■ MLS 4G

Banks, Paulette Ann

Credo, Hillary
Delfin, Amanda Mae
Deluso, Gaye Rhianna
Kipte, Keala
 INTRODUCTION
Hillary Credo
 Steroid Hormones
■ Are not packaged, but synthesized and immediately released.

■ Are all derived from the same parent compound: cholesterol.

■ Enzymes which produce steroid hormones from cholesterol are located in mitochondria and
smooth ER.

■ Steroids are lipid soluble and thus are freely permeable to membranes so are not stored in cells.

8/17/2018
Steroid Hormones
■ Are not water soluble so have to be carried in the blood complexed to specific binding globulins.

■ Corticosteroid binding globulin carries cortisol.

■ Sex steroid binding globulin carries testosterone and estradiol.

■ In some cases a steroid is secreted by one cell and is converted to the active steroid by the
target cell: (androgen is secreted by the gonad and converted into estrogen in the brain).

8/17/2018
Steroid Hormones
■ Steroid hormones play important roles in:
- carbohydrate regulation (glucocorticoids)
- mineral balance (mineralocorticoids)
- reproductive functions (gonadal steroids)

■ Steroids also play roles in inflammatory responses, stress responses, bone metabolism,
cardiovascular fitness, behavior, cognition, and mood.

8/17/2018
Structure of Steroid Hormones
■ Steroids are lipophilic
molecules.

■ All steroids, except calcitriol,

have
cyclopentanoperhydrophen
anthrene structure
(sterane).

8/17/2018
■ Are all derived from the
same parent compound:
cholesterol.

8/17/2018
General Steps In Steroid Determination

■ Hydrolysis using either an acid or an enzyme

■ Extraction

■ Purification

■ Estimation

8/17/2018
Estimation of Steroids

■ Fluorometric method

■ Chromatographic method (gas or high performance chromatography)

■ HPLC (considered as reference method)

■ Colorimetric method
 Porter-Silber reaction for corticosteroids or 17-hydroxycorticosteroids
 Zimmerman reaction for ketogenic steroids or 17-ketosteroids
 Kober reaction for estrogen

8/17/2018
Hormones of the Adrenal Cortex
by: Paulette Ann Banks
■ The major hormones, aldosterone, cortisol, and dehydroepiandrosterone sulfate (DHEAS), are
uniquely synthesized from a common precursor by cells located in one of the three functionally
distinct zonal layers of the adrenal cortex.
■ These zonal layers are:
■ Zona glomerulosa/ G-zone
■ Zona fasciculata/ F-zone
■ Zona reticularis/ R-zone
Histologically, the adrenal cortex is layered into three different zones. From the
outside, These are the:
■ Zona glomerulosa
■ Zona fasciculata
■ Zona reticularis
Each of these layers is specialized to produce a particular set of steroid
hormones. These hormones are the
■ Mineralocorticoids
■ Glucorticoids
■ Androgens or sex hormones respectively
Mineralocorticoids

■ These hormones are produced by the Zona Glumerulosa. These hormones are
involved in electrolyte and water balance. The major mineralocorticoid is the
hormone aldosterone (critical for sodium retention (volume), potassium, and
acid-base homeostasis).
G-zone

■ Aldosterone is inactivated and excreted by formation of glucoronides (via conjugation with

glucoronic acid) in the liver. The levels of aldosterone in the blood is regulated primarily by
the renin-angiotensin-aldosterone (RAA) system.
■ To a lesser extent, the pituitary ACTH could also trigger the release of aldosterone from
the zona glomerulosa. The rest of the mineralocorticods have a minor role in electrolyte and
water balance. They are usually labeled as weak mineralocorticoids.
■ 11-deoxycorticosterone (DOC) and 11-deoxycortisol which are metabolites in the synthesis
of aldosterone.
Regulation of Aldosterone Secretion from the
Adrenal Cortex
F-Zone
■ Zona fasciclata cells synthesize glucorticoids, such as cortisol and corticosterone critical to
blood glucose homeostasis and blood pressure.
■ Fasciculata cells are cords of clear cells, with a high cytoplasmic-to-nuclear ratio and lipids
laden with “foamy” cytoplasm
■ The fasciculata also generate androgen precursors, DHEA, which is sulfated in the innermost
zona reticularis.
Glucorticoids

