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REPLAY: Biomarker Testing for Mental Health with Dr. Adrian Lopresti and Dr. Sinan Ali

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REPLAY: Biomarker Testing for Mental Health with Dr. Adrian Lopresti and Dr. Sinan Ali

Dr. Sinan Ali, academic and researcher in stress physiology and hormone analysis, joins our ambassador Dr. Adrian Lopresti to discuss the use of hormone and biomarker analysis for the management of mental health conditions. Together they discuss the benefits (and the down-side) of using blood, urine, salivary and hair testing techniques and their application to provide further insight into a patient’s clinical presentation and to support clinical management. 

Sinan discusses current research supporting the use of cortisol, BDNF, oxytocin, corticotropin-releasing hormone and many other hormones for hormonal and mental health conditions and others that present exciting opportunities for future research in this area of functional pathology including malonyl dialdehyde, interleukin-6 and CRP. 

Covered in this episode

[00:32] Welcoming Dr. Sinan Ali
[01:41] How testing informs clinical opinion
[05:59] Can you diagnose depression using  biomarkers?
[09:40] Inflammatory cytokines and other up and coming biomarkers
[14:37] The challenges of measuring neurotransmitters
[20:25] Measuring cortisol and melatonin for sleep disturbances and insomnia
[26:30] How to choose the right sample - blood, saliva, urine, or hair - for what you want to measure
[41:40] Other things that will affect test results
[46:48] Thanking Sinan and closing remarks

Key takeaways 

  • Pathology testing of biomarkers and hormones can provide insight and support the clinical management of mental health conditions. 
  • Testing can be used to complement the work practitioners do and treatment options through the assessment of symptoms and the use of DSM-5 for diagnosis. 
  • Neurotransmitters can be tested using serum tests, or urine tests that identify metabolites of the neurotransmitters in the urine. 
  • Testing of adrenal function can provide insight into the production of neurotransmitters. 
  • Sleep disturbance biomarkers can include cortisol and melatonin with testing to be done at specific times of the day in accordance with the circadian rhythm.  
  • Salivary testing is less invasive and may provide insight into levels of bioavailable, bioactive steroid hormones that are not bound to carrier proteins in the blood. 
  • 90-95% of the total amount of a steroid hormone is bound to a binding protein to support solubility in blood. The remaining 5% is considered to be the free, bioactive form of the steroid hormone. By measuring cortisol and other steroid hormones in saliva, only the free, bioactive hormone is recorded. Blood hormone measurements include both the free and the bound hormone. 
  • The free-index of a hormone is the total measurement of hormone (blood) and the measured binding capacity within it. The Coulomb equation can identify the free hormone levels, which is likely to indicate the same result as a salivary test, suggesting the benefits of a saliva test. 
  • Topical supplementation levels may be measured in salivary testing, but are unlikely to be identifiable in blood tests.  
  • Urine may be a good measure for metabolites .
  • Some biomarkers measured through saliva may be flow-rate dependent. Steroid hormones are not flow-rate dependent. Larger protein biomarkers have a different entry route than hormones into the saliva are flow-rate dependent. 
  • Hair-hormone analysis provides insight into hormone levels deposited in the hair matrix for a specific period of time based on the length of hair from the scalp as hair grows 1cm each month. This allows for clinical assessment of hormones over a defined period of time, or a chronic measure. 
  • Inflammatory causes for mental health conditions may be supported through the identification of inflammatory markers including CRP. 
  • Exercise prior to testing is to be considered a stressor and may impact test results. Alcohol should not be consumed in the 12 hour period leading up to hormone testing. Similarly, abstaining from smoking a couple of hours prior to hormone testing is advised. Eating prior to hormone testing should be minimised and kept to low glycaemic index foods to minimise the impact glucose and insulin has on the levels of neurotransmitters testing. 
  • Hormone testing in saliva including cortisol, testosterone and oestradiol are stable. Hair hormone samples are stable for extended periods of time. 

Resources Discussed in this episode

Dr. Sinan Ali
Australasian College of Health and Wellness
SALPATH Functional Pathology
Saliva Flow Rate
Study: The Cortisol Assessment List (CoAL) A tool to systematically document and evaluate cortisol assessment in blood, urine and saliva (Laufner, et al., 2021)

*Correction: In the introduction to the episode, Adrian says that Dr. Gupta is the Associate Dean of the Australasian College of Health and Wellness. He is currently the former Associate Dean.


Adrian: Hi, and welcome to FX Medicine where we bring you the latest and evidence-based integrative, functional, and complementary medicine. I'm Dr. Adrian Lopresti, and joining us today is academic researcher Dr. Sinan Ali

Sinan's research in the area of stress physiology and hormone analysis has been widely published. He's the former Associate Dean of the Australasian College of Health and Wellness and founder of My Hormones and Me and SALPATH Functional Pathology, which provide hormone analysis to university researchers, integrative-medicine practitioners, and aesthetics clinics. 

