps us reach a healthy old age.

LDL cholesterol is not a reliable guide to the state of cholesterol flux unless TG/HDL (and HbA1c) are factored in as well. LDL may increase when cholesterol is being removed or in states where it is not being taken up by cells.

Reverse cholesterol transport can remove prodigious amounts of cholesterol from the body during weight loss.

Excessive triglycerides due to insulin resistance can impair reverse cholesterol transport, as can smoking.

Various nutritional factors found in whole food diets have been found to assist in reverse cholesterol transport (including phospholipids, CLA, and polyphenols).

HDL in the high (if not the “extremely high”) range usually correlates with efficient reverse cholesterol transport and has benefits for cardiovascular health, inflammation, antioxidant status etc, but people with HDL outside (higher or lower than) the ideal range can be equally healthy if their overall metabolic health (insulin sensitivity) is good.

The TG/HDL ratio is a good measure of insulin sensitivity, and if excessive can be improved by lowering excessive insulin levels. A low carb diet, intermittent fasting, exercise, or weight loss are all effective ways to correct the TG/HDL ratio.



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[30] Barter PJ, Rye K-A. HDL cholesterol concentration or HDL function: which matters? European Heart Journal. 2017; 0: 1–3

[31] Bathum L, Depont Christensen R, Engers Pedersen L, Lyngsie Pedersen P, Larsen J, Nexøe J. Association of lipoprotein levels with mortality in subjects aged 50 + without previous diabetes or cardiovascular disease: A population-based register study. Scandinavian Journal of Primary Health Care. 2013;31(3):172-180. doi:10.3109/02813432.2013.824157.

[32] Mente A, Dehghan M, Rangarajan S et al. Association of dietary nutrients with blood lipids and blood pressure in 18 countries: a cross-sectional analysis from the PURE study.
Lancet Diabetes Endocrinol 2017 Published Online August 29, 2017
PURE lipids and BP

The Track Your Plaque Guide to At-Home Laboratory Testing


The Track Your Plaque Guide to At-Home Laboratory Testing is meant help the user understand the significance of the blood and salivary testing provided through the Track Your Plaque program. It is not meant to diagnose any condition, nor is it intended to provide a treatment program; this can only be provided by a healthcare provider familiar with your health situation.

For “normal” or “reference range” values for each test, please refer to the “Reference Range” cited with the return of your test results.

Cardiometabolic Profile (Total cholesterol, LDL cholesterol, triglycerides, c-reactive protein, HbA1C) Blood spot only

LDL cholesterol

LDL cholesterol is meant to reflect the amount of cholesterol present in the low- density fraction of lipoprotein blood particles (as opposed to those in the high- density fraction, or HDL). It is the basis for most conventional predictions of heart disease risk, since it has been statistically connected to occurrence of heart attack and is recoverable from atherosclerotic plaque when examined.

LDL is often called “bad” cholesterol, since the higher the LDL, the greater the likelihood of cardiovascular events like heart attack, an observation documented repeatedly from the Framingham Study to other populations (Kannel WB et al 1979; Kannel WB 1995; ATP-III 2001). Despite the controversies the drug industry has created by its overenthusiastic marketing of the LDL-reducing statin drugs, reduction of LDL cholesterol, whether with statin drugs, diet, fibers like oat bran or ground flaxseed, or other strategies has been confidently tied to reduction in heart attack (Selwyn AP 2007).

In the Track Your Plaque program, we aim for an LDL of 60 mg/dl or less, a level consistent with maximal reduction of cardiovascular risk and coronary plaque (Ballantyne CM et al 2008).


ATP III. Evaluation, and Treatment of High Blood Cholesterol in Adults (Adult Treatment Panel III). Executive Summary of the Third Report of the National Cholesterol Education Program (NCEP) expert panel on detection. JAMA. 2001;285(19):2486-97.

Ballantyne CM, Raichlen JS, Nicholls SJ et al for the ASTEROID Investigators. Effect of Rosuvastatin Therapy on Coronary Artery Stenoses Assessed by Quantitative Coronary Angiography: A Study to Evaluate the Effect of Rosuvastatin on Intravascular Ultrasound-Derived Coronary Atheroma Burden. Circulation 2008;117:2458-66.

Kannel WB, Castelli WP, Gordon T. Cholesterol in the prediction of atherosclerotic disease. New perspectives based on the Framingham Study. Ann Intern Med 1979 Jan;90(1):85−91.

Kannel WB. Range of serum cholesterol values in the population developing coronary artery disease. Am J Cardiol 1995 Sep 28;76(9):69C-77C.

Selwyn AP. Antiatherosclerotic effects of statins: LDL versus non-LDL effects. Curr Atheroscler Rep 2007 Oct;9(4):281−85.


The liver produces a class of particles called “very low-density lipoproteins,” or VLDL, comprised principally of triglycerides. VLDL production is fueled by poor insulin responses triggered by sedentary life, obesity, and over-reliance on foods that increase blood sugar (Adiels M et al 2008). Increased triglycerides (and VLDL) signal increased risk for heart disease (McBride P 2008).