■ These hormones are produced by the zona fasciculata. Like

mineralocorticoids, glucorticoids are made up of 21 carbon atoms.
Specifically, glucorticoids are 21-carbon-steroids with a hydroxyl group in
carbon-17. As such they are most often referred to as 17-hydroxy-
corticosteroids (17OHCS).
■ They are involved in glucose metabolism by increasing gluconeogenic activity
and protein breakdown. These group of steroids are also
immunosuppressive and anti-inflammatory.
■ The major glucorticoid in humans is cortisol. The release of cortisol by the zona-
fasciculata exerts a negative feedback to ACTH production from the anterior
pituitary. That is the release of cortisol is largely controlled by the hypothalamo-
pituitary-adrenal cortex axis.

5-10%
■ Aside from ACTH control levels of cortisol in the blood is also controlled by diurnal rhythm.
■ The diurnal pattern is due to circardian patterns of ACTH release.
■ Cortisol levels are usually ↑ between 4:00-8:00 AM. They are ↓ the rest of the day. In normal
sleep-wake schedule.
■ ACTH is lowest shortly after midnight. Stress is also an important factor for the release of cortisol.
Among the stressful stimuli for cortisol release are surgical trauma, pyrogens, hypoglycemia, and
hemorrhage
Regulation of Cortisol Secretion from the Adrenal
Cortex
Cortisone

■ Is a less important glucorticoid. Both cortisol and cortisone may be inactivated by conversion to
tetrahydro derivatives via reduction reactions. Examples of tetrahydro derivatives are
tetrahydrocortisol, tetrahydrocortisone, allotetrahydrocortisol, etc.
■ The excretion of cortisol is via urine after conjugation with glucoronides and sulfates in the liver
facilitating their excretion (i.e.. making them water-soluble). Cortisol secretion is controlled
mainly by the hypothalamic-pituitary-adrenal system (i.e, Cortisol-Releasing Hormone from
hypothalamus and ACTH from pituitary).
Sex Hormones

■ These hormones are produced by the zona reticularis of the adrenal cortex. Sex hormones
produced by the adrenal cortex are also responsible for the development of secondary sexual
characteristics of humans.
■ These include androgens (19-C steroids with saturated A rings) and estrogens (18-C steroids
with unsaturated A rings).
■ The major androgen produced by the adrenal cortex is the dehydroepiandrosterone (DHEA)
SUMMARY:
Hormone Target tissue Response
Mineralocorticoids Kidneys; to lesser degree, Increase rate or sodium
(aldosterone) intestine and sweat transport into body,
glands increase rate of
potassium excretion;
secondarily favor water
retention

Glucorticoids (cortisol) Most tissues (e.g., liver, Increase fat and protein
fat, skeletal muscle, breakdown; increase
immune tissues) blood nutrient levels;
inhibit inflammation and
immune response

Adrenal androgens Most tissues Insignificant in males;

increase female sexual
drive; growth of pubic
and axillary hair
 Laboratory Measurement of
Adrenal Cortical Steroid
Hormones
-Keala Karess Kipte
 Adrenal cortex function is usually assessed by measuring the levels
of the steroid hormones produced by the gland as well as the levels
of their metabolites.
 Either plasma or urine sample may be utilized.
Parameters Commonly measured:

 Urinary Corticosteroids
Plasma ACTH
Plasma Cortisol
Urinary Free Cortisol
Total 17-Ketosteroids
■ Urinary Corticosteroids
 Commonly referred to as 17-Hydroxycorticosteroids (17-OHCS)

 Derived from cortisol or part of the pathway for glucocorticoids.