All right, we're very excited to have you here today, Sinan, and welcome to FX Medicine. Thanks for joining us today.

Sinan: Well, thank you very much, Adrian. And I am excited to be here. And thank you for this opportunity to have a chat all things measurement with you.

Adrian: Yeah, absolutely. So, today we're going to be discussing what the different testing options are for pathology testing in mental-health conditions, as well as different biomarkers that we can look for in terms of guiding us with regards to our assessments and treatments. So, hopefully, we'll be able to provide some useful information for our listeners today.

But first thing, I suppose what I wanted to do first Sinan, is just talk a bit about how testing can be used to complement some of the work that practitioners are doing. So, typically, I know that assessment's primarily done through assessments of symptoms, as a practitioner myself and other practitioners, they’ll know that what we generally do is, when we see somebody with depression or anxiety, we'll ask about their symptoms. 
Many psychologists and psychiatrists, for example, we use the DSM-5 to help them come up with a diagnosis. But, obviously, one thing that is available to us is the testing and using testing to help us guide us with regards to treatment and so forth. Can you tell us a little about how testing can be used to complement some of the traditional interviews and diagnostic assessments that we do?

Sinan: Yeah. I think, from a testing point of view, testing is very important in order to help to inform your clinical opinions. However, I'll put a caveat right in there in that testing is not there to replace what you do as a clinician. So, it's simply there to guide you to help along.

Having said that, the testing can, in some instances, and I've had some personal experience with this, not in the mental-health realm, but testing can be helpful in that it does bring up issues that have not been diagnosed clinically, for one reason or another, largely because the client does not mention them, does not think of them to be important enough. But we've seen hormonal measurements in some people that go right against what the client was there to see. Naturopathy, for example, we've been able to shed some further light. But certainly the testing is not there to replace what is happening in the clinic world but to inform, to help along, to guide, to provide some numbers perhaps that could be useful.

In terms of mental health, there's not a lot of functional pathology testing that informs mental health. We know that hormone imbalances have negative impacts for a lot of people in the physical and the mental-health arena. So, hormone imbalances can influence anxiety, and tiredness, and irritability, weight fluctuations, etc., etc., etc.

Many disease conditions are multi-factorial. So, in many cases, there's not a single biomarker that you could look for to inform or to determine the treatment and there are many many different conditions that can affect the level of a single biomarker. So, where do you go from that? I mean, we know that low cortisol can be caused from PTSD and chronic fatigue syndrome, etc., etc. So, it's a matter of "Take you pick," as it were.
So, also, when these biomarkers change, are they the cause of a particular type of condition, a particular type of illness, or do they result from that illness? And we can talk about adrenal fatigue as an example of that, but I'll leave that there for a somewhat quick answer.

Adrian: Yeah, absolutely. Yeah, certainly, I think that's the thing, there's been, in terms of a diagnosis, obviously, there's more and more research to look at different biomarkers and see whether people with depression or anxiety have elevated or disturbances in different biomarkers. 

But at the moment, in terms of an assessment or a diagnosis, we really have to rely on our assessments, our interview, the use of questionnaires to diagnose somebody with depression or anxiety. And certainly, as you mentioned, research is showing, and we're guided by the research, that maybe there's some imbalances in some of them. 

But there isn't a biomarker there that all people with depression or all people with anxiety, and correct me wrong, that, if you have an elevated level of cortisol, for example, or low cortisol, not everybody with depression or anxiety has elevated cortisol or local or low cortisol, do they?

Sinan: Absolutely. There are many many different conditions that will cause an increase in cortisol, that will cause a decrease in the level of cortisol. So, you can't pinpoint it to a depressive state and you can't pinpoint, even if you had a biomarker X that was up or down in depression, then there are many many different forms of what we generally classify as depression. And that's your area of expertise, not mine. But they have not been dissected out in terms of the different classifications of depression, and these are the biomarker changes that you would see in each one of them. So, I guess we're at the early stages for that. There's certainly a lot of research done in the area. But to bring that to the clinical world, it's going to take some more time and a little bit more understanding, at this point in time. 

But are there markers that we could look for? There certainly are markers that we could look for. And in major depressive states, major depression, MDD, there's certainly markers like BDNF. And we know that decreased BDNF leads to the pathophysiology of major depressive disorder. And that's possibly due to increased cortisol levels and increased glucocorticoid activation in the hippocampus, which has structural effects on the hippocampus atrophy and the like and that can lead to a decrease in BDNF.