Some people have high triglycerides due to genetic factors (such as a deficient form of the enzyme, lipoprotein lipase, that clears triglycerides from the blood). In this situation, triglycerides can range as high as several thousand mg/dl. Far more commonly, triglycerides are high (100 mg/dl to 500 mg/dl) due to excess weight, indulgence in processed carbohydrates, and resistance to insulin (metabolic syndrome), the very same triggers for VLDL.

When VLDL particles in the blood come into contact with LDL and HDL particles, triglycerides from VLDL are shared with LDL and HDL particles, which also become bloated with triglycerides as a result. Triglyceride-loaded LDL and HDL are a ready target for enzymes in the blood and liver that reconfigure them into smaller versions, small LDL and small HDL. Small LDL and HDL are undesirable particles that powerfully stimulate growth of atherosclerotic plaque in the heart’s arteries and elsewhere. Thus, excess triglycerides and VLDL lurk behind the creation of small LDL and small HDL (Berneis KK, Krauss RM 2002). This process begins at a triglyceride level as low as 45 mg/dl, becomes progressively worse with increasing levels of triglycerides, substantial with triglycerides >150 mg/dl.

Increased triglycerides are also a feature of the metabolic syndrome, or pre- diabetes, and diabetes (Adiels M et al 2008). Increased triglycerides from VLDL, along with low HDL and small LDL are very common. This is because the poor insulin responses in these conditions cause the liver to produce VLDL particles without restraint (Verges B 2005). The result: a doubling or tripling of the risk for heart attack (Depres JP et al 2008).

In the Track Your Plaque program, we aim to maintain triglycerides at 60 mg/dl or less, a level that is associated with maximal suppression of triglyceride- containing lipoproteins (Otvos J 1999).


Adiels M, Olofsson SO, Taskinen MR, Boren J. Overproduction of very low-density lipoproteins is the hallmark of the dyslipidemia in the metabolic syndrome. Arterioscler Thromb Vasc Biol 2008 Jul;28(7):1225-36.

Berneis KK, Krauss RM. Metabolic origins and clinical significance of LDL heterogeneity. J Lipid Res 2002 (Sept);43:1363-79.

Depres JP, Cartier A, Cote M, Arsenault BJ. The concept of cardiometabolic risk: Bridging the fields of diabetology and cardiology. Ann Med 2008;40(7):514-23.

McBride P. Triglycerides and risk for coronary artery disease. Curr Atheroscler Rep 2008 Oct;10(5):386-90.

Otvos J. Measurement of triglyceride-rich lipoproteins by nuclear magnetic resonance spectroscopy. Clin Cardiiol 1999 Jun;22(6 Suppl):II21-7.

Verges B. New insight into the pathophysiology of lipid abnormalities in type 2 diabetes. Diabetes Metab 2005 Nov;31(5):429-39.

C-reactive protein

C-reactive protein (CRP) has emerged as a practical measure of low-grade, hidden inflammation. The new “high-sensitivity” method of measure permits detection of inflammation at levels below the threshold of perception. Studies have shown that the higher your CRP, the brighter the flame of inflammation is burning in the body, and the greater your risk for various illnesses, including cancer, diabetes, glaucoma, macular degeneration, carotid disease, depression, and dementia (Il’yasova D et al 2005; Bluher M et al 2005; Toker S 2005).

Active inflammatory blood cells anywhere in the body generate increased levels of the signaling protein, IL-6. Excess fat cells also cause increased IL-6. IL-6, in turn, activates liver production of CRP.

If there’s an obvious source of inflammation like pneumonia, flu, or a healing wound, then CRP can be dramatically elevated, often 100 mg/l or higher. However, in the absence of active, overt inflammation, levels from 0.5 to 10 mg/l can indicate that inflammation is smoldering somewhere in the body and risk for heart attack increases two- or three-fold, regardless of LDL cholesterol levels (Pearson TS et al 2003). When high CRP occurs in the company of small LDL particle size, heart attack risk is 7-fold greater (St-Pierre AC et al 2003). The Track Your Plaque program is designed to keep CRP at low levels below 0.5 mg/L.


Bluher M, Fasshauer M, Tonjes A, Kratzsch J, Schon MR, Paschke R. Association of interleukin- 6, C-reactive protein, interleukin-10, and adiponectin plasma concentrations with measures of obesity, insulin sensitivity and glucose metabolism. Exp Clin Endocrinol Diabetes 2005 Oct;113(9):534–37.

Il’yasova D, Colbert LHG, Harris TB, Newman AB, Bauer DC, Satterfield S, Kritchevsky SB. Circulating levels of inflammatory markers and cancer risk in the healthy aging and body composition cohort. Cancer Epidemiol Biomarkers Prev 2005 Oct;14(10):2413–18.

Pearson TA, Mensah GA, Alexander RW et al. Markers of inflammation and cardiovascular disease: application to clinical and public health practice: a statement for healthcare professionals from the Centers for Disease Control and Prevention and the American Heart Association. Circulation 2003;107:400–511.