 Measurement of urinary corticosteroids may also include

derivatives of aldosterone especially if aldosterone levels are very
high.
Method for Measuring Urinary Corticosteroids

Porter-Silber Reaction
- 17-OHCS are allowed to react with phenylhydrazine (DNPH)-sulfuric
acid reagent to form a yellow derivative measured at 410 nm.

- Levels are increased in hyperadrenalism and decreased in

hypoadrenalism.
 Plasma ACTH

 Measurement of adrenocorticotrophic hormone (ACTH) in the

blood is used to differentiate between primary and secondary
adrenal insufficiency
 Difference between Primary and Secondary Adrenal
Insufficiency
Primary hypocortisolism Secondary hypocortisolism

Plasma Cortisol ↓ ↓

Plasma ACTH ↑ ↓
Pathways in Synthesis of Steroid Hormones
Important Precautions to be Observed in Measuring Plasma ACTH

1.Don’t use glass when collecting blood

2.Collect between 8:00 – 10:00 AM
3.Store samples at -20ᵒC when there is delay.
4.Normally, plasma ACTH does not go beyond 50 ng/L. In stressful conditions,
however, levels as high as 500 ng/L may be observed.
Other Applications of the Measurement of ACTH levels in the Blood

1.Establishing a diagnosis of Cushing’s syndrome characterized by a

decrease ACTH level
2.Establishing a diagnosis of adrenal adenomas or carcinomas where
ACTH is also decreased.
3.Determining the adequacy of cortisol replacement in congenital
adrenal hyperplasia
 Plasma Cortisol
- Used to evaluate adrenal stimulation or suppression tests.
- The methods used in measuring plasma cortisol include:
- Immunoassay
- Competitive protein binding assay
- Fluorometry
- HPLC
 Urinary Free Cortisol
- Used as adjunct in the diagnosis of adrenal cortical disease

- A 24 hour urine specimen is the best specimen for this purpose

- HPLC is preferred method

 Total 17-Ketosteroids
-Also referred as 17- ketogenic steroids
- Usually include adrosterone, dehydroepiandrosterone (DHEA) and other
androgens except testosterone and dihydroxytestosterone
- Detection of 17-ketosteroids is based on the Zimmerman Reaction.
-This is a reaction between 17-ketosteroids and the reagent m-
dinitrobenzene in alcoholic KOH to form a reddish purple dintrobenzene measured at 520nm.
Testosterone and dihydroxy testosterone are not detected by the reaction.
CUSHING SYNDROME
■Hillary Credo
CUSHING SYNDROME

■ Increase in plasma

■ Increase in urine cortisol and its metabolites

Most Consistent Findings in this condition are:

■ Excessive and persistent production of cortisol

measured as serum cortisol (increased), urinary free cortisol (increased), and

urinary 17-OHCS (increased)

8/17/2018
Most Consistent Findings in this condition are:

■ Loss of the usual circadian rythm of ACTH and cortisol

■ Loss of suppression of cortisol production by administration of the synthetic

glucocorticoid dexamethasone (an inhibitor of ACTH)

8/17/2018
THE TWO CATEGORIES OF CUSHING SYNDROME

■ Cushing Disease

- This is characterizd by increased cortisol production due to excessive development

and activity of the pituitary gland.

■ Cushing Syndrome
- There is increased cortisol production due to tumors hich produce either excessive
ACTH or cortisol.

8/17/2018
ADDISON’S DISEASE
(Adrenal Insufficiency/ Hypoadrenalism)
Gaye Rhianna Deluso
Addison’s dse or Adrenal Insufficiency

■Occurs from an autoimmune process

■Patients have deficiencies in both
glucocorticoids and mineralocorticoids
■↓ Aldosterone
■↓ plasma cortisol
■↓ urine free cortisol
■↓ 17-OHCS (17-hydroxycorticosteroids)
Causes of HYPOADRENALISM
■ Defect in adrenal gland
■ Defect in hypothalamus or pituitary
If the defect is in the……
■ADRENAL CORTEX – no change in plasma or
urine cortisol before and after challenge

