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Download the Estrogen Interpretive Guide here

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By: Stacy Hinz, MS

There are many reasons to use 24-hour comprehensive urinary testing in assessing hormones and metabolites in the steroidogenic pathways, but most practitioners choose comprehensive urinary hormone testing for the following reasons:

  • Urinary hormones are a direct measurement of the active free hormone available to the tissue receptors. In serum, the amount of hormone available to the tissue receptor is estimated based on binding calculations and population assumptions.1,2
  • By testing over a larger time period and normalizing daily hormone fluctuations, urinary hormone testing prevents false highs and lows that can show up when testing in more immediate, moment-in-time methods of detection like serum and saliva.
  • Urinary hormone testing provides a more in-depth clinical picture due to the ability to measure hormone metabolites along the steroidogenic pathway. These metabolites indicate how the primary hormones are affected by enzymes and cofactors between hormone synthesis and hormone elimination.

This deeper dive into the clinical utility of urinary hormones and metabolites will be the focus of this article. As the primary hormones and hormone metabolites move along the steroidogenic pathway, urinary testing can trace imbalances that are contributing to common clinical issues by assessing key indicators along the pathway. The result is a clearer clinical picture and greater ability to monitor subsequent therapies and lifestyle changes.

This article will describe some of the benefits of urinary hormone testing and will highlight specific cases where common clinical findings, (often difficult to identify in serum or salivary testing) were easily uncovered, and even predicted, by measuring primary hormones and their metabolites in aging adults.

Progesterone Metabolites

Why one dose does not fit all

It is a common clinical question when prescribing progesterone to different patients with similar progesterone levels; why do some still suffer from anxiety and insomnia, while some are too sedated or tired, and some respond as expected? The answer lies in the way the patient metabolizes progesterone. Progesterone metabolizes to 5a-pregnanediol and 5b-pregnanediol at varying levels. The amount of each is dependent upon the amount of 5-alpha or 5-beta-reductase activity.3–5 By examining the metabolites of progesterone and assessing other metabolism pathways of 5-alpha-reductase activity, practitioners can predict how a patient may respond to progesterone therapy and can also modify the pathways of progesterone metabolism for the best clinical response.

Total Estrogen Analysis vs Estradiol Alone

Uncovering estrogen dominance in patients with low estradiol

Aromatase activity is responsible for converting androstenedione to estrone as well as testosterone to estradiol. In younger adults, estradiol is the primary estrogen produced. However, as patients age, estrone becomes the primary estrogen produced.1 This transition from more estradiol to more estrone presents an issue when measuring and monitoring estrogen in aging patients because many practitioners only use estradiol to monitor estrogen levels and aging patients can falsely appear estrogen deficient when estradiol is the only estrogen measured. Measuring only estradiol is not advisable when deciding if premenopausal woman, or even postmenopausal woman, need estrogen therapy because many aging women with estrogen dominance or elevated estrogens can still have low estradiol levels. Placing this category of women on estrogen therapy could potentially lead to worsening symptoms and even endometrial cancer risk.2,6,7 Additionally, metabolites of estrone and estradiol can indicate certain risk factors and often have estrogenic strength as well, so it is important to know the total effect E1, E2, E3 and their metabolites are having on the target tissues.

Physicians Lab provides a Total Estrogen Load in each of its urinary hormone testing profiles (Figure 1), so it is easy to assess total estrogen effect in the patient. Each estrogen is weighted by its expected binding affinity at the estrogen receptor and is also presented in a helpful pie chart that compares expected relative levels with the levels observed in the patient.  In men, estrogen is assessed vs testosterone, and in women, estrogen is assessed vs progesterone to identify estrogen dominance and uncover any imbalanced between these metabolic pathways.

Figure 1: Total Estrogen Load comparison between the patient results (actual) and population expectations (expected)

Aromatase Activity

Pathways to estradiol and estrone

Aromatase activity converts testosterone to estradiol or androstenedione to estrone. While increased aromatase activity can be treated with an aromatase inhibitor, aromatase inhibitors can dramatically decrease estrogen and result in heart issues as well as declining brain and bone health.3,7,8 Monitoring and understanding the pathways of aromatase can assist practitioners in identifying areas of improvement with or without aromatase inhibitors. Aromatase activity is easily monitored in urine.

Aromatase to Estradiol

In this male patient (Figure 2), we can see increased aromatase activity indicated in several areas.

  • Estradiol is dominating the total estrogen load and is higher than expected, in relative ratio to the other estrogen components of the total.
    • This means that aromatase activity is predominantly aromatizing testosterone to estradiol
  • His elevated testosterone/estrogen ratio with decreased metabolism to downstream metabolites shows that testosterone is metabolizing toward estradiol more often and with more preference than downstream metabolites of testosterone

Testosterone must be metabolized and eliminated. There are essentially two pathways for this: the 5a-reductase pathway (creating testosterone metabolites) or the aromatase pathway (creating estradiol). When testosterone is elevated, often aromatase activity will often increase, and the level of expected testosterone metabolites will decrease (which is the case with this patient). When this happens, the ratio between testosterone and estrogen will indicate estrogen dominance. This means that aromatase activity is creating an imbalance between testosterone and estrogen. Aromatase activity and 5a-reductase activity are often negatively correlated, because when 5a-reductase goes up, there is less testosterone available for aromatase activity, and vice versa. However, this is not always the case. Sometimes when 5a-reductase activity is low, testosterone will start to pool and appear elevated due to the inability to metabolize downstream and the excess testosterone will not aromatize. Therefore, we examine the ratios of testosterone to estrogen and testosterone to its downstream metabolites in order to determine which endpoint of testosterone is dominating.

  • Symptoms of estrogen dominance
    • Although the testosterone is elevated, the patient is struggling with symptoms that mimic testosterone deficiency because estrogen is having a greater effect at the tissues. This is common in patients with estrogen dominance.

Not every male with elevated aromatase activity will have high levels of testosterone with elevated estrogen (like the patient described here), so it is important to have multiple measures with steroid pathway metabolites when interpreting and assessing aromatase activity pathways.

By examining the pathways and measuring multiple points along these pathways of hormone synthesis and metabolism, it becomes clear where certain pathways may have accelerated and others may have decreased.  

Figure 2: Male patient with increased aromatase activity leading to estrogen dominance, low testosterone metabolism and a testosterone/estrogen ratio leaning toward estrogen.

