Accession # 00526588

Male Sample Report

123 A Street
Sometown, CA 90266

Ordering Provider: DOB: 1967-08-09 Collection Times:

Precision Analytical Age: 54 2022-06-10 02:00AM
2022-06-10 07:00AM
Sex: Male 2022-06-10 09:00AM
2022-06-10 06:00PM
2022-06-10 11:00PM

Hormone Testing Summary

Key (how to read the results): Sex Hormones

Testosterone
Age Range
25
patient
0.8 19 18-25 50-115
low limit high limit
result 0.5 2.2 115 26-40 40-95
41-60 30-80
Estradiol(E2) Testosterone
>60 25-60

Adrenal Hormones See pages 4 and 5 for a more complete breakdown of adrenal hormones

Total DHEA Production

300
1000 5500
Age Range 1072
(ng/mg)

Daily Free Cortisol Pattern 20-39 3000-5500

240 40-60 2000-4000
>60 1000-2500 Total DHEA Production
(DHEAS + Etiocholanolone + Androsterone)
Cortisol

Hi
180 gh
Ra
n ge
Lim
it
120
75 4550
102 5838
Patient Values
60 300 10000

Lo w Ra ng
e Limit 24hr Free Cortisol cortisol Metabolized Cortisol (THF+THE)
0 (A+B+C+D) metabolism (Total Cortisol Production)
Waking (A) Morning (B) Afternoon (C) Night (D)

Free cortisol best reflects tissue levels. Metabolized cortisol best reflects total cortisol production.

The following videos (whic h c an also be found on the website under the listed names along with others) may aid your understanding:
DUTCH Complete Overview Estrogen Tutorial Male Androgen Tutorial Cortisol Tutorial
PLEA SE BE SURE TO REA D BELOW FOR A NY SPECIFIC LA B COMMENTS. More detailed comments can be found on page 9.

Precision Analytical (Dawn Huo, Ph.D., Lab Director) Male Sample Report Page 1 of 15
3138 Rivergate Street #301C FINAL REPORT CLIA Lic. #38D2047310
McMinnville, OR 97128 06/20/2022 DutchTest.com
 Accession # 00526588
Male Sample Report
123 A Street
Sometown, CA 90266

Sex Hormones and Metabolites

Ordering Provider: DOB: 1967-08-09 Collection Times:
Precision Analytical Age: 54 2022-06-10 02:00AM
2022-06-10 07:00AM
Sex: Male 2022-06-10 09:00AM
2022-06-10 06:00PM
2022-06-10 11:00PM

Category Test Result Units Normal Range

Progesterone Metabolites (Urine)
b-Pregnanediol Below range 66.0 ng/mg 75 - 400
a-Pregnanediol Below range 10.0 ng/mg 20 - 130
Estrogens and Metabolites (Urine)
Estrone(E1) Below range 3.7 ng/mg 4 - 16
Estradiol(E2) Low end of range 0.8 ng/mg 0.5 - 2.2
Estriol(E3) Within range 4.6 ng/mg 2-8
2-OH-E1 Within range 1.6 ng/mg 0 - 5.9
4-OH-E1 Within range 0.2 ng/mg 0 - 0.8
16-OH-E1 Within range 0.68 ng/mg 0 - 1.2
2-Methoxy-E1 Within range 0.7 ng/mg 0 - 2.8
2-OH-E2 Low end of range 0.08 ng/mg 0 - 0.6
4-OH-E2 Within range 0.09 ng/mg 0 - 0.3
Total Estrogen Low end of range 12.4 ng/mg 10 - 34
Androgens and Metabolites (Urine)
DHEA-S Low end of range 30.3 ng/mg 30 - 1500
Androsterone Below range 364.0 ng/mg 500 - 3000
Etiocholanolone Within range 678.0 ng/mg 400 - 1500
Testosterone Below range 19.1 ng/mg 25 - 115
5a-DHT Below range 3.07 ng/mg 5 - 25
5a-Androstanediol Low end of range 30.7 ng/mg 30 - 250
5b-Androstanediol Low end of range 67.2 ng/mg 40 - 250
Epi-Testosterone Low end of range 28.9 ng/mg 25 - 115

Precision Analytical (Dawn Huo, Ph.D., Lab Director) Male Sample Report Page 2 of 15
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McMinnville, OR 97128 06/20/2022 DutchTest.com
 Hormone metabolite results from the previous page are presented here as they are found in the
steroid cascade. See the Provider Comments for more information on how to read the results.

Age-Dependent Ranges
Androgens
Age DHEA-S
20-39 150-1500
Pregnenolone
40-60 60-800
30 >60 30-300
30
Etiocholanolone Androsterone
1500 20-39 800-1500 20-39 1500-3000
DHEA 40-60 600-1200 40-60 1000-2000
DHEA-S >60 400-1000 >60 500-1000

5ß-androstanediol 5α-androstanediol
20-39 70-250 20-39 60-250
40-60 55-210 40-60 50-180
Androstenedione 25
19 >60 40-150 >60 30-130
5α
5ß

ar 115 Testosterone 5α-DHT

om
at 18-25 35-115 20-39 9-25
as 26-40 30-95 40-60 7-20
e Testosterone
41-60 25-80 >60 5-16
ar
om >60 20-60
at
as
400 1500 500 3000 e
678 364

