Exam #3 - Adv Clinical Chemistry

Ch. 14, 16-17, 24-25

2 pituitary hormones that are necessary for life & the rest “just for fun” (6)

• TSH, ACTH

• “fun”: hGH, LH, FSH, PRL, ADH, oxytocin

Hypothalamic releasing factors (5)

• Corticotropin Releasing Factor (CRF)

• Thyrotropin-releasing hormone (TRH)

• Growth hormone-releasing hormone (GHRH)

• Somatostatin

• Gonadotrophin-releasing hormone (GnRH)

Anterior pituitary hormones (6)

• TSH = Thyroid Stimulating Hormone

• ACTH = Adrenocorticotropic Hormone

• hGH = human Growth Hormone

• LH = Luteinizing hormone

• FSH = Follicle Stimulating Hormone

• PRL = Prolactin

Posterior pituitary hormones (2) & Pituitary trophic hormones (function)

• oxytocin & antidiuretic hormone (Arginine Vasopressin)

• “feed” and increase size of target cell in end-organs to maintain their normal size, mass, and function

Negative feedback loop (analogy)

• works like thermistor in house thermometer: control output of a furnace

  • analogy: hormone level is like temp; warm = high circulating, cold = low circulating

  • pituitary secreting hormone is like furnace generating heat; furnace turns on when cold & off when warm

• pituitary turns on (secrete stimulating hormone) when temp is cold

• pituitary turns off secretion when temp is warm

• temp in well controlled room fluctuates with time as a sine curve

General rules of endocrinology practice (6)

• A gland is not secreting hormone when it should → kick start it

• A gland is secreting too much hormone → suppress it

• Stimulation & Suppression Tests are used in the setting of thyroid & adrenal endocrinopathies

• Primary disease: problem is confined within gland

• Secondary disease: problem is in pituitary function

• Tertiary disease: problem in hypothalamic function

Hypothalamus & Pituitary: Circulation (input, anterior & posterior)

• master integrator of input from higher CNS

• input: pain, stress, cold, light/dark cycles

• hypothalamic-hypophyseal portal circulation collects blood from capillaries of hypothalamus → plexus of veins surrounding pituitary stalk → directs blood into anterior pituitary gland

• hypothalamic hormones are carried down from neurons of Supraoptic & PV Nuclei → directly into posterior pituitary

Hypothalamic releasing factor: Releasing location, measure, CRF, TRH

• not released directly into peripheral circulation; released into a portal circulation that supplies anterior pituitary

• released directly by neurons into posterior pituitary (PP)

• Difficultly measured as blood draining hypothalamus must be sampled in Inferior Petrosal Sinus (invasive)

• CRF: stimulates the release of ACTH by corticotrophs in anterior pituitary (AP)

• TRH: stimulates release of TSH by thyrotrophs in AP

Disorders of pituitary (2)

• Trauma/injury/radiation usually causes hypopituitarism: panhypopituitarism involves loss of stimulatory hormones from the AP & PP

• Adenomas:

  • Non-functioning: do not secrete hormones

  • Functioning: usually secrete GH & PRL, rarely TSH, ACTH, FSH, LH from the anterior pituitary

Thyroid function: Specialization, feedback loop players, follicular cells, protein bound

• specialized for endocrine hormone production

• feedback loop: pituitary thyroid stimulating hormone (TSH/thryotropin) & triiodothyronine (T3)

• follicular cells (FC) express receptor for TSH → promotes thyrocytes’ growth & biosynthetic functions

• thyroid hormones extensively protein bound (99.9%): thyroid binding globulin (TBC) & Albumin; free thyroid hormone is important to feedback loops & disease

Thyroid function (FC, Clcitonin, thyroglobulin, iodinases, C cells)

• FC produce thyroxine (T4) & smaller amounts of triiodothyronine (T3)

  • calcitonin: tumor marker of medullary thyroid cancer

  • thyroglobulin: post-treatment tumor marker of residual thyroid cancer

• extrathyroidal T4 converted to T3 by peripheral iodinases

• T3 binds to nuclear receptor regulating + / - genes

• thyroid contains parafollicular/C cells: produce calcitonin (inhibits bone resorption)

Life-sustaining action of thyroid hormones (development, regulation, modulation, FC)

• fetal & childhood growth & CNS development

• regulate HR, myocardial contraction & relaxation, GI motility, renal clearance, weight & lipid metabolism

• modulation of BMR, energy expenditure, O2 consumption, heat generation, metabolism

• FC: synthesize hormonal precursor protein thyroglobulin (TG) & concentrate iodide intracellular from circulation

Dietary iodine intake (adults, pregnant women, deficiency, <50)

• 150 μg for adults, 200 μg for pregnant and lactating women, & 90 μg for children

  • the kidneys excrete most iodide

  • urinary iodide excretion: index of dietary intake

• Dietary iodine deficiency: daily iodine intake < 100 μg/d

• iodide intake < 50 μg/d: normal-sized thyroid cannot sustain adequate hormone production → gland enlargement (goiter) & hypothyroidism

Thyroid hormone synthesis (symporter, use of iodine, uptake, requirements, other tissues)

• Thyrocytes express Na-iodide symporter (spans the cells’ basal membranes and actively transports iodide from the blood)

• thyroid gland uses only a fraction of the iodide supplied to it for hormone synthesis, the remainder returns to the extracellular fluid pool.

