Lab Testing Made Simple

Monitoring Topical Hormone Therapy in Saliva - Understanding the Ranges

2011-06-10T08:44:00.000-07:00

If you've used saliva testing for topical hormones, you know that it can be a challenge. Why? Because "normal" levels of hormones like progesterone are supraphysiological, even at modest doses. Why is this? That has been covered in a previous blog, and if you're new to this topic it may be worth your time to read. To make sense of topical hormones, you need to know what women's levels typically are given the particular dosage of interest. ZRT Laboratory can provide this data and it will help you to make sense of salivary results.

You can find a more comprehensive listing of progesterone, estradiol, and testosterone (men) here. For the purpose of this blog, let's just consider progesterone.

- You need to have tested 12 or 24 hours after the last dose (ranges for other time points are not available) and select the appropriate graph.

- Find the dose used (x-axis) and move vertically until you find the patient's result (y-axis).

This will tell you how your patients results compare to what is expected given this particular scenario. The data has been generated from tens of thousands of saliva results from individuals using hormonal supplementation. The line in the middle represents the average (median) patient's result on the relevant dosage. The top and bottom lines make a reference range for the particular dosage of interest. The top line represents the 80th percentile, and the bottom line represents the 20th percentile. This effectively gives a reference range for all dosage found on the x-axis at either 12 or 24 hours after supplementation. Values significantly higher than the 80th percentile may be due to sample contamination. Note: being within the reference range for a particular dosage is, in no way, an endorsement of that particular supplementation regiment.

Here is an example for your consideration:

Topical Progesterone, 12 hours:

Topical Progesterone, 24 hours:
Please see ZRT Laboratory Ranges & Dosage for more information

Salivary cortisol testing

2011-03-23T19:12:00.000-07:00

Lena Edwards, MD, internist, Balance Health & Wellness Center, discusses salivary cortisol testing which is increasingly being used in the diagnosis and treatment of patients with hypothalamic pituitary adrenal dysfunction. The pattern of cortisol release is important and salivary testing allows this to be analyzed.

Watch the video: http://www.thedoctorschannel.com/video/4137.html

Your Spit May Hold Key to Predicting Burnout

2011-02-24T09:53:00.000-08:00

By Wallace Immen
from CTV News
2/22/2011

Go ahead: Spit if you feel frustrated about your job. What your saliva reveals could alert doctors to whether you’re at risk of burnout at work, according to new Canadian research.

And testing saliva could also help people with symptoms of burnout avoid being put on medication that might actually make the condition worse, said Robert-Paul Juster, a doctoral student at McGill University in Montreal who helped design the research.

A clue that someone is suffering burnout is lowered levels of cortisol, often referred to as the “stress hormone” because it is secreted when we feel anxious or agitated. But if we are under continual stress, our bodies can shut down production of the hormone rather than try to keep up with the constant demand.

“We wanted to … find a simple way to find low levels of the hormone showing up in people who have not yet had problems, and how that may predict risk of burnout,” Mr. Juster said.

Normally, cortisol tends to spike in the morning as people wake up, which is the body’s way of revving up after a night’s sleep. Levels usually decline during the day. “But we find that people with high stress don’t have that boost of cortisol in the morning,” Mr. Juster said. “They report feeling exhausted in the morning, even though they’ve had a full night’s sleep.”

Burnout, clinical depression, or anxiety-related issues in the workplace affect at least 10 per cent of North Americans and Europeans, according to estimates prepared by the International Labour Organization.

The Montreal research included a random sample of 30 middle-aged workers in a variety of professions. They took samples of their saliva at home and at work a total of five times a day; using a questionnaire, they also rated their stress levels and any physical symptoms they were experiencing.

What Testing to Use? by guest blogger - Jim Paoletti, Pharmacist

2011-02-10T15:16:00.000-08:00

Jim Paoletti, Pharmacist, FAARFM, talks about his preferred methods of laboratory testing, and when to use saliva and blood spot testing.

UPDATE: IOM's Report: Vitamin D Daily Intake Recommendation

2011-02-04T07:18:00.000-08:00

The Institute of Medicine of the National Academies offered up new recommendations for daily intake of vitamin D and calcium late last year. The new Recommended Dietary Allowance (RDA) is 600 IU/day. They define the RDA as “levels of intake that are likely to meet the needs of about 97.5% of the population.” The Upper Level Intake is listed as 4,000 IU/day, which is an increase.

These conclusions are in sharp contrast to the vitamin D zealots of the world, the most aggressive of whom are calling for 5,000-10,000 IU/day to achieve the health benefits of vitamin D sufficiency. I happened to be at the CDC recently for a meeting with some folks very involved in vitamin D research. These are fairly conservative research types (not to be confused with the vitamin D zealots, which I am one) and the group of renowned epidemiologists was disappointed with the ruling, to say the least. The difference of opinion between the vitamin D research world and the IOM is vast, so let’s break this down, so you can make some reasonable decisions for you, your family, and your patients.