Etiocholanolone Androsterone
Testosterone
5α 3.7 0.8 4.6
5ß

4.0 16.0 0.5 2.2 2.0 8.0

CYP3A4
Estrone(E1) Estradiol(E2) Estriol(E3)
Less Androgenic
Metabolites primary estrogens (E1, E2, E3)
5.00
3.07
CY

25.00
P3
A4

5a-DHT
Estrogens

0.68
1.20
0.00
40
67 30
31 16-OH-E1 Phase 1 Estrogen Metabolism Ratios
CYP
CYP1A1 (protective pathway)

1 B1

250 250

5b-Androstanediol 5a-Androstanediol

Patient 2-OH 4-OH 16-OH

5ß preference 5α Preference Percentages 64% 8.1% 27.9%
(androgenic)
Expected 60-80% 7.5-11% 13-30%
0.2 Percentages (2-OH) (4-OH) (16-OH)
5α-Reductase Activity
0.8
5α -metabolism makes androgens more potent, most 0.0
notably 5α -DHT is the most potent testosterone metabolite 4-OH-E1

Glutathione detox

Low High QUINONE

COMT
0.7 1.6 (reactive)

2.8 methylation 5.9

0.0 0.0
Methylation-activity 2-Methoxy-E1 2-OH-E1
2-Methoxy/2-OH
Methylation detox
4-OH-E1

If not detoxified, 4-OH-E1 can

bind to and damage DNA

Precision Analytical (Dawn Huo, Ph.D., Lab Director) Male Sample Report Page 3 of 15
3138 Rivergate Street #301C FINAL REPORT CLIA Lic. #38D2047310
McMinnville, OR 97128 06/20/2022 DutchTest.com
 Accession # 00526588
Male Sample Report
123 A Street
Sometown, CA 90266

Adrenal
Ordering Provider: DOB: 1967-08-09 Collection Times:
Precision Analytical Age: 54 2022-06-10 02:00AM
2022-06-10 07:00AM
Sex: Male 2022-06-10 09:00AM
2022-06-10 06:00PM
2022-06-10 11:00PM

Category Test Result Units Normal Range

Creatinine (Urine)
Creatinine A (Waking) Within range 0.7 mg/ml 0.3 - 3
Creatinine B (Morning) Within range 1.41 mg/ml 0.3 - 3
Creatinine C (Afternoon) Within range 0.34 mg/ml 0.3 - 3
Creatinine D (Night) Within range 1.24 mg/ml 0.3 - 3
Daily Free Cortisol and Cortisone (Urine)
Cortisol A (Waking) Low end of range 23.9 ng/mg 13 - 80
Cortisol B (Morning) Within range 67.0 ng/mg 35 - 180
Cortisol C (Afternoon) Below range 7.7 ng/mg 10 - 45
Cortisol D (Night) Low end of range 3.6 ng/mg 0 - 20
Cortisone A (Waking) Within range 64.2 ng/mg 40 - 160
Cortisone B (Morning) Within range 147.1 ng/mg 80 - 240
Cortisone C (Afternoon) Low end of range 47.2 ng/mg 40 - 130
Cortisone D (Night) Within range 18.9 ng/mg 0 - 70
24hr Free Cortisol Low end of range 102.0 ng/mg 75 - 300
24hr Free Cortisone Low end of range 277.0 ng/mg 220 - 550
Cortisol Metabolites and DHEA-S (Urine)
a-Tetrahydrocortisol (a-THF) Below range 119.0 ng/mg 175 - 700
b-Tetrahydrocortisol (b-THF) Low end of range 2168.0 ng/mg 1750 - 4000
b-Tetrahydrocortisone (b-THE) Within range 3551.0 ng/mg 2350 - 5800
Metabolized Cortisol (THF+THE) Within range 5837.9 ng/mg 4550 - 10000
DHEA-S Low end of range 30.0 ng/mg 30 - 1500

Precision Analytical (Dawn Huo, Ph.D., Lab Director) Male Sample Report Page 4 of 15
3138 Rivergate Street #301C FINAL REPORT CLIA Lic. #38D2047310
McMinnville, OR 97128 06/20/2022 DutchTest.com
 ST R ESS

Stress (or inflammation)

Hypothalamus Total DHEA Production
CRH

causes the brain to release ACTH,

which stimulates the adrenal glands Age Range
Pituitary 20-39 3000-5500
to make hormones
40-60 2000-4000
Pineal >60 1000-2500

10
23
ACTH

85

Melatonin* (Waking) 1000

1072 5500

Total DHEA Production

Adrenal Gland DH EA (DHEAS + Etiocholanolone + Androsterone)

4550
5838
10000

Metabolized Cortisol (THF+THE)

Cor Cortisol Metabolism (Total Cortisol Production)
tis ol
Cir
cu
lat
ing

More cortisone More cortisol

Fre

metabolites (THE) metabolites (THF)

eC

NOTE: This 11b-HSD index measures the balance of cortisol and cortisone metabolites
ort

which best reflects the overall balance of active cortisol and inactive cortisone systemically.
iso

400 300
(ng/mg)
Cortisone (ng/mg)