Aromatase to Estrone

In a similar example, aromatase activity to estrone is the conversion of androstenedione to estrone (instead of testosterone to estradiol), so there is a different mechanism to examine when aromatase activity results in increased estrone. Androstenedione originates from DHEA and progesterone pathways of metabolism. As mentioned previously, estrone is the dominant estrogen in aging men and women; but what mechanisms cause estrone to increase and estradiol to decrease in aging patients?

Figure 3: Total estrogen load in two patients with increased aromatase activity; (left) aromatase to estradiol and (right) aromatase to estrone

Testosterone to estradiol conversion happens at the sex organs in younger adults whereas androstenedione conversion to estrone happens in adipose tissue and adrenals as well as the sex organs.9 As patients age, the ovaries and testicles decline in function causing lower testosterone production, increased androstenedione production and increased conversion of androstenedione to estrone vs. testosterone.8  Androstenedione is the predominate steroid hormone produced by the postmenopausal ovary in women and aging patients often have inflammatory responses and other issues causing the adrenals to produce more androstenedione as well. The presence of more androstenedione is not the only reason for increasing levels of estrone – the declining expression of certain enzymes required for testosterone production in the steroidogenic pathway pushes metabolism of androstenedione directly to estrone instead of testosterone.10

Androstenedione is primarily a product of DHEA but can also be created from progesterone, so when increased aromatase activity results in estrone, it is important to check both DHEA and progesterone levels (especially in patients taking DHEA or progesterone supplementation).

When deciding how to treat patients with increased aromatase activity, there are several options to consider. Assessing the multiple markers along the pathway, as well as examining which pathway of metabolism seems to be predominant, is crucial. Increasing testosterone, decreasing testosterone, increasing DHEA, decreasing DHEA, decreasing progesterone, or inhibiting aromatase activity are all options. However, choosing the best options requires an understanding of the pathway of metabolism.

Hypothyroidism and Estrogens

Linking aromatase activity, thyroid activity and cortisol metabolism

The conversion of androstenedione to estrone opens the topic of how hypothyroidism affects estrogen levels. Hypothyroid decreases the rate of metabolism to androstenedione, thereby decreasing the amount of estrone produced.11,12 This can cause irregular periods in premenopausal women and can dramatically reduce the total estrogen load in postmenopausal women who primarily produce estrone from androstenedione so hypothyroidism can decrease estrone production. Did you know that hypothyroidism is often caused by estrogen excess/dominance? The human body relies so much upon maintaining balance that excess estrogen can cause hypothyroidism, which then has the ability to reduce estrogen production. This is one of many mechanisms that reminds us how crucial balance is, how the body tries to correct itself and why it is so important to know which pathways of metabolism are being affected before attempting to achieve and maintain balance during therapy.

When examining urinary hormones in the HPA-Axis, low metabolism of cortisol is a good first indicator of hypothyroidism.11 As you can see in the same male patient from Figure 2, estrogen dominance is combined with low metabolism of cortisol (Figure 3). While estrogen dominance is not the only thing that can cause hypothyroidism, we can certainly see that this patient’s elevated testosterone, low testosterone/estrogen ratio, and decreased 5a-reductase activity are clear indicators of increased aromatase activity. This pattern is almost always associated with the reduction in cortisol metabolism, which indicates hypothyroidism, and shows why elevated aromatase activity is almost always associated with insulin resistance.13 Simply reducing testosterone and prescribing an aromatase inhibitor may benefit the patient as long as all of the balancing measures are closely monitored during treatment.

Figure 4: Cortisol to metabolite ratio indicating decreased metabolism of cortisol; usually associated with hypothyroidism

Male Testosterone Pellet Therapy Issues

Avoiding 2nd and 3rd pellet disappointment in men

How many times do male patients feel a significant improvement in energy, libido and overall wellbeing after the first testosterone pellet, only to be faced with declining benefits in the 2nd and 3rd pellet insertion?  Monitoring testosterone in serum can show misleading moderate testosterone results and tracking estrogen levels in serum can mask increased aromatase activity. The male patient described had this experience due to elevated levels of testosterone and elevated levels of aromatase activity. The patient was experiencing symptoms of low androgens, even though the testosterone levels were adequate in serum. The patient insisted that he was ready for a higher dose of testosterone, and when the practitioner measured serum the testosterone levels justified a slight increase. Unfortunately for this patient, the free testosterone levels in urine would have indicated elevated testosterone, but the calculated serum testosterone levels (not a direct measurement of free) indicated room for more therapy. This patient already had some symptoms of testosterone excess such as acne, hair loss, irritability and aggression, but the patient was more concerned about the reoccurrence of other symptoms like erectile dysfunction, depression, anxiety, fatigue, and weight gain which he associated with low testosterone. In fact, these additional symptoms were associated with excess estrogen caused by increased aromatase activity. What appeared to be declining effects of testosterone, turned out to be increasing effects of estrogen with symptoms of testosterone excess. Additionally, the increased aromatase activity triggered lower cortisol metabolism (associated with hypothyroidism) accompanied by hypothyroid symptoms, which can also mimic low testosterone. Although the serum testing and the patient’s symptoms appeared to warrant higher levels of testosterone therapy, urinary hormone testing showed that this patient needed a different approach to reach and maintain balance between testosterone and estrogen for the best clinical outcome.

Therapy:

  • Using an aromatase inhibitor to reduce the amount of estrogen produced and help with energy levels and sexual function.
  • Supporting Phase I and Phase II detoxification of estrogens to help eliminate excess estrogen down the most favorable pathways of metabolism and mitigate the risks associated with elevated levels of estrogen in men.
  • Reducing testosterone therapy addressing acne, hair loss, and mood.
  • The reduction in aromatase activity and detoxification of excess estrogen should improve the sluggish cortisol metabolism and help reverse the signs of hypothyroidism. However, a healthy-thyroid lifestyle and/or thyroid support will also help the transition back to balance

Metabolic Syndrome, Type II Diabetes and the HPA-Axis

Addressing imbalances in metabolic pathways

As already mentioned, low metabolism of cortisol is a good first indicator of hypothyroidism. This makes sense when you consider the connection between cortisol, insulin resistance and thyroid. However, when we see elevated cortisol metabolism, we experience insulin resistance issues as well.13,14 For example; this patient appears to have low cortisol levels throughout the day with an unexpected increase in the bedtime sample (Figure 5).  We confirm, through cortisone results, that the cortisone has a similar trend but at higher levels than cortisol, which is a first clue of increased metabolism.  When we look at the cortisol metabolites, they appear elevated and the cortisol:cortisol metabolite ratio appears low. That indicates high cortisol metabolism, so we can see that even though the cortisol levels appear predominantly low, the adrenal glands are pumping out cortisol and this patient is metabolizing that cortisol at elevated rates. This pattern is very typical of someone who is at a high risk of Type II diabetes.13 In fact, new studies indicate that the pattern of elevated cortisol metabolism combined with an evening increase in diurnal cortisol was used to predict the onset of Type II diabetes over the course of 9 years with over 70% accuracy (refs supporting these numbers).  