First, let me just say that this is NOT the result of some new study. All of the amazing studies over the past decade on the correlations between vitamin D levels and various diseases are as legitimate today as they were before this release. This is simply the IOM’s attempt to digest all of the studies and make public health recommendations. It is important to understand the public health philosophy at play here. This approach really has little to do with individualized medicine. Consider the following statements the IOM makes:

“An important aspect of Dietary Reference Intake (DRI) development is its grounding in public health applications and the concept of distributions of risk. This approach may appear strange to some and may be disconcerting to those with a clinical orientation who are familiar with the medical model in which the goal is to treat the patient in the most efficacious manner to enhance a positive outcome. This report [is] therefore in contrast to a medical model approach.”

If you’re slow like me you may need to read that again, but let me highlight one point – this report is “in contrast to a medical approach.” A “medical approach” is defined as one “in which the goal is to treat the patient in the most efficacious manner to enhance a positive outcome.”

The above is a HUGE distinction to me. It doesn’t mean I ignore the results of this recommendation or become outraged, but we have to realize that if you are involved in individualized medicine (through a physician or simply in dealing with yourself), the philosophical approach taken here is admittedly different, but this isn’t the most important observation from these recommendations.

The IOM has concluded from their review that there is insufficient evidence to promote vitamin D with respect to its protective properties against anything not related to bone health. That means that all of the interesting and compelling epidemiological studies showing decreased risk for cancers, autoimmune disease, etc did not impress the group enough to affect their recommendations. I personally am compelled by the epidemiology and find it very relevant that risks for some of the most common cancers are reduced by higher levels of vitamin D. Levels that imply protection from colon cancer to autoimmune diseases (to name a few) are realized at levels that greatly exceed what this committee considers acceptable. Remember vitamin D levels required to achieve bone health is considerably lower than the amount it takes to achieve protection against other D-related conditions. If the IOM reached their tipping point and conceded that these other issues are relevant to their recommendations, they would likely be making major jumps in their recommendations.

You can see from their statement below that this committee considers 12 ng/ml to be sufficient for most of the population.

“This committee’s review of data suggests that persons are at risk of deficiency at serum 25OHD levels of below 12 ng/mL. Some, but not all, persons are potentially at risk for inadequacy at serum 25OHD levels from 12 to less than 20 ng/mL. Practically all persons are sufficient at levels of 20 ng/mL and above. Serum concentrations of 25)HD above 30 ng/mL are not associated with increased benefit. There may be reason for concern at serum 25OHD
levels above 50 ng/mL."

Remember to read the above statement through the filter that this committee only considers vitamin D status to be relevant with respect to issues of bone health. If we want to prevent only rickets and osteomalacia, I agree with the above statement. There has been a huge increase in concern for vitamin D levels over the past decade. This increase is largely due to the compelling nature of the epidemiological studies that show the preventive power of vitamin D (the relationship to bone health has been known for almost 100 years). The IOM is simply not impressed by these same studies. If overall risk reduction is the goal, I believe the evidence is plenty strong to encourage us to keep our levels well above 30 ng/mL. Some experts think 40-60 ng/mL is a fine range and I take no issue with that.

If 40 ng/mL is good, 60 or 80 ng/mL should be even better, right? What about the concern over folks with higher levels? This is a relevant discussion and I think moderation is a reasonable approach. Personally, I do not subscribe to the theory that we should be getting the general population to levels above 60 ng/mL. I do, however, agree that there may be benefits to these levels. Colon cancer and common flue/cold bugs may well be positively affected by high levels. There may also be some increased risks from these levels and we should not ignore this possibility. The IOM brings up increased risk for pancreatic cancer as an example. This strikes me as partially a good point and partially hypocritical. There is a study that has linked higher levels of vitamin D to pancreatic cancer risk. This should be considered. At the same time, if you are going to let one study of that type sway you away from higher levels, shouldn't you also consider the weight of the evidence for the protective benefits of having higher levels? The IOM has considered all of the benefit evidence as not sufficiently strong yet the single study on pancreatic cancer (there are many on colon cancer benefits) is sufficiently strong to create concern.

At the end of the day, this is about risk assessment and cost-benefit analysis. Having higher levels of vitamin D might be associated with slightly higher risk for a few diseases/conditions. It is also associated with significantly lower risk for a fairly lengthy list of etiologies. If 40-60 ng/mL is your goal after considering all of the arguments, the IOM recommendations aren't simply a bit too low, they are entirely irrelevant.

A common sense approach makes sense to me. Common sense tells me that the body begins to use negative feedback to keep vitamin D production reasonable if there is great sun exposure. You simply can't get toxic from sun exposure. Individuals in the sun regularly find their levels routinely above 50 ng/mL. Are we to assume that groups like the IOM will soon be recommending that people be cautious of being overly active outdoors so as not to increase vitamin D to potentially dangerous levels? That doesn't pass the common sense test.

Finally, a note about the new upper limits:

Their upper limits (4,000IU/day) are intended to serve as a lifetime public health measure for free-living, unmonitored population. Unmonitored?

Individual responses to a particular dose of vitamin D differ by a factor of 8! When several people all took the same does of vitamin D, some folks had their levels go up by on 4 ng/mL, whereas some went up by 32 ng/mL. The assumption made by IOM that 4,000IU is the upper max is assuming that nobody is testing their levels. I submit that with testing this is irrelevant. If you are taking 4-10,000IU/day, you should be testing your levels because you could end up anywhere from 15 ng/mL to over 100 ng/mL depending on your individual response.