St-Pierre AC, Bergeron J, Pirro M, Cantin B, Dagenais GR, Despres JP, Lamarche B; Quebec Cardiovascular Study. Effect of plasma C-reactive protein levels in modulating the risk of coronary heart disease associated with small, dense, low-density lipoproteins in men (The Quebec Cardiovascular Study). Am J Cardiol 2003;91:555-58.

Toker S, Shirom A, Shapira I, Berliner S, Melamed S. The association between burnout, depression, anxiety, and inflammation biomarkers: C-reactive protein and fibrinogen in men and women. J Occup Health Pscyhol 2005 Oct;10(4):344–62.

Hemoglobin A1C (HbA1C)

Blood sugar (glucose) modifies proteins in the bloodstream, a process called “glycosylation.” One of the proteins susceptible to glycosylation is hemoglobin contained in red blood cells. If blood sugar is increased, there will be an increased percentage of glycosylated hemoglobin within red blood cells, also known as hemoglobin A1C, or HbA1C.

Because the lifespan of a red blood cell is approximately 120 days, HbA1c provides an assessment of blood glucose over the same period. (Any condition that modifies the lifespan of red blood cells, such as some forms of anemia, can therefore alter the HbA1C; HBA1C must then be interpreted with caution.)

HbA1C can be used to calculate an “Average Glucose” (AG) for the preceding 120-day period (Nathan DM et al 2008):

AGmg/dl = 28.7 × A1C − 46.7

The Track Your Plaque nutritional program is designed to help maintain HbA1C at 5.0% or less, corresponding to an average glucose of 96.8 mg/dl or less.


Nathan DM, Kuenen J, Borg R et al. Translating the A1C assay into estimated average glucose values. Diabetes Care 2008 August;31(8):1473-78.

Blood spot or salivary

The human adrenal gland produces approximately 10 mg of cortisol per day with a predictable circadian pattern. The first daily surge occurs during the first hour of wakefulness, followed by several lesser surges during the course of the day; lowest levels occur in early sleep at 12 am to 3 am. Stressful situations can also trigger cortisol surges, regardless of time of day. Depression, anxiety, caregiver stress, unemployment and other stress life situations can chronically increase cortisol levels. Increased levels of evening cortisol can be associated with unhappy marriage, depression, and social isolation (McEwen BS 1998).

Cortisol influences a multitude of body processes, though its major effect is to maintain fluid and blood pressure. Severe deficiency of cortisol is called Addison’s disease and is marked by severe dehydration and drops in blood pressure (Debono M et al 2009).

Cortisol is also at the center of a heated debate. At one end are proponents of the idea that stress, sleep deprivation, and unhealthy diets all lead to increased levels of cortisol, the “hormone of stress.” Others argue that the cortisol stress response is a relatively short-lived phenomenon followed by a chronic lack of cortisol, called “adrenal fatigue.” Proponents of this concept site the following symptoms as potentially due to adrenal fatigue:

  1. Brain fog, mental cloudiness, depression
  2. Low thyroid function
  3. Low blood sugar
  4. Morning and mid-afternoon fatigue
  5. Fragmented sleep
  6. Low blood pressure
  7. Impaired immune function
  8. Salt cravings

Both situations can be explored through assessment of cortisol levels. Because of the circadian variation in cortisol levels, 2-4 cortisol values at different times of the day are advised by authorities.

Because obtaining several blood measurements through the course of the day is difficult, salivary cortisol assessment has proven to be a useful means to assess repeated levels (Castro M et al 2000). Salivary cortisol levels provide a measure that correlates best with free, or unbound, cortisol blood levels.


Castro M, Elias PCL, Martinelli CE et al. Salivary cortisol as a tool for physiological studies and diagnostic strategies. Braz J Med Biol Res 2000(Oct);33(10):1171-75.

Debono M, Ross RJ, Newell-Price J. Inadequacies of glucocorticoid replacement and improvements by physiological circadian therapy. Eur J Endocrinol 2009;160:719-29.

McEwen BS. Protective and damaging effects of stress mediators. N Engl J Med 1998;338:171- 79.

Blood spot or salivary

Dehydroepiandrosterone, or DHEA, is a hormone secreted by the adrenal gland in large quantities during our 20s and 30s, followed by a decline of approximately 20% per decade. By age 70, DHEA blood levels in both men and women are at <25% of youthful levels (Kroboth PD et al 1999).

DHEA occurs principally as the sulfated derivative, DHEA-S, in the blood. DHEA- S is the form usually measured to assess DHEA status.

DHEA administration to adults over age 40 has been shown to enhance both physical vigor and emotional well-being (Wolkowitz OM 1999). Beyond the increase in DHEA blood levels, DHEA supplementation increases testosterone and triggers a modest rise in effective growth hormone levels (Morales 1997). Most studies documenting physical and emotional benefits of DHEA have examined doses between 10 and 50 mg per day, the quantity required to restore youthful DHEA-S blood levels in the majority of people.

