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Pituitary

Presented by Dr. Moyassar A.Zaki Professor of Chemical Pathology Medical Research Institute Alexandria University PITUITARY HORMONES

Pituitary gland is divided into two main lobes: anterior (adenohypophysis) and posterior (neurohypophysis) lobes each secreting different types of hormones.

ANTERIOR PITUITARY HORMONES (A.P.) gland, under the control of , secretes a number of trophic (tropic) hormones that regulate growth, functions of other endocrine glands and influence metabolic reactions in other target tissues. There are mainly five types of cells in the A.P. gland: • Somatotrophic cells secrete growth (GH) (Somatotropin) . • Mammotrophic (lactotrophic) cells secrete (PRL). • Thyrotrophic cells secrete stimulating hormone (TSH) (Thyrotropin) . • Corticotrophic cells secrete adrenocorticotrophic hormone (ACTH) (Corticotropin) . • Gonadotrophic cells produce two related hormones (): (LH) and follicle stimulating hormone (FSH). PRL and GH are closely related acting on non-endocrine target tissues , unlike the other hormones which act primarily on direct endocrine organs and are largely regulated by the production of their respective target glands. NB: Other cell types secrete uncharacterized pituitary hormones such as tissue- specific growth factors and endothelial cell growth factors. Difference between the terminologies “tropic”and “trophic”: : Tropic hormones are hormones that have other endocrine glands as their target. Trophic hormone is a hormone that has a growth effect, hyperplasia or hypertrophy, on the tissue it is stimulating.

(1) Corticotropin and related Adrenocorticotropic hormone (ACTH) is derived from (POMC) by . In the corticotrophs (Pituitary gland) through the action of subtilisin-like proprotein convertase PC1/3, POMC is first cleaved into two fragments: the 22 kDa pro-ACTH fragment and beta- (amino acids 42-134), whose function remains poorly understood. Next, PC1/3 releases ACTH (amino acids 1-39) from pro- ACTH. The resulting N-terminal fragment is further cleaved to pro-gamma-MSH (a.k.a. N-POC) and a joining (JP). ACTH is not further cleaved in corticotrophs.

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POMC and its precursor relationship to ACTH, β-LP, MSH, and the

In the hypothalamus, skin, and melanotrophs further processing of N-POC, ACTH, and beta-lipotropin occurs. The action of PC2 in the hypothalamus, skin, and melanotrophs of the intermediate lobe of the pituitary is to release gamma-MSH from N-POC; alpha-melanocyte stimulating hormone (alpha- MSH; amino acids 1-13) and corticotropin-like intermediate lobe peptide (CLIP, amino acids 18-39) from ACTH; and gamma-lipotropin (amino acids 42-101) and beta-endorphin (amino acids 104- 134) from beta-lipotropin. Last, beta-MSH (amino acids 84-101) is derived from gamma-lipotropin via PC2. a) ACTH (corticotropin): (39 a.a. peptide) Its primary effect is to stimulate secretion of glucocorticoids (mainly), mineralocorticoids and androgenic steroids (to a lesser extent) from the adrenal cortex by binding to a G- coupled cell membrane receptor through a cAMP mediated mechanism. It also stimulates adrenal cell growth, , , release, GH secretion and enhances and transport into muscles. Normal adult plasma ACTH level has a diurnal rhythm (lowest level in the evening = 5 – 45 pg/ml and peak level early in the morning = 10 – 80 pg/ml). This rhythm is independent of sleep. b) β -lipotropin and β-endorphin: In the anterior lobe, they are secreted in an equimolar ratio to ACTH in response to all types of stimuli, since both molecules are present in the same precursor as ACTH. However, they have a slower metabolic clearance. β-lipotropin serves as a precursor for β-endorphin. Pituitary endorphins are inactive as they are acetylated (unlike those secreted in CNS that act as neurotransmitters).

c) Melanocyte-stimulating hormones (MSH): types: α-, β- & γ-MSH. In the anterior pituitary gland , the pro-opiomelanocortin (POMC) molecule produces α- & β-MSH . In the intermediate lobe , if present, the pro-ACTH also produces γ-

- 2 - MSH. These smaller peptides can be found in human foetus, but only exist in trace amount in adults. Skin pigmentation found in ACTH-secreting tumors and adrenal insufficiencies are probably due to the α-MSH activity of excess ACTH.