So, certainly, we could measure BDNF. And we do measure BDNF not as a routine but we do a lot of work in measuring BDNF for researchers. We could certainly transfer that to the clinical world and start measuring salivary BDNF. But then, when you start measuring salivary BDNF, is that BDNF of hippocampal origin? 

Because most of the research shows a decrease in hippocampal BDNF. So, if we measure salivary BDNF, do we know its origin? Is it coming from the hippocampus? Is it coming from the peripheral cellular regions? Does BDNF cross the blood-brain barrier at all? We don't know the answer to those sorts of questions. And BDNF changes are also there in other psychotic disorders. And getting back to the age-old question, is that the cause or the result of?

Adrian: Can you just tell us what BDNF is?

Sinan: Brain-derived neurotrophic factor, which is involved in neuronal function, neurogenesis, and the like. So, the more BDNF you have, the better neuronal function you're likely to have. So...

Adrian: Thank you.

Sinan: ...the less neurotropic factor decreases in major depressive disorder, yeah.

Adrian: Yes. Okay.

Sinan: That's not the only one, there's others that could be measured that are measured quite routinely in the research world, various cytokines, in particular the inflammatory cytokines, interleukin 6, interleukin-1, TNF alpha. There's the anti-inflammatory cytokines, interleukin 4, 10, and 13, that are measured. So, you're looking for a balance between these inflammatory and pro-inflammatory cytokines together with CRP, which is your measure of inflammation, systemic inflammation.

And there's correlations in depressive states with various changes in the concentration of these cytokines. And we know that these cytokines can cross the blood-brain barrier and have an effect on the brain. So, it has effects on glutamate and monoamine transmission, so, things like your serotonin, norepinephrine, epinephrine. So you get less serotonin in the brain, has effects on synaptic remodelling, it has various effects on neurogenesis, and so forth and so on. And all of these, of course, have behavioural responses, behavioural effects. So, cytokines is one of those areas that is very very heavily studied in research.

Adrian: All right. So, you’ve mentioned, obviously we know that there's a link, a relationship between inflammation and depression, so, you mentioned that measuring CRP you've got different cytokines, the pro-inflammatory and the anti-inflammatory cytokines, and maybe looking at the balance between that. You've mentioned brain-derived neurotrophic factor, or BDNF, which then may be important around neuroplasticity. So, sorry, I cut you off, you were about to mention something else, what was that?

Sinan: The story goes on and on...

Adrian: Yeah, there's even some research around oxytocin too, isn't there?

Sinan: There's research around oxytocin, there's research around corticotropin-releasing hormone from the hypothalamus, CRH. From the pituitary, there's vasopressin, or antidiuretic hormone, these all potential markers. And exciting new potential markers, oxidative stress markers. 

So, obviously, you can look at generation of free radicals, you can look at the antioxidant potential in the body. You can look at an exciting new one, which is a product of lipid peroxidation, which is malonyl dialdehyde, MD, which is now starting to gain traction as a reliable marker for major depressive disorder. You can look at various other biomarkers, superoxide dismutase, again, part of the oxidative process that's been looked at, it increases in serum with major depressive disorder. 

Some of these biomarkers are more reliable than others. And I think a mark of whether they are reliable or not is to see whether they consistently change back to what is thought to be their native levels, original levels, with antidepressant treatments or any other form of treatment that resolves depression. Do some of these biomarkers actually change? 

Adrian: Yeah.

Sinan: And that, by and large, has been, in the research world, being used to determine whether they are good candidates as biomarkers or not. But yeah, I think things BDNF, I think CRP, interleukin 6, and malondialdehyde are likely to be candidates that would be used by even functional pathologies in the future.

Adrian: Yeah, okay. All right, so, we've got then, obviously, your inflammatory markers, your oxidative-stress markers that you've mentioned there, and then, obviously, your BDNF, your neurotrophic factors there. And then, obviously, the one that's commonly people are probably a bit more aware of is cortisol too, that would be another option for people, wouldn't it?

Sinan: Absolutely. Cortisol is pretty much at the centre of all of the testing. I would put my house on that cortisol is probably the most widely used marker coming out of functional pathology.

Adrian: What about neurotransmitters? Can you test neurotransmitters through blood or urine or whatever?

Sinan: Neurotransmitters can certainly be tested in blood. You can look at the metabolic products in urine. They can certainly be done, but there are questions of the reliability. Because when you're looking at neurotransmitters in terms of mental health, they are in the central nervous system. And where they are used is in the central nervous system. 

So, how do we get access to these neurotransmitters, that are in the central nervous system, by measuring blood? Yes, some of those neurotransmitters do end up in the circulation but we have to think about if we are measuring neurotransmitters such as norepinephrine or epinephrine, we really need to think about their major source. And the major source is the adrenal medulla because the medullary cells of the adrenal gland are modified postganglionic neurons, and that's why they're capable of producing classical neurotransmitters that circulate in blood and have major effects.