We know that abdominal obesity is linked to both Type II diabetes and elevated cortisol metabolism. We also know that Type II diabetes is one of the qualifiers for metabolic syndrome, so it makes sense that a the warning signs of type II diabetes, insulin resistance and metabolic syndrome can all be identified by examining the cortisol metabolites and the anabolic:catabolic ratio. It should also make sense that if we can identify these warning signs in urine, there are likely other metabolic issues that can be addressed by measuring, achieving and maintaining the delicate balance between the anabolic and catabolic metabolism pathways.

Figure 5: Patient with increased cortisol metabolism despite a lower diurnal trend of daily free cortisol. This pattern of elevated even cortisol with increased cortisol metabolism and elevated cortisol metabolites is a predictor or Type II diabetes without intervention.

The Anabolic vs Catabolic Ratio

Measuring, achieving and maintaining metabolic balance in the HPA-axis

The anabolic to catabolic ratio compares HPA-Axis balance between 17-ketosteroids (mostly DHEA metabolites) and 17-hydroxysteroids (mostly cortisol metabolites). The easiest way to understand the anabolic/catabolic ratio is to view it as a balance between creating energy (through catabolic processes) and using that energy to build (through anabolic processes).

In order to build and maintain tissues, the body requires energy. This energy is created by breaking down (catabolizing) energy storage sources such as carbohydrates, fats and proteins.15  If the catabolic pathways are balanced with the anabolic pathways, then we end up with the perfect amount of energy generated by the catabolic pathway and utilized by the anabolic pathway.16 When the catabolic pathway is higher than the anabolic pathway, we end up with more stored energy – often in the form of fat – and run the risk of Type II diabetes and metabolic syndrome.17 Although we focus a large deal on low anabolic/catabolic ratios where cortisol metabolism and 17-hydroxysteroids are dominating over anabolic metabolism and 17-ketosteroids, there are plenty of cases where the anabolic pathways dominate the catabolic pathways as well. While this can be less common, it is in no way less harmful to the patient. For these patients, they have the ability to build but they lack the energy to do so. When the anabolic pathway is higher than the catabolic pathway, patients are often hypoglycemic, underweight, fatigued and require more caffeine. Due to craving carbohydrates, a percentage of patients may be overweight with muscle loss because their energy sources are more carbohydrate and muscle than fat. These are generalizations for understanding the anabolic to catabolic ratio, but it points out how balance is best.

Elevated Cortisol Metabolism

When cortisol metabolism is increased, this will often result in elevated 17-hydroysteroids and an imbalance in the anabolic to catabolic ratio leaning toward catabolic.  When 17-hydroxysteroids increase, balancing the ratio should include slowing the rate of adrenal output – even when diurnal cortisols appear normal or low, because elevated 17-hydroxysteroids directly correlate to increased adrenal output. Temporarily increasing DHEA is also a valid consideration and allows for balance to be achieved between anabolic and catabolic while the patient works on lifestyle improvements to lower cortisol production. Increasing DHEA, even just temporarily, often works well because an imbalance (leaning toward catabolic) can make lifestyle changes, such as weight loss, nearly impossible due to the fact that elevated cortisol metabolites are driving energy storage and weight gain. DHEA therapy can also increase 5-alpha-reductase activity which is often helpful in reducing cortisol metabolism because decreased 5a-reductase is often associated with increased cortisol metabolism as well. Although increasing DHEA is not always an option, DHEA replacement is usually a good option when an imbalance exists due to low 17-ketosteroids and decreased 5a-reductase activity.

Lower 17-ketosteroids

Understanding where the decreased 17-ketosteroids are stemming from in the pathway is important in choosing therapeutic options. To accomplish this, the 17-ketosteroids are displayed in a pie chart in the report allowing for the comparison between the patient results (actual) and the expected results in each individual ketosteroid. Through this graphic, accelerated or sluggish pathway mechanisms can be quickly identified. For example, the patient in Figure 6 has an anabolic to catabolic ratio that is leaning toward the catabolic side. The results indicate both elevated catabolic metabolites and low anabolic metabolites combining to produce an extremely imbalanced ratio. The root cause of the extremely low 17-ketosteroids is due to low DHEA. The cause of the increased cortisol metabolites is not obesity (the patient is only 120 lbs.), but rather metabolism down the 5b-reductase pathway at a much higher rate than expected, which is also associated with increased cortisol metabolism.

Figure 6: Patient with catabolic dominance due to both increased 17-hydroxysteroid (associated with increased 5-beta-reductase activity) and decreased 17-ketosteroids (associated with low DHEA).

In these cases, we can often increase DHEA, which also increases 5a-reductase activity,7,17 and consider slowing 5β-reductase activity using licorice.18 One other benefit of licorice is that it will slow the rate at which back-door androgen metabolism occurs,10 (which is not an issue in this patient) and will assist in sending DHEA metabolism down the 17-ketosteroid pathway. We may also see increases in testosterone and estrogen as DHEA and 5a-reductase activity increase. The end result will be more balance between the anabolic and catabolic in the ratio, both by increasing the anabolic side through DHEA supplementation and decreasing the catabolic side by calming the adrenals and reducing 5b-reductase activity.

CONCLUSION

Physicians Lab’s comprehensive report was designed to take some of the guesswork out of interpreting results and treating patients. The totals and ratios are a good start and the dynamic text contained in these reports can often tell you exactly what is happening because the text is based on the results of each patient.

Our primary focus is to deliver the most accurate results with the highest amount of clinical information and our comprehensive report containing results-driven text allows us to transfer this information directly to the report as the scientific community gains more insight into these pathways.

REFERENCES

1.        Roos J, Johnson S, Weddell S, et al. Monitoring the menstrual cycle: Comparison of urinary and serum reproductive hormones referenced to true ovulation. Eur J Contracept Reprod Heal Care. 2015;20(6):438-450. doi:10.3109/13625187.2015.1048331

2.        Maskarinec G, Beckford F, Morimoto Y, Franke AA, Stanczyk FZ. Association of estrogen measurements in serum and urine of premenopausal women. Biomark Med. 2015;9(5):417-424. doi:10.2217/bmm.15.10

3.        de Ronde W, de Jong FH. Aromatase inhibitors in men: Effects and therapeutic options. Reprod Biol Endocrinol. 2011;9(1):93. doi:10.1186/1477-7827-9-93

4.        Starka L, Hampl R, Bicikova M, Jelinek R, Doskovil M. Observations on the biological activity of epitestosterone. Physiol Res. 1991;40(3):317-326.