(2) Gonadotropins and related glycoproteins They are glycoproteins made up of identical α-subunits and unique β-subunits (has a different a.a. sequence) responsible for their specific biological activity. a)LH and FSH (gonadotropins): Functions of LH: Females: -It induces in the presence of certain amount of FSH -It starts the formation of and stimulates production by the corpus luteum. Males: It stimulates testicular interstitial cell (Leydig cells) function and results in enhanced production of . Functions of FSH : Females: -It stimulates ovarian follicular growth and ovum maturation -In the presence of LH, it promotes secretion by the mature follicles in the . Males: It stimulates testicular growth and spermatogenesis (with testosterone). Hypothalamic regulation of gonadotropins secretion: Two hypothalamic centres for controlling the level exist: -Tonic centre: controls the continuous basal gonadotropin level and is responsible for the negative feedback regulation of steroids. -Cyclic centre: induces the mid cycle peak (ovulatory) of gonadotropins in response to the positive feedback of the raised estrogen. Inhibin (a glycoprotein produced in the ovary and testis) : has an inhibitory feedback on FSH secretion. The LH and FSH release in normal adult is pulsatile every 1-2 hours. LH & FSH plasma levels: higher in adult female at the mid and after menopause and lower in children. Reference Intervals LH (IU/L) FSH (IU/L) Adult males 1.2 – 7.8 1.4 – 15.4 Females Pre menopausal • Follicular phase 1.7 – 15 1.4 – 10 • Mid cycle phase 22 – 57 6 – 17 • Luteal phase 0.6 – 16 1 – 9 Post menopausal 14.2 – 52.3 19.3 – 101

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- 4 - b)TSH (thyrotropin): (glycoprotein, made up of α and β subunits). Biological effects: (Its effects on thyroid gland are largely analogous to those of ACTH on the adrenal cortex). -It increases synthesis and release of thyroid hormone by binding to cell membrane receptors through cAMP mediated mechanism. -It also stimulates the growth of thyroid cells by stimulation of RNA and protein synthesis. Normal adult (21 – 54 yrs) TSH plasma level: 0.4 - 4.2 µIU/ml. Its secretion is pulsatile and shows a diurnal rhythm of slightly higher level at night (2 – 4 am) and lower level at afternoon (5 – 6 pm). The circadian rhythm of TSH is lost during illness.

(3) Somatomammotropic hormones a) (Somatotropin) (GH): a 21.5 KDa single chain polypeptide hormone (191 a.a. with 2 intramolecular disulphide bridges). It is structurally similar to prolactin and to human (HPL) with which it has overlapping biological activities. 4–5 modified forms of GH exist, representing about 10% of the total GH secreted. Biological actions of GH: 1-Anabolic in most tissues (except in lipocytes as it cause lipolysis): GH is important for linear growth of bone and cartilage, as well as cardiac and renal tissues growth (either direct effect or indirect via IGFs). 2-Regulator of metabolic processes, e.g. synthesis of new proteins, lipolysis, and glucose utilization. (Acute ↑GH: ↓ blood glucose / chronic ↑GH: ↑ glycogenolysis & ↓ glucose uptake and utilization). 3-Hormonal effects: GH stimulates the production of insulin, , and . Normal adult basal plasma GH level: 2–5 ng/ml with several secretory episodes that occur about 3 hours after meals . Its level is higher in female during childbearing period. Females have higher basal level (due to estrogen sensitization of the hypothalamus to other GH stimuli) but lower pulse peaks. The secretion of GH is pulsatile : The majority of GH secretion occurs about 1 – 1½ hours after the onset of sleep and is associated with sleep stage 3 and 4 (GH level reaches its peak during the period of deepest sleep). GH secretion changes dramatically with age: • During the 1st few days of life it is extremely high. • By the 2nd week of life it decreases. • During pruberty its level is higher than that of adulthood. • After the 4th decade there is gradual and progressive decrease in GH secretion and response to the releasing stimuli. ( It has been suggested that the age-related decline in stage 4 sleep may account for decline in GH level seen with aging). Insulin-like growth factors (IGFs): Most of the GH effects are mediated by GH-dependent growth factors called IGFs (formerly called ). GH binds to a specific cell-membrane receptor of many tissues, including cartilage, to stimulate production of these factors. The most important of these factors is IGF-1 (-C). The systemic form of IGF-1 originates mainly from the . It may also be synthesized and act locally in tissues. Cell growth and cell differentiation require both GH and IGF-1. Serum IGF-1