Daily Free Cortisone Pattern Daily Free Cortisol Pattern

320 240
Cortisol

Hig Hi
240 hR 180 gh
a ng Ra
eL n
imit ge
Lim
it
160 Patient Values 120

Patient Values
80 60
Lo w Ra ng
e Limit
Lo w Ra ng
e Limit
0 0
Waking (A) Morning (B) Afternoon (C) Night (D) Waking (A) Morning (B) Afternoon (C) Night (D)

e interconv ert
C ortis on (11b-
l and HS D
tis o )
Co r
220 75
277 102
550 300

24hr Free Cortisone 24hr Free Cortisol

(A+B+C+D) (A+B+C+D)

The first value reported (Waking "A") for c ortisol is intended to represent the "overnight" period. When patients sleep through the night, they
c ollec t just one sample. In this c ase, the patient woke during the night and c ollec ted (see the top of the report for the times c ollec ted). We c all
this value "A1" and the value from the sample c ollec ted at waking "A2." These values are used to c reate a "time-weighted average" to c reate the
"A" value. However, there was no measurable c ortisol found in the A2 sample, so the A1 sample has been used as the A sample for all values. This
c ortisol value represents the time between bed and the middle of the night c ollec tion and not the entirety of the overnight period.

Precision Analytical (Dawn Huo, Ph.D., Lab Director) Male Sample Report Page 5 of 15
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McMinnville, OR 97128 06/20/2022 DutchTest.com
 Accession # 00526588
Male Sample Report
123 A Street
Sometown, CA 90266

Organic Acid Tests (OATs)

Ordering Provider: DOB: 1967-08-09 Collection Times:
Precision Analytical Age: 54 2022-06-10 02:00AM
2022-06-10 07:00AM
Sex: Male 2022-06-10 09:00AM
2022-06-10 06:00PM
2022-06-10 11:00PM

Category Test Result Units Normal Range

Nutritional Organic Acids
Vitamin B12 Marker (may be deficient if high) - (Urine)
Methylmalonate (MMA) Within range 1.99 ug/mg 0 - 3.5
Vitamin B6 Markers (may be deficient if high) - (Urine)
Xanthurenate Within range 0.57 ug/mg 0.2 - 1.9
Kynurenate Within range 3.89 ug/mg 1 - 6.6
Biotin Marker (may be deficient if high) - (Urine)
b-Hydroxyisovalerate Within range 8.5 ug/mg 0 - 18
Glutathione Marker (may be deficient if low or high) - (Urine)
Pyroglutamate Within range 53.6 ug/mg 38 - 83
Gut Marker (potential gut putrefaction or dysbiosis if high) - (Urine)
Indican Within range 34.1 ug/mg 0 - 131
Neuro-related Markers
Dopamine Metabolite - (Urine)
Homovanillate (HVA) Low end of range 4.4 ug/mg 4 - 16
Norepinephrine/Epinephrine Metabolite - (Urine)
Vanilmandelate (VMA) Within range 4.7 ug/mg 2.5 - 7.5
Neuroinflammation Marker - (Urine)
Quinolinate Within range 9.1 ug/mg 0 - 12.5
Additional Markers
Melatonin (*measured as 6-OH-Melatonin-Sulfate) - (Urine)
Melatonin* (Waking) Low end of range 23.4 ng/mg 10 - 85
Oxidative Stress / DNA Damage, measured as 8-Hydroxy-2-deoxyguanosine (8-OHdG) - (Urine)
8-OHdG (Waking) Within range 4.52 ng/mg 0 - 8.8

Precision Analytical (Dawn Huo, Ph.D., Lab Director) Male Sample Report Page 6 of 15
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 Clinical Support Overview
Thank you for choosing DUTCH for your functional endocrinology testing needs! We know you have many options
to choose from when it comes to functional endocrinology evaluation, and we strive to offer the best value, the
most up-to-date testing parameters and reference ranges, and the greatest clinical support to ensure the most
accurate results.

Please take a moment to read through the Clinical Support Overview below. These comments are specific to the
patient’s lab results. They detail the most recent research pertaining to the hormone metabolites, treatment
considerations, and follow-up recommendations. These comments are intended for educational purposes only.
Specific treatment should be managed by a healthcare provider. To view the steroid pathway chart, click here
Steroid Pathway Chart

Alert comments:

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 How to read the DUTCH report
This report is not intended to treat, cure or diagnose any specific diseases. The graphic dutch dials in this report
are intended for quick and easy evaluation of which hormones are out of range. Results below the left star are
shaded yellow and are below range (left). Results between the stars and shaded green are within the reference
range (middle). Results beyond the second star and shaded red are above the reference range (right). Some of
these hormones also change with age, and the age-dependent ranges provided should also be considered.

In a few places on the graphical pages, you will see fan-style gauges. For sex hormones, you will see one for the
balance between 5a/5b metabolism as well as methylation. For adrenal hormones, you will see one to
represent the balance between cortisol and cortisone metabolites. These indexes simply look at the
ratio of hormones for a preference. An average or "normal" ratio between the two metabolites (or
groups of metabolites) will give a result in the middle (as shown here). If the ratio between the
metabolites measured is "low" the gauge will lean to the left and similarly to the right if the ratio is higher than
normal.

Patient or Sample Comments

Throughout the provider comments you may find some comments specific to your situation or results. These
comments will be found in this section or within another section as appropriate. Comments in other sections that
are specific to your case will be in bold.

The patient reports significant symptoms of estrogen deficiency.

