Testing and Laboratory Science

Jay H Mead MD FASCPFounder and Medical Director, Labrix LLC

Saliva is the solution

Why?

Disease vs. Dysfunction

• Diabetes• Auto-immune diseases• Hypothyroidism• Arthritis• Coronary artery disease• Cancer• Neurodegenerative disease

Allopathic Medicine utilizes laboratory to diagnose (end organ) disease:

Functional medicine utilizes laboratory to detect earlier warning signs:

• Adrenal dysfunction• Insulin resistance• Metabolic syndrome• Estrogen dominance• Chronic Fatigue• Suboptimal thyroid• Muscle weakness and

pain• Inflammation

Reference Ranges: determined by science or statistics?

Reference Ranges do NOT equal Normal ranges

“Treat the patient not the laboratory”

Reference Ranges• TSH 0.5 – 5.0

• Some studies have recommended an upper range of 2.0-2.5.

• “Pollution” of the reference range pool. It is likely that the current upper limit of the population reference range is skewed by the inclusion of persons with occult/functional thyroid dysfunction

• Estradiol (postmenopause) 28 – 112• Testosterone male 240 – 950• You have to think for yourself when you interpret

laboratory tests if you want to make a difference.

Paolo G, et al. A prospective study of endogenous estrogens and breast cancer in postmenopausal women. J Natl Cancer Inst. 1995; 87: 190-97.Groschl M. Current status of salivary hormone analysis. Clin Chem. 2008; 54: 1759-69. Carey BJ, et al. A study to evaluate serum and urinary hormone levels following short and long term administration of two regimens of progesterone cream in postmenopausal women. Br J Obstet Gynaecol. 2000; 107: 722-726.

Measure Saliva Serum Urine

Free (unbound) active portion of

hormone

Yes No Yes and No (mainly metabolites)

Daily production from organ

Yes Yes Yes

& monitors topical hormones

Yes No No

& monitors injectables

Yes Yes Yes

& PO hormones Yes Yes Yes

Serum advantages

•Familiarity•Drawn along with other “blood tests”

•Measures organ production•Monitor injectable or PO hormone replacement

Serum disadvantages

• Free levels are expensive and technically difficult• Ineffective when used to monitor topical hormone

replacement• Stressful: requires needles• Limited to venipuncture access• Inconvenient to the patient• Will not be sensitive enough to detect functional

distress and conditions

Hofman LF. Human saliva as a diagnostic specimen. J Nutr. 2001; 131: 1621S-55S.Freshney IR. Serum-free media. In: Culture of Animal Cells. United Kingdom: John Wiley & Sons; 2005.

Free vs. bound hormone

• Most hormones are bound to carrier systems (97%), e.g. SHBG, TBG, CBG

• Methods to separate free from bound• Ultracentrifugation• Equilibrium dialysis• Gel separation• Solvent extraction• Until Salivary testing…we had to settle for a calculation

Speroff L, Fritz MA. Clinical Gynecologic Endocrinology and Infertility. 7th ed. Philadelphia, PA: Lippincott Williams and Wilkins; 2005.The Merck Manual. Endocrine Disorders. Available at: http://www.merckmanuals.com/professional/endocrine_and_metabolic_disorders/principles_of_endocrinology/endocrine_disorders.html. Accessed October 15, 2013.

Free Androgen Index (FAI)

• FAI = total testosterone (ngm/dl)/SHBG nmol/l)x 0.0347

•Expected range= 0.7 to 1.0• http://www.issam.ch/freetesto.htm

ISSAM International Society for the Study of the Aging Male http://www.issam.ch. Accessed October 15, 2013.

Serum Disadvantages:Venipuncture causes rapid rise in serum ACTH

• Serum cortisol and ACTH compared on 9 subjects before and after venipuncture.

• ACTH increased within 90 seconds following venipuncture.

• Cortisol levels increased within 4 minutes.