5.        Hiipakka RA, Zhang HZ, Dai W, Dai Q, Liao S. Structure-activity relationships for inhibition of human 5α-reductases by polyphenols. Biochem Pharmacol. 2002;63(6):1165-1176. doi:10.1016/S0006-2952(02)00848-1

6.        Lu LJW, Cree M, Josyula S, Nagamani M, Grady JJ, Anderson KE. Increased urinary excretion of 2-hydroxyestrone but not 16α- hydroxyestrone in premenopausal women during a soya diet containing isoflavones. Cancer Res. 2000;60(5):1299-1305.

7.        McCann SE, Edge SB, Hicks DG, et al. A pilot study comparing the effect of flaxseed, aromatase inhibitor, and the combination on breast tumor biomarkers. Nutr Cancer. 2014;66(4):566-575. doi:10.1080/01635581.2014.894097

8.        Leder BZ, Rohrer JL, Rubin SD, Gallo J, Longcope C. Effects of aromatase inhibition in elderly men with low or borderline-low serum testosterone levels. J Clin Endocrinol Metab. 2004;89(3):1174-1180. doi:10.1210/jc.2003-031467

9.        Strauss JF. Some new thoughts on the pathophysiology and genetics of polycystic ovary syndrome. Ann N Y Acad Sci. 2003;997:42-48. doi:10.1196/annals.1290.005

10.      Fiandalo M V., Wilton J, Mohler JL. Roles for the backdoor pathway of androgen metabolism in prostate cancer response to castration and drug treatment. Int J Biol Sci. 2014;10(6):596-601. doi:10.7150/ijbs.8780

11.      Hoshiro M, Ohno Y, Masaki H, Iwase H, Aoki N. Comprehensive study of urinary cortisol metabolites in hyperthyroid and hypothyroid patients. Clin Endocrinol (Oxf). 2006;64(1):37-45. doi:10.1111/j.1365-2265.2005.02412.x

12.      Taniyama M, Honma K, Ban Y. Urinary cortisol metabolites in the assessment of peripheral thyroid hormone action: Application for diagnosis of resistance to thyroid hormone. Thyroid. 1993;3(3):229-233. doi:10.1089/thy.1993.3.229

13.      Westerbacka J, Yki-Järvinen H, Vehkavaara S, et al. Body Fat Distribution and Cortisol Metabolism in Healthy Men: Enhanced 5β-Reductase and Lower Cortisol/Cortisone Metabolite Ratios in Men with Fatty Liver. J Clin Endocrinol Metab. 2003;88(10):4924-4931. doi:10.1210/jc.2003-030596

14.      Declue TJ, Shah SC, Marchese M, Malone JI. Insulin resistance and hyperinsulinemia induce hyperandrogenism in a young type B insulin-resistant female. J Clin Endocrinol Metab. 1991;72(6):1308-1311. doi:10.1210/jcem-72-6-1308

15.      Mueller MB, Tuan RS. Anabolic/Catabolic balance in pathogenesis of osteoarthritis: identifying molecular targets. PM R. 2011;3(6 Suppl 1):S3-S11. doi:10.1016/j.pmrj.2011.05.009

16.      Tzanis G, Dimopoulos S, Agapitou V, Nanas S. Exercise intolerance in chronic heart failure: The role of cortisol and the catabolic state. Curr Heart Fail Rep. 2014;11(1):70-79. doi:10.1007/s11897-013-0177-1

17.      Ueshiba H, Shimizu Y, Hiroi N, et al. Decreased steroidogenic enzyme 17,20-lyase and increased 17-hydroxylase activities in type 2 diabetes mellitus. Eur J Endocrinol. 2002;146(3):375-380. doi:10.1530/eje.0.1460375

18.      Ferrari P, Sansonnens A, Dick B, Frey FJ. In vivo 11β-HSD-2 activity: Variability, salt-sensitivity, and effect of licorice. Hypertension. 2001;38(6):1330-1336. doi:10.1161/hy1101.096112

Co-authored by Clifford Morris, PhD and Thomas Kent

Paradigm of Artificial Intelligence

Technology moves only in one direction—forward. In general, the observation of this fact is welcome. After all, the ability to develop and apply technology is a defining aspect of our species. This fact becomes unwelcome when outcome-driven industries trade their lots for technological efficiency. In medicine, the efficiency economy has resulted in doctors spending less time with patients. This is highlighted by an average first appointment time of only twelve minutes.1 Before we continue, it is important to acknowledge that the gains we have made in efficiency are important to total patient outcomes, as more efficiency equates to more treated patients. Instead, we are concerned with individual patient outcomes.

For too long, medicine, enabled by the worst aspects of technological advancement, was trending in the direction of cookie-cutter solutions to the unique needs of individual patients. When pondering how to rehumanize medicine, AI is often the last thought on one’s mind. Yet, the current data may lead one to the opposite conclusion. AI seems to be able to give us this lost time back, allowing for doctors to give patients the time they truly need. The extra human interaction time is not just beneficial for the patients, but for the physicians as well. A study conducted at the University of Colorado showed that taking the computer out of the exam room and supporting doctors with human medical assistants significantly reduced burnout rates.2 If AI could use its data-processing ability to help with the more clerical work nurses do, it could permit nurses more time with both doctors and patients. The placement of AI in a clinical setting may facilitate more one on one time between doctors and patients, leading to a reduction in nosocomial infections and hospital readmissions as well.1,3 This could save the health care industry millions, while providing superior patient care. All that is needed is for someone to commit to the first push.