Over the past 40 years, DHEA has been studied in a number of applications, some successful, some not. Among the successes DHEA has yielded:

  • DHEA may slow progression of osteoporosis in postmenopausal women (Baulieu EE et al 2000), and increase bone density (Labrie F et al 1997).
  • DHEA improves sexual dysfunction and libido in women over age 60 (Baulieu EE et al 2000).
  • DHEA may improve erectile dysfunction in men who have low DHEA levels (Reiter WJ et al 2001).
  • DHEA alleviates depressive symptoms such as inability to cope, worrying, lack of motivation, and sadness (Bloch M et al 1999).
  • DHEA improves symptoms of chronic fatigue syndrome (Cleare AJ 2003).
  • In people with adrenal failure whose adrenal glands produce little or noDHEA, DHEA replacement (50 mg per day) improves mood, physical energy and performance, and bone density when used along with standard hormone replacement therapy (e.g., cortisol; Arlt W et al 1999).

Interestingly, most studies of DHEA in athletic performance have raised questions over its benefits for this purpose, but DHEA remains banned in Olympic athletes.

Beyond increasing DHEA and DHEA-S levels, DHEA supplementation increases testosterone and estradiol modestly in females, with little to no change in males. DHEA has little effect on progesterone (Stomati 2000).

Though difficult to quantify, DHEA replacement helps many people feel better: greater physical stamina, brighter outlook, more “get up and go.” Problems seem less overwhelming and “lows” tend not to be as low. Though there are clinical data to support these “soft” effects, they are inconsistent. In our experience, people who start out with sluggishness, low energy, and a negative outlook, accompanied by a low DHEA-S level, are the most likely to experience positive results with DHEA replacement.

Occasionally, DHEA supplementation will bring out aggressive behavior, such as short-temperedness, intolerance, and impatience. A reduction in dose usually resolves this issue (e.g., reduce from 50 mg to 25 mg per day).

Beyond enhanced energy and stamina, in the Track Your Plaque program we monitor DHEA levels when DHEA supplementation is used to reduce lipoprotein(a).


Arlt W, Callies F, van Vlijmen JC et al. Dehydroepiandrosterone replacement in women with adrenal insufficiency. N Engl J Med 1999;341:1013–20.

Baulieu EE, Thomas G, Legrain S et al. Dehydroepiandrosterone (DHEA), DHEA sulfate, and aging:contribution of the DHEAge Study to a sociobiomedical issue. Proc Nat Acad Sci 2000 Apr 11;97(8):4279–84.

Bloch M, Schmidt PJ, Danaceau MA, Adams LF, Rubinow DR. Dehydroepiandrosterone treatment of midlife dysthymia. Biol Psychiatry 1999Jun 15;45(12):1533–41.

Cleare AJ. The neuroendocrinology of chronic fatigue syndrome. Endocr Rev 2003 Apr;24(2):236–52.

Kroboth PD, Salek FS, Pittenger AL, Fabian TJ, Frye RF. DHEA and DHEA-S: a review. J Clin Pharmacol 1999;39:327–48.

Reiter WJ, Schatzl G, Mark I, Zeiner A, Pycha A, Marberger M. Dehydroepiandrosterone in the treatment of erectile dysfunction in patients with different organic etiologies. Urol Res 2001 Aug;29(4):278–81.

Stomati M, Monteleone P, Casarosa E et al. Six-month oral dehydroepiandrosterone supplementation in early and late postmenopause. Gynecol Endocrinol 2000 Oct;14(5):342-63. Wolkowitz OM, Reus VI, Keebler A et al. Double-blind treatment of major depression with dehydroepiandrosterone. Am J Psychiatry 1999 Apr;156(4):646-9.

Blood spot or salivary

There are three principal human estrogens: estradiol, estrone, and estriol. In addition to being responsible for female characteristics such as breast development, female brain development, and maturation of the female reproductive organs, estrogens also accelerate metabolism, increase bone density, and affect vascular health.

Assessment of estrogen levels become most important in the perimenopausal and menopausal periods in females, when estrogen levels fall substantially, resulting in many (though not all; see Progesterone) of the phenomena of menopause, including weight gain, hot flashes, vaginal dryness, and bone thinning (demineralization; NIH Consensus Statement 2005).

Estrogen assessment can be important in males, also, especially during the period during and after the “andropause,” the period in males after age 40, when testosterone levels begin to decline; increased estrogens can therefore exert outsized effects as testosterone effects recede (Amore M et al 2009).

Estrogen replacement in menopausal females is at the center of a controversy. Large-scale clinical studies, such as HERS and the Women’s Health Initiative, employed non-human mixtures of estrogens (ACOG Committee Opinion 2008); large studies using human estrogens (and progesterone) have not been performed, though a vocal and anecdotal grassroots effort have made a strong case for the superiority of so-called “bio-identical” hormone use (Holtorf K 2009).


ACOG Committee Opinion No. 420, November 2008: hormone therapy and heart disease. Obstet Gynecol 2008 Nov;112(5):1189-92.

Amore M, Scarlatti F, Quarta AL, Tagariello P. Partial androgen deficiency, depression and testosterone treatment in aging men. Aging Clin Exp Res 2009 Feb;21(1):1-8.