The patient reported significant fatigue in both the AM and PM.

Androgen Metabolism

Androgen Metabolites: DHEA

DHEA and androstenedione are made almost exclusively by the adrenal gland (although a smaller amount is
made in the testes). These hormones appear in urine as DHEA-S (DHEA-Sulfate), androsterone and
etiocholanolone.

DHEA peaks for men in their 20’s with a slow decline expected with age. DHEA mainly circulates throughout the
body as DHEA-s, with interconversion to active DHEA as it reaches various tissues. DHEA is a weak androgen
and will predominately convert to androstenedione, which will then convert to testosterone or aromatize to
estrone. DHEA-s is made by sulfation, has a much longer half-life than DHEA and lacks a diurnal rhythm, which is
why it is considered the best way to assess DHEA levels in the body. DHEA-s levels can be affected both by the
total production as well as by the body’s ability to sulfate DHEA.

The best way to assess the total production of DHEA is to add up these three metabolites. As DHEA production
decreases quite significantly with age, we provide the age-dependent ranges.

The Total DHEA Production (page 1) was about 1,072ng/mg which is within the overall range but
is below the range for the patient's age-dependent range. This implies that the adrenal glands
are not producing appropriate DHEA levels for the patient's age. Low DHEA is associated with
depression, diabetes, heart disease, inflammation and immune disorders. It can be caused by
hypothyroidism. It can cause fatigue, low mood and low libido. Supplementing DHEA in women
often raises both testosterone and estrogen, which may or may not be desirable here. DHEA
may increase with adaptogens such as maca and rhodiola, which improve overall adrenal
output.

Androgen Metabolites: Testosterone

The DUTCH test measures the total of testosterone glucuronide and testosterone sulfate. These conjugates of
testosterone are formed mostly from bioavailable testosterone that undergoes phase 2 metabolism to make it
Precision Analytical (Dawn Huo, Ph.D., Lab Director) Male Sample Report Page 8 of 15
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 ready for urine excretion.

Testosterone glucuronide is mostly made by the UGT2B17 enzyme, which also makes the glucuronide forms of
5a-DHT and 5b-androstanediol. Genetic variants of this enzyme reduce the urinary levels of these hormones
without affecting serum levels. The genetic variants of UGT2B17 vary in the population from 7-80% (variation
dependent on genetic ancestry, with the highest rates in those of Asian descent). Heterozygous individuals show
milder reductions in urinary testosterone than homozygous. For this reason, low and very low levels of urinary
testosterone should be confirmed with serum testing before treatment is applied. Serum testing can include free
and total testosterone and SHBG.

The testes make most of the male's testosterone. Levels tend to be their highest at around 20 years of age and
start to decline when men get into their 30's. Levels continue to drop as men age. Testosterone is needed for
building bones and muscle mass, regulating body fat distribution and in the production of sperm and red blood
cells. Testosterone is also important for libido and downstream production of modest amounts of estrogen.

Age dependent ranges are provided for all androgens as some decline is seen with age. Testosterone levels in
healthy men vary widely so it is suggested that these ranges be interpreted with caution and consideration of
symptoms. In addition, because estrogen also supports libido, erections and healthy weight management,
estrogen levels should be considered along with the testosterone levels when assessing symptoms.

Andogen Metabolites: 5a-reductase versus 5b-reductase

5a-reductase converts testosterone into 5a-DHT (DHT), which is even more potent (~3x) than testosterone.
High levels of DHT can lead to symptoms associated with too much testosterone (thinning scalp hair, acne, etc.)
and may also be associated with prostate issues in older men. However, 5aDHT plays an integral role in
supporting bone, muscle and connective tissue integrity and improving brain health through the upregulation of
dopamine, which can improve mood and libido.
Metabolites created down the 5b-pathway are significantly less androgenic than their 5a counterparts.

The fan-style gauge below the hormones shows the 5a or 5b preference based on the balance between
etiocholanolone (5b) and androsterone (5a) as well as 5a-androstanediol and 5b-androstanediol. The gauge
shows the relative ratio of 5a to 5b products but does not express the absolute value of DHT or if 5a-reductase
inhibition is or is not indicated. Consider symptoms and look at the total androgen levels if high androgen
symptoms are a concern.