The Role of AI in the Clinical Lab Field

Although AI may not be prevalent in the clinical laboratory sector of medicine yet, saying that it is non-existent altogether is far from the truth. For example, AI is already prevalent in the laboratory setting. A great example of this is AI in the Liquid Chromatography Mass Spectrometry (LC-MS) field.4 LC-MS is a great tool used to measure various compounds in the human body, including everything from hormone levels to trace metals. One of the ways AI has already integrated with LC-MS is how it cuts down on the rate limiting steps of LC-MS, which more often than not are sample prep and LC separations. One system that Physicians Lab has made use of is parallel processing using SCIEX MPX 2.0 High Throughput System. This system can couple parallel runs with one LCMS instrument, resulting in twice the speed with no loss to accuracy. It can do this by staggering two runs either using the same method, or different methods entirely. What really makes this system great is its ability to automatically detect carryover and inject solvent blanks to clean the instrument. The system will then continue its analyzing, while automatically reinjecting samples that may be affected by the carryover. It will also flag high concentration without user input, allowing for easy detection of possibly faulty samples. This allows it to operate without users from startup to shut down. Some of the other ways that it can be used to increase efficiency are by using integrated network features to work on anything from streamlining management to increased throughput. Physicians Lab is also taking advantage of AI systems by incorporating the ASCENT software from Indigo Bioautomation. This software uses smart algorithms and machine learning to automatically integrate the LCMS peaks and turn them into results – a tedious job normally done by people. A major issue in laboratories using LC-MS is that the result interpretation of peaks is highly user-subjective and dependent on their attention, training, and behaviors.5 Using ASCENT eliminates user-to-use variability, improves workflow speed, and allows the users to focus only on problematic result interpretations. The result is a highly dependable and consistent result interpretation system, as well as a better utilized staff. Finally, just about every process from plate and sample vial changing to gradient formation can be automated, allowing for fast and accurate results. AI has allowed all of these changes, and it has shown what the power of a machine that can adjust to new circumstances can do. These features can almost double lab work speeds in some cases, allowing doctors to diagnose quicker and attack the problems faster.

AI has played a large role in advancing LC-MS instrumentation, but that is not the only aspect of clinical laboratory life that AI has enhanced. All AI-based lab software works through a term known as “computational pathology”. This means that through the use of visuals and machine learning, the AI makes the product of the data more useful and easily understood for practicing physicians. One of the ways this is achieved is through wearables that can measure blood glucose levels, heart rate, and temperature, as well as other factors. This data can be uploaded to a mass cloud. Another way AI could easily influence lab settings is through tumor detection.4 Tumor detection is generally achieved by analyzing set genes that are already associated with tumor growth, and comparing the genes that are mutated in patients with tumors. This allows for personalized therapy, depending on what tumors the patient has. AI will use machine learning along with traditional techniques for more accurate tumor identification and diagnostic process. Also, the ability to integrate pre-existing algorithms with machines gives them the ability to have a greater pattern recognition than even the best doctors.

The Greater Extent of AI in Healthcare.

AI in the lab will also help doctors design more accurate treatment paths by analyzing real-time data. The data constructed from laboratories are generally broken up into three sectors: patients about to be diagnosed, patients previously diagnosed who are responding to treatment, and patients who have been diagnosed that are not responding to treatments. If one could take this data and integrate it into AI, you could get more effective treatments to patients in a shorter time span. For example, instead of going with a primary treatment that works 80% of the time, AI may catch something that would instead recommend a less-used course of action that would be more effective. So instead of going through a trial and error of multiple treatments, AI could help find the better solution. As well, if the first line therapy fails, this could provide a faster way to reach the second line therapy. In order for AI to truly reach its maximum potential, there must be a shift in thinking. Instead of relying solely on past data, doctors would be able to rely on real-time, ever-changing data to get the best results possible.

So with AI becoming prevalent in the clinical lab, how does this translate into doctor-to-patient interaction? Theoretically, the AI would use one of two ways to treat a patient in a clinic.1 The first being the flowchart method. This is where a doctor would essentially transfer all of their data from past interviews of patients and the results into the AI. The AI would then have this knowledge, and be able to ask patients questions and form conclusions in an “If x, then y” format. There are two main issues with this method, the first being the sheer amount of data that needs to be integrated. The second is the AI’s inability to detect when patients may be hiding something. For example, someone lying to get painkillers. This could be where cohesiveness steps into play, as you could have the AI ask questions, with the doctor overriding some of the patients answers. The second method is known as database learning. This requires the machine to be shown the same image over and over again, until it can memorize that image. This could be useful if a doctor sees a mole that he’s not sure is a melanoma or not. The doctor could then go to the computer for a second, and possibly more accurate, opinion. Some of this AI is already here. There is already basic flowchart AI, robotic surgical systems, AI therapy, and AI scheduling programs. AI is bound to be even more integrated into clinical medicine as time marches on. One example of this is Stanford University’s Program in AI-Assisted Care (PAC). One of these programs allows seniors who live alone to be able to reach immediate help if needed, along with monitoring behavioral and movement patterns to spot irregularities. Other examples of future AI in the medical field involve “Molly”, a virtual nurse that can monitor and follow up with patients. This could free up an immense amount of doctors’ time. Not to mention that although certain AI has already been able to help schedule, there are more advanced AI’s which will be able to adapt to the struggles of a busy ward, freeing up more time for nurses.

Although AI may be originally thought of as a bane to physicians and the remaining human aspect of medicine, this is far from the truth. Throughout the continued advancement of AI in the clinical lab, and the newer yet fascinating use of AI in the clinic itself, medicine has a chance to regain its once human form. The abilities of AI to help with anything from scheduling to diagnostics should reduce the combination of doctor’s stress levels, burnout, and hospital readmission rate. This, in turn, should give the doctors much more one on one time with patients, drastically increasing the quality of patient care. The re-humanization of medicine starts not with man, but with the machine.

References:

1.        Topol EJ. High-performance medicine: the convergence of human and artificial intelligence. Nat Med. 2019. doi:10.1038/s41591-018-0300-7

2.        Wright AA, Katz IT. Beyond burnout – Redesigning care to restore meaning and sanity for physicians. N Engl J Med. 2018. doi:10.1056/NEJMp1716845

3.        Naugler C, Church DL. Automation and artificial intelligence in the clinical laboratory. Crit Rev Clin Lab Sci. 2019. doi:10.1080/10408363.2018.1561640

4.        Workman TE, Hirezi M, Trujillo-Rivera E, et al. A Novel Deep Learning Pipeline to Analyze Temporal Clinical Data. Proc – 2018 IEEE Int Conf Big Data, Big Data 2018. 2019:2879-2883. doi:10.1109/BigData.2018.8622099

5.        Gaona García PA, Montenegro Marin CE, Gaona García EE. Model of Learning Objects Exchange between LCMS Platforms through Intelligent Agents1. Ing y Univ. 2015. doi:10.11144/javeriana.iyu19-2.mloe

By: Mari Salazar, PA, MMS

Patient education. We all know it’s important, we’d like to do more of it, but it’s time consuming and, frankly, some of us aren’t good at it. Some patients like a lot of it, while some patients feel overwhelmed and are turned off. And when you add to this the fact that we are asking for saliva, blood, urine, stool and money, it’s no surprise patients may feel that this type of medicine is not for them and decide to just double up on their antidepressants!