• normal fractional uptake of iodide is approximately 10-30% after 24 hours

• Thyroid hormone synthesis requires: NIS, TG, & enzyme thyroid peroxidase (TPO) all be present, functional, and uninhibited

• Salivary, gastric, & breast tissues: express NIS & concentrate iodide less than thyroid, but these tissues do not organify or store iodide

Thyroid hormone synthesis with iodide steps (6)

1. Iodide trapping: Active transport of iodide across the basement membrane into the thyroid cell with Na+

2. Oxidation of iodide (TPO) & iodination of tyrosyl residues in TG

3. Coupling: Linking pairs of iodotyrosine molecules within TG → form iodothyronines T3 and T4

4. Pinocytosis & then proteolysis of TG with release of free iodothyronines & iodotyrosines into the circulation

5. Deiodination of iodotyrosines within the thyroid cell (with conservation & reuse of the liberated iodide)

6. Intrathyroidal 5′-deiodination of T4 to T3

TPO: Function, TSH, drug inhibitors)

• membrane-bound glycoprotein (MW 102 kD) containing a heme moiety: catalyzes iodide oxidation & covalent linkage of iodine to the tyrosine residues of TG

• TPO gene expression is stimulated by TSH

• The thiocarbamide drugs (methimazole, carbimazole, and propylthiouracil (PTU)) are competitive inhibitors of TPO

  • Resulting ability to block thyroid hormone synthesis useful in treatment of hyperthyroidism

Thyroid hormones: T3 & T4 Transport (free vs bound, factors affecting TBG levels)

• T3 & T4 are extensively protein bound by albumin or TGB

• 0.01% free and biologically active with thyroid hormone nuclear receptors & feedback loops

• pregnancy, estrogen-secreting tumors, & estrogen therapy all increase sailic acid content of TBG molecule → decreased metabolic clearance & elevated serum TBG levels

Factors that decrease TBG levels

• nephrotic syndrome & protein-losing enteropathy

• liver failure

• major systemic illness due to cleavage by leukocyte proteases & reduction in TBG’s binding affinity for thyroid hormones

  • both lower serum total thyroid hormone

Thyroid hormones: TRH Feedback loop (location, gene expression, AP, related to TSH)

• TRH found: other portions of hypothalamus, brain, spinal cord (functions as neurotransmitter)

• TRH gene expression is (-) regulated by thyroid hormone (T3 delivered by circulation & arising from T4 deiodination in peptrigergic neurons)

• Anterior pituitary: TRH binds to specific membrane receptor located on thyrotropes → stimulates synthesis & release of TSH

• TRH-stimulated TSH secretion is pulsatile

Thyroid hormones: TSH (structure)

• 28-kD glycoprotein composed of alpha & beta subunits that are noncovalently linked

• alpha subunit: common to 2 pituitary glycoproteins, FSH, LH, & placental hCG

• beta subunit: unique for each glycoprotein hormone, conferring specific binding properties & bio activity

TSH: Controls, stimulates, intact, detection

• TSH controls thyroid cell growth & hormone production by binding to specific TSH receptor

• TSH stimulates: changes in thyroid cell morph, growth, iodine metabolism, O2 uptake glucose oxidation in thyrocytes, & synthesis and secrete thyroid hormones

• intact TSH & the isolated alpha subunit both present in circulating blood

• TSH detectable by immunoassay in concentrations: 0.5-4 mU/L & 0.5-2 microgram/L

TSH levels: Primary hypothyroidism, thyrotoxicosis, half-life, high/low T3/T4

• serum TSH level is increased in primary hypothyroidism & decreased in thyrotoxicosis (endogenous or excessive oral intake of thyroid hormones)

• plasma half-life of TSH: 30 minutes

• TSH synthesis & release inhibited by high serum levels of T3 & T4

• TSH synthesis & release stimulated by low levels of thyroid hormone

TSH: 2 factors & Thyrotoxicosis on synthesis & release

• 2 major factors that control synthesis & release of TSH:

  • T3 level within tyrotroph cells (regulate mRNA expression, TSH translation, hormone’s release)

  • TRH (control posttransitional glycosylation & release)

• thyrotoxicosis (hyperthyroidism) can suppress serum TSH levels beneath limits of assay detection & recovery of normal TSH secretion

  • may require wks or months after restoration of normal thyroid hormone levels

Thyroid hormones: Genomics (2 mechanisms, TRs to TREs, TRs other function)

• thyroid hormones exert actions through 2 mechanisms:

  • genomic actions effected through T3 interactions with nuclear receptors, regulate gene activity

  • nongenomic actions mediated by T3 & T4 interactions with enzymes, glucose transporters, nitrochontrial, & membrane proteins

• TRs bind to TREs (typically paired), specific oligonucleotide sequences

• TRs also function as heterodimers with receptors for other transcription factors (such as retinoid X & retinoic acid receptors)

Thyroid hormones: Genomics (TREs location, + regulated genes, TRE bound)

• TREs generally located upstream of transcription start site for coding regions of thyroid hormone (responsive genes)

• in (+) regulated genes: unbound TRs interact with corepressors & silence mediator for retinoic & thyroid hormone receptors (SMRT) → repress basal transcription by recruiting histone deacetylases that alter nearby chromatin structure

• when TRs bound by T3: corepressor complexes released & T3-bound TRs associate with coactivator complexes (promote local histone acetylation)

Thyroid hormones: Physiology (transcriptional effects, 12)

• transcriptional effects of T3 have a lag time of hours or days to achieve full effect

• fetal development: brain development & skeletal maturation

• O2 consumption & free radical formation

• heat production: increased basal metabolic rate, Na/K ATPase activity (heat/cold intolerance in thyroid disease)

• Cardiovascular: increase in cardiac output, contractility, sensitivity to epinephrine

• Sympathetic nervous system: increased adrenergic sensitivity

• Pulmonary: hypoventilation in hypothyroidism; weakened respiratory muscles in hyperthyroidism

• Hematopoietic: potentiates activity of EPO; increases 2,3-BPG

• Gastrointestinal: gut motility

• Skeletal: bone turnover

• Neuromuscular Effects

• Lipid and Carbohydrate Metabolism: cholesterol synthesis and degradation

• Endocrine: growth, development, puberty

Thyroid: Testing guide (screening, 1 change, free levels)

• screen for thyroid disease begins with TSH & total/free T4 levels

• 1 fold change in thyroid hormone → several fold changes in TSH

• free hormone levels are important especially in pregnancy (high TBG) & liver disease (low TBG)

RR: Serum T4, free T4, serum T3, free T3, serum thyrotropin, thyroxine-binding globulin