I think the efforts by the IOM have clearly been very costly, based on the number of folks involved and the thoroughness of the review and it is somewhat regrettable that the scope was so narrow.

For more consideration, please read this viewpoint.

Vitamin D

2011-02-02T18:04:00.000-08:00

Listen to Mark Newman's radio interview with Daniel Davis on Beyond 50 radio station, on the importance of Vitamin D. Click here.

New York State Certification for Saliva Testing

2010-06-23T09:30:00.000-07:00

We’re really happy to report that ZRT is now certified to perform saliva testing in the state of New York!

New York has its own certification process, which is different from other agencies that oversee labs, such as CLIA. When surveying the lay of the land in NY four years ago, ZRT had two different options in terms of its approach with the State of New York. We took a path that made the journey longer, but we are very glad we did it the way we did. We are now working towards getting bloodspot testing certified in New York.

I thought I would give you a bit of a recap of the process – and some inherent challenges – in getting through this process. Due to some of the internal standards we have established for our testing protocols, it was more difficult for ZRT to be approved by New York.

At the core of the issue is that there are essentially three types of testing systems that can be used by labs to test salivary hormones:
1. FDA “approved” (the FDA prefers the word “cleared”): These have been cleared for sale by the FDA, some reviewed by the FDA (510k) and some not (exempt)
2. Modified FDA approved tests: These are approved, but the lab has modified the procedure to (presumably) improve performance.
3. Tests NOT approved by the FDA: These tests would be considered “research only”

If a lab decides to use only FDA approved tests (#1 above), New York gives the lab a pass on the validation data for that particular test. In this scenario, labs are only required to notify NY that they are participating in a particular test, but the actual performance characteristics of the test are not reviewed. As a laboratory with decades of experience developing laboratory tests for saliva, ZRT has always made a commitment to the highest standards in our testing. As a result, we were coming at all of this with the second scenario noted above as our situation, since we use all FDA cleared products, but have customized our procedures to create what we believe is improved performance and accuracy for our testing.

New York also has a completely separate team that deals with modified methods, so not only are the requirements and requests considerably robust, but the need for patience becomes paramount. While we were not willing to compromise our core testing protocols and standards, we did have to meticulously maneuver to make changes to some aspects of our methods to meet some of the specific requirements of the special laboratory validation unit in NY. As a result of this commitment, the complexity of this issue, and the inherent ‘back-and-forth’ nature of the process, it took nearly four years to complete.

We are very glad to have gone through the process and we have actually been able to implement some improvements to our own quality assurance program, thanks to suggestions made by NY. ZRT is constantly looking for ways to improve the reliability of our testing, and a process like this was another opportunity to put this commitment ‘to the test’. We believe that our judgment in “how” to conduct saliva testing is world-class, and going through this process in addition to other regulatory exercises continues to keep us at the top of our game. We now look forward to providing the physicians and patients of New York the same industry leading standards that the rest of the country has been enjoying for more than ten years.

The 2/16 Ratio...does it really matter?

2010-05-26T10:53:00.000-07:00

I keep rewriting this blog because I think there is a HUGE misunderstanding on the clinical validity of this particular test, but I don’t want to be overly negative in my approach. This is difficult because at the end of the day, I can’t escape the fact that there seem to be too many individuals and groups with a vested interest in the continued use of this test, but supporting it with pretty thin evidence. What test am I talking about? I am talking about the urine test for the ratio of the estrogen metabolites 2-hydroxyestrogens and 16-hydroxyestrone.

Why do people do this test?

The 2:16 ratio is sold as something that can tell you your relative risk of developing breast cancer.

How does this test assess your risk of breast cancer?

In short, 2-hydroxyestrogens are “good” and 16-hydroxyestrone is “bad.” Estrogens start out as estrone or estradiol and are metabolized either by 2-hydroxylation or 16-hydroxylation. Looking at the ratio of those two options will then give some valuable insight into how “good” a person’s individual metabolism is, and there are some nutritional interventions that can improve this metabolism.

Sounds good, so what’s wrong with this?

Let me be brief and then expand on this. The concept of the 2/16 ratio was developed before we knew how estrogens caused cancer. Now we know much more about the mechanisms involved and guess what? The 16-hydroxyestrone is just not that important. Again, I’ll expand on this, but estrogen causes cancer by 1) promoting growth and 2) by damaging DNA by making DNA-adducts. The 16-hydroxyestrogens don’t make DNA-adducts AT ALL! That leaves only growth promotion. In terms of growth promotion, the 16-hydroxyestrogens are >5X less potent than estradiol.

What about all the studies that show the test’s predictive power in terms of breast cancer risk?