I ask myself “what is my job here, with THIS particular patient?”, and the answer is always –help them feel better physically, mentally, and emotionally. This almost never works when we overwhelm them with information that is confusing without actionable steps for improvement.

So, what I’ve learned after a lot of trial and error is to not over-do it with labs in the beginning. Order what will give the most valuable and actionable information first. Let the patient see the value in the tests you pick and how much better they feel with the treatment plan you customized based on those labs and symptom profile they present with.

Physician’s Lab 24 Hour Urinary Metabolite test is one of the labs with the most valuable information I do on my patients. But we’ve all heard, “I went to another office for hormones and I didn’t have to do this urine test!”  How do we overcome this push back?  Here is one of my little scripts…..

“We are optimizing your hormones so that you feel better physically, mentally, and emotionally not to mention improve the way you age. We want to do this responsibly because all these hormones need to get broken-down/metabolized. Some of us do this well, and some of us don’t. Certain things happen when we don’t metabolize our hormones well. It could be the reason you don’t feel as great as your friends do on BHRT, and it could even give us an idea of how at-risk you are for breast, uterine, ovarian, or prostate cancer.

The beauty of this is that even if you are an at-risk person, there are certain things we can do to make us healthier metabolizers and not only reduce our risk of certain cancers, but make your experience with BHRT an amazing one.”


Good Metabolizers

  • – Prevents bone loss
  • – Natural antidepressant
  • – Improves insulin sensitivity
  • – Decreases fatigue
  • – Maintains elasticity in arteries
  • – Increases skin collagen
  • – Prevents Alzheimer’s disease
  • – Reduces cataracts
  • – Maintains memory
  • – Works as an antioxidant

Bad Metabolizers

  • – Menopausal symptoms
  • – PMS
  • – Heavy bleeding
  • – Tender breasts
  • – Cancers: Prostate/Breast/Ovarian/Uterine

So, when I see that a particular patient is not metabolizing their estrogens down the healthier 2-OH pathway, here are some general changes in lifestyle that should be implemented.

1. Decrease exposure to fake estrogens (xenoestrogens).

Avoiding all of these is impossible, but being aware of those we can avoid can make a huge impact on your health. Read your labels! Ingredients to stay away from as much as possible:

(EWG.org makes picking products easier without having to read labels)

  • – DEA (DIETHANOLAMINE), MEA (MONOETHANOLAMINE), TEA (TRIETHANOLAMINE)
  • – PARABANS PRESERVATIVES (METHYL, PRPOYL, BUTYL, ISOBUTYL, and  ETHYL)
  • – MINERAL OIL: PETROLATUM, PETROLEUM JELLY (LIQUID PARAFFINUM, PARAFFIN OIL, PARAFFIN WAX, POSH MINERAL OIL)
  • – PROPYLENE GLYCOL/ BUTYLENE GLYCOL
  • – SILICONE DERIVED EMMOLIENTS (DIMETHICONE, DIMETHICONE COPOLYOL, CYCLOMETHICONE)
  • – TALC
  • – DIBUTYL PHTHALATE
  • – 1,4-DIOXANE
  • – BHT (BUTILATED HYDROXYTOLUENE), BHA (BUTILATED HYDROXYANISOLE)
  • – BENZALKONIUM CHLORIDE and BENZETHONIUM CHLORIDE
  • – TRICLOSAN and TRICLOCARBAN
  • – PARFUM
  • – Air fresheners, deodorizers, fabric softeners, scented candles, body perfumes
  • – Fabric detergents, dishwashing detergents, clothing softeners, clothing, cosmetic and personal care products

2. Promote detoxification

  • – We detox through foods, supplements, exercise, and sleep!
  • – Fresh, whole, unprocessed, organic, colorful, HIGH FIBER, nuts, seeds, omega 3’s, herbs, fermented products, cabbage, cauliflower, broccoli, high fish consumption (Sardines)
  • – Soluble fiber and low glycemic load
  • – Increase green leafy vegetables (10-12 servings)
  • – Green tea
  • – Berries
  • – Flax
  • – Chia seeds

3. Enhance elimination (poop)

  • Daily bowel movements are essential. We rid our bodies of estrogens when we poop. If you’re constipated, you’ll actually reabsorb the estrogen that was supposed to be eliminated, and it gets circulated again throughout the body, causing more havoc.

4. Decrease insulin stimulation

  • This means you should eliminate sugars and processed carbs, as well as reduce the frequency of eating. Every time we put something in our mouths, we spike insulin. If we strive to eat 3 (ideally 2) meals a day with no snacking in between, we will stabilize and lower our insulin leading to fat loss and better hormones.

5. Targeted supplementation: Based on individual labs and symptoms

  • – Reduced glutathione
  • – NAC
  • – Lipoic acid
  • – Whey protein
  • – Magnesium
  • – Vitamin C
  • – Silymarin
  • – Pantothenic acid
  • – SAMe
  • – DHEA
  • – DIM
  • – Vitamins Bs 6,9,12

6. Lifestyle modifications

  • Stress management: healthy levels of cortisol will keep your hormones at happier levels, as well as allow weight loss and detoxification. Stress needs to be managed. Make it a priority. Go out in nature, get natural sunlight, yoga, meditation, exercise, sleep.

7. Address digestion

  • Optimize gut health with the 5 R program

8. Exercise/movement

  • We know all the benefits of exercise. Get on a routine that works for you and stick to it. Our lives are more sedentary than what we were made for. You need to move for proper muscle mass as well as lymphatic flow (which keeps you healthy and helps prevent cancer)! High intensity Interval Training (HIT) is what we recommend.

9. Sleep

  • Improve your sleep habits! All of the effort made to eat the right things and exercise will not pay off unless you get good quality sleep.  This means get in bed earlier, use blue-light-blocking glasses if you’ll be on your phone or watching TV, do not eat right before bed, put cell phone on airplane mode or keep out of the room (EMFs), and bedroom should be as dark as possible (lights from TV, phone, and window interfere with your production of melatonin, which is needed for healthy sleep).

10. Mind-body-spirit connection

  • Whatever your spiritual practice is, make sure you have reconnected and aligned with that.

11. Support

  • We are social beings. Connection with friends and family is of utmost importance in a world where we have replaced the rewards of real relationships (the “reward” I am talking about is actually production of feel good hormones that connection and touch provide) with social media. Put your phones down and reconnect with those you care about.