• serum T4: 4.6-12 ug/dL

• free thyroxine (FT4): 0.7-1.9 ng/dL

• serum T3: 80-180 ng/dL

• free T3 (FT3): 230-619 pg/dL

• serum TSH: 0.5-6 uU/mL

• TBG: 12-20 ug/dL T4 + 1.8 ugm

Hypothyroidism: Classifications & most common cause

• Primary (most common), Secondary (pituitary TSH deficiency), Tertiary (hypothalamic TRH deficiency), Peripheral thyroid hormone resistance

• Most common cause: autoimmunity

  • chronic lymphocytic thyroiditis (Hashimoto’s)

  • Autobody panels: serum anti-TPO, blocking TSI (Grave’s/Perry) or less sensitive anti-TG

Hyperthyroidism/Thryotoxicosis (thyrotoxic crisis, ademona, goiter, treatment)

• thyrotoxic crisis (thyroid storm): acute exacerbation of all the symptoms & signs of thyrotoxicosis, often as a syndrome that may be life-threatening

• toxic adenoma: functioning adenoma hypersecreting T3 & T4

• Toxic multinodular Goiter (Plummer disease)

• treatment: methimazole (TPO inhibitor) surgery, radioactive iodine, propanolol & other beta blockers

Grave’s disease (what, disorder, ophthalmopathy, treatment)

• Primary hyperthyroidism: most common form of thyrotoxicosis (females 5x more likely)

• autoimmune disorder associated with TSIs: thyroid stim immunoglobulins (autoantibodies) that activate TSH receptor → oversecretion of T4 & T3

• thyroid -associated Ophthalmopathy: immune system (targeting the TSH receptor) attacks tissues around the eyes → releases cytokines that cause orbital tissues to swell & expand—through increased fat and muscle size—which pushes the eyeballs forward

• treatment: methizamole (TPO inhibitor) surgery, radioactive iodine, propanolol & other beta blockers

Thyroid testing guide: Classical presentation (hypothyroidism, hyperthyroidism, goiter, autoantibodies)

• hypothyroidism: fatigue, cold intolerance, weight gain, constipation, dry skin, myalgia, menstrual irregularities, bradycardia, hypertension, delayed relaxation phase of deep tendon reflexes (myxedema coma)

• Hyperthyroidism: anxiety, emotional lability, weakness, tremor, palpitations, heat intolerance, increased perspiration, & weight loss despite a normal or increased appetite (thyroid storm/thyrotoxicosis)

• Goiter: caused by prolonged elevated TSH levels

• Most thyroid disease is caused by autoantibodies, causing either immune thyroiditis (Hashimoto’s) or Graves’ disease (thyroid stimulating immunoglobulins)

Thyroid testing guide: Classical presentation (hypothyroidism, hyperthyroidism, subclinical hypothyroidism)

• hypothyroidism: elevated TSH, low free T4; most patients with Hashimoto’s show elevated TPO (thyroid peroxidase) autoantibodies

• hyperthyroidism: patients with primary hyperthyroidism have low TSH; most with overtly hyperthyroidism (<0.5 mU/L)

  • TSH levels suppressed by intact feedback loop in elevated thyroid hormone levels

  • diagnosis confirm by radioiodine uptake

• Subclinical hypothyroidism: elevated TSH, normal T4; pituitary working overtime to maintain thyroid hormone levels

Thyroid cancer (routine tests, biopsy, prevalence, thyroglobulin, thyrogen stim, calcitonin)

• routine lab tests not reliable alone → radioiodine uptake with ultrasonography & other imaging techniques

• biopsy: histology (fine needle aspiration)

• cancer has higher than avg prevalence in Los Almost & Rio Arriba counties

• thyroglobulin: tumor marker for follow up treatment for recurring thyroid cancer

• thyrogen stim test: instead of withdrawal, synthetic TSH can be given to stim thyroid & unmask residual cancer

• Calcitonin: tumor marker for medullary TCa

Glucocorticoids & adrenal androgens (adrenal cortex, immunomodulatory, feedback loop)

• adrenal cortex produces cortisol, aldosterone, & adrenal androgens

• suppressing inflammation and immunity in the short term while potentially contributing to immune dysregulation with chronic stress

• feedback loop involves ACTH (pituitary) & cortisol

  • Hypothalamus → CRH → Pituitary.

  • Pituitary → ACTH → Adrenal Glands.

  • Adrenal Glands → Cortisol is released into the blood

  • High Cortisol feeds back to SHUT OFF the Pituitary (ACTH) & Brain (CRH).

  • When cortisol drops, the loop starts again

Glucocorticoids & adrenal androgens: ACTH, protein-bound, salivary

• ACTH secreted as part of preopiomelanocortin (POMC) precursor with melanocyte stimulating hormones

• Cortisol is extensively protein bound (CBG)

• Salivary cortisol is best estimate of free cortisol level

Glucocorticoids & Adrenal androgens: Cortisol overproduction, Primary Adrenal insufficiency, Congenital adrenal hyperplasia

• Cortisol overproduction: Cushing’s syndrome (from any cause including stress)/Cushing’s Disease (from pituitary ACTH)

• Primary adrenal insufficiency: Addison’s disease

• Congenital adrenal hyperplasia: 21-hydroxylase deficiency

Biosynthesis of Adrenal androgens & Cortisol: Where, conversion, sTAR, products

• Steroid biosynthesis in the zona fasciculata and zona reticularis of the adrenal cortex

• conversion of cholesterol to pregnenolone is the rate-limiting step in adrenal steroidogenesis & major site of ACTH action in the adrenal

• steroidogenic acute regulatory protein (StAR; mitochondrial phosphoprotein) enhances cholesterol transport from the outer to the inner mitochondrial membrane

• The major secretory products are DHEA, sulfate, androstenedione, corticosterone, and cortisol

Biosynthesis of Adrenal androgens & cortisol: Zona glomerulosa vs fasciculata & reticularis

• zona glomerulosa produces aldosterone & constitutes about 15% of adult cortical volume,

• deficient in 17α-hydroxylase activity → cannot produce cortisol or androgens

• zonae fasciculata and reticularis are regulated by ACTH; excess or deficiency of this hormone alters their structure & function

  • both zones atrophy when ACTH is deficient

  • ACTH is in excess, hyperplasia & hypertrophy of these zones occur

Biosynthesis of Adrenal androgens & cortisol: Pregnenolone, 17α, Lyase, Sulfate, Androstenedione

1. Cholesterol into mitochondria → P450scc/CYP11A1 cleaves 6-C side chain → pregnenolone

2. 17α-Hydroxylase activity: Converts Pregnenolone to 17-OH-Pregnenolone.