• Conclusion: ACTH and cortisol levels may be greatly altered by the stress of venesection. Plasma ACTH measure after a difficult venesection in a subject with poor venous access may be misleading.

Meeran K, Hattersley A, Mould G, Bloom SR. Venipuncture causes rapid rise in plasma ACTH. Br J Clin Pract. 1993; 47: 246-7.

Urine advantages

• No needles• Stable• Readily measures metabolites• 2/4/16 OH estrone ratio• Monitor injectable or PO hormone

replacement

Cooke DG, et al. Validation of a reference ELISA for estrone glucuronide using urine samples normalized by dilution to a constant rate of urine production. Steroids. 2007; 72: 580-91.

Urine disadvantages

• Does not measure actual hormone levels, only metabolites…

• Spot urines are subject to troughs and peaks• 24 hr. collections have a poor compliance (40%

missed samples)• Inconvenient to the patient• INEFFECTIVE when used to monitor topical

hormone replacement

Klee GG. Maximizing efficacy of endocrine tests: importance of decision-focused testing strategies and appropriate patient preparation. Clin Chem. 1999; 45: 1323-30.

Saliva advantages

• Diagnose• Guide Treatment• Monitor• Simple• Stress-free• Noninvasive (no needles)• More convenient for

patient and practitionerVining RF, et al. Salivary cortisol: A better measure of adrenal cortical function than serum cortisol. Ann Clin Biochem. 1983; 20: 329-35.Gozansky WS, et al. Salivary cortisol determined by enzyme immunoassay is preferable to serum total cortisol for assessment of dynamic hypothalamic-pituitary-adrenal axis activity. Clin Endocrinol (Oxf). 2005; 63: 336-41.Galbois A, et al. Assessment of adrenal function in cirrhotic patients: salivary cortisol should be preferred. J Hepatol. 2010; 52: 839-45.

Saliva advantages

• Optimized for collection any time of day/month, any place

• Hormones stable in saliva for prolonged period of time• Convenient shipment by USPS, UPS or FedX• More representative of bioavailable level of steroid

hormones

• Only Effective method to accurately measure topical, trans-vaginal, rectal suppositories, or sublingual hormone supplementation and PO, SQ and IM therapy

Groschl M. Current status of salivary hormone analysis. Clin Chem. 2008; 54: 1759-69.O'Leary P, et al. Salivary, but not serum or urinary levels of progesterone are elevated after topical application of progesterone cream to pre- and postmenopausal women. Clin Endocrinol. 2000; 53: 615-20.

Saliva limitations

• Restricted to non-polar lipid soluble molecules, e.g. hormones…Limited number of tests (assays) available

• Technically more challenging: need 10-20x sensitivity

• Saliva can be contaminated with topical hormones on lips or hands

• Spurious results with periodontal disease—the barrier to polar molecules is breached.

Lewis JG. Steroid analysis in saliva: an overview. Clin Biochem Rev. 2006; 27: 139-46.

Salivary cortisol: a better measure of adrenal cortical function than serum• Saliva reflects bio-available cortisol and is

unaffected by CBG level • CBG rises with BCP and during pregnancy

• Stress free and easy to collect • Lends itself to multiple samples • Routine serum samples at 0900 and 1700 do not

accurately reflect adrenal dysfunction• IV cortisol injection shows salivary rise within 5

minutesVining RF, et al. Salivary cortisol: A better measure of adrenal cortical function than serum cortisol. Ann Clin Biochem. 1983; 20: 329-35.Ahn R, et al. Salivary cortisol and DHEA levels in the Korea population: Age-related differences, diurnal rhythm and correlations with serum levels. Yonsei Med J. 2007; 48: 379-88.Gozansky WS, et al. Salivary cortisol determined by enzyme immunoassay is preferable to serum total cortisol for assessment of dynamic hypothalamic-pituitary-adrenal axis activity. Clin Endocrinol. 2005; 63: 336-41.