- 5 - concentration is influenced by age, degree of sexual maturation and nutritional status (decreased in acute or chronic protein calorie malnutrition). NB: Other human growth factors include : IGF-2 (somatomedin A), platelet-derived (PDGF), and (EGF).

b) Prolactin(PRL)(lactotroph): (198 a.a. peptide). Sites of action: Prolactin receptors are present on cell membrane of its target tissues, e.g. (nipple), liver, , , testis and . Biological actions: In lactating females: Its main role is to enhance milk production in breast tissues which is already primed by estrogen and progesterone. Suckling is the main physiologic stimulus for prolactin secretion →↑ maternal plasma PRL within minutes. NB: PRL is not required for normal in non-pregnant females. During , it results in further breast tissue growth and milk protein formation (in conjunction with estrogen, progesterone and placental lactogen). Following parturition, the abrupt decrease in estogen and progesterone allows the unopposed action of prolactin to permit initiation of . Several months after parturition, serum prolactin level returns to normal even with continuation of lactation. The actual milk let-down reflex is mediated by the release of rather than prolactin. In non- lactating females, males and children : its actual function is not fully understood. It may be important in: -Control of osmolality -Subcutaneous fat metabolism -Calcium, and carbohydrate metabolism -Fetal lung development -Steroidogenesis (adrenal androgen production) -Immunomodulation. PRL acts at the hypothalamus to inhibit GnRH secretion . Inhibition of GnRH results in decreased LH and FSH release from the anterior pituitary. 1. In females: this leads to a decrease in estrogen and progesterone synthesis and secretion by the ovaries and a failure of ovarian follicular maturation (ovulation). 2. In males, this results in decreased testicular production and synthesis of testosterone and a halt in spermatogenesis. Stimuli for PRL release: Suckling –Labor & delivery –Stress –ACTH – PRL itself (+ve feedback loop) – Suppression of secretion – TRH (also known as PRF: prolactin releasing factor). Normal adult plasma PRL level : In non lactating female and in male it does not exceed 25 ng/ml (4–23 & 3–15 ng/ml respectively). Its level has no significant variation during menstrual cycle, but its level decreases at menopause. During pregnancy , its level rises continuously till full term as a result of estrogen stimulation (95–500ng/ml).This is largely due to an increase in number of PRL- secreting cells and can be associated with doubling or even greater increase in pituitary gland size. PRL levels fall back to baseline about 3 weeks postpartum in women who are not breastfeeding. In nursing mothers basal PRL levels remain moderately elevated and with episodic bursts in secretion in response to suckling. Its release from the A.P. gland is episodic and it has a diurnal rhythm : Low level at midday and highest level shortly after the onset of sleep.

- 6 - Regulation of Anterior Pituitary Hormones (1) Hypothalamic hormones Many anterior pituitary trophic hormones (THs) (e.g., ACTH, TSH, GH, LH, FSH) are regulated by hypothalamic releasing hormones (HRHs). Releasing hormones secreted by the hypothalamus reach the pituitary via the hypothalamic-pituitary portal system (HPPS). Six different hypothalamic hormones or factors (4 releasing- and 2 inhibitory) control anterior pituitary gland function: • Corticotropin releasing hormone(CRH) • Thyrotropin releasing hormone (TRH) (also called PRF) • Growth hormone releasing hormone (GHRH) • Gonadotropin releasing hormone (GnRH) • Growth hormone-inhibiting hormone (GHIH) (): also called somatotropin release-inhibiting factor [SRIF]. • Prolactin inhibiting hormone (PIF) (Dopamine) Remarks: -Most of the A.P. hormones are primarily controlled by stimulatory effect (e.g ACTH, LH, FSH, TSH and GH). -Prolactin is regulated by an inhibitory effect only. -GH is unique in having an additional inhibitory regulation. The major neurotransmitter systems utilized for intracellular communication within the central nervous system consist of monoamines and peptides. These neurotransmitters can influence the hypothalamic hormone-secreting neurons. Examples of biogenic amines : Catecholamines (dopamine, & epinephrine), indolamines (serotonin & ), acetylcholine, γ-amino-butyric acid (GABA) and histamine. Examples of the : endorphin- peptides ( β-endorphin, & ). GHRH : Stimulated by catecholamines, indolamines and endorphins. Inhibited by melatonin. TRH and GnRH : Stimulated by catecholamines. Inhibited by serotonin and endorphins. CRH : Stimulated by acetylcholine and serotonin. Inhibited by norepinephrine, enkephalins and melatonin. Basal and episodic secretion, diurnal rhythm and nocturnal release of A.P. hormones are all considered to be secondary to CNS events that are mediated through the hypothalamic hormones.