So, when you get a flight-and-fight response in stress, the flight-fight response is a result of that those neurotransmitters that are circulating that are of adrenal medulla origin rather than central nervous system origin. Yes, there are other neurotransmitters such as serotonin, etc., that are mainly in the central nervous system, yes, they are also found in the blood, but comes a question of their origin. Because the medulla can also produce serotonin.

And if you really want to take the story a little bit further, we've got to think of the the largest organ in the body. And the largest organ in the body is, obviously, the skin. And we know that the skin is a receptor, so it responds to many of the hormones, it responds to neurotransmitters. But 15 years ago, 18 years ago or so, we discovered that the skin is also capable of producing hormones and producing neurotransmitters. Now, we presume that a lot of those actions are local but, having said that, there is very little stopping those hormones, very little stopping those neurotransmitters that are producing the largest organ of the body from ending up in the systemic circulation. 

Adrian: Okay.

Sinan: So, there's an additional level of complexity that comes into the system, which is the origin. 

So, when I'm measuring anything in any fluid in any part of the body, I really need to question, "What is the origin of the biomarker that I'm measuring? What is it likely to be the most?" And the functional pathology arena is full of examples of measuring hormones in saliva, measuring hormones in blood. And you get elevated hormones in one fluid, such as saliva, but not in another. 

So, if you're taking topical hormones, for example, topical progesterone, topical oestradiol, testosterone, you will be able to measure a high concentration of them as they cross through the skin and end up in saliva. But if you measure their blood concentration, you don't see an increase in any of these hormones.

So, a clinician needs to understand this. We as researchers, we as a laboratory, need to understand this so that we're not overdosing patients on things like topical hormones because, if you measure it in blood, you really need to overdose quite significantly in order to get sufficient concentrations that end up in blood. But you see it in saliva where still you see very high concentrations of these topical hormones in a medium that we do a lot of measures on, which is in hair, and the source of where these hormones are coming from.

Adrian: I certainly want to discuss the different options for taking saliva samples. So, I'll certainly go back to that. 

So, just with then what you've mentioned there, obviously, there's all these different markers that we can test for, but, as practitioners and as researchers, we also need to question, "Well, you can measure different neurotransmitters or metabolites of neurotransmitters, but is it actually a reflection of what's going on in the central nervous system?" 

So, I suppose, if you want to measure neurotransmitters, you've actually got to try to get into the brain or use spinal fluid or things like concentrations in spinal fluid that would probably more accurately predict neurotransmitter concentrations, would that be right?

Sinan: Absolutely. So, if you're measuring concentrations in the spinal fluid, then that would be a much greater measure because you know you're confined to what's happening in the central nervous system there.

Adrian: Yeah. Okay

Sinan: But that's not a test that's going to be widely used outside of an emergency type department

Adrian: Yeah, no. Exactly.

Now, so, you've mentioned some with depression, I suspect a lot of those markers you could potentially use for somebody experiencing stress or anxiety. What about if somebody was suffering from insomnia or sleep disturbances, is there any particular markers there that people could look at?

Sinan: In terms of sleep, there really isn't a lot there. A couple of the hormones that immediately spring to mind, obviously, with sleep is melatonin and is cortisol. But when we talk about sleep, there's different types of sleep problems from acute sleep deficits to chronic sleep deficits to obstructive sleep apnea, circadian disruptions. And I think, when we generally think about sleep problems, we generally think of circadian disruptions. So, people not getting a restful sleep, people waking up, people can wake up from obstructive sleep apnea, for example. Now, they're very very hard to diagnose outside of the clinical situation. As a biomarker, we can look at melatonin. As a biomarker, we can look at cortisol.

Adrian: Yeah.

Sinan: And both of these have quite distinct circadian patterns where we know when the peak should start, we know when the peak should peak, and we know at what rate roughly those peaks decline and what their levels are throughout the 24 hours. So, we can measure both of those, we can measure cortisol at regular intervals, we can measure melatonin at regular intervals to determine whether there are circadian disruptions there, whether they are shifts in the peak.

So, for example, if you look at cortisol, then we all should be peaking somewhere around when the sun rises, and within half an hour on either side of that. But certainly when you wake up, half an hour post waking, you should have your maximum cortisol level.

Now, if you look at individuals who are shift workers, so, rather than getting up as we normally do at sun up to start our day, they are sleeping through that period and they are working at night time. So, you're looking at roughly a 12-hour shift in their level of activity. And when you measure the circadian pattern in cortisol of these people, what you generally find is that, rather than having a circadian pattern that is tied in to the day-night cycle, the light cycle, that shifts towards the right 2 to 3 hours. 