Holtorf K. The bioidentical hormone debate: are bioidentical hormones (estradiol, estriol, and progesterone) safer or more efficacious than commonly used synthetic versions in hormone replacement therapy? Postgrad Med 2009 Jan;121(1):73-85.

NIH State-of-the-Science Conference Statement on management of menopause-related symptoms. NIH Consens State Sci Statements. 2005 Mar 21-23;22(1):1-38.

Blood spot only

The fundamental defect in metabolic syndrome, pre-diabetes, and type II diabetes is poor responsiveness to insulin, often called “insulin resistance.” Because the pancreas attempts to maintain blood sugars within a narrow normal range, insulin output increases to overcome the “resistance” to insulin’s effects. Blood level of insulin therefore increases (Lann D et al 2008).

Fasting insulin provides a means to assess the body’s responsiveness or resistance to insulin. Low levels signal normal insulin responsiveness; high levels signal reduced insulin responsiveness. However, if insulin resistance has been present for an extended period, usually several years, then the pancreas loses its capacity to manufacture insulin and insulin blood levels will drop (and may be indistinguishable from normal); at this point, blood sugars will increase above the normal range. Pre-diabetes or diabetes then develops.

Higher insulin levels have been shown to correlate with greater likelihood of progression of heart scan (coronary calcium) scores (Lee KK et al 2009). Increased blood levels of insulin have also been associated with 12% greater risk for cardiovascular events (Sarwar N et al 2007).

Inhe Track Your Plaque program, we maintain fasting insulin levels at 10 mIU or less.


Lann D, Gallagher E, leroith D. Insulin resistance and the metabolic syndrome. Minerva Med 2008 Jun;99(3):253-62.

Lee KK, Fortmann SP, Fair JM et al. Insulin resistance independently predicts the progression of coronary artery calcification. Am Heart J 2009 May;157(5):939-45.

Sarwar N, Sattar N, Gudnason V, Danish J. Circulating concentrations of insulin markers and coronary heart disease: a quantitative review of 19 Western prospective studies Eur H J. 2007 Oct;28(20):2491-97.

Blood spot or salivary

Progesterone is the hormone responsible for many uniquely female characteristics. Some call progesterone the “forgotten female hormone,” lost in the shadow of better-known estrogen. As you age, like estrogen, progesterone levels decrease.

Progesterone levels are 10–100 fold greater during pregnancy. Progesterone is the progestational hormone, so named because it helps prepare the uterus and associated organs for pregnancy. The “blue” feeling that follows delivery that many women experience, sometimes severe enough to be labeled “postpartum depression,” is suspected to be due to the abrupt 95% drop in progesterone after delivery.

Progesterone levels begin to decline as early as the late 30s with an abrupt drop through the menopause. Symptoms associated with declining progesterone include difficulty sleeping through the night, poor energy, foggy thinking, and irritability. Many women also experience bloating and weight gain. As a rule, all women in the menopausal period have very low levels of progesterone; levels during the transitional years towards menopause, “perimenopause,” can vary from woman to woman.

As with estrogens, clinical studies have employed synthetic forms of progesterone, “progestins,” which have been associated with increased cardiovascular risk. Many authorities speculate that natural human, “bio- identical,” progesterone does not share the adverse effects of progestin, though outcome data are lacking (Hermsmeyer RK et al 2008; L’hermite M et al 2008). Anecdotal experience with progesterone has suggested that the natural human form is superior for providing relief of symptoms of progesterone deficiency.


Hermsmeyer RK, Thompson TL, Pohost GM, Kaski JC. Cardiovascular effects of medroxyprogesterone acetate and progesterone: a case of mistaken identity? Nat Clin Pract Cardiovasc Med 2008 Jul;5(7):387-95.

Holtorf K. The bioidentical hormone debate: are bioidentical hormones (estradiol, estriol, and progesterone) safer or more efficacious than commonly used synthetic versions in hormone replacement therapy? Postgrad Med 2009 Jan;121(1):73-85.

L’hermite M, Simoncini T, Fuller S, Genazzani AR. Could transdermal estradiol + progesterone be a safer postmenopausal HRT? A review. Maturitas 2008 Jul-Aug;60(3-4):185-201.

Prostate specific antigen (PSA) Blood spot only

PSA is a protein produced by the prostate gland in men and detectable in the blood. It is typically measured as a screening test for prostate cancer. It is also used to track results after prostate removal for cancer.

One limitation of PSA testing is that other conditions can influence PSA levels, including factors that reduce PSA, such as obesity and anti-inflammatory agents (aspirin, ibuprofen, etc.), and those that increase PSA, such as prostate inflammation or infection (prostatitis) and benign enlargement of the prostate (“benign prostatic hypertrophy,” or BPH). For this reason, most authorities recommend repeating a suspicious level to confirm before action is taken.


Fleshner NE, Lawrentschuk N. Risk of developing prostate cancer in the future: overview of prognostic biomarkers. Urology 2009 May;73(5 Suppl):S21-7.