3. 17,20-Lyase activity: Cleaves the side-chain of 17-OH-Pregnenolone to produce Dehydroepiandrosterone (DHEA). DHEA is a weak androgen and a major precursor.

4. Sulfation: DHEA converted to DHEA-Sulfate (DHEA-S) by the enzyme SULT2A1. DHEA-S is the most abundant steroid in the blood, a stable reservoir.

5. Androstenedione: DHEA is converted to Androstenedione by the enzyme 3β-HSD (in the adrenal). Androstenedione is a direct precursor to testosterone and estrone

Biosynthesis of Cortisol (4)

1. Pregnenolone → (via CYP17A1) → 17-OH-Pregnenolone.

2. 17-OH-Pregnenolone → (via 3β-HSD) → 17-OH-Progesterone.

3. 21-Hydroxylation: 17-OH-Progesterone → (via CYP21A2) → 11-Deoxycortisol.

4. 11β-Hydroxylation (Final Step): 11-Deoxycortisol → (via CYP11B1) → Cortisol.

Circadian rhythm related to ACTH & Cortisol secretion

• peak in early morning, nadir b/w 12a-4a & PM cortisol interpreted in context of time of day

• rhythm changed by: physical stresses (major illness, surgery, trauma, or starvation), psychologic stress (severe anxiety, endogenous depression, the manic phase of manic-depressive psychosis), CNS & pituitary disorders (Cushing syndrome), liver disease, and other conditions that affect cortisol metabolism (chronic renal failure & alcoholism)

RR: Cortisol & ACTH (8a, 4p, & during sleep)

• C at 8a: 10-20 μg/mL

• C at 4p: 3-10 μg/mL

• C during sleep: <5 μg/mL

• ACTH at 8a: 10-50 pg/mL

• ACTH at 4p: < 20 pg/mL

• ACTH during sleep: 5-10 pg/mL

Adrenocorticotropic hormone (ACTH): regulated, administration, chronic stimulation & deficiency

• regulated by the hypothalamus & CNS via neurotransmitters, corticotropin-releasing hormone (CRH) & arginine vasopressin (ADH)

• administration → rapid synthesis & secretion of steroids (plasma levels of these hormones rise within minutes)

• Chronic ACTH stimulation → adrenocortical hyperplasia & hypertrophy;

• ACTH deficiency → decreased steroidogenesis & accompanied by adrenocortical atrophy, decreased gland weight, and decreased protein and nucleic acid content

Adrenocorticotropic hormone (ACTH): Stresses, cortisol secretion, 3 mechanisms

• Plasma ACTH & cortisol are secreted within minutes following the onset of stresses → abolish circadian periodicity if the stress is prolonged

• Cortisol secretion is closely regulated by ACTH, & plasma cortisol levels parallel those of ACTH

• 3 mechanisms of neuroendocrine control:

  • Episodic secretion & the circadian rhythm of ACTH

  • Stress responsiveness of the hypothalamic-pituitary-adrenal (HPA) axis

  • Feedback inhibition by cortisol of ACTH secretion

Adrenocorticotropic hormone (ACTH): IL-1 & Cortisol

• Interleukin-1 (IL-1) stimulates ACTH secretion

• Cortisol inhibits IL-1 synthesis

Cortisol transport: Percentages, free cortisol, CBG, antibody, salivary cortisol

• 10% of the circulating cortisol is free, 75% is bound to CBG, and the remainder is bound to albumin

• The plasma free cortisol level: 1 μg/dL; this bio active cortisol is regulated by ACTH

• Corticosteroid-binding globulin (CBG) is produced by the liver, and binds cortisol with high affinity

• CBG in plasma cortisol-binding capacity: 25 μg/dL.

  • [Total plasma cortisol] rise above this level → free concentration rapidly increases & exceeds usual fraction of 10% of the total cortisol

• There isn’t a “good” antibody to measure free cortisol levels

• Salivary cortisol: an estimate of the free cortisol level

Cortisol transport: Factors affecting CBG levels

• CBG levels are increased in high-estrogen states (pregnancy; estrogen or oral contraceptive use), hyperthyroidism, diabetes, certain hematologic disorders, and on a genetic basis.

• CBG concentrations are decreased in familial CBG deficiency, hypothyroidism, and protein deficiency states such as severe liver disease or nephrotic syndrome.

Cortisol activity: Glucocorticoid receptor & Liver (3)

• GR: glucocorticoid (nuclear steroid) receptor, modulates gene expression in target cells

• Liver:

  • Increased expression of gluconeogenic enzymes, phosphoenolpyruvate carboxykinase, glucose-6-phosphatase, & fructose-2,6-bisphosphatase

  • Maintains plasma glucose during fasting (anti-hypoglycemic action)

  • Increases plasma glucose during stress (hyperglycemic action)

Cortisol activity: Adipose Cells, Skeletal muscle, Pancreas, Skin

• adipose: permissive for lipolytic signals (GH) → elevated plasma FFA to fuel gluconeogenesis

• skeletal muscle: degrade fibrillar muscle → amino acid substrates for gluconeogenesis

• pancreas: inhibit insulin secretion

• skin: Glucocorticoids in excess inhibit fibroblasts → loss of collagen & connective tissue → thinning of the skin, easy bruising, stria formation, & poor wound healing

Cortisol activity: Immune system (3) & Mood (3)

• immune: Inhibits monocyte proliferation and antigen presentation; decreased production of IL-1, IL-6, and TNFα

  • Demargination of neutrophils by suppressing the expression of adhesion molecules

  • Inhibition of inflammation by inhibiting PLA₂, → inhibiting production of leukotrienes & prostaglandins; suppresses COX-2 expression