Once again, there will be an expansive look at this later (many of you may not want to yawn through that). When you actually look at the studies, you’ll find two things. First, studies that are in favor of this test almost all come from one research group, which may have some connection to the sale of the test kits with which the test is performed (conflict of interest with financial incentives). There are plenty of studies which show this test to have no significance, and these are from groups with fewer vested interests in the outcomes. Second, studies that support the use of this test show a strong correlation between breast cancer and low levels of 2-hydroxyestrogens, and/or the 2/16 ratio; however, the correlation to 16-hydroxyestrone is usually much weaker and oftentimes nonexistent. These studies almost all ignore the 4-hydroxyestrogens because the group that sells the test kits for the 2 and 16-hydroxyestrogens don’t have an immunoassay for the 4-hydroxyestrogens (you need mass spectrometry to test these compounds). Yes, the ratio is significant, but only because of the 2, not because of the 16-hydroxyestrogens.

Before I get into some very interesting (some of you will disagree that it is interesting and you can stop after reading this paragraph) details of these points, WHAT IS THE BOTTOM LINE?

Now we know how estrogens cause cancer: First – they promote cell proliferation, so the strongest estrogens will have the strongest effects. Guess what parameter has the very strongest correlation to breast cancer… absolute levels of estradiol, the strongest estrogen. The increased risk, if there was any, from 16-hydroxyestrone would be from this mechanism and it is 5X weaker than estradiol. 16-hydroxyestrone has a weak influence on this effect, much weaker than estradiol.

Second – they create DNA-adducts which essentially damages the DNA. This can lead to cancerous growth. Don’t miss this: Only 2-hydroxyestrogens and 4-hydroxyestrogens make these DNA adducts, and only the 4-hydroxyestrogen-DNA adducts are harmful. 16-hydroxyestrone has zero effect here.

The reason the 16-hydroxy metabolism has some significance is likely because it parallels the 4-hydroxy metabolism. If you shift your metabolism away from the 16 and towards the 2-hydroxyestrogens with indole-3-carbinol supplements, or by eating cruciferous vegetables, you are also shifting metabolism away from the 4-hydroxyestrogens. I think research will eventually show that the very slight significance of 16-hydroxyestrone is simply coincidental in that if it is high, the 4-hydroxyestrogens are also likely high.

Lastly, Ercole Cavalieri’s group has performed some very fascinating research that concludes that the levels of all of these estrogen metabolites are not very significant. Rather it is the amount of the 4-hydroxyestrogens that end up reaching into the DNA and plucking out a purine (adenine and guanine) base pair to create the DNA adduct that is significant. This forces the DNA to repair and creates an opportunity for an error, which can lead to cancerous growth. Remember we knew nothing about this mechanism when the theory of the 2/16 ratio was postulated. The 2:16 needs to be abandoned in favor of more significant testing. The problem? Nobody has figured out how to test the DNA adducts in a way that can be commercialized. You’ll have to wait. In the meanwhile, don’t throw your good money after a marginally significant test. If you do use the test, the only thing that really matters is the absolute levels of the 2-hydroxyestrogens from the data I’ve reviewed. And remember, if you’re using a lab that is testing only the 2 and the 16 (and not other metabolites), they are probably using an immunoassay. Studies have shown that immunoassays are terribly inaccurate for these metabolites, particularly at low levels. Other metabolites, like the methoxyestrogens, may prove to be more significant than the 16-hydroxyestrone, but definitive research is still pending.

The following is substantial data that shows a significant difference between breast cancer groups and controls. This type of distinction is worthy of your consideration and possibly your patients money. You won’t see anything near this level of distinction in 2:16 research. The subject of the graph below will be the topic for the second part of this blog, so you’ll have to stay tuned to see what parameters/metabolites they are actually measuring. I will tell you ahead of time that it is NOT something currently commercially available.

Now for the gory details. This is actually really fascinating stuff, so keep reading…

How Does Estrogen Cause Cancer?

1. Estrogen promotes growth, so the more potent estrogens are more problematic with respect to potential cancer growth.
2. Certain estrogen metabolites create adducts (fuse together with) with pieces of DNA (specifically Guanine and Adenine). This causes potential replication errors and can lead to cancer.

The two main pathways for estradiol are the 2-hydroxylation and the 16-hydroxylation pathways. These were the original focus. One must be the “good” way and the other the “bad.” As it turns out, the ratio of these two pathways (the 2:16 hydroxyestrogen ratio, thus the popular test) does correlate in some studies to breast cancer incidence. In many studies there is actually no correlation, but don’t expect those selling the test to bring those up. If you eat cruciferous vegetables or take indole-3-carbinol it will actually shift this metabolism. Why? It doesn’t do anything to the 16-hydroxy pathway, but it does induce the enzyme that sends estrogens preferentially down the 2-hydroxy pathway. As a result, more estradiol heads down the 2-OH road and consequently less heads down the 16-OH road. This is a good thing. So, why isn’t the test of the ratio a good thing?

The fact that the 16-OH is going down some is mere coincidence. Yes, the 2-hydroxyestrogens are relatively “good” metabolites, but the bigger issue is ignored in many of these studies. If you favor the 2-OH and disfavor the 16-OH, you are also disfavoring one other minor pathway – the 4-OH pathway. The 4-hydroxyestrogens are minor constituents in that there is much less of them than the others. But, they pack a punch because they form DNA adducts. The 16-OH does not.