Salivary cortisol: a better measure of adrenal cortical function than serum

Additional references

• Aardal-Eriksson E, et al. Salivary cortisol--an alternative to serum cortisol determinations in dynamic function tests. Clin Chem Lab Med. 1998; 36: 215-22.

• Crewther BT, et al. Monitoring salivary testosterone and cortisol concentrations across an international sports competition: data comparison using two enzyme immunoassays and two sample preparations. Clin Biochem. 2012; Nov 30: Epub.

• Gozansky WS, et al. Salivary cortisol determined by enzyme immunoassay is preferable to serum total cortisol for assessment of dynamic hypothalamic-pituitary-adrenal axis activity. Clin Endocrinol. 2005; 63: 336-41.

• Tackett JL, et al. J Pers Disord 2014 Feb;28(1):25-39. doi: 10.1521/pedi.2014.28.1.25

Many studies have “proven” that transdermal progesterone is ineffective

• Authors randomized 27 estradiol exposed (Climara 0.100 weekly) postmenopausal women into transdermal progesterone 16 mg, 32 mg or 64 mg groups.

• Serum levels and endometrial biopsies were monitored. • Conclusion: The use of transdermal progesterone for 14

days over three cycles, even at concentrations as high as 64 mg daily, did not increase circulation blood progesterone concentrations sufficiently to induce any evidence of secretory effect in the endometrium.

Wren BG, et al. Micronised transdermal progesterone and endometrial response. Lancet. 1999; 354: 1447-48.

Free vs. Bound Hormones• Endogenous hormones are secreted from the ovaries, testes or adrenals

wrapped in protein envelopes called SHBG, CBG, etc. so that they can be transported in the serum.

• These protein bound hormones are not fully biologically active.• The more important and relevant hormone levels are the 1-10% that are

unbound, and thus biologically active• Saliva contains only biologically active hormones• When progesterone is absorbed through the skin, it is not coated with a protein

carrier, but is rather carried in blood’s fatty components, such a red blood cell membranes.

• Only when absorbed progesterone exceeds the carrying capacity of the fatty material in the blood will it “slop over” into the serum.

• Thus, when serum levels of transdermal progesterone are seen to rise, it is probably a sign of overdosing.

• Conversely, transdermal progesterone absorption is quick and efficient, showing up in saliva in just a few hours, indicating that it is well absorbed and available to cells in a biologically active form.

Lee, John MD. What Your Doctor May Not Tell You About Menopause. New York: Warner Wellness, 1996. Print.

Why is saliva testing optimal for monitoring topical hormones?Transport and delivery of steroids from bloodstream to salivary glands

S

SS

S

S

S

Salivary Gland

red blood cell

carrier protein (SHBG, CBG, albumin)

steroid hormone

capillary

S

S

S

S

S

S

S

S

S

SALIVA

Groschl M. Current status of salivary hormone analysis. Clin Chem. 2008; 54: 1759-69.Groschl M. The physiological role of hormones in saliva. Bioessays. 2009; 31: 843-52.Cook CJ. Rapid noninvasive measurement of hormones in transdermal exudate and saliva. Physiol Behav. 2002; 75: 169-81.

Salivary levels (but not serum or urinary levels) of progesterone are elevated after topical application of progesterone cream to pre- and postmenopausal women

• Topical progesterone 64 mg single dose to 6 pre and 6 postmenopausal women.

• Monitored serum progesterone and urine metabolite (pregnanediol-3-glucuronide)

• Saliva level’s change was remarkable, showing appreciable levels within 30 minutes

• Conclusion: “Saliva levels show tissue delivery. No statistically significant change in the serum or urine levels.”

O'Leary P, et al. Salivary, but not serum or urinary levels of progesterone are elevated after topical application of progesterone cream to pre- and postmenopausal women. Clin Endocrinol. 2000; 53: 615-20.