• Mood: Eucortisolemia maintains emotional balance, Increases appetite, Suppression of REM sleep

Cortisol activity: Thyroid & Bone (4)

• thyroid function: TSH synthesis & release are inhibited by glucocorticoids, & TSH responsiveness to TRH is frequently subnormal

• Bone:

  • Glucocorticoids directly inhibit bone formation → decreasing osteoblast proliferation & the synthesis of RNA, protein, collagen, and hyaluronate

    • → markedly reduce intestinal calcium absorption → lower serum calcium → promotes a state of secondary hyperparathyroidism to maintain the serum calcium within the normal range

  • supraphysiologic doses of glucocorticoids stimulate bone resorption (via activation of the receptor activator of nuclear factor kappa B (RANK)-ligand/RANK signaling that is osteoclastogenic) → osteolysis & increased biochemical markers of bone turnover

  • Glucocorticoid-induced osteoporosis

Adrenal androgens: Precursors & Conversions

• Androstenedione, DHEA, & DHEA sulfate: precursors for peripheral conversion to the active androgenic hormones, testosterone (T) & dihydrotestosterone (DHT)

• DHEA sulfate secreted from the adrenal → DHEA → in peripheral tissues to androstenedione (immediate precursor of the active androgens)

Adrenal androgens: Effects in Males (2) vs Females (4)

• Males:

  • adrenal androstenedione → T (< 5% of the production rate of this hormone) → physiologic effect is negligible

  • adult males: excessive adrenal androgen secretion has no clinical consequences; boys: premature penile enlargement & early development of secondary sexual characteristics (Hercules)

• Females

  • adrenal substantially: total androgen production by the peripheral conversion of androstenedione to T

  • follicular phase of the menstrual cycle: adrenal precursors account for 2/3 of T production and 1/2 of DHT production

  • During midcycle, the ovarian contribution increases, & the adrenal precursors account for only 40% of T production

  • abnormal adrenal function: Cushing syndrome, adrenal carcinoma, and congenital adrenal hyperplasia → excessive secretion of adrenal androgens, & their peripheral conversion to T results in androgen excess

ACTH & Cortisol Expected values: Plasma ACTH (1), Plasma Cortisol (6)

• ACTH: diagnosis of pituitary-adrenal dysfunction: IRMA or ICMA is 9-52 pg/mL

• Cortisol:

  • With radioimmunoassay 8a: 3-20 μg/dL & average 10-12 μg/dL.

  • 4p: half of morning values

  • 10p-2a: < 3 μg/dL

  • Stress: increases in patients who are acutely ill, during surgery, & following trauma (> 40-60 μg/dL)

  • [Total plasma cortisol] elevated w increased CBG-binding capacity (commonly when circulating estrogen levels are high eg, during pregnancy): 2-3x higher than normal

  • [Total plasma cortisol] increased in severe anxiety, endogenous depression, starvation, anorexia nervosa, alcoholism, & chronic kidney disease

Cortisol deficiency: Addison’s, etiology, affects, clinical presentations, test

• Addison’s disease = adrenal insufficiency

• Autoimmune etiology: involved in polyendocrine autoimmune syndromes (with pancreas and thyroid)

• Affects all adrenal steroids including mineralocorticoids (aldosterone) & androgens

• Hypoglycemia, hyperkalemia, hyponatremia. nausea, hyperpigmentation

• Cosyntropin stimulation test useful

  • Cosyntropin is hR ACTH fragment with biological activity toward adrenal receptor

  • Low dose stimulation (1μg subQ injection) → cortisol peak of > 18 μg/dL in 1-2 hrs

Primary Adrenal insufficiency (Addison’s disease)

• Deficient adrenal production of glucocorticoids or mineralocorticoids → adrenocortical insufficiency

  • consequence of destruction or dysfunction of the cortex (primary adrenocortical insufficiency/Addison disease) or secondary to deficient pituitary ACTH secretion (secondary adrenocortical insufficiency)

• Glucocorticoid therapy is the most common cause of secondary adrenocortical insufficiency

Primary Adrenal insufficiency: Cortisol & Mineralocorticoid deficiency, Chronic primary adrenal sufficiency

• Cortisol deficiency causes weakness, fatigue, anorexia, nausea and vomiting, hypotension, hyponatremia, and hypoglycemia.

• Mineralocorticoid deficiency produces renal sodium wasting & potassium retention → severe dehydration, hypotension, hyponatremia, hyperkalemia, & acidosis.

• Chronic primary adrenocortical insufficiency: symptoms are hyperpigmentation, weakness & fatigue, weight loss, anorexia, & GI disturbances

Primary adrenal insufficiency: Acute adrenal crisis, Secondary adrenocortical insufficiency

• Acute adrenal crisis: state of acute adrenocortical insufficiency & occurs in patients with Addison's disease who are exposed to the stress of infection, trauma, surgery, or dehydration due to salt deprivation, vomiting, or diarrhea

• Secondary adrenocortical insufficiency: ACTH deficiency result of exogenous glucocorticoid therapy

  • Pituitary or hypothalamic tumors, and their treatment, are the most common causes of naturally occurring pituitary ACTH hyposecretion

Congenital Adrenal Hyperplasia (CAH): Deficiency, virilization, severe forms, detection

• P450c21, 21α-hydroxylase deficiency accounts for 95% of CAH

• Virilization of fetus due to androgen excess

  • Exacerbated by high ACTH (loss of negative feedback)

  • Clitoromegaly

  • Females may require surgery to correct fused labia; Males: “Hercules”

• Severe forms are salt-wasting (why?)

• Detected by neonatal testing of (elevated) 17-beta-hydroxyprogesterone levels

Rare forms of CAH (4)

• 11-Beta hydroxylase deficiency: no symptoms associated with adrenal crisis, but are subject to others (HTN) due to salt retention and ambiguous genitalia in females.

• 17a-hydroxylase deficiency: ambiguous external genitalia in males & lack of pubertal development or menstrual cycles (amenorrhea) in females.