Initial research focused on the 2-OH and 16-OH for two reasons. First, they were the “major” metabolites. Secondly, there are easy immunoassay tests for the 2-OH and 16-OH metabolites and NOT for the 4-OH metabolites (you need expensive and sophisticated mass spectrometry for that).

Question: If you had three doors in your house and one was blocked by three menacing looking toddlers (think 2-hydroxyestrogens), another by two cranky looking elderly ladies with forks (16-hydroxyestrone), and the third by just one fit looking man with automatic weapons (4-hydroxyestrognes), which would you consider most threatening? Dumb question, yes… but it is analogous. The 2-OH metabolites are prevalent but so innocuous, we actually consider them “good.” While the 16-hydroxyestrone may have some slight negative effect, it pales in comparison to the threat of the 4-hydroxyestrogens even though the former is more abundant than the latter.

The above schematic shows the “bad” estrogen metabolism. Estrone (or estradiol) is hydroxylated by P450-1B1 at the 4-position. COMT can come to the rescue and methylate the 4-hydroxyestrogen to the harmless methoxyestrogens, or it can proceed to the quinone. It is this quinone metabolite that can reach in and steal a purine from your DNA and cause real problems. The 2-hydroxyestrogens also form DNA adducts, but they are more reversible and don’t seem to be a major player in the cancer equation.

To be clear, the 16-hydroxyestrogens DO NOT have the capacity to form DNA adducts – not even a little. This leaves only estrogenic promotion of cellular growth as the mechanism by which the 16-hydroxyestrogens would likely be problematic. While this metabolite is more estrogenic than many metabolites, it is five times less potent than estradiol. One thing often overlooked in all this metabolite research is that the absolute levels of estradiol are actually one of the biggest “risk factors” for breast cancer.

If you consider what I’ve just written and look again at all the research promoting the idea of the 2:16 ratio you likely will come to the conclusion that it is simply the favoring of the 2 and not the disfavoring of the 16 pathway that is really critical. There are many studies that conclude that the 2:16 ratio is not significant. There are also many studies that conclude that the amount of the 2-hydroxyestrogens is significant, while the amount of 16-hydroxyestrogens is not. These studies happen to also conclude that the ratio is significant, but it is driven by the 2 and not the 16-hydroxyestrogens.

Consider the following data from a paper where they are trying to tease 2:16 significance out of the data. They are looking at Asian women born in the West to those in Asian countries. They are attempting to look at the effects of Western diets on the 2:16 ratio. Take a look at the first example – premenopausal Chinese women. The ratio (1.96 and 1.70 respectively) is higher for women in China (East) than for those in the USA (West). However, the difference for the 16-OHE1 is only 2% (8.02 vs. 8.2). 90% of the shift in the ratio is because of the change in the 2-OHE1. If you move to the far right to the postmenopausal Chinese women, the16-OHE1 goes in the wrong direction by 17% (3.63 in Western Chinese women compared to 4.31), but the ratio still goes in the right direction. It is all about the 2-hydroxy…the 16-hydroxy is essentially irrelevant.

Studies like this are a complete waste of time without looking at the 4-hydroxymetabolites. Why don’t they look at that crucial metabolite? This group (which has resulted in most of the studies showing usefulness in the 2:16 ratio) only uses immunoassays for their analysis. There is no immunoassay for the 4-hydroxyestrogens. You need mass spectrometry to test these metabolites. If you want to find studies that conclude that the 2:16 ratio is significant, just add the name Bradlow to your search. His group has put out many studies on this topic. To be clear, I don’t know if there is a financial connection between Bradlow’s group and the company that sells these kits, but I can’t figure out why else they would always measure the 2 and the 16 and never the other relevant metabolites.

Consider this sad study. Why sad? Because they went to all the trouble to look at genetic variations in the enzymes that send estrogens down the 4-hydroxy pathways, but they didn’t bother to measure the 4-hydroxyestrogen metabolites. Why? It’s Bradlow’s group, and they only measure the 2 and 16.

Please see the abstract for the following study: Variants in estrogen metabolism and biosynthesis genes and urinary estrogen metabolites in women with a family history of breast cancer for my next point. They are studying deviant genetic enzymes which metabolize estrone and estradiol to create the 4-hydroxyestrogens. Having a deviation in this enzyme (follow this because it’s potentially confusing but important) probably means that there is more of the 4-hydroxyestrogen metabolite created. Yes, this is a worthwhile research topic, but not if you don’t bother to measure the other metabolites involved. It would be quite interesting to repeat many of the studies where they looked at the 2:16 ratio and include the other estrogen metabolites AND their DNA adducts.

Below you will find some information on the 2:16 ratio from the Women’s Health Initiative (WHI). It may look interesting initially, but what it really shows is that if you compare cases (cancer) to controls, there is almost no distinction between the two in any of the four groups. It does show that the ratio goes up with hormone therapy (HT), but there is no difference between those who get cancer and those who don’t.

Please see the following four abstracts and note that this ratio is not significant. However, the last two abstracts conclude there is a significant correlation between a high 2:16 ratio and decreased incidences of breast cancer.