Sex Hormone-Binding Globulin (SHBG) Blood spot only

SHBG is the blood protein carrier that transports sex hormones, estrogen and testosterone. The fraction of estrogen or testosterone carried by SHBG is called the “bound” fraction and serves as a repository for hormone storage. The fraction of estrogen or testosterone not carried on SHBG is called “unbound” or “free;” this represents the active fraction and is generally no more than 1-2% of total hormone present.

An increase in the pool of SHBG can effectively decrease the free fraction of hormone; conversely, a decrease in SHBG frees up more hormone. Thus, SHBG levels influence the amount of free hormone available. SHBG levels are increased (thereby reducing available hormone) in situations including hyperthyroidism (overactive thyroid), liver disease, and calorie deprivation. SHBG levels are decreased (thereby increasing available hormone) in hypothyroidism (underactive thyroid) and obesity.

However, fluctuations in SHBG levels are not the only determinant of free hormone levels. For this reason, a gauge of free or “bioavailable” testosterone can be obtained by calculating the Free Androgen Index (FAI). The Free Androgen Index (FAI) is calculated as follows:

FAI = Total Testosterone / SHBG

(FAI is sometimes modified by a “correction factor,” e.g., multiply by 100.)

Lower SHBG and higher FAI values in females may serve as markers for increased coronary risk (Lambrinoudaki I et al 2006;Sutton-Tyrrell K et al 2005). In both males and females, higher FAI correlates with features of the metabolic syndrome (Pugeat M et al 1995).


Lambrinoudaki I, Christodoulakos G, Rizos D et al. Endogenous sex hormones and risk factors for atherosclerosis in healthy Greek postmenopausal women. Eur J Endocrinol 2006;154(6):907- 16.

Pugeat M, Moulin P, Cousin P et al. Interrelations between sex hormone-binding globulin (SHBG), plasma lipoproteins and cardiovascular risk. J Steroid Biochem Mol Biol 1995 Jun;53(1- 6):567-72.

Sutton-Tyrrell K, Wildman RP, Matthews KA et al for the SWAN Investigators. Sex Hormone– Binding Globulin and the Free Androgen Index Are Related to Cardiovascular Risk Factors in Multiethnic Premenopausal and Perimenopausal Women Enrolled in the Study of Women Across the Nation (SWAN) Circulation 2005;111:1242-49.

Blood spot or salivary

Testosterone levels peak in a male’s teens and twenties during their reproductive prime. That’s also the period of a man’s greatest physical capacity, muscle mass, physical energy, libido, and stamina.

Starting at age 30, testosterone levels diminish gradually. By the time a man reaches his 70s, testosterone has dropped to low levels. Diminishing testosterone levels lead to loss of muscle mass, increased body fat, and reduced libido. Mood disruptions are prominent, with deeper swings into blue, depressed feelings, struggles with feeling beaten and overwhelmed, and fatigue. Reduced concentration, irritability, passivity, loss of interest in activities, and even hypochondria can also result.

These changes become perceptible after a man passes beyond his mid-40’s. Some call this time the “male menopause” or “andropause.” Though not as visible as a woman’s transition to menopause since there’s no particular external cue like cessation of menses, most men simply dismiss the changes as “getting old.”

Studies have shown that the lower the starting blood testosterone level, the greater the benefits of testosterone replacement. With rare exceptions, few men before age 40 will benefit from testosterone, as they maintain healthy levels.

The rate of testosterone decline varies from one male to another. One 50-year old man, for instance, might have a blood level of 390 pg/ml, and another 50-year old could have a level of 50 pg/ml. The second man enjoys greater benefits because of the lower starting value.

From a heart health standpoint, potential benefits of testosterone replacement in men with lower starting levels include:

  • Reduction in vascular tone and endothelial dysfunction—Testosterone increases production of the natural arterial dilator, nitric oxide, and suppresses growth of smooth muscle cells in arteries (a constituent of plaque; Khalil RA 2005).
  • Improvement in abnormal resistance to insulin—The essential phenomenon behind pre-diabetes and metabolic syndrome (Marin P et al 1992; Simon D et al 2001).
  • Reduction in inflammatory proteins—Levels of tumor necrosis factor and interleukins, in particular, are considerably reduced (Malkin CJ et al 2004).Men with coronary disease have been shown to be more likely to have low testosterone levels. In one study, a marked deficiency of testosterone was found in 25% of men with overt coronary disease (e.g., history of heart attack or procedures; Malkin CJ et al 2004).

    Most males obtain optimal results with testosterone replacementenhanced well-being, physical stamina, and libidowith restoration of blood testosterone to levels that reproduce the level most men have in their mid- to late-30s.

    An assessment of total testosterone blood levels provides a starting point for assessing testosterone status. A more in-depth assessment can be made by adding free testosterone, bioavailable testosterone, and dihydrotestosterone (not yet available through blood spot testing). A Free Androgen index can be calculated if a SHBG level is available (see SHBG, above).