By: Clifford Morris, Ph.D.
Chief Chemist and Research Scientist

Introduction

The largest failure of the biomedical research enterprise over the past 50 years has been its inability to adequately address Alzheimer’s disease. There are currently four FDA-approved medications for the relief of Alzheimer’s symptoms; three of which are acetylcholine esterase inhibitors, and the other is memantine. However, there exists no pharmaceutical intervention that arrests or reverses the disease. Moreover, a meta-analysis recently revealed that patients diagnosed with Alzheimer’s receiving acetylcholine esterase inhibitors showed an increase in cognitive decline compared to placebo.1 Since 2003, every disease-modifying agent has failed in Phase II or III trials.2 Alzheimer’s is the sixth leading cause of death in the United States, afflicting more than 5.8 million Americans.3 Between 2000 and 2017, heart disease mortality dropped nearly 9%, while Alzheimer’s mortality increased 145% (all ages).3 Taken together, this data reveals a grim outlook for the future of progression towards traditional disease-modifying therapies. For these reasons, Alzheimer’s risk reduction and prevention efforts have gained traction. Current Alzheimer’s prevention protocols seek to reduce the area under the curve of a function of Alzheimer’s risk factors. We can think of Alzheimer’s risk as being a function of certain variables, some of which are modifiable while others are non-modifiable. Modifiable risk factors include insulin resistance, obesity, inflammation, and smoking, whereas non-modifiable risk factors are age, sex, and family history. The figure below is meant to depict the difference between an at-risk individual who has taken measures to reduce his/her risk for Alzheimer’s versus one who has not.

The Interplay Between Hormones and Alzheimer’s Disease

One of the most notable non-modifiable disease risk factors is sex: women are two times more likely to die of Alzheimer’s than are men.3–5 Estrogen depletion in postmenopausal women is thought to be associated with this risk.4,5 In 2002, The Cache County Study revealed that women who have used hormone replacement therapy (HRT) for 10 years or more are no more likely to develop Alzheimer’s then are men. However, there was no suggestion of risk reduction if used for less than 10 years.6 These findings highlight the importance of identifying at-risk individuals and starting treatment early. More recently, some studies have suggested that long term use of hormone replacement therapy may actually increase Alzheimer’s risk in women.7 Thus, the story here is complicated, and there is still much to uncover. However, the majority consensus is that hormone replacement therapy may reduce Alzheimer’s risk for certain individuals.6,8,9 Estrogen has been found to play many roles in the human body, most notably in females. It has a unique property of being synthesized directly in the brain; this leads to many of its neuroprotective and neurodegenerative effects. It also plays a role in growth, differentiation, and sexual development.10 Most recently, Estrogen has been implicated with hormone replacement therapy in an attempt to treat Alzheimer’s.10–13 Although HRT can involve many hormones, the ones that most seem to be implicated in impacting Alzheimer’s disease are estrogen and progesterone. The reason for estrogen and progesterone having such a large implication in Alzheimer’s is their perceived sex bias. It has been suggested, although not all studies agree, that women are more likely to develop Alzheimer’s, and it is likely that Alzheimer’s dementia-inducing and deteriorating effects also occur more rapidly in women. There are numerous reasons for this, but perhaps one of the more pronounced ones is the gene APOE-4. APOE-4 is known to be a huge risk marker for Alzheimer’s and plays a role in the development of amyloid plaque deposition. However, it has been shown that being positive for APOE-4 plays a much larger role in cognitive decline in women than it does in men.  This could be due to the extra amounts of estrogen in women, and that is why HRT has been viewed as a possible way to help treat Alzheimer’s. The two most common forms of HRT used for Alzheimer’s are estrogen-only therapy and estrogen in combination with progesterone. Studies have found that younger women (aged 50-63 years) who had undergone hormone therapy were significantly less likely to develop Alzheimer’s, while HRT showed no such effect for older women.10,12 However, in the Women’s Health Initiative trial, women who were on active treatment with estrogen plus progesterone had nearly a twice as likely chance of getting Alzheimer’s with serious dementia, while the women on only estrogen were nearly half as likely to get Alzheimer’s with full-fledged dementia. Data like this has led to a hypothesis known as the critical window hypothesis, where HRT (especially estrogen and progesterone) are only effective for a short amount of time in post-menopausal women. This seems to indicate that the critical window for estrogen therapy would be starting younger and using for a longer amount of time. This window, however, does differ from the window associated with HRT that combines estrogen and progesterone. Women using combination HRT for under a year revealed an elevated risk, but women using combination HRT for between 1-3 years had a lower risk. This not only backs up the critical window hypothesis but also seems to suggest that it changes depending on which hormones are used. That being said, women are not the only ones effected by Alzheimer’s; men also are, but generally at a lower rate. If estrogen decline was found to be a large factor in the development of Alzheimer’s, then that may help explain why men are not affected as much. Serum estrogen rates have actually been found to be higher in elderly males than in post-menopausal women. This is because men constantly make testosterone and then convert that testosterone (albeit at a low rate of 0.2%) to estrogen, providing them with what may be increased protection. Further, another reason that men might be more well protected is the drop-off found in women’s estrogen levels post menopause is not nearly as severe for elderly men despite reduced testosterone production. While it has been shown that estrogen effects Alzheimer’s, it is unknown in exactly what way it does. While HRT may be a promising field that can help with Alzheimer’s, finding out what the critical window is for each person and which set of hormones to use remains a challenge. In the future, a more appropriate form of treatment may be personalized HRT based on the patients’ medical history combined with genetic testing to see if they have an increased risk for Alzheimer’s disease. Physicians Lab offers comprehensive urinary hormone testing to help assess hormone levels and biomarkers that may be critical in developing a therapeutic solution for Alzheimer’s disease prevention.