Neurotransmitter effect on hormonal secretion by the anterior pituitary gland

- 7 - (2) Feedback regulations : The functional relationship between the anterior pituitary gland and its target glands is based on feedback regulation; primarily negative feedback regulation: a) Long feedback loop: It occurs from the target gland to the hypothalamus and/or the anterior pituitary gland. Its effect is typically opposite to that of the initial stimulus. b) Short feedback loop: It comprises the anterior pituitary gland and the hypothalamus, by which the pituitary hormones act on the hypothalamus to inhibit its own secretion. c) Ultra short feedback mechanism: involves the anterior pituitary hormone feeding back at the anterior pituitary.

Remarks: In some circumstances, secretion of anterior pituitary hormones are regulated by a positive feedback mechanism, where the target hormone produces stimulation of the initial hormone, (e.g. the positive feedback of high estrogen results in the mid- cycle surge of LH and FSH, resulting in induction of ovulation). Stress is a potent-stimulus for ACTH release (overrides the feedback inhibition). Stress can also increase GH and prolactin secretion. Role of cytokines in axis modulation: The chemical mediators released by inflammatory cells (cytokines) anticipate in altering the control mechanisms associated with the neuroendocrine axis. Modulation of the feedback loop between the hypothalamic-pituitary-adrenal axis by cytokines, such as Interleukins (1L-1 and IL-6) released as a result of infection or stress has been shown to diminish the immune system. The concept of a hypothalamic-pituitary- adrenal-immune axis has been evolving for some time, and now substantial evidence is available to support the concept that cytokines do modulate the responsiveness of the hypothalamic-pituitary-adrenal axis under certain physiological response situations, such as infection or stress.

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Determination Of Anterior Pituitary Hormones Specimen needed for A.P. hormone estimation 1) Serum is the specimen of choice in most hormones (except ACTH which requires EDTA plasma). Some may use heparinized plasma but values obtained are method dependent for each hormonal assay. 2) The patient should be overnight fasting (diet affects GH and PRL) and must be in a complete physical and mental resting state for about 1/2 hr before sampling. 3) Avoid hemolyzed, lipemic, and/or icteric specimens: particularly in LH/FSH determinations. 4) For ACTH: the specimen must be collected under prechilled conditions in polystyrene tube (ACTH adsorbs to glass surface), centrifuged at 4°C, transferred into a new plastic or eppendorf tube and stored at -20°C or lower till time of analysis. 5) Some of the hormones have pulsatile nature of secretion (e.g. PRL, GH, LH/FSH), so it is preferable to estimate their level in a pooled specimen (taken at fixed intervals for a certain period of time. 6) A protease inhibitor (e.g. aprotinin or trasylol) can be added during sampling to delay the degradation of hormones like ACTH. 7) Some hormones have a diurnal rhythm (GH, PRL, TSH and ACTH) or their levels change with menstrual cycle (Gn) therefore the time of taking the sample should be recorded. 8) Age and full history should be taken from the patients about any medication taken and smoking habits since both could affect serum levels of some hormones. 9) Use of antioxidants (eg. mercaptoethanol) that inhibits oxidation of ACTH. 10) Storage of the serum sample: at 2 to 8°C if not to be tested within 8 hours for GH, 24 hours for PRL, 5 days for TSH, and 2 weeks for LH/FSH. If specimens must be stored for longer periods (months), they should be frozen at −20°C or colder. 11) In urinary gonadotropins assay: timed urine specimens should not contain preservatives. Storage at or below −20°C is recommended. 12) Some hormones may need an extraction step prior to their estimation, e.g. ACTH and IGF-1. Nowadays non extraction kits exist for determination of many hormones and hormone like substances (growth factors).