    If feelings of sadness, bloating, and weight gain are present, an estradiol level might considered. Estradiol is the form of estrogen that can be increased, particularly in overweight men and increases risk for heart disease. Weight loss can correct elevated estradiol, as can prescription “aromatase inhibitors,” such as Arimidex®. A nutritional supplement, chrysin (usual dose, 1000 mg per day), has been shown to reduce estradiol levels; however, there’s little supportive data documenting its effectiveness and safety.


Khalil RA. Sex hormones as potential modulators of vascular function in hypertension. Hypertension 2005 August;46(2):249–54.alkin CJ, Pugh Pj, Jones RD et al. The effect of testosterone replacement on endogenous inflammatory cytokines and lipid profiles in hypogonadal men. J Clin Endocrinol Metab 2004;89:3313–18.

Marin P, Holmang S, Jonsson L et al. The effects of testosterone treatment on body composition and metabolism in middle-aged obese men. Int J Obes Relat Metab Disord 1992;16:991–97.

Simon D, Charles MA, Lahlou N et al. Androgen therapy improves insulin sensitivity and decreases leptin level in healthy adult men with low plasma total testosterone: a 3–-month randomized placebo-controlled trial. Diabetes Care 2001;24:2149–51.


Thyroid testing:
TSH, free T3, free T4, antithyroglobulin antibody Blood spot only

The thyroid gland is a butterfly-shaped gland located on the front of the neck just beneath the surface of the skin. The thyroid produces thyroid hormones:

T3 (triiodothyronine), the most active hormone. The thyroid produces no more than 20% of bodily T3 requirements; most T3 develops from conversion of T4 to T3 that occurs in other tissues. The preferred method to assess T3 status is to measure the free, unbound fraction, called free T3.

T4 (tetraiodothyronine or thyroxine)80% of hormone produced by the thyroid is T4. T4 is converted to the active form, T3, via the action of deiodinase enzymes that remove one iodine atom. The preferred method to assess T4 status is to measure the free, unbound fraction, called free T4.

TSH (Thyroid Stimulating Hormone or thyrotropin) is the pituitary gland hormone that signals the thyroid to produce thyroid hormones and maintain T4 and T3 levels. If thyroid hormone levels are low, TSH will increase in an effort to increase thyroid hormone production; if thyroid hormone levels are high, TSH will decrease. In the 25,000-participant HUNT Study, cardiovascular mortality began to increase with TSH of 1.4 mIU, with 70% increased (relative) risk with TSH of 2.5-3.5 mIU (Åsvold BO et al 2008).

Thyroid Peroxidase Antibody (TPO Ab): Thyroid peroxidase (TPO) is an enzyme involved in production of thyroid hormones. Autoimmune conditions result in antibodies targeting TPO. Approximately 90% of sufferers of Hashimoto’s thyroiditis will test positive for elevated TPO antibodies (Carlé A et al 2006).

Free T3, free T4, and TSH are the basic lab tests usually obtained when an assessment of thyroid function is required. Antibody assessments, such as thyroid peroxidase antibody, can be added if the question of an inflammatory condition of the thyroid is suspected.

Hypothyroidism, in which thyroid production of T3 and T4 falls below the body’s needs, is by far the most common abnormal condition affecting the thyroid. Hypothyroidism can trigger numerous symptoms:

  • Reduced energy, fatigue, increased sleep
  • Feeling inappropriately cold; reduced sweating
  • Dry, itchy skin
  • Dry, brittle, thinning hair
  • Weight gain without apparent cause or more-than-usual difficulty losingweight
  • Impaired short-term memory, slower thinking
  • Muscle cramps, joint aches
  • Constipation
  • Puffiness or swelling around the eyes, hands, ankles, and feet
  • Heavier and/or more frequent menstrual periods, worse premenstrualsymptoms
  • Depression, sadness, apathy
  • Abnormally slow heart rate (<60 beats per minute)
  • Iron deficiency anemia, low ferritin (an iron storage protein)Symptoms can be vague, often not present at all. Levels of thyroid hormones, free T3 and T4, along with pituitary hormone, TSH, are therefore used to confirm the diagnosis. Low body temperature (oral temperature below 97.3° F immediately upon arising) can also suggest low thyroid.

    Excessive levels of thyroid hormone, hyperthyroidism, can also occur, though much less commonly. Hyperthyroidism can account for anxiousness, unexplained weight loss, muscle weakness, and fast heart rate. Hyperthyroidism is generally caused by inflammation of the thyroid gland, such as Hashimoto’s thyroiditis or Grave’s autoimmune thyroiditis.

    While most laboratories cite a TSH range of 0.5-5.5 mIU as normal, in the Track Your Plaque program, we aim for TSH less than 1.0 mIU. We also aim to keep free T4 and free T3 in the upper half of the reference range.


    Åsvold BO, Bjøro T, Nilsen TI et al. Thyrotropin levels and risk of fatal coronary heart disease (The HUNT Study) Arch Intern Med 2008;168(8):855-60.

    Carlé A, Laurberg P, Knudsen N et al. Thyroid peroxidase and thyroglobulin auto-antibodies in patients with newly diagnosed overt hypothyroidism. Autoimmunity 2006 Sep;39(6):497-503.