• 3-Beta-hydroxysteroid dehydrogenase deficiency: ambiguous genitalia in males and females. In both genders it can lead to salt-wasting.

• StAR (Steroidogenic Acute Regulatory Protein) Congenital lipoid adrenal hyperplasia: may cause early death due to adrenal crisis

  • Males have ambiguous genitalia. Both males and females, if they survive, would likely be infertile.

Cortisol excess: Cushing syndrome vs disease, ectopic ATCH, signs, detection

• Cushing’s syndrome = cortisol excess from any cause

• Cushing’s disease = cortisol excess from pituitary adenoma

• Ectopic ACTH syndrome: paraneoplastic syndrome

• Classical signs: striae, supraclavicular & other fat deposits (buffalo hump, moon-face)

  • Delayed wound healing & impaired immune response

• Plasma ACTH, salivary and 24 h urinary cortisol measurements are useful to detect excessive secretion

Dexamethasone suppression test

• test may be informative for Cushing’s syndrome

  • Dexamethasone has potent negative feedback on ACTH release

  • Low dose: 8a cortisol value in normal is < 2 μg/dL, AM cortisol > 14 = suspected Cushing’s syndrome

  • High dose: 8 mg, most people with Cushing’s disease suppress, those with ectopic ACTH syndrome do not

Cushing’s syndrome & disease: Excess, therapy, spontaneous, classification

• Chronic glucocorticoid excess → constellation of symptoms & physical features known as Cushing syndrome

• It is most commonly iatrogenic, resulting from chronic glucocorticoid therapy

• Spontaneous Cushing syndrome: abnormalities of the pituitary or adrenal gland or a consequence of ACTH or CRH secretion by nonpituitary tumors (ectopic ACTH syndrome; ectopic CRH syndrome)

• Cushing disease is defined as the specific type of Cushing syndrome due to excessive pituitary ACTH secretion from a pituitary tumor

• Classified as ACTH-dependent/ACTH-independent

Expected diurnal variation in Cortisol (1), ACTH (3), Testosterone (1)

• Cortisol: highest level in early morning → steep decline → gradual decline through evening

  • Cushing’s syndrome: high levels in the evening (late-night salivary cortisol)

• ACTH: just like cortisol but with sharper spikes

  • Addison's: High ACTH due to lack of cortisol feedback

  • Secondary Adrenal Insufficiency (Pituitary failure): Low or "inappropriately normal" ACTH.

  • Cushing's Differential: An elevated or high-normal ACTH at 8a suggests ACTH-dependent Cushing's (pituitary or ectopic tumor); suppressed ACTH suggests ACTH-independent (adrenal tumor)

• testosterone: just like cortisol but ~20-35% more in morning

  • diurnal variation diminishes with age: flatter curve

Adrenal medullary hormones: Catecholamines, norepinephrine, pheochromocytoma

• Catecholamines: Neuroendocrine hormones (Epinephrine & Norepinephrine)

• Metabolites are Norepinephrine and Normetanephrine (Metanephrines)

• Pheochromocytoma:

  • Neuroendocrine tumor overproduces catecholamines & causes severe HTN

  • Adrenals are difficultly imaged

  • Plasma & urine metanephrine measurements are sensitive & specific for pheochromocytoma

  • Adrenal ‘incidentalomas’ are commonly found, incidentally

Ovarian development & function: 6 weeks, secretion, LH & FSH, puberty

• At 6 weeks gestation, fetus is sexually bi-potential

• Estrogen secretion begins in fetal ovary

• Fetal LH & FSH peak midterm & fall to low levels at birth

• Puberty: hypothalamic GnRH stimulates increases in LH & FSH (including nocturnal rise in LH)

Ovary feedback loops: FSH/LH, low/high estradiol, progesterone

• FSH/LH release from anterior pituitary induced by GnRH

• Follicle-stimulating hormone (FSH): Stimulates maturation & growth of GF

• Luteinizing hormone (LH): Stimulates ovulation & Induces progesterone secretion (peak) by ovary

• Low [estradiol] during menstruation & mid-follicular phase: Provides (-) feedback to GnRH, FSH & LH secretion

• High [estradiol] midcycle: Provides (+) feed forward for FSH & LH secretion

• Progesterone: Peak in mid-luteal phase provides (-) feedback to GnRH,
  FSH & LH secretion

Estrogens: Estradiol (3), types of receptors (3)

• Estradiol has many beneficial effects on:

  • Vascular endothelium (& heart): lower rate of CVD in women until menopause

  • Maintenance of bone & bone mineral density (BMD): Single most powerful determinant of BMD in men & women

  • Muscle & CNS

• At least 2-3 types of estrogen receptors are expressed in men and women

  • Estrogen receptor alpha (ESR-1): nuclear steroid receptor transactivating proliferation signals

  • Estrogen receptor beta (ESR-2): nuclear steroid receptor transactivating differentiation signals and may be activated by an androgen metabolite as its primary ligand

  • GPR30? a G-protein coupled receptor – Discovered here in 2007 Go Lobos! Yet highly controversial

Ovarian hormones: Androgens, Activins, Relaxin

• Androgens: androstenedione, dehydroandrostenedione, testosterone & dihydrotestosterone (DHT)

  • Important for female libido

  • Excess secretion causes hirsutism & extreme cases virilization

• Activins: induce FSH secretion & steroidogenesis

• Relaxin: prepares for delivery by loosening the muscles & ligaments in pelvis

Ovarian hormones: Inhibins A & B (folliculostatin; function, FSH, A vs B, secretion)

• inhibits FSH

• FSH stimulates the secretion of inhibins from the granulosa cells of the ovarian follicles in the ovaries

• Inhibin B reaches a peak in the early- to mid-follicular phase, & 2nd peak at ovulation

• Inhibin A reaches its peak in the mid-luteal phase

• Inhibin secretion is diminished by GnRH & enhanced by insulin-like growth factor-1

Androgens: Male sex differentiation, andropause

• Weeks 6-7: Bipotential fetus develops testes due to SRY gene on Y chromosome.

• Week 10: Leydig cells produce androgens (testosterone) under hCG influence.