- 9 - Methods for estimation of A.P. hormones 1. Bioassays 2. Receptor assays 3. Immunoassays (most commonly used in practice)

Methods of hormone estimation 1.Bioassays: They are not used routinely. They are used in comparing the biological potency and immunoreactivity of hormone fragments and its synthetic peptides in research works, in calibrating reference material and in elucidating the disease etiology. They are either in vivo or in vitro bioassays. 2.Receptor assays: They are also of limited use in routine determination. They have greater specificity and are much simpler than bioassays. However, they are less sensitive than the immunoassays and the enzymes present in the specimen may degrade the receptor or destroy the labeled tracer. The complexity and lability of receptor preparation make the use of this method limited to the determination of the biological function of a hormone. 3.Immunoassays: They are used routinely. They are either : a. Competitive binding assays (either under equilibrium or non-equilibrium condition). They can be either: -Isotopic (radiolabeled): Radioimmunoassay (RIA). -Non isotopic labeled: competitive EIA. b. Immunometric assays: (The sandwich assay) they have better sensitivity and specificity. They use either mono-or polyclonal antibodies. They may be: -Isotopic (radiolabeled) : RIA – Immunoradiometric (IRMA) -Non-isotopic : 1-ELISA (enzyme labeled) 2-Immunofluorometric (fluorescent labeled) (IFA) 3-Immunochemiluminescence (chemiluminescent labeled) (CLIA: ChemiLuminescent ImmunoAssays). Remarks: -They are available as manual commercial kits or in automated systems. -All these methods have better precision, expanded working range, faster reaction time, better sensitivity and higher specificity than bioassays & receptor assays.

- 10 - HORMONES The posterior pituitary gland releases two hormones: arginine (AVP) and oxytocin. Both hormones are octapeptides. They are synthesized in special neurosecretory cells in the hypothalamus where they are transported through the pituitary stalk "hypothalamo-hypophyseal tract" to be stored in special neural elements in the posterior pituitary gland called pituicytes. The neurosecreotory cells in the hypothalamus responsible for their synthesis are: • → (ADH + 1/6 oxytocin) • Paraventricular nucleus → (Oxytocin + 1/6 ADH) Both hormones are synthesized as prohormones in conjunction with their specific carrier proteins called I and II (for oxytocin and AVP; respectively) has been used as a marker for oxytocin release.

Oxytocin hormone It is present in both males and females, but its physiological effects are found only in females. Stimuli for release of oxytocin hormone: 1. Suckling is the primary stimulator for oxytocin secretion. 2. Visual or auditory stimuli from the baby. 3. Uterine contraction and cervical distention during labor. 4. Hypertonicity of body fluids appears also to stimulate its secretion. Factors that inhibit oxytocin secretion: 1. Psychological and emotional factors (pain, anxiety, fear). 2. Alcohol intake. Hormonal regulation of oxytocin secretion: 1. Estrogen enhances its secretion directly while progesterone inhibits it: via increasing uterine sensitivity (in the former) and decreasing its sensitivity (in the latter) to oxytocin. 2. (an ovarian peptide that suppresses the uterine contraction and relaxes the pelvic connective tissues during parturition) suppresses oxytocin release. Oxytocin biological activities: In females: 1. It stimulates the uterine contraction, only in estrogen primed uterus, leading to expulsion of the and during labor. However, the initiation of labor is oxytocin-independent. The total absence of oxytocin does not prevent parturition, although prolonged labor occurs. It is a useful therapeutic agent for improving the labor quality 2. Oxytocin stimulates contraction of myoepithelial cells surrounding the terminal acinar lobules that expel their milk into the lobular ducts. In males: It increases ejection of sperm into semen, in response to stimulation of the reproductive organs. Other functions of oxytocin: 1. It retains some antidiuretic activity under some disorders (but not under physiological condition). 2. It causes relaxation of vascular leading to decrease in blood pressure.