So, they'll actually shift, well, in Australia, in Sydney, at the moment, it would be somewhere around 7:00 to 7:30 in the morning, but if you're a night-shift worker and consistently working, I’m not just talking about doing a couple of shifts here, a couple of shifts there.

Adrian: Yeah.

Sinan: It takes some time for the circadian pattern to actually move, as you would experience when you have jet lag, about three, four days quite significantly and about seven, eight days to totally get over jet lag. There's a shift from 7:00 to somewhere around 9:00 to 10:00 is the shift of their cortisol peak.

Adrian: Okay, okay.

Sinan: So, we can measure those shifts in circadian pattern and that would be some indication of sleep disturbances that people might be having. As a biomarker, that's pretty much all we have at our disposal.

Adrian: I suppose, with melatonin, that's something that people, I think, need to consider, and I'm not sure whether it's considered a lot. Obviously, we know that light will affect melatonin. So, if, for example, somebody tests their melatonin and they're been sitting in a room with lots of light and they've been sitting there for half an hour and then they collect the melatonin and then they do a post assessment and, the second time around, it's been dark, the lights have been down, is that going to affect the melatonin levels?

Sinan: Oh, absolutely, absolutely. So, melatonin is pretty much low during daylight hours and it starts to increase a couple of hours before nightfall, a couple of hours before you actually start to wind down and go to bed. So, if you're going to bed consistently around 9:00, 10:00, for example, your level of melatonin is going to start to increase a couple of hours before that. Your melatonin will not peak until you're well and truly asleep. And currently, your melatonin will peak somewhere around 3:00 or 4:00 in the morning, I suggest because it's...well, somewhere around 3:00 and 4:00 in the morning. And then, as soon as you wake up, it's gone again.
So, there is no point in making a comparison, melatonin levels, if you, as we are doing right now, sitting in a room in daylight with lights on and taking a sample and then taking a sample sometime at night and make that comparison, well, the nighttime sample is going to be higher but only if the lights are dim or there's no lights. Because the minute you have light, your melatonin levels will plummet again.

Adrian: Wow. All right. So, there are things that people, obviously, need to consider.

Sinan: Yes.

Adrian: Now, obviously, you can measure via blood, you can measure via saliva, you can measure via urine, and now you can also measure via hair. I know you do a fair bit of hair testing too. So, why would we choose one sample over another? How do clinicians decide, "Yep, let's go with hair. Let's go to saliva.” What do they measure and what's different about them?
Sinan: So, it really depends on what you want to measure. Traditionally, I suppose, pathology type biomarkers have been measured from blood. And, by and large, if you go to your local GP, by and large, they're measured still out of blood. 

So, if you wanted to get cortisol done, and there's only two reasons why you would get cortisol done through a GP, if you have suspected Cushing's or suspected Addison's disease, I think Medicare doesn't cover it for much beyond that, it would be a blood measurement. So, why would you use blood versus that of saliva, because we can also measure cortisol, and many of the other hormones, in saliva? 

Well, there's one difference between blood and saliva. When you're talking about hormones, and I'm not talking about all biomarkers here, if you have a protein biomarker, if you have a fairly large protein biomarker, definitely, consider blood for its measure. But when you're looking at fairly small molecules, like these steroid hormones, when you're looking at molecules that are hydrophobic in nature, that is fat-loving in nature, such as the steroid hormones, they are derived from cholesterol and they are fat-soluble or hydrophobic, I would certainly think of measuring them outside of the blood. 

Why? Because in blood we have a large number of proteins that bind to the steroid hormones. And I'll give you an example of the hormones that we tend to measure in pathology: cortisol, oestradiol, testosterone, progesterone. All of these steroid hormones do not circulate in what is known as a free and bioactive form. They circulate bound to specific binding proteins. In the case of cortisol, it circulates bound to CBG, or corticosteroid-binding globulin. In the case of testosterone and oestradiol, they circulate bound to SHBG, which is sex-hormone-binding globulin.

And the reason why they do that is to keep them in the high concentrations that we do find them, or relatively high concentrations that we do find them in blood, and to keep them solubilised in blood. Don't forget, these are hydrophobic chemicals that are in a hydrophilic environment, in watery environment. So, they need to be solubilised. So, one way you can solubilise them, one way you can keep them in the reservoir which is the blood, is to bind them to a binding protein. 

Ninety to 95% of the total amount of steroid hormone, i.e. cortisol, testosterone, oestradiol that you have, is bound up. And the 5% or so is in the free form. And it is thought, and I'll put a caveat to that later on, but it is thought that the 5%, the free form, is the bioavailable and therefore, the bioactive component.