You will also see levels of epi-testosterone, which is not androgenic like testosterone. It happens to be produced
in about the same concentrations as testosterone (this is an approximate relationship). This can be helpful when
assessing the validity of urinary testosterone testing in an individual patient. If epi-testosterone is much higher
than testosterone, serum testosterone assessment should considered before initiated therapy for low
testosterone. Epi-testosterone is suppressed when exogenous testosterone is given, which can serve as a proxy
for assessing endogenous testosterone production which can be obscured by the exogenous hormone
administration.
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 Estrogen Metabolism
When evaluating estrogen levels, it is important to assess the following:
The status (low, normal or high?) of estrogen production:
Levels of the primary estrogen, estradiol (the strongest estrogen), as well as "total estrogens" may be
considered.
Phase I Metabolism:
Estrogen is metabolized (primarily by the liver) down three phase I pathways. The 2-OH pathway is considered
the safest because of the anti-cancer properties of 2-OH metabolites. Conversely, the 4-OH pathway is
considered the most genotoxic as its metabolites can create reactive products that damage DNA. The third
pathway, 16-OH creates the most estrogenic of the metabolites (although still considerably less estrogenic than
estradiol) - 16-OH-E1.
When evaluating phase I metabolism, it may be important to look at the ratios of the three metabolites to see
which pathways are preferred relative to one another. It may also be important to compare these metabolites to
the levels of the parent hormones (E1, E2). If the ratios of the three metabolites are favorable but overall levels
of metabolites are much lower than E1 and E2, this may imply sluggish phase I clearance of estrogens, which
can contribute to high levels of E1 and E2.
The pie chart will assist you in comparing the three pathway options of phase I metabolism compared to what is
"normal." 2-OH metabolism can be increased by using products containing D.I.M. or I-3-C. These compounds are
found (or created from) in cruciferous vegetables and are known for promoting this pathway.
Methylation (part of Phase II Metabolism) of estrogens:
After phase I metabolism, both 4-OH and 2-OH (not 16-OH) estrogens can be deactivated and eliminated by
methylation. The methylation-activity index shows the patient's ratio of 2-Methoxy-E1 / 2-OH-E1 compared to
what is expected. Low methylation can be caused by low levels of nutrients needed for methylation and/or
genetic abnormalities (COMT, MTHFR). The COMT enzyme responsible for methylation requires magnesium and
methyl donors. Deficiencies in folate or vitamin B6 or B12 can cause low levels of methyl donors. MTHFR genetic
defects can make it more difficult for patients to make sufficient methyl donors. Genetic defects in COMT can
make methylation poor even in the presence of adequate methyl donors.

For this patient 2-Methoxy-E2 was not reportable due to an interfering substance or instrument
irregularity for this compound.

Progesterone Metabolism

Male progesterone is synthesized in the testes and, to a lesser degree, in the adrenal glands. It’s role in men’s
health is not well understood, although progesterone is known to be involved in sperm activation. In healthy
men, progesterone is positively correlated to markers of inflammation.

Metabolites of progesterone are measured in urine, including 5b-pregnanediol and 5a-pregnanediol. 5b-
pregnanediol is inactive in the body but is the major metabolite of progesterone. 5a-pregnanediol is often a
metabolite of more interest, as it can cross the blood brain barrier and up-regulate GABA activity and is
considered neuroprotective to the brain. Both taken together represent the major metabolic end points for
progesterone and can be used to represent total progesterone production.

The progesterone metabolites are low. In males, the significance of progesterone is not well
understood. Low progesterone might be an artifact of low overall testicular or adrenal hormone
production. Treatment might include support for testosterone such as maca, ashwagandha,
Tribulus, fenugreek, zinc and vitamin C.

DUTCH Adrenal
The HPA-Axis refers to the communication and interaction between the hypothalamus (H) and pituitary (P) in the
brain down to the adrenal glands (A) that sit on top of your kidneys. When cortisol is needed in the body, the
hypothalamus releases cortisol releasing hormone (CRH) and the pituitary responds by releasing
adrenocorticotropic releasing hormone (ACTH), which is the signal to the adrenal gland to release cortisol, DHEA
and DHEA-s. It is these adrenal hormones that are assessed on the DUTCH test to understand the patient’s HPA
axis.
The cortisol awakening response is a complex interaction between the HPA axis and the hippocampus, where
ACTH normally surges right after waking leading to the day’s highest levels of cortisol. This signal is considered
by researchers to be separate from the regular circadian rhythm (the smooth transition from lower cortisol at
night to modestly higher cortisol in the morning) and to reflect the person’s anticipation of stress during the day,
some psychosocial factors such as depression or anxiety and their metabolic state. The waking surge in cortisol
helps with energy, focus, morning blood sugar and immune regulation.
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 As the day progresses, ACTH declines and subsequent cortisol decreases throughout the day, so it is low at
night for sleep. This cycle starts over the next morning.
Free cortisol provides negative feedback to CRH & ACTH. When free cortisol is too low, ACTH will surge. ACTH will
also surge when a physical or psychological stressor occurs.
Only a small fraction of cortisol is "free" and bioactive. The “free” cortisol is what the person feels in terms of
energy and focus. Free cortisol is also what feeds back to the hypothalamus and pituitary gland for ACTH and
cortisol regulation. The free cortisol daily pattern is very useful for understanding cortisol and its interaction with
the patient’s symptoms throughout the day. However, because only a fraction of the cortisol is bioactive, when
considering treatments that affect the whole HPA axis, including DHEA, it is essential to measure metabolized
cortisol to get a bigger picture.
In urine, we can measure both the total metabolized cortisol (THF) and total metabolized cortisone (THE)
excreted throughout the day. These two components better represent the total cortisol production from the
adrenal glands than the free cortisol alone. Outside of the HPA axis, metabolism of cortisol occurs with the help
of thyroid hormone in the liver. A significant amount of cortisol is also metabolized in adipose tissue.

To best determine total adrenal production of cortisol throughout the day it is important to assess both
metabolized cortisol and free cortisol.