After 22 days

http://health.allrefer.com/pictures-images/skin-layers.html

Papillary dermis - right beneath the squamous layer - has capillaries

How does hormone get to the target without showing in the serum?

Hitching a ride on a lipid containing vehicle

RBC’s: The hormone “taxi” service

The permeability of the human red cell membrane to steroid sex hormones

• Sex hormones estrone, estradiol, estriol, progesterone, and testosterone.

• Affinities of hormone binding to cells and hormone indicate that as much as 15-35% of the total hormone content in whole blood is confined to red cells.

• We suggest that red cells function as carriers of sex hormones in the bloodstream in a manner similar to that of albumin.

Koefoed P, Brahm J. The permeability of the human red cell membrane to steroid sex hormones. Biochim Biophys Acta. 1994; 1195: 55-62.Hayahsi T, Yamada T. Association of bioavailable estradiol levels and testosterone levels with serum albumin levels in elderly men. Aging Male. 2008; 11: 63-70.

Routine handling of serum sample

Hormone attached to RBC membrane, discarded

Why topically applied hormone is not detected in serum or urine…

• Steroid hormones are lipid soluble, so diffuse freely through the cell membrane and associate with an intra-cytoplasmic receptors

• Receptor-hormone complex enters the nucleus• Complex attaches to appropriate region of genome• Genome is activated to produce messenger RNA• RNA enters cytoplasm and is translated into enzymes,

which alters metabolism• The initial receptor hormone complex is then

catabolized within the cell by cellular organelles—never to be seen again

Speroff L, Fritz MA. Clinical Gynecologic Endocrinology and Infertility. 7th ed. Philadelphia, PA: Lippincott Williams and Wilkins; 2005.

Basic steroid hormone physiology

Why topically applied hormone is not detected in serum…Basic steroid hormone physiology

Speroff L, Fritz MA. Clinical Gynecologic Endocrinology and Infertility. 7th ed. Philadelphia, PA: Lippincott Williams and Wilkins; 2005.

Why isn’t transdermal hormone reflected in serum levels…summary• Transdermal hormones are free and largely are not

bound to protein carriers or, • Weak bond with RBCs, so quickly transferred into

cells• RBCs discarded when serum is tested• Hormones are catabolized within the cell by cellular

organelles—never to be seen again• Thus venous blood or urine are not good matrixes to

test

But what about the endometrium?Topical progesterone cream: an alternative progestin in hormone replacement therapy.

• 20 women completed a 1 year randomized, controlled, cross-over study comparing conjugated equine estrogen (Premarin, 0.625mg) paired with progesterone cream (Pro-Gest, 40mg) vs. conjugated equine estrogen paired with medroxyprogesterone acetate (Prempro).

• Endometrial biopsies were performed at the end of each 6 month arm of the study. No hyperplasia was found in either group.

• Incidence of spotting was similar in both groups. Participants preferred the progesterone cream composition (77% vs 5%, p<0.001).

Leonetti HB, Anasti JN, Landes J. Topical progesterone cream: an alternative progestin in hormone replacement therapy. Obstet & Gynecol 2003; 101(4 Suppl.):85.

Studies supporting salivary testing

• Salivary progesterone can be used as a practical noninvasive sampling alternative for the assessment of normal luteal function. Zorn JR, McDonough PG, Nessman C, Janssens Y, Cedard L. Salivary progesterone as an index of the luteal function. Fertil Steril. 1984; 41: 248-53. Ishikawa M, et al. The clinical usefulness of salivary progesterone measurement for the evaluation of the corpus luteum function. Gynecol ObstetInvest. 2002; 53: 32-37.

• Salivary progesterone showed a significant correlation compared with serum with the added advantages of convenience and reduced stress (no needles)Webley GE, Edwards R. Direct assay for progesterone in saliva: comparison with a direct serum assay. Ann Clin Biochem. 1985; 22: 579-85.Stanczyk FZ, et al. Percutaneous administration of progesterone: blood levels and endometrial protection. Menopause. 2005; 12: 232-7.