When you measure cortisol in blood, you are measuring the total amount of cortisol in blood, not just the 5% which is the bioactive component. When you measure cortisol, and oestrogen, and testosterone, and progesterone in saliva, only the free hormone gets into saliva, so, you're measuring the bioavailable and, therefore the bioactive.

Adrian: So, that's probably some of the reasoning behind the differences. So, if you get a blood test and a saliva test, you might get discrepancy in the results. And that's because one's measuring total and the other one's measuring free.

Sinan: Yeah. So, you can ask for a free index. So, in order to get the free index, you need to first measure the total, then you need to measure the capacity of the binding protein that is there. And from the capacity of the binding protein, there's an equation called the Coulomb's equation that you plug the numbers into and you will get the free component of the hormone. It's doing two measurements and then putting that into an equation to get what, essentially, you would get from saliva anyway.

And saliva is a much easier measure. I certainly would not go to blood measurements if I'm doing any experiments whatsoever, or any measurements with cortisol, because there's an enormous number of people, including myself, who are needle-phobic. And the minute I think about going to give blood, my cortisol level is going to be up anyway. It's going to be a pointless measure, in my opinion. Whereas, it's so much easier just to drool in a tube with a bit of saliva that can be measured.

So, that would be the reason why you would measure out of saliva instead of blood. And as we've said before, if you're doing any form of treatment that requires topical supplementation, then again there's a number of studies showing that the concentration of those topical hormones can be measured in saliva but they're not detectable in blood.

Now, it could be that they're not detectable, and I've read some and heard some arguments saying that the red blood cells absorb them, so forth and so on, I think what happens is that they're just simply absorbed by the binding proteins. Now, these binding proteins are fairly high-affinity but there's also a low-affinity binding protein, and that's albumin. And 55% of the total protein content in your blood is albumin. And it can bind to these hormones, and it does so. Yes, it's low affinity and, therefore, it's regarded as being a free form, but nonetheless albumin combined to the hormones. And you may not be able to really readily be able to measure them. So, you could use urine, if you're looking at any of the metabolites, you would definitely need to use urine, but again, from a simplicity point of view, saliva is the way to go. 

The only thing you need to watch out with saliva, and these are the pros and cons that I'll throw in here and there, the only thing that you need to watch out with saliva is the flow rate. In some of the biomarkers that you measure, they will be flow-rate-dependent. Things like your steroid hormones, they're not flow-rate-dependent at all but, if you're looking at larger protein biomarkers, which have a different route of entry into the salivary fluid, they are likely to be flow-rate-dependent. So, you need to be careful with that.

So, blood, saliva, urine. And we also do quite a bit of hair analysis now. And hair is quite unique in all of these measures in that we can get a historical measurement from hair-hormone analysis. Hair grows roughly 1 centimetre per month and hormones are deposited and they are trapped in the matrix of the hair. And what we can do is we can extract them from the hair and we can measure them. 

Now, we can extract the first centimetre, the second centimetre, the third centimetre. We can extract 6 centimetres, whatever is the paradigm that the client wishes. And we can extract those hormones and measure them and we can say, "Okay, two months ago, this was what your cortisol was doing. Twelve months ago, this is what you cortisol was doing." So, that's part of the advantage of doing things in hair.

The other advantage of doing things in hair, Adrian, is this. If I'm taking a measurement from blood, if I'm taking a measurement from saliva, I am getting the measure, or the level of hormones, at the time of sampling. So, it's a one-off at the time of sampling. And they could be elevated, or they could be decreased for whatever reason. And we won't go into that at this point in time but, for whatever reason, they could be elevated. 
Whereas in hair, we don't see those elevations because hormones are deposited continually into the hair matrix, so, you get this averaging effect that goes on. 

So, whereas we can pick up quite nicely a circadian pattern in cortisol in saliva, we can't pick that up at all in hair because we pick up all the highs, we pick up all the lows, as an average, and we get an average value of all of hair.

Adrian: So, just a chronic measure?

Sinan: A chronic measure is one way of looking at them.

Adrian: Or an overall measure.

Sinan: There was a study done back in September, 2021, so, not long ago, that looked at all these different measures from blood, saliva, urine, hair and looked at the advantages and disadvantages of them and their reliability. And they were able to determine that chronic stress determination, by far the most reliable measure, resulted from hair cortisol analysis. 

Adrain: Oh, wow.

Sinan: So, it's quite a good study to do.

Now, we do, as I said, quite a bit of hair work for researchers but we also have a number of practitioners that have taken up looking at hair cortisol. And that's the only one that we do with the practitioners at the moment. And that's because we've done so many, we've done thousands upon thousands upon thousands of hair samples. So, we've got quite a set of data from which we can draw our norms and therefore, be able to utilise hair cortisol measurements. 

Adrian: Wow.