Dietary Approaches for Alzheimer’s Disease

As our appreciation for the complexity of Alzheimer’s pathology has evolved, we have begun to view the disease through several different lenses (e.g., the amyloid hypothesis, mitochondrial disfunction, and cerebral hypoperfusion).  An emerging viewpoint is Alzheimer’s as an inflammatory disease.14 Studies have shown that Alzheimer’s patients present with elevated serum levels of inflammatory markers, such as COX, TNF-α, and IL-6.15–18 These inflammatory markers have shown the ability to disrupt amyloid clearance  and combat signaling pathways responsible for cell survival.16,17 Because of the relationship between Alzheimer’s and inflammation, anti-inflammatory drugs show promise in reducing Alzheimer’s risk. Long term use of non-steroidal anti-inflammatory drugs (NSAIDs) is associated with reduced risk of Alzheimer’s disease.15 Studies suggest that unselective COX inhibitors are more potent than selective inhibitors in terms of risk reduction.19 Further, dietary and lifestyle changes associated with reduced inflammation show promise in Alzheimer’s risk reduction. Specific dietary changes may involve avoiding high-carbohydrate, high-calorie meals that are associated with elevated serum levels of IL-6.15

In regards to lifestyle changes, Matthew Walker has devoted a large part of his career to understanding the relationship between sleep disruption and Alzheimer’s. His findings overlap well with the inflammatory hypothesis and suggest that high quality sleep may be critical in preventing the onset of Alzheimer’s disease.20 Despite many positive discoveries to help with the treatment of Alzheimer’s, a cure still remains unknown. This has led individuals to study other areas outside of traditional pharmacology, such as diet and nutritional supplementation. Although a majority of trend diets come and go, there is evidence to support a few diets that may help reduce the incidence of the Alzheimer’s disease: the Mediterranean diet, DASH (Dietary Approaches to Stop Hypertension), and MIND (Mediterranean-DASH Intervention for Neurodegenerative Delay). Studies have shown that the DASH diet and Mediterranean diet were both effective in reducing Alzheimer’s risk.21 The DASH and the Mediterranean diet were originally designed to help with cardiovascular health, while the MIND diet is actually focused on reducing Alzheimer’s risk. Data shows that the MIND diet has close to a 50% chance of being an effective preventative action in regard to Alzheimer’s disease. These studies make sense as these diets are high in foods known as AGE (advanced glycation end products).2,21 Low AGE foods are considered to be good and include fish, fruit, legumes, and certain carbohydrates. These are foods that are rich in Omega-3’s, DHA’s (docosahexaenoic acid), and EPA’s (eicosapentaenoic acid), as well as B and D vitamins. Low AGE foods tend to be prevalent in the MIND diet, furthering the belief that MIND diet can help prevent Alzheimer’s. Diet is not the only non-traditional means used to combat Alzheimer’s either; natural supplements have also been in the mix as well. There are a lot of supplements that have been tested, but only a few were found to have clinical relevance. Huperzine A, Ginkgo biloba, Coral calcium, Coenzyme Q10, and Caprylic acid were all found to be ineffective by the Alzheimer’s Association. Despite these being touted by celebrities, they have failed to display real evidence in working in phase three trials. However, not all hope is lost as far as alternative therapies go, but larger scale studies will be needed to provide conclusive evidence in Alzheimer’s prevention.

The Unknown Factors

Another neuropathological feature of Alzheimer’s is oxidative stress. Postmortem examination of the Alzheimer’s brain reveals increased lipid peroxidation, protein and DNA oxidation, and evidence of impaired mitochondrial function.15,22 Alzheimer’s risk reduction with respect to reactive oxygen species (ROS) generation is primarily concerned with mediating homocysteine levels. Epidemiological studies have associated homocysteine, a known ROS stimulator, with the onset of Alzheimer’s disease.23 B vitamins are required to metabolize homocysteine to either methionine or cysteine. In 2007, a double-blind, placebo-controlled trial showed that extended use of vitamin B9 is associated with significantly slower cognitive decline versus placebo.24 It is important to note that a significant fraction of Hispanics and Caucasians may suffer from an MTHFR polymorphism that is linked to hyperhomocysteinemia.25 Such at-risk individuals are important to identify and treat appropriately as early as possible. There are a multitude of unknown and co-dependent factors resulting in the presentation of Alzheimer’s disease. The nebulous mechanisms in which these all act in concert are a testament to the complexity of the disease, and it will take significantly more time and resources until we fully understand these discreet processes.

In Conclusion

We have reviewed some of the most trafficked avenues of Alzheimer’s risk reduction and sought to understand Alzheimer’s as a multifaceted malady. Alzheimer’s risk is a function of many variables, some modifiable and others non-modifiable. Clinical action towards addressing each variable is unique. Some risks may be alleviated with pharmaceutical intervention, while others may require lifestyle and dietary changes.

References

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(2)        Galvin, J. E. Prevention of Alzheimer’s Disease: Lessons Learned and Applied. J. Am. Geriatr. Soc. 2017, 65 (10), 2128–2133.

(3)        Alzheimer’s Association. 2019 Alzheimer’s Disease Facts and Figures. Alzheimer’s Dement. 2019, 15 (3), 321–387.

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(5)        Medeiros, A. D. M.; Silva, R. H. Sex Differences in Alzheimer’s Disease: Where Do We Stand? J. Alzheimer’s Dis. 2019, 67 (1), 35–60.

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(9)        Seshadri, S.; Zornberg, G. L.; Derby, L. E.; Myers, M. W.; Jick, H.; Drachman, D. A. Postmenopausal Estrogen Replacement Therapy and the Risk of Alzheimer Disease. Arch. Neurol. 2001, 58 (3), 435–440.

(10)      Henderson, V. W. Alzheimer’s Disease: Review of Hormone Therapy Trials and Implications for Treatment and Prevention after Menopause. Journal of Steroid Biochemistry and Molecular Biology. 2014.

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(13)      Janicki, S. C.; Schupf, N. Hormonal Influences on Cognition and Risk for Alzheimer’s Disease. Current Neurology and Neuroscience Reports. 2010.

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(16)      Álvarez, A.; Cacabelos, R.; Sanpedro, C.; García-Fantini, M.; Aleixandre, M. Serum TNF-Alpha Levels Are Increased and Correlate Negatively with Free IGF-I in Alzheimer Disease. Neurobiol. Aging 2007, 28 (4), 533–536.

(17)      Hüll, M.; Strauss, S.; Berger, M.; Volk, B.; Bauer, J. The Participation of Interleukin-6, a Stress-Inducible Cytokine, in the Pathogenesis of Alzheimer’s Disease. Behav. Brain Res. 1996, 78 (1), 37–41.

(18)      Heneka, M. T.; O’Banion, M. K.; Terwel, D.; Kummer, M. P. Neuroinflammatory Processes in Alzheimer’s Disease. J. Neural Transm. 2010, 117 (8), 919–947.

(19)      Gasparini, L.; Ongini, E.; Wenk, G. Non-Steroidal Anti-Inflammatory Drugs (NSAIDs) in Alzheimer’s Disease: Old and New Mechanisms of Action. J. Neurochem. 2004, 91 (3), 521–536.

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