When evaluating cortisol levels, it is important to assess the following:

The daily pattern of free cortisol throughout the day, looking for low and high levels:
Abnormal results should be considered along with related symptoms. Remember that with urine results, the
“waking” sample reflects the night’s total for free cortisol. The sample collected two hours after waking captures
the cortisol awakening response, which is typically the time with the most cortisol secretion.
The sum of the free cortisol as an expression of the overall tissue cortisol exposure:
This total of four free cortisol measurements is the best way to assess the total of free cortisol throughout the
day, and this result correlates reasonably well to a true 24-hour urine free cortisol. Do be aware that this
measurement does not consider transitory shifts in cortisol in the late morning or early afternoon. This number
is calculated from the simple addition of the 4 points, so if a single point is very high or very low, it may skew the
number up or down especially if it is the morning “B” point, as it is weighted more heavily in the reference range.
The total level of cortisol metabolites:
We call this calculation "Metabolized Cortisol" which is the sum of a-THF, b-THF and b-THE (the most abundant
cortisol metabolites). While free cortisol is the best assessment for tissue levels of cortisol, it only represents 1-
3% of the total produced. The total metabolized cortisol best represents the total glandular output of cortisol for
the day, closer to 80% of the total produced.

Free cortisol levels are on the lower side of the reference range. Levels of metabolized cortisol
confirm that overall cortisol production is reasonable, and the actual diurnal pattern of free
cortisol should be examined to further examine cortisol production.

A potential preference for cortisol or cortisone (the inactive form):

To determine total systemic preference for cortisol or cortisone, it is best to look at which metabolite
predominates (THF or THE?). This preference can be seen in the fan style gauge. This is known as the 11b-HSD
index. The enzyme 11b-HSD II converts cortisol to cortisone in the kidneys, saliva gland and colon. 11b-HSD I is
more active in the liver, fat cells and the periphery and is responsible for reactivating cortisone to cortisol. Both
are then metabolized by 5a-reductase to become tetrahydrocortisol (THF) and tetrahydrocortisone (THE)
respectively. We can see more cortisol or cortisone in difference metabolic conditions. If one is favored over
another, read below for potential causes and treatment considerations.

Nutritional Organic Acids

Organic acids are the metabolic byproducts of cellular activity in the body. Organic acid production varies by the
individual and can be influenced by foods, environmental toxins, medications or supplements, nutrient status,
genetics and more. Organic acids begin to build up when a nutrient cofactor or mineral is not present for a
specific reaction to occur. As a response, byproducts (organic acids) build up and can be measured in urine.
On the DUTCH test, the organic acids we measure were chosen due to their specific roles in the metabolism and
function of enzymes required for hormone and adrenal health and function. As industry standard dictates, the
organic acids are measured from the waking sample.

Methylmalonate (MMA)
Methylmalonic acid is a metabolic byproduct of the Citric Acid Cycle (Krebs cycle). Methylmalonic acid requires
adenosylcobalamin for conversion to succinyl-CoA and onto ATP synthesis. If someone does not absorb enough
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 B12 from their diet due to low B12-rich food consumption, low stomach acid, has an autoimmune disorder
impacting Intrinsic Factor in the gut (required for B12 absorption), or has an MUT enzyme SNP (required for
conversion of MMA to Succinyl coA, dependent on adenosylcobalamin) then MMA will build up. Vitamin B12 is
required for COMT activity (estrogen methylation, dopamine breakdown) and PNMT activity (the enzyme that
takes norepinephrine to epinephrine), but is also critical for memory, energy production (ATP synthesis), gait and
more. When MMA is high, consider supporting B12 through foods, digestive support or supplementation.

Xanthurenate & Kynurenate

Xanthurenate and kynurenate are metabolic byproducts in the production of tryptophan to NAD in the liver. If
either xanthurenate or kynurenate build up in the urine, it can indicate a need for vitamin B6. This need is
amplified if BOTH markers are elevated, and often indicates a more severe deficiency of vitamin B6. Vitamin B6
is critical as a co-factor to over 100 important reactions that occur in the human body and is stored in the
highest concentration in muscle tissue.
Tryptophan is converted to NAD by the liver and one of the steps in this pathway requires B6. When B6 is
insufficient, xanthurenate is made instead. Xanthurenate can also bind to iron and create a complex that
increases DNA oxidative damage resulting in higher 8-OHdG levels. If both the xanthurenate and 8OhdG levels
are elevated, there is likely an antioxidant insufficiency.
Kynurenate may also become elevated when patients are B6 deficient because of a different, possibly less B6
dependent pathway. While there is always some tryptophan going down the kynurenine pathway towards NAD,
and possibly xanthurenate, this process is up regulated by inflammation, estrogen and cortisol elevations. If
levels of estrogen or cortisol are high, it may exacerbate kynurenic acid and increase the need for vitamin B6.
As the Xanthurenate and Kynurenate pathways lead to biomarkers with other influence in the body, elevations in
these markers may not always agree.

b-Hydroxyisovalerate
b-Hydroxyisovalerate is made when the body is deficient in biotin. This marker has an inverse relationship with
biotin, therefore elevated levels represent deficiencies in biotin. Biotin is an important cofactor in mitochondrial
function, metabolism of fatty acids, glucose, and protein, as well as ROS production. Biotin deficiency has similar
symptoms as other B-vitamin deficiencies but is most often associated with hair loss. Factors that influence
biotin levels include inadequate dietary intake, long-term and high-dose B5 supplementation, dysbiosis/gut
health, antibiotic use, medications, and biotinidase deficiency.