Sinan: And they do bring up some very nice data. We had a client from London who sent us six years worth of hair, and we were able to extract on a three monthly basis from six years, were able to extract and measure cortisol. And again, someone from London, we don't know her, we have no idea of the clinical history, but we were able to show and drive a nice graph of changes of…and I think she had about three dips, over the last six years, or over the period of the six years that we measured. And we sent her the data and she wrote back to us and said, "I've got tears in my eyes because I've kept meticulous records of my symptomology because no one could diagnose me with anything, and certainly we didn't diagnose her with anything, we just helped her see that, whenever she had those symptoms, that there were dips in her cortisol level. So, the three dips that we did find in her case, for example, coincided quite nicely with her symptomology, whatever that symptomology was for her. 

Adrain: Wow.

Sinan: So, that's the utility, I guess, of these measures. And the other utility of them is that hormones do get trapped in the hair matrix. So, they're not going anywhere, they're not going to be cleared.

Adrian: Yeah.

Sinan: So, we can measure them quite nicely, quite easily. And we've had instances...and I've had a couple of recent ones where we were able to measure very very high levels, in fact, levels that were off the scale. So, in one instance, they were way way above the normal reference range. In another instance, they were so high that we just could not measure it. I mean, we could if we went and diluted etc., etc., but there was no point, it was just very very high. And these were both individuals. Well, one was taking topical pregnenolone, for example, which is converted to progesterone, as well as taking topical progesterone because her levels were very very low. And that was very easy to measure.

But the one that I really liked was an individual who, for all intents and purposes, was trying to fall pregnant. And without giving too much away, she couldn't. And when we measured her progesterone, it was through the roof. And when I reported that, the clinician said, "No, no, you've got it wrong. She's not on a supplement, she's not on anything. In fact, she's trying to fall pregnant." So, we redid it. And we always run controls, and everyone else that was in that same room was quite normal. And we redid it and got exactly the same result two weeks later. It ends up that her husband, who was trying to slow down his hair loss, was on a progesterone cream.

Adrian: Ah, wow.

Sinan: So he's taking the progesterone cream and she's getting a dose because, every time she touched, she got a dose. And if he took it at night and he's lying and she's lying next to him, she's getting a dose.

Adrian: Wow, it's amazing.

Sinan: No wonder she was not falling pregnant. Now, if we did saliva, and we didn't do saliva on her, but, if we did saliva, then we wouldn't pick up those huge levels because it's being continually cleared. So, that's the utility of the hair. If you're bald, then we don't have that option.

Adrian: Yeah, it's not an option. Well, so, really then, we could just keep going for a long time. And there's lots of questions I have in my head that I'd to ask you but, obviously, we're just running out of time. 

But just to summarise, it looks then, basically, you've got a number of different options that practitioners could look at in terms of assessing, if somebody's coming in with depression or anxiety or sleep-related problems. 

So, there needs to be, firstly, the question that they need to ask themselves is, "Which markers do I think may be useful? Is it cortisol? Is it BDNF? Is it inflammatory markers? Is oxidative stress markers?" And that's where, I suppose, the clinical assessment can help guide the clinician to go, "Okay, what do I think might be contributing to some of the symptoms? So, I think maybe there's an inflammatory role here," and then you can, obviously, then choose your inflammatory type markers. So, you've got that.

Sinan: Absolutely.

Adrian: Then you've got to make a decision around, "Which sample do I use? Do I use hair? Do I use saliva? Do I use blood? Do I use urine?" 
And then you've also mentioned that things timing, and light, and, I suspect, things exercise, and the food we'll eat, and whether you brush your teeth, or whether you wash your hair, and colour your hair, and all those things would probably affect some of the results too. They're all things that need to be considered. Am I correct in saying that too?

Sinan: Absolutely. So, the timing and a lot of these things do have circadian patterns. So, the timing is important. Exercise is a form of stressor, and obviously, we need to avoid exercise when we're looking at providing samples. 

Alcohol is a big no-no. So try to avoid drinking alcohol for a 12-hour period or so before you provide a sample. The longer, the better, I suppose. Again, the same with smoking but not so much. So, you don't need to stop smoking for 12 hours. Generally, if you stop smoking for a couple of hours and provide a saliva, that's fine. 

Eating is a big one. So, we ask that people don't eat for an hour at least before they provide a sample. And if you are going to eat, try and keep it low GI because high sugary foods or high-glycemic-index foods can really mess around with your glucose levels, your insulin levels, which then can affect your neurotransmitter levels which can affect your cortisol level, for example. So, we need to think about all that. 

With hair, there's not a lot that affects the level of cortisol or testosterone, oestradiol in hair. So, washing it, dying it makes very little difference. Even if you've got topical hormones that you put on the outside, because we remove all of that before we extract from hair anyway. So, it tends to be a fairly good measure. These things are very very stable. 