• After exogenous T administration, saliva and serum T rose abruptly and in parallel. This study demonstrates that salivary T levels may be used as an index of free serum T. Wang C, et al. J Clin Endocrinol Metab. 1981 Nov;53(5):1021-4

Sex steroid hormones may fluctuate ‘wildly’ throughout the day

Saliva samples taken every 10 minutes

IBL-Hamburg. Saliva Diagnostics. Available at: http://www.ibl-international.com/en_us/saliva-diagnostics. Accessibility verifiedOctober 15, 2013. Gandia A, et al. Salivary estradiol as a marker of the biological response to induction of ovulation. In Kirschbaum C, et al. (eds): Assessment of Hormones and Drugs in Saliva in Biobehavioral Research. Seattle; Hofgrefe & Huber Publishers: 1992.Stanczyk FZ, et al. Limitations of direct estradiol and testosterone immunoassay kits. Steroids. 2003; 68: 1173-78.

Hours

Labrix Saliva Testing Advantages The Pooled Tube• When test kits arrive at our lab a sample from

each timed sample is put into a fifth tube –‘the pooled tube’.

• When we test the sex hormones: (estrogens, progesterone, testosterone and DHEA) we do so from the pooled tube giving us an “average” of that hormone’s levels throughout the day.

• This process yields significantly more accurate and clinically useful results.

• We were the first and only major saliva laboratory in the USA to employ this methodology

Saliva: Recap• Clinically proven for over 30 years• Saliva testing measures

“bioavailable” steroid hormone levels• Not limited to the hormone bound to carrier proteins • Measuring active hormones, not metabolites

• Collection is easy to do in patients of all ages, locations and levels of wellness.

• Saliva testing can be used for clinical diagnosis and to monitor any route of BHRT administration

• Saliva testing is the only effective method to monitor topical or trans-mucosal BHRT dosing

• Saliva testing is convenient for monitoring cortisol throughout the day.

Neurotransmitter testing

• HPLC Triple Quadrupole MS/MS: the most accurate methodology for measuring neurotransmitters

• Serotonin, GABA, glutamate, dopamine, norepinephrene and epinephrine

• Urine testing has been employed clinically for over 10 years in US; 20 years in Europe

Hughes JW, et al. Depression and anxiety symptoms are related to increased 24-hour urinary norepinephrine excretion among healthy middle-aged women. J Psychosom Res. 2004; 57: 353-358.Chekhonin VP, et al. Catecholamines and their metabolites in the brain and urine of rats with experimental Parkinson’s disease. Bull Exp Biol Med. 2000 130: 805-809.Lynn-Bullock CP, et al. The effect of oral 5-HTP administration on 5-HTP and 5-HT immunoreactivity in monoaminergic brain regions of rats. J Chem Neuroanat. 2004; 27: 129-138.Struys EA, et al. Determination of the GABA analogue succinic semialdehyde in urine and CSF by dinitrophenylhydrazine derviatizationand liquid chromatography-tandem mass spectrometry: Application to SSADH deficiency. J Inherit Metabol Dis. 2005; 28: 913-20.

Levels of serotonin in plasma and urine are significantly but less strongly correlated with levels in CSF.

Audhya T, et al. Correlation of serotonin levels in CSF, platelets, plasma and urine. Biochim Biophys Acta. 2012; 1820: 1496-501.

evidence suggests that neurotransmitters excreted in the urine may have a place in clinical practice as a biomarker of nervous system function to effectively assess disturbances and monitor treatment efficacy.

Marc DT, et al. Neurotransmitters excreted in the urine as biomarkers of nervous system activity: validity and clinical applicability. Neurosci Biobehav Rev. 2011; 35: 635-44.

Urine neurotransmitter summary

• Industry Standard in Functional Medicine

• Simple collection• Cost effective • Decades of use in Europe• Clinically correlates with diagnosis and

treatment