- 11 - Arginine vasopressin /antidiuretic hormone (ADH) Vasopressin (AVP), as oxytocin, comes from a common precursor gene, and the prohormone peptide is transported in neurosecretory granules to the posteiror pituitary gland. During transport, it is cleaved to neurophysin II and AVP. There are two pools of AVP-containing neurosecretory granules in the posterior pituitary gland: • One adjacent to the cell membrane, available for immediate release and a • Second storage pool away from cell membrane. Release of AVP hormone in response to hypothalamic stimulus occurs by an exocytotic process involving fusion of the neurosecretory granules with plasma membrane. Calcium influx activates the exocytotic process. Function of AVP (ADH): It exerts major physiologic effects on the collecting tubules of the nephron and also on tone. 1. The major physiologic function of ADH (AVP) is regulation of water permeability of the collecting tubules and ascending limb of loop of Henle. 2. ADH induces a generalized vasoconstriction that lead to rise in the arterial blood pressure. 3. It also augments the action of CRH in stimulation of corticotropin release. There are three distinct receptors for mediating AVP effects: V1a receptors: expressed in arterial smooth muscle cells. ADH binding to the V1a receptor stimulates the secretion of vascular endothelial growth factor (VEGF). The V1a receptor may also affect platelet aggregation, coagulation factor release, and glycogenolysis by its expression on platelets and hepatocytes. ADH is believed to play an important role in the maintenance of arterial blood pressure during blood loss beside the main player RAAS. This effect is regulated by V1 receptors, through the action of Phosphatidyl inositol-3 Phosphate /Ca 2+ . V1b receptors (V3 receptor): expressed in the CNS. In this way, ADH can release corticotropin to aid in the response to stress. The V1b receptor also has been reported to be expressed in islet cells, influencing insulin secretion. V2 receptors: expressed on collecting duct cells and the medullary part of the ascending loop of Henle. The V2 receptors have a greater affinity for AVP than V1 do, a mechanism mediated by cAMP and calcium. They bind ADH with resultant activation of a G-protein coupled system, with ATP being converted to cAMP via adenylate cyclase with protein kinase-A activation. This leads to translocation of aquaporin-2 water channels from an intracellular pool to the apical plasma membrane, allowing free water uptake by cells of the collecting duct. On the basolateral plasma membrane aquaporin-3 and aquaporin-4 water channels, free water then leaves these cells.

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- 13 - Regulation of ADH release: Stimulators of ADH release: 1. Plasma osmolality is the predominant regulator for ADH secretion. Osmoreceptors in hypothalamus respond to changes in plasma osmolality. A 2% increase in ECF osmolality causes shrinkage of the osmoreceptors and stimulation of ADH release. A plasma osmolality above 280 mOsm/kg is considered the osmotic threshold for ADH release. 2. The baroreceptors (mechanoreceptors that sense any change in vascular wall tension in both arterial and venous circulation) respond to alteration in blood volume, i.e. decrease in blood volume (10% ↓ in blood vol.) and/or arterial pressure will stimulate ADH secretion. 3. Other non-osmotic stimuli: e.g. sleep, emotional stress, exercise, physical trauma, pain, nicotine, caffeine, catecholamines, -II, opiates, , anesthetics and barbiturates. 4. Age: →→→ ↑ ADH secretion → water retention & hyponatremia. Inhibitors of ADH release: 1. Atrial (ANP): provides a negative feedback for ADH release. 2. Drugs: e.g. alcohol, phenytoin and glucocorticoids also inhibit ADH release. Remark: Responses involving ADH, thirst and kidney are coordinated to maintain the plasma osmolality in healthy subjects within narrow range (284-295 mOsm/Kg).

- 14 - METHODS OF ADH AND OXYTOCIN ESTIMATION Sampling precautions: 1. Blood specimens for ADH should be collected into prechilled tubes containing EDTA as an anticoagulant. 2. Most procedures recommend that specimens be delivered to the laboratory on ice and centrifuged at 4 °C within 30 minutes of collection. 3. The plasma is then removed and stored or shipped frozen at −20 °C until analysis is performed. 4. Random urine specimens may be collected without preservatives; alternatively, complete 24 hour urine specimens may be collected in 10 mL of 6 mol/L hydrochloric acid. 5. Significant deterioration of ADH occurs after prolonged storage. Methods for determination: Most of the immunoassays used (RIA – ELISA) require a preliminary extraction procedure to concentrate the minute amount of the hormone and to remove the non specific interfering substances. Such purification can involve the use of: • Chemicals : acetone, petroleum ether, or ethanol • Column chromatography (using ODS C 18 column) Reference ranges for plasma ADH & oxytocin hormones • Normal ADH level: • Adult Plasma (in relation to Osmolality): <1.5 pg/mL (at osmolality from 270-280 mOsm/kg). • Urine (random specimen) : 1 – 112 pg/mL • Normal plasma oxytocin level: • Male and non pregnant female: 1.0-1.9 µU/mL. • Females in 2 nd stage of labor: 3.1 – 5.3 µU/mL.

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