• Prenatal: Sertoli cells secrete Müllerian Inhibiting Hormone (MIH), causing regression of female reproductive structures.

• Andropause: Gradual, age-related decline in testosterone secretion in some men.

Androgens: LH, FSH, inhibin, GnRH

• LH → Stimulates Leydig cells → Testosterone production (≈95% of androgens).

• FSH + Testosterone → Activate Sertoli cells → Support spermatogenesis.

• Sertoli cells also produce inhibin, which with testosterone provides negative feedback on pituitary/hypothalamus (GnRH).

• GnRH is released in pulsatile manner with a nocturnal surge.

Androgens: Transport, diurnal rhythm, regulation, FSH action, Androgen receptor

• Transport: Testosterone is 95% protein-bound (45% to SHBG, 50% to albumin); only 5% is free and active.

• Diurnal Rhythm: Highest in early morning, lowest around midnight.

• Regulation: Testosterone and estradiol provide negative feedback to inhibit Leydig cell production (via LH suppression).

• FSH Action: Acts on Sertoli cells to boost protein synthesis, inhibin secretion, and androgen receptor (AR) expression.

• Androgen Receptor Effect: Activated ARs trigger synthesis of FSH receptors on Sertoli cells, enhancing FSH sensitivity.

Androgens: Inhibin, intracellular metabolism, risks of excess T, anabolic, lupron

• Inhibin Function: Secreted by Sertoli cells to provide specific negative feedback on pituitary FSH secretion.

• Intracellular Metabolism: Testosterone is converted to more potent DHT or to estradiol (E2) in tissues like fat.

• Risks of Excess (T/DHT): Polycythemia (↑ HCT), hyperviscosity, prostate enlargement, and aggressive behavior.

• Anabolic Steroid Side Effect: Suppresses the HPT axis → testicular atrophy, impotence, and mood/behavior disturbances.

• Lupron: A GnRH agonist used to suppress sex hormone production; treats prostate cancer, endometriosis, precocious puberty, and is used for chemical castration.

Sex steroids Endocrinopathies: Hypogonadism, PCOS

• Hypogonadotropic hypogonadism: deficiencies of FSH and LH

• Hypergonatropic hypogonadism: ovarian failure (elevated FSH ± LH) example: Turner’s syndrome (XO)

• Polycystic Ovarian Syndrome (PCOS): characterized by infertility, anovulation, hyperglycemia, hyperlipidemia, hirsutism, treated with metformin

Growth hormone: Somatotropin (AP hormones, size, insulin, T&E, feedback loop)

• Anterior pituitary hormones necessary for appropriate growth: TSH,
  ACTH, FSH, LH, GH

• Somatotrophs > 1/3 mass of pituitary: synthesize & release GH

• Pancreatic hormone necessary for appropriate growth: Insulin

• Steroid hormones necessary for appropriate growth: T&E

• Feedback loop involves hypothalamic GHRH (+ regulator), Ghrelin (+
  regulator), Somatostatin (- regulator) & other factors

GH: Bio activity, surrogate marker, assay

• Biological activity:

  • Insulin antagonist: promotes gluconeogenesis & lipolysis

  • Anabolic effect on muscle increasing Pi & N uptake

  • Induces Insulin-Like Growth Factor-1 (IGF-1, old name

• Somatomedin C) synthesized by the liver

• IGF-1 is a good surrogate marker

• IGF-1 assay is preferred method assessing deficiency or excess GH secretion & marker of overall protein nutritional status

GH: test, deficiency, in excess

• Preferred test for autonomous GH secretion by pituitary adenoma:
  75g oral glucose load suppression – adenomas are not suppressed

• GH deficiency: stimulation tests using insulin-induced hypoglycemia
  (older) or GHRH + L-arginine/L-arginine + L-DOPA

• Acromegaly/Gigantism in excess

Prolactin: Uniqueness, stimulate, increases seen, excess

• Unique pituitary stress hormone

  • Does not target other primary endocrine organs

  • Secretion is under tonic inhibition by dopamine (neurotransmitter/neuroendocrine hormone)

  • Promotes lactation and suppresses ovulation (via suppression of FSH & LH secretion and action)

• TRH, estradiol stimulate prolactin secretion

• Increases seen in pituitary adenoma (Prolactinoma), renal failure, PCOS, after exercise

• Excess causes hypogonadism, gynecomastia & infertility, and galactorrhea (rarely in men)

RR Prolactin & Testosterone

• Prolactin in M: 4-15 ng/mL

• Prolactin in F: 5-23 ng/mL

• Prolactin > 150 usually = prolactinoma

• Testosterone M adult: 240-950 ng/dL

• Testosterone F adult: 8-60 ng/dL

Pituitary ademonas

• Functioning PAs: secrete hormones: most common are Prolactin secreting, followed by GH and TSH

• Non-functioning PAs: do not secrete hormones

• Surgery is often needed to preserve eyesight (PAs press on optic chiasma causing double-vision)

• Surgical removal of pituitary masses can cause DI followed by SIADH (biphasic) & DI followed by SIADH, followed in turn by DI (triphasic)

• Copeptin: secreted along with ADH & longer circulating half-life than ADH

  • Is the “C-peptide” of ADH

Chromatography: Forms, TLC, HPLC, GC/GCMS

• All forms feature a stationary and mobile phase, but bio samples with drugs or other lipophilic compounds must 1st be extracted into an organic solvent

• Thin layer chromatography (TLC): stationary phase is usually silica gel or aluminum oxide attached to inert support; mobile phase is an organic solvent

• Liquid chromatography (HPLC): silica or hydroxyapatite media and polymer resins: stationary phase is a column packed with beads coated with silica or hydroxyapatite media and polymeric resins such as polystyrene divinylbenzene

• Gas chromatography GC/GCMS: Stationary phase is a long capillary column coil packed with immobilized porous polymers or alumina, mobile phase is a gas (H2) carrying vaporized compounds of interest

Chromatography: Principle, compound separation, standards, retention time (Rf)

• Principle of chromatography: compounds can be separated with high resolution according to whether they have high, low or intermediate affinities for either the stationary or mobile phase (or both)