1. Estrogen metabolites and the risk of breast cancer in women.

2. Circulating estrogen metabolites and risk for breast cancer in premenopausal women.

3. Urinary 2-hydroxyestrone/16alpha-hydroxyestrone ratio and family history of breast cancer in premenopausal women.

An observation about the above paper… it is used by labs who sell the 2:16 test in the reference section because the title leads you to assume that it finds significance in the ratio. In fact, the findings show zero significance. I guess those that sell the test are trusting you won’t actually read the papers or the abstracts.

4. Urinary 2-hydroxyestrone/16alpha-hydroxyestrone ratio and risk of breast cancer in postmenopausal women.

Two things to note about the next two studies, which concluded there was a correlation between the 2:16 ratio and cancer rates, 1) it is Bradlow’s group for both studies and 2) the first study showed correlation in premenopausal women, but not in postmenopausal women (there was actually a correlation in the wrong direction for postmenopausal). The second study found just the opposite – no correlation in premenopausal women and a slight one in postmenopausal women. I put the actual data from the second study below the abstract. It is really insignificant as the difference between the ratio of cancer patients and controls is zero in premenopausal women and miniscule in postmenopausal women.

1. Estrogen metabolism and risk of breast cancer: a prospective study of the 2:16alpha-hydroxyestrone ratio in premenopausal and postmenopausal women.
2. Do urinary oestrogen metabolites predict breast cancer? Guernsey III cohort follow-up.

Here is the data from the above study:

I am somewhat confused about how the above paper reached a point of significance that it merited publication. In my next blog we’ll look at some information about estrogen metabolism that DOES have a lot of significance. Here’s a sneak peak…Do you wonder what parameter showed such stark contrast between those who have cancer (or high risk of cancer) and controls? We’ll explore this next time.

The Facts:Topical Hormones and Lab Results (Part 2)

2010-02-03T15:58:00.001-08:00

First, we should all admit, with a dose of humility, that this topic is not yet completely understood. We don’t understand how hormones can be picked up from the skin, delivered to the tissue, and not significantly increase serum levels. This can be observed via animal studies. In the graph below, the white bar represents where radioactively labeled progesterone was found when administered to a rat’s skin. It also shows that the tissue (uterus, lung, etc) receives more of the hormone than is found in the plasma.

Is this phenomenon true only for progesterone? Consider the following for estradiol:

The makers of Evamist, a topical estradiol spray, boast its effects (reduced hot flashes, etc.) are exerted without raising levels of estradiol significantly. Curious, no? They also report serum levels as lower than those of a patch, such as Vivelle (see the middle bar graphs below). The sprays contain 1.5mg of topical estradiol, which is a lot of estrogen. One could conclude that it’s poorly absorbed since serum levels don’t go up. Or, one could look at the saliva and blood spot values (see the other two sets of bars from ZRT data) and conclude that the tissues are in fact being flooded with estradiol, but it’s not being detected in serum. ZRT data illustrates that a dose of 0.1mg estradiol reduces hot flashes more than any higher dose on average. Think about that…0.1mg, that’s 15 times less estradiol than you would get with Evamist. The big problem with testing serum is when a doctor attempts to accomplish “hormone restoration” with topical products and push levels back to premenopausal levels, they would have to use around 8-20mg estradiol (ever wonder where the Wiley protocol came from?). The Evamist advertisement states the dose is 1.5mg, and the estradiol delivery rate is 0.021mg/day? I would guess this might be a presumption based on serum levels. The actual dosage at the cellular level is probably much closer to 1.5mg.

The above advertisement would have you believe that Evamist has 1.53mg per daily dosage and Vivelle has 0.585mg (intended for an entire week of use), yet the daily estradiol delivery is thought to be lower for Evamist???? This only makes sense if you’re testing with serum and getting a serious underestimation of hormone delivery.

Let me sum up what I’ve just said about Evamist:
• Serum levels do not increase above baseline with 1.5mg/day
• Saliva levels go up >10X
• Blood spot levels go up >8X
• Symptoms are relieved

I have personally talked with women taking even less estradiol than 1.5mg topically who said they felt pregnant. I don’t think we yet have every piece to this puzzle. What we do know is that serum levels of hormones don’t track with reality for topical hormone application.

Saliva levels go up in a dose-dependent manner with topical supplementation (see graphs at the end). The question is, “should you shoot for higher supplementation ranges or physiological ranges?” The answer may depend on the hormone…more on this later.

Notice in the graph below that: a) levels go up in a dose-dependent manner; b) hot flash symptom severity is optimized at a very small dose (0.1mg daily). So, why do so many use such high dosage (1.5-8mg)? This has likely occurred from people trying to achieve physiological serum levels while using topical hormones.

With topical progesterone, again results are dose-dependent. However, even at low dosages results are “supraphysiological.” We’ll discuss this at a later time. In terms of over-dosing, the fact that 64mg doesn’t raise serum levels AT ALL, should allow us to safely use 20-30mg (which is usually effective) without too much concern for toxicity.

The Facts:Topical Hormones and Lab Results (Part 1)

2010-01-18T10:12:00.000-08:00

Happy New Year everyone! I am writing this half awake because while Vitamin D may help prevent the flu, it didn’t prevent my daughter from getting a double ear infection while we were on vacation. Needless to say, sleep was in short supply during my Christmas break, which was otherwise quite enjoyable.