Vitamin D: 25-hydroxy vitamin D Blood spot only

Vitamin D is proving to be among the most exciting phenomena in health uncovered in the past 50 years. This everyday vitamin, ignored for years as just something we got from milk, packs health benefits that have far exceeded anyone’s expectations.

Among the fascinating effects recently identified:

  • Blood pressure—People deficient in vitamin D are more likely to have high blood pressure; vitamin D supplementation reduces blood pressure with the same effectiveness as prescription medication. One study demonstrated 10–20 mmHg drop in blood pressure in men with pre- diabetes when vitamin D was supplemented (Pfeifer M et al 2001; Lind L et al 1995).
  • Anti-cancer effects—Epidemiologic studies demonstrate reduced likelihood of cancer, particularly breast, prostate and colorectal cancer, in people with higher blood levels of vitamin D (Vieth R 1999).
  • Anti-inflammatory effects—Recent studies demonstrate reduced inflammation (C-reactive protein, CRP, and matrix metalloproteinase, MMP). Reductions in CRP of 60% or more have been documented (Timms PM et al 2002).
  • Anti-diabetic effects—Several studies have shown that vitamin D administration reduces blood sugar and increases sensitivity to insulin. Improvement in insulin sensitivity triggers a cascade of benefits, including beneficial effects on lipoproteins (reduced triglycerides, increased HDL) (Zitterman A 2006).
  • Osteoporosis prevention—Vitamin D is sorely neglected in this area. Replacement to healthy levels substantially increases bone density more effectively than calcium supplementation. Intestinal absorption of calcium more than doubles when sufficient vitamin D is present (Holick MF 2006).New studies are showing that the dose required to achieve a healthy blood level of vitamin D in most adults is around 4000 units per day in the absence of sun exposure (Vieth R et al 2001). That’s 10 times the recommended Institute of Medicine’s Adequate Intake.

    People from the northern U.S. (Massachusetts, New York, Pennsylvania, Wisconsin, Michigan, the Dakotas, etc.), Canada, or northern Europe, are likely to be deficient, though many people from southern climates are also deficient. If you’re like most Americans, you get sun sporadically in summer (weekends) and virtually none from September to April. Dark-skinned races are at greater risk of vitamin D deficiency, since melanin pigment in skin acts as a natural sun screen.

Dark-skinned individuals require five times longer sun exposure to obtain the same amount of vitamin D as a fair-skinned person. African-Americans, for this reason, are among the most vitamin D deficient of all.

The only way to know your vitamin D status is to measure the blood level of 25- hydroxy vitamin D (not to be confused with 1,25-di-OH-vitamin D, a related metabolite that reflects kidney function). While some authorities argue that a minimum 25-OH-vitamin D3 level of 30 ng/ml, or 75 nmol/l, be achieved (Vieth R et al 2001), in the Track Your Plaque program we aim for a blood level of 60-70 ng/ml (150-175 nmol/L).

In young people, 15 minutes of sun exposure in midday, wearing shorts and t- shirt to expose skin surface area, may provide sufficient vitamin D. The ability to activate vitamin D in the skin is lost as we age, such that a dark tan in our 60s and 70s can conceal severe vitamin D deficiency. Vitamin D supplementation is therefore required as we age.

Personally, I take 5,000 units in the late fall, winter, and early spring, and then I vary doses the rest of the time depending on sun exposure. I also have my 25(OH)D level checked twice a year, once in the early spring and again in the early fall. My 10 year-old daughter takes 2,000 units a day in the
winter months and my three-year-old takes 1,000 units a day in the winter.

Dr. John Cannell The Vitamin D Council

Anyone with kidney disease, cancer, glandular disorders, or a history of high calcium blood levels should only take vitamin D with medical supervision.


Holick MF. The role of vitamin D for bone health and fracture prevention. Curr Osteoporos Rep 2006 Sep;4(3):96–102

Lind L, Hanni A, Lithell H et al. Vitamin D is related to blood pressure and other cardiovascular risk factors in middle-aged men. Am J Hypertens 1995 Sep;8(9):894–01.

Pfeifer M, Begerow B, Minne HW et al. Effects of a short-term vitamin D3 and calcium supplementation on blood pressure and parathyroid hormone levels in elderly women. J Clin Endcrinol Metab 2001;86:1633–37.

Timms PM, Mannan N, Hitman GA, Nooonan K, Mills PG et al. Circulating MMP9, vitamin D and variation in the TIMP-1 response with VDR genotype: mechanisms for inflammatory damage in chronic disorders? Q J Med 2002;95:787–96.

Vieth R. Vitamin D supplementation, 25-hydroyvitamin D concentrations, and safety. Am J Clin Nutr 1999;69:842–56.

Vieth R, Chan P-C, MacFarlane GD. Efficacy and safety of vitamin D3 intake exceeding the lowest observed adverse effect levels. Am J Clin Nutr 2001;73:288–94.

Zittermann A. Vitamin D and disease prevention with special reference to cardiovascular disease. Prog Biophys Mol Biol 2006 Sep;92(1):39–48.