So, when you look at hormones in saliva - cortisol, testosterone, oestradiol, they tend to be fairly stable. There's not a lot that can happen to them in saliva. In fact, when you look at the level of cortisol in saliva, if I get a sample today and I leave it on the bench for two weeks and re-measure it, there'll be no change in the level of cortisol at all.

Adrian: Okay.

Sinan: So, it's very very stable. In hair, obviously, they're stable for years and years and years. But we do ask that people freeze them before they send them to us. If you're doing melatonin, obviously, light exposure is very very important for that. 

So, I think clinicians need to have an appreciation for all of these things. And you may not know the ins and outs and the intricacies of all this. And I think this is where clinicians researchers can pick up a phone and call people myself and have a chat. “I'm looking at this patient or I'm looking at this study that I want to conduct, what are some of the pitfalls? What are the some of the things that I need to do and what are some of the things I need to avoid?” 

So, you need to be aware of all of these things. And it's only a matter of chatting with whichever laboratory you choose to do your work. If you're doing research, if you're a clinician, again, talk to the laboratory that you choose to do the work.

Adrian: And I know I've used you. But certainly, Sinan, you've done a lot of testing for me in our studies. So, when I often pick up the phone and have a conversation with you to help me...

Sinan: Yeah, absolutely.

Adrian: ...come up with ideas around the intricacies of testing. So, I'd certainly encourage practitioners, if they're interested in doing testing and they're not quite sure which tests and how to do it, to pick up the phone and to contact, obviously, the laboratory and, certainly, contact you. 

We’ve run out of time, I could keep going. I think, yeah, this is a couple of days course really that could really be done for people. So, certainly, Sinan, thank you very much for coming on the show today and really just taking us through the various pathology tested options that we have at our disposal to help assess and, I suppose, also with our treatment of mental-health problems, such as anxiety and depression and sleep problems. And, obviously...

Sinan: Yeah, my pleasure, my pleasure, Adrian. And I think we're in an exciting stage, we're at the early stages of biomeasurements for mental health, but certainly look out in the future for BDNF, look out in the future for malondialdehyde and interleukin 6 measurements in terms of measuring chromogranin a and something else that we haven't talked about. But we won't add anything more to what we have talked about.

Adrian: Yeah.

Sinan: Maybe at another podcast.

Adrian: Absolutely, absolutely. Certainly, we so often rely on our patients' symptoms, but through our conversations today, we can see that there's a wide range of biomarkers that we could test to help gain further insights about what's happening from a biochemical level. 

So, I think for a lot of the information that you provided us, there's lots of questions that practitioners will have but, hopefully, they've taken something away and they understand the potential benefits but also the potential pitfalls and cautions that they need to really consider when doing testing. So, thank you very much, Sinan.

Sinan: Yeah, absolutely. And if I can just add, Adrian, that when you look through various laboratories' websites and they don't offer a certain test that you'd like, again, we're only too happy to oblige. And I've had clinicians call me and say, "Look, I really want to measure this. What do you think?" And we've done enough measurement of different sorts of biomarkers that we have sufficient data that we can look at some degree of normative data that we can compare the general population against. 

But, as a clinician, we cannot replace your clinical judgement of what you see in your patients, we can only aid in that. And if there's anything you guys out there would to like measure that isn't on offer, most labs are very very happy to oblige, including mine.

Adrian: Brilliant. Thanks, Sinan. Well, thank you, everyone, for listening today. Don't forget that you can find all the show notes, transcripts, and other resources from today's episode on the FX Medicine website. I'm Dr. Adrian Lopresti, and thanks for joining us. We'll see you next time.

About Dr. Sinan Ali

Dr Sinan Ali completed a Bachelor of Science and PhD within the School of Biological Sciences at Macquarie University, and shortly thereafter joined the department as a full time teaching and research academic from 1992 - 2012. Sinan taught human physiology and ran a research program in stress physiology, in particular looking at the role of binding proteins in steroid hormone action. While supervising Honours, Masters and PhD students in his laboratory and set up and provided a hormone analysis service to other researchers.

After Macquarie University Sinan became Associate Dean at the Australasian College of Health and Wellness (ACHW), helping the college attain higher education status and writing their inaugural degree in Clinical Aesthetics. Sinan left full-time employment end of 2017 but continues as a sessional academic lecturing in physiology and pathophysiology and academic consultant at the College. Sinan is also a board member of the Australian Spinal Research Foundation (ASRF) and is chair of their research committee (2014-present). In 2012 Sinan established SALPATH Functional Pathology which provides hormone analysis services to University researchers, integrative health practitioners, and direct to the public through My Hormones and Me website.


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