I’ve been meaning to discuss the issue of topical hormones for quite some time. No matter what type of lab testing you use, you’ve probably experienced unusual results with topical hormones. Serum results are typically low in these cases, even with relatively high doses, while saliva results are high. This dichotomy has led some physicians to abandon topical hormones even if they work clinically.

This is a fascinating topic in which you will definitely want to pay attention. The following is what we know about topical application of hormone:

• Tissue uptake is accomplished with topical hormone
- Chang KJ. De Lignieres B. Fertile Sterility 63: 785-791, 1995
- This study showed progesterone levels went up in women’s breast tissue about 100-fold when topical progesterone was applied
• Progesterone exerts its anti-proliferative effects on the uterus
- Leonetti. Fertility & Sterility 2003
- This study showed that 30mg reduced endometrial proliferation 90% and 80mg stopped proliferation completely
• Saliva levels go up…WAY UP… with topical supplementation
- Even at low doses (10mg progesterone in women, 5mg testosterone in men) levels are supraphysiological 24 hours after application
- Looking at increasing dosages, saliva levels increase in a linear, dose-dependent manner (this is important, this not contamination)
• Serum levels go essentially nowhere even with relatively high doses of hormone
- Carey (British J. Ob & Gyn 2000) showed no statistically significant increase in serum progesterone at 20 and 40mg
- O’Leary (Clin. End. 2000) showed no increase at 64mg of topical progesterone
- The above studies also showed no increase in urinary levels
• Blood spot levels increase to physiological levels at physiological dosages
- Progesterone levels raise from <2ng/ml>

A picture is worth 1000 words, but it takes about that many words to describe this picture accurately. Remember, blood spot and serum correlate if you are NOT taking topical hormones. Yet, at dosages of 20, 40, and 64mg, serum (in red) doesn’t go up AT ALL. This would lead to a conclusion that topical hormones don’t work, but notice a premenopausal level of progesterone (20ng/ml) in blood spot is achieved with 30mg. Which is correct? Leonetti showed (in orange) that with just 30mg, endometrial proliferation was greatly reduced, implying that blood spot is providing a more accurate account of what’s occurring at tissue level.

Saliva results can be confusing. I multiplied them by ten to get them on the same scale. Remember, saliva values are usually 50 times lower than blood levels. While the saliva line is below the blood spot line, it is actually way above physiological levels. The point is that saliva and blood spot both increase in a dose-dependent manner, as does the effect of topical progesterone. Serum and urine levels do not.

What are we to make of all this? Stay tuned for my next blog…

Testosterone Testing in Saliva - Part 2

2009-10-26T15:42:00.000-07:00

There have been a few negative publications on testing testosterone in saliva lately. The North American Menopause Society (NAMS) published a paper in which testosterone was tested in serum and saliva. The conclusion was that “salivary testosterone does not correlate strongly with…measurements from the serum samples.” Interestingly, it was also NAMS (North American Menopause Society) who concluded that for salivary estradiol, as compared to serum measurements, “the correlation … is nearly zero.”

Here’s what we know about testosterone and estradiol in saliva:

• In some papers saliva and serum correlate. In some, they don’t quite so well. Are we to believe that at one point in time there is an agreement between saliva and serum, and at other times there is not? Perhaps it has to do with the phases of the moon or which political party is in office during the study. Perhaps the same cosmic force that keeps the Cubs losing makes some researchers studies turn out differently.

• There are two reasons why saliva wouldn’t correlate with serum.

1. Topical Supplementation (this is a long story that we’ll get into some other day), but suffice to say that serum testing severely underestimates for creams.

2. The test isn’t accurate. This is very significant for testosterone and estradiol in saliva (and even in serum). Some assays are sensitive enough to measure high level samples, but not at low concentrations. If you use a test that isn’t good enough to measure low-level samples and then conclude that saliva and serum don’t match, have you really accomplished anything? Consider the following for estradiol:

Observations:
• Results are accurate
• There is a 2-3 fold difference between days 1-5 and days 19-21… there should be
• Serum Correlation was good!
• Conclusion: Saliva Testing Works

Accurate Work = Good Saliva Testing

Observations:
• Results at day 1 are 5 X higher than above (inaccurate)
• There is no difference between days 1-5 and days 19-21… there should be
• Serum Correlation was Poor
• Conclusion: Saliva Testing Does NOT Work

Inaccurate Work = Bad Saliva Testing

There are two sets of results in the literature. These both propose to show women’s estradiol (estrogen) levels throughout their cycle. I recreated the data here.

Wong used a method that is FAR more sensitive than Chatterton (I’ll spare you the details on the difference between a 3H extracted RIA and a ‘direct’ 125I RIA but Wong’s method is far more sensitive).

Chatterton got results that are 3-5 times higher than Wong. Wong says salivary estradiol correlates to serum (r>0.9) and Chatterton says it doesn’t (negative r-value). Are we to believe that in one group of women there was terrific agreement between saliva and serum levels and not in the other? No, reality is Chatterton’s test has so much background “noise” (that’s why his levels are so much higher). Remember, the method has to be accurate and reproducible before anything else matters and for testosterone and estradiol, not all methods are good enough…some are really poor.