■HYPOTHALAMUS / PITUITARY GLAND -

↑plasma and urine cortisol
HORMONES OF THE
GONADS
Amanda Mae A. Delfin
MLS4G
■Responsible for regulating the reproductive processes

■Menstrual cycle: Ovaries & anterior pituitary

■Gonadotropins: Luteinizing hormone (LH) & Follicle-stimulating hormone (FSH)

47
PHASES OF THE MENSTRUAL CYCLE: (1/3)

1. Follicular or Proliferative phase

■ Day 1 to 13
■ FSH: Stimulates the ovarian follicle to develop
■ Estrogen: stimulates the ovarian follicle to proliferate

48
PHASES OF THE MENSTRUAL CYCLE: (2/3)

2. Ovulatory phase:
■Day 13: Surge of estrogen in the blood (estradiol) then;
■Creates a positive feedback to the adenohypophysis to release FSH and
LH
■Mid-cycle peak of the gonadotropins
■Help in the release of the ovum from the mature ovarian follicle
■LH: neutralizes the action of a peptide oocyte maturation inhibitor
■FSH: stimulates the production of glycosaminoglycans which mucify the
environment and disperse the cumulus oophorus and induces enzymes that
catalyze the final breakdown of the follicular wall

49
PHASES OF THE MENSTRUAL CYCLE:
(3/3)
3. Luteal or Secretory phase:

50
51
Laboratory measurement
of gonadal steroids
Progesterone
PROGESTERONE

■ Ovary
■ Adrenal cortex: precursor for the formation of other adrenal cortical steroids
■ Placenta: takes over the role of the corpus luteum in the synthesis of progesterone

53
■ Decreased in:
• early stages of the menstrual cycle,
• later stages of the menstrual cycle &;
• towards the end of the 28-day period

■ Increased in:
• mid-cycle &;
• early part of the luteal ohasee
Once released in the circulation:

■ Cortisol-binding globulin (CBG): minor amount

■ Free hormone: major amount
FUNCTIONS:

■ Promote growth of the endometrium for the implantation of the fertilized ovum
■ Exert a negative feedback control on the synthesis and release of LH and FSH
THREE IMPORTANT GROUPS:

1. Pegnanediones
2. Pregnanolone
3. Pregnandediols: major metabolite
DETERMINATION OF PROGESTERONE
LEVELS
■ Anovulary cycles
■ Absence of clinical rise in pregnanediols excretion in the urine
■ Immunoassay
 ESTROGENS
Gaye Rhianna Deluso
MAJOR ESTROGEN IN THE HUMAN FEMALE:
During menstrual cycle, two peaks of
estradiol could be seen namely:
■During midcycle peak of LH (about day 13)
■During progesterone peak (about day 21)
FUNCTIONS OF ESTROGEN
■ Assist in regulating FSH and LH release by the
anterior pituitary
■ Control the development of the endometrium in
conjunction with progesterone
■ Trigger the process of menstruation through a drop
in its level at the end of the cycle
KOBER REACTION
■ Involves heating the urine in a strong aqueous
sulphuric acid containing hydroquinone
■ Form reddish brown color
ANDROGENS
■ Synthesized by testis and small amounts are
contributed by the adrenal cortex.
■ These includes testosterone, androstenedione, and
dehydroepiandrosterone (DHEA)
■ Major androgen produced by the testis is
testosterone
TESTOSTERONE
■Highest at about 7:00 am
■Lowest at about 8:00 pm
■Formed from cholesterol
■Enhanced by binding of LH testicular
Leydig’s cell receptor
FUNCTIONS OF TESTOSTERONE
■ Facilitate the development of secondary male
sexual chracteristics
■ Enhance protein synthesis leading to growth in
both skeletal muscle and bone (especially during
puberty)
■ Involved in spermatogenesis
DIHYDROTESTOSTERONE
■Most of it comes from androstenedione
■Major androgen in skin, prostate, seminal
vesicle and epidydimis
■ END