• Compounds are separated into spots (TLC) or rings (liquid/gas) as they are pumped or carried through the stationary phase that can be detected visually (thin-layer) or by other detection techniques (N/P, flame ionization, mass spectroscopy)

• Rf (how long is compound characteristically retained on stationary phase in minutes) & help ID unknown compounds

• Standards are run for comparison of retention times & help calculate [unknown]

Thin-layer chromatography (5)

• Lanes 1, 2, 3, and 4 are preloaded standards, extracted urine sample disc fits into the black circles

• Toxi-Lab A and B TLC “plates” are placed in jar where a small amount of solvent is wicked up to their tops

• Plates are developed in various solution stages (I-IV) and subjected to UV radiation

• Match to standard (¡patrones!) at each stage enables qualitative ID

• Relative migration factor Rf: ratio of how far drug/metabolite migrates as compared with solvent at top of plate

Mass spectroscopy: Components, steps, tandem

• ion source: molecules will be ionized (charged)

• mass analyzer: filter & separate molecules based on mass using electrical currents

• detector: detect current that is produced by ionized molecule(s) & transform it into a mass

• 5 steps: ionization, acceleration, deflection, detection, data analysis/libraries

• tandem mass spec: used to reduce changes of getting interfering compounds that would pollute signal of molecule of interest

Biochemical & Physiological changes in Pregnancy (10)

• Expansion of plasma volume by 85% of pre-conception values

• 10% decrease in ECF osmolality at end of term

• Dilutional but not absolute decrease in RBC/PCV

• Modulation of the Renin-Angiotensin-Aldosterone Axis by Relaxin & hCG resulting in increased ADH secretion, sodium and water retention

• Progesterone causes K+ retention

• Increased GFR helps balance sodium from increase in aldosterone

• Increased demand for and risk of iron/iodine deficiency (as well as protein, calories, calcium, etc.)

• 2-fold increase in calcium absorption

• Kisspeptin from hypothalamus rises 10,000 x in plasma: stimulator of GnRH with antimetastatic activity

• Rise in TRH causes increased total thyroid hormone

Human chorionic gonadotropin (hCG): blastocyst, urine test, diagnosis, placenta

• Glycoprotein hormone produced by blastocyst that will develop into a placenta

• Urine test is more sensitive for detection of early pregnancy as compared with serum

• Used to establish “diagnosis” of pregnancy (as early as 6-12 days post-fertilization)

• hCG is hyperglycosylated before placenta forms, & not reactive with POC testing kits

hCG: during term, peaks, double time, low, cross reactivity, estrogen

• Peaks at 10 weeks gestation, declines by midterm to a lower plateau

• doubling time (in serum) in 1st trimester (2-3 days) is informative in ectopic pregnancy

• levels remain low in ectopic pregnancy (do not meet doubling time criterion – typically ‘flat-line’)

• hCG cross reactivity with TSH receptor

• Primary estrogen secreted in pregnancy is estriol

Routine testing during pregnancy: Urinalysis (3)

• screen for pre-eclampsia, gestational diabetes, infection

• pre-eclampsia triad: HTN, edema, proteinuria

• gestational diabetes: test for GDM at 24-26 weeks for pregnant women without prior diabetes

Anencephaly & Spina bifida: Risk factor, screening, age, AFP

• risk factor: folate status with genetic factors

• all women should be offered a screen @ 15-20 weeks gestation

• Knowing precise gestational age is important in interpreting results

• AFP is elevated in NTCDs (& hepatocellular cancer), & low in Down syndrome

Anencephaly & Spina bifida: MSAFP (what, concentration, rising, fetal)

• Alpha-fetoprotein measured first in maternal serum (MSAFP), in amniotic fluid, & fetal plasma (in MoM – multiples of the median)

• MSAFP concentration < in amniotic fluid or fetal plasma

• MSAFP rises in early pregnancy, peaks between 28 and 32 weeks of gestation, & then falls

• Fetal plasma alpha-fetoprotein peaks between 10-13 weeks of gestation → declines exponentially from 14-32 weeks → falls dramatically near term

Non-invasive Prenatal testing for Trisomies (NIPT)

• AKA: cell free DNA screening (cf-DNA) for:

  • Trisomy 21: Down Syndrome

  • Trisomy 18: Edwards Syndrome

  • Trisomy 13: Patau Syndrome

  • Determines gender of fetus & X/Y chromosomal abnormalities

• Ratio of cell-free DNA from fetus & mom compared to determine trisomy

Amniotic fluid: Amniocentesis, volume, determine fetal age

• Indications for amniocentesis: fetal distress, fetal maturity
  assessment, confirmation of prenatal maternal screening result
  using cell free placental DNA in maternal blood

• Volume: 800-1200 mL at third trimester

• Determination of fetal age: creatinine values before 36 weeks (1.5-2.0 mg/dL), after 36 weeks (> 2.0 mg/dL)

Differentiation of amniotic fluid from maternal urine to r/o premature rupture of membranes (PROM) (4)

• Creatinine levels are much lower in amniotic fluid as compared with urine

• Fern test

• pH (amniotic fluid 7.1 to 7.3, normal vaginal fluid 4.5 to 6.0)

• Biomarkers: PAMG-1 (placental alpha-macroglobulin), IGFBP-1/PP12
  (insulin-like growth factor binding protein-1/placental protein 12),
  AFP(alphafetoprotein) + IGFBP-1

Fetal lung maturity (FLM): Maturity, surfactant, L/S, PG, detection, index

• fetal lung matures late during the course of pregnancy

• Pulmonary surfactant phospholipid (made by type II pneumocytes) after week 35 of pregnancy & is necessary to keep alveoli from collapsing (atelectasis)

• Lecithin-sphingomyelin ratio (L/S ratio): indicator of FLM (> 2.0)

• Phosphatidyl glycerol (PG): indicator of FLM. Delayed in maternal DM

• Thin-layer chromatography detection

• Foam stability index (shake up and watch bubbles) & lamellar bodies (uses
  PLT channel from automated cell counters)