Pyroglutamate
Pyroglutamate is an intermediate in glutathione recycling and production. Glutathione requires the amino acids
cysteine, glycine and glutamate for production. If the body cannot convert pyroglutamate forward to glutathione,
it will show up elevated in the urine. High pyroglutamate is an established marker for glutathione deficiency.
Remember that glutathione is one of the most potent antioxidants in the human body and is especially
important in getting rid of toxins including the reactive quinone species formed by 4-OH-E1 and 4-OH-E2. This
reactive species can damage DNA if not detoxified by either methylation or glutathione.
Some have reported that low pyroglutamate may also be indicative of a need for glutathione; however, this is
not established in the scientific literature.
Note: Pyroglutamate in the urine can also be elevated with Italian cheese consumption. Italian Cheeses
(parmesan, etc.) may transiently increase pyroglutamate because they use a thermophilic lactobacilli to
ripen the cheese- which our gut breaks down into pyroglutamate. This is not clinically significant and only
reflects that they ate this style of cheese (if applicable).

Indican
Indican is a byproduct of tryptophan putrefaction by microbes in the gut. Accumulated levels of indican in the
urine suggest higher levels of tryptophan putrefaction from gastrointestinal dysbiosis or malabsorption.
Production of indican occurs when tryptophan creates indoles in the colon. No other endogenous indoles are
metabolized in this way, so when we see indican in the urine, it is directly related to gut production and a direct
reflection of gut health. When there is concern of dysbiosis, there may be poor metabolism of sex hormones
(including estrogen) along with chronic low-grade inflammation that can impact cortisol production and
metabolism. This test is not diagnostic but generally warrants further testing to rule out gut dysbiosis.

Vegetarian and vegan style eating may influence results as these diets have less protein generally, therefore
elevated levels are likely stronger suggestions of gut dysbiosis. The amount of indican present does not
correlate to the degree of dysbiosis but merely shows that dysbiosis is present. Common causes of high indican
include malabsorption of protein as a result of low stomach acid, poor pancreatic function, Celiac disease, the
overgrowth of anerobic bacteria in the colon, small intestinal bacterial overgrowth (SIBO), medications that
reduce protein absorption (like proton pump inhibitors or other antacids or H2 blockers), and constipation.
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 Neuro-related Organic Acids
Neurotransmitters are chemical signals produced by neurons in tissues throughout the body that act as
chemical messengers that influence mood, cortisol, heart rate, appetite, muscle contraction, sleep and more.
Measuring neurotransmitters directly is difficult because of their instability, and their direct urinary
measurements are controversial with respect to how well they reflect the body’s level of these neuro-hormones.

Each of the neurotransmitters assessed on the DUTCH test (dopamine, norepinephrine/epinephrine) can be
assessed indirectly by measuring their urine metabolites (HVA and VMA respectively). While these metabolites
are not a perfect reflection of what is going on in the brain, the scientific literature does affirm their use for a
good representation of overall levels of these neurotransmitters in the body.

Homovanillate (HVA)
Homovanillate (HVA) is the primary metabolite of dopamine, a brain and adrenal neurotransmitter that comes
from tyrosine (with BH4 and iron as co-factors). Dopamine goes on to create norepinephrine and epinephrine
(adrenaline).

Low levels of dopamine are associated with depression, addictions, cravings, apathy, pleasure seeking
behaviors, increased sleepiness, impulsivity, tremors, low motivation fatigue and low mood.
High levels of dopamine are associated with agitation, insomnia, mania, hyperactivity, hyper-focus, high stress,
anxiety and addictions/cravings/pleasure seeking (to maintain high levels).
High HVA can be caused by the use of the following supplements, foods or medications within 72 hours of
collecting urine samples: tyrosine, phenylalanine, mucuna, quercetin, bananas, avocados as well as parkinson's
medications. If these are being used, the HVA on the DUTCH test may not accurately reflect circulating
dopamine levels and should be disregarded.

Vanilmandelate (VMA)
Vanilmandelate (VMA) is the primary metabolite of norepinephrine and epinephrine (adrenaline). The adrenal
gland makes cortisol and DHEA (from the adrenal cortex) as well as norepinephrine and epinephrine (from the
adrenal medulla). When adrenal hormone output is low, VMA levels may be low. If HVA levels are significantly
higher than VMA, there may be a conversion problem from dopamine to norepinephrine. This case can be
caused by a copper or vitamin C deficiency.
The enzymes COMT (methylation of catechols) and MAO are needed to make HVA and VMA from dopamine and
norepinephrine respectively. If these enzymes are not working properly, HVA and/or VMA may be low in urine,
when circulating levels of dopamine and/or norepinephrine/epinephrine may not be low.

Low levels of norepinephrine/epinephrine are associated with addictions, cravings, fatigue, low blood pressure,
low muscle tone, intolerance to exercise, depression, and loss of alertness.

High levels of norepinephrine and epinephrine are associated with feelings of stress, aggression, violence,
impatience, anxiety, panic, excess worry/hypervigilance, insomnia, paranoia, increasing tingling/burning, loss of
memory, pain sensitivity, high blood pressure and heart palpitations.