Let’s look at this one more way. Chatterton’s numbers have a much higher background level that obscures the specific estradiol binding sites in the test and raises the “apparent” estradiol concentration throughout the menstrual cycle. This causes the background to increase about 5-fold. To really compare them, let’s just divide by 5 (20% of original values) to make them “the same” and see what that does.

Observations:
• Bloating the numbers by a factor of 5 obscures the critical differences between the three phases of the menstrual cycle.
• Only the accurate test clearly distinguishes the ‘low,’ (day1-8) ‘normal,’ (21-23) and ‘high’ (day 14, ovulation) phases of the cycle.

Wong’s results look exactly as what you would see in a text book or from serum results. It’s subtle, but the lack of distinction between days 1-5 and days 21-23 in the other test means you can’t tell the difference between “low” and “normal.” You can see why there is no serum correlation and no clinically useful information using the inaccurate test. You can also see why a group like NAMS might pick the inaccurate method to conclude that saliva testing “doesn’t work” if it is politically expedient for them to reach such a conclusion.

Whether you are talking about estradiol or testosterone, study conclusions need to always be taken with a grain of salt. It would be better if studies like Chatterton’s were never done. Instead we have an inaccurate method reaching negative conclusions about saliva testing.

Fortunately for ZRT Laboratory, we have our own data and we can correlate our actual testing to clinical parameters. My last blog on testosterone showed lots of great data which should increase confidence in using such a test. Here is a little bit of data for estradiol to show its clinical utility. Remember these figures are from actual patient data tested over the past 10 years. This is a lot of information, but just scroll down through it, read the bullet points, and see if it isn’t impressive clinical utility for a test that some would have you believe is not relevant.

• Here we see results from nearly 50,000 women, and we can see the expected relationship between estradiol levels and age.

• We can see a nice correlation between levels of estradiol and the severity (self-reported) of their hot flashes and also vaginal dryness.

• We know that a higher BMI means more adipose tissue. More adipose tissue means more aromatase, which will convert testosterone to estradiol. We can see this relationship strongly from levels measured in premenopausal women. The graph looks much the same (except lower levels of course) with postmenopausal women.

• One criticism of testing is that there is no relationship between the hormones taken and the clinical levels in the patients. This strongly refutes that position. First, with oral estradiol…

• Even more impressive data with topical hormone. Not only do we see very nice dose-dependent results, but we also know how severe these women's hot flashes were (we collect that information on our requisition forms). You can see two hugely important things.
1. Dosages of 0.1 mg get most women up and into the premenopausal range, and
2. Low dosages do a very good job of relieving symptoms.

If you are using 1-4 mg of topical estradiol, this begs the question, why? The answer to that will have to wait for next time. In short, topically measured hormones do not increase serum levels. This has lead to many physicians who are monitoring topical hormones to overdosing.

The above data shows tremendous clinical utility for salivary estradiol. ZRT’s assay for estradiol is uniquely sensitive, which is critically important for this test. Don’t assume that this type of data would fall out of another lab’s estradiol test. It only will if it is accurate. Many of the tests available are not optimally accurate, so if you use a different lab, ask for this type of data to instill confidence in using the test. As Chatterton showed, just because samples are measured and numbers are given doesn’t necessarily mean that the numbers have a high degree of clinical relevance.

Testosterone Testing in Saliva - Part 1

2009-10-26T15:16:00.000-07:00

The following is some data I mined from the ZRT Laboratory database of literally over 1 million people tested for salivary hormones over the past 10 years. This data clearly substantiates the clinical utility of measuring testosterone in saliva, in contrast to recent publications that cast doubt on the relevance of testing testosterone in saliva. Here’s some data to show that it does indeed work if it is done accurately and reproducibly.

• We can see that correlation of testosterone with age makes sense: levels are dropping off as women age, just as we would expect to see in serum measurements.

• You can also see a VERY strong correlation between levels of testosterone and symptoms of excess testosterone (excess facial/body hair). Women who complain of these symptoms have much higher levels than women who do not complain of these symptoms.

• Here we can see that as women take topical testosterone, levels go up in a dose-dependent manner. This is HUGE if you plan to use saliva testing to monitor supplementation. The recent North American Menopause Society (NAMS) publication stated that they saw no increase in salivary levels with topical supplementation(1). We see otherwise.

• Note the expected values are different if you test at 12 hours or 24 hours.

• Have people test at 12 or 24 hours, so we can give expected values for that particular situation. Topical testosterone ranges are not currently on the ZRT web site for women, but they will be soon.

• In men we can also see the linear, dose-dependent relationship between salivary levels and the amount of topical testosterone taken.

The recent publication from Menopause showed little clinical utility for measuring testosterone in saliva. The data in their publication showed almost no correlation with serum. I will address this issue in the next installment.

(1). Flyckt, Rebecca, James Liu, Heidi Frasure, Kathryn Weksleman et al. "Comparison of salivary versus serum testosterone levels in postmenopausal women receiving transdermal testosterone supplementation versus placebo." Menopause. 2009; 16(4): 680-688.