Quinolinate (QA)
Quinolinate is a neurotoxin derived from tryptophan. Elevated quinolinate is seen in brain and nerve tissue
damage, especially in disorders such as Alzheimer’s disease, Parkinson’s disease, Huntington’s disease, motor
neuron diseases, multiple sclerosis, epilepsy, amyotrophic lateral sclerosis, and major depressive disorder. We
can also see elevated quinolinate due to low serotonin and need for vitamin B3 (niacin). The causes of elevated
quinolinate include neuroinflammation, general inflammation, infection, phthalate exposure, and/or oral
tryptophan use.

Melatonin (measured as 6-OHMS)

Melatonin is considered one of our sleep hormones. It is made predominately by the pineal gland in response to
darkness and is stimulated by melanocyte stimulating hormone (MSH). A low MSH is associated with insomnia
and an increased perception of pain. Mold exposure can inhibit MSH as well. The majority of our melatonin
production comes from the pineal gland, but melatonin is also made in the gut, and to a lesser extent in the
bone marrow, lymphocytes, epithelial cells and mast cells.

The DUTCH test uses the waking (A) sample to test melatonin. The urine sample given on waking reflects
overnight hormone production and metabolism. This sample can be used to assess melatonin throughout
the night. When patients take a middle of the night urine sample, a large amount of data strongly
suggests that the waking sample alone still correlates best to overnight melatonin production, so the
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 waking sample is still used for the DUTCH melatonin result.

8-OHdG (8-Hydroxy-2-deoxyguanosine)
8-OHdG (8-Hydroxy-2-deoxyguanosine) is a marker for estimating DNA damage due to oxidative stress (from
ROS creation). 8-OHdG is considered pro-mutagenic and is a biomarker for various cancer and degenerative
disease initiation and promotion states. It can be increased by chronic inflammation, increased cell turnover,
chronic stress, hypertension, hyperglycemia/pre-diabetes/diabetes, kidney disease, IBD, chronic skin conditions
(psoriasis/eczema), depression, atherosclerosis, chronic liver disease, Parkinson's (increasing levels with
worsening stages), Diabetic neuropathy, COPD, bladder cancer, or insomnia (to name a few). Studies have
shown higher levels in patients with breast and prostate cancers. When levels are elevated it may be prudent to
eliminate or reduce any causes and increase the consumption of antioxidant containing foods and/or
supplements.

Urine Hormone Testing - General Information

What is actually measured in urine? In blood, most hormones are bound to binding proteins. A small fraction of
the total hormone levels are "free" and unbound such that they are active hormones. These free hormones are
not found readily in urine except for cortisol and cortisone (because they are much more water soluble than, for
example, testosterone). As such, free cortisol and cortisone can be measured in urine and it is this
measurement that nearly all urinary cortisol research is based upon. In the DUTCH Adrenal Profile the diurnal
patterns of free cortisol and cortisone are measured by LC-MS/MS.

All other hormones measured (cortisol metabolites, DHEA, and all sex hormones) are excreted in urine
predominately after the addition of a glucuronide or sulfate group (to increase water solubility for excretion). As
an example, Tajic (Natural Sciences, 1968 publication) found that of the testosterone found in urine, 57-80% was
testosterone-glucuronide, 14-42% was testosterone-sulfate, and negligible amounts (<1% for most) was free
testosterone. The most likely source of free sex hormones in urine is from contamination from hormonal
supplements. To eliminate this potential, we remove free hormones from conjugates. The glucuronides and
sulfates are then broken off of the parent hormones, and the measurement is made. These measurements
reflect the bioavailable amount of hormone in most cases as it is only the free, nonprotein-bound fraction in
blood/tissue that is available for phase II metabolism (glucuronidation and sulfation) and subsequent urine
excretion.
Disclaimer: the filter paper used for sample collection is designed for blood collection, so it is technically
considered "research only" for urine collection. Its proper use for urine collection has been thoroughly validated.

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 Reference Range Determination (last updated 07.01.2022)
We aim to make the reference ranges for our DUTCH tests as clinically appropriate and useful as possible. This
includes the testing of thousands of healthy individuals and combing through the data to exclude those that are
not considered “healthy” or “normal” with respect to a particular hormone. As an example, we only use a
premenopausal woman’s data for estrogen range determination if the associated progesterone result is within
the luteal range (days 19-21 when progesterone should be at its peak). We exclude women on birth control or
with any conditions that may be related to estrogen production. Over time the database of results for reference
ranges has grown quite large. This has allowed us to refine some of the ranges to optimize for clinical utility. The
manner in which a metabolite’s range is determined can be different depending on the nature of the metabolite.
For example, it would not make clinical sense to tell a patient they are deficient in the carcinogenic estrogen
metabolite, 4-OH-E1 therefore the lower range limit for this metabolite is set to zero for both men and women.
Modestly elevated testosterone is associated with unwanted symptoms in women more so than in men, so the
high range limit is set at the 80th percentile in women and the 90th percentile for men. Note: the 90th percentile
is defined as a result higher than 90% (9 out of 10) of a healthy population.
Classic reference ranges for disease determination are usually calculated by determining the average value and
adding and subtracting two standard deviations from the average, which defines 95% of the population as being
“normal.” When testing cortisol, for example, these types of two standard deviation ranges are effective for
determining if a patient might have Addison’s (very low cortisol) or Cushing’s (very high cortisol) Disease. Our
ranges are set more tightly to be optimally used for Functional Medicine practices.

Below you will find a description of the range for each test:

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