1
Q
What are the different assay types
A
Colourimetric
- Colour change
Turbidometric
- Cloudiness
Fluorometric
- Light excitement
Immunoassay
- Antibody/antigen binding
- RIA / ELISA
2
Q
Describe colorimetry
A
Chemical composition of solution can affect colour
As chemical reaction progresses degree of colour change can be related to concentration of substrate
Absorbance of light of specific wavelength for colour of interest can be used in calculation for concentration of substrate
3
Q
What are the components of an immunoassay
A
Antibodies from animal (polyclonal or monoclonal) that react with hormone in species of interest
Tracer - enzyme or radioactive tag on molecule or antibody
Detection systems - colour, light, radiation
Separation - separate tracer signals that have reacted with hormone from those that haven’t
4
Q
Describe process of an ELISA test
A
5
Q
Describe an RIA test
A
6
Q
What is chemiluminescence detection system?
A
Enzyme induced light emission rather than colour change
Common in non-specialist reference labs
7
Q
What is validation & its components
A
Validation = check the test can give correct results
Precision = how closely will the results match if we keep repeating the test
Accuracy = how far away from the real result is the tested result
8
Q
What are the components of packaging for blood tests
A
UN3373 - P650:
<50ml
3 packaging components:
- primary receptacle
- secondary packaging
- outer packing
- leak proof
- secondary and outer packing rigid
- absorbent material between primary and secondary layers
9
Q
How can lab tests be used for diagnosis
A
Reference intervals - what we expect in healthy animals
Interpretative thresholds - diagnostic cut-offs
Positive/negative tests
10
Q
Define sensitivity
A
proportion of animals with disease that yield positive test result
high sensitivity = good screening test
11
Q
Define specificity
A
proportion of animals that dont have disease that would yield negative test result
high specificity = good confirmatory test
12
Q
What groups of animals are required for testing sensitivity & specificity
A
Diseased - used to derive sensitivity
Healthy - used to derive specificity
13
Q
What are the consequences of a bad sensitivity test
A
higher false negatives
Diagnosis missed (-ve result)
May re-present, be referred
Outbreak may worsen in epidemic
Costs (financial, life, welfare, emotional)
14
Q
What are the consequences of a bad specificity test
A
higher false positives
Diagnosis when disease is absent (+ve result)
Unnecessary life long therapy (e.g. endocrine)
Unnecessary euthanasia
Costs (financial, life, welfare, emotional)
15
Q
What is prevalence/pre-test probability
A
Proportion of animals in tested population that have condition
You can affect pre-test probability in animals you choose to test:
- Widespread healthy population screen for infectious disease – low pre-test probability
- Only testing animals for disease that have several relevant clinical circumstances – high pre-test probability
16
Q
How does pre-test probability affect predicted values
A
As Prevalence goes up so does positive predictive value
As Prevalence falls negative predictive value goes up
17
Q
What are positive and negative predicted values
A
PPV = the proportion of positive results that are actually affected cases
NPV = proportion of negative results that are actually non-affected cases
18
Q
Describe positive predicted values
A
The proportion of positive results that are actually affected cases
Tell you if a positive result is reliable
Specificity has the greatest influence on PPV
- High specificity tests have good PPV even at low prevalence
Confidence to confirm
19
Q
Describe negative predicted values
A
The proportion of negative results that are actually non-affected cases
Sensitivity has the greatest influence on NPV
- High sensitivity tests have good NPV even when prevalence is high
Confidence to rule out
20
Q
Why are endocrine tests not very good and how can they be improved?
A
Endocrine systems respond to the environment physiologically in way that looks similar to pathology of the endocrine system
- e.g., Increased cortisol when stressed and increased cortisol in hyperadrenocorticism
Improved by using dynamic endocrine function tests:
- stimulate hormone production - test for hypofunction
- suppress hormone production - test for hyperfunction
21
Q
Which test(s) is best to screen for hypothyroidism and which is best to confirm hypothyroidism
A
screen = Total T4
confirm = Free T4 & cTSH
22
Q
What is the difference between plasma & serum
A
Plasma - liquid portion of blood that still contains clotting factors
Serum - liquid portion of blood that lack clotting factors due to the clotting process
23
Q
Why might you need a serum sample
A
Chemical constituents in circulation (clinical chemistry)
(not fibrinogen)
Antibodies (serology)
24
Q
How is serum collected and what tubes can be used
A
Coagulation cascade proceeds, centrifuged and liquid supernatant removed from cells
Tubes:
- plain glass/plastic
- tubes containing clotting activators
- tubes containing gel that forms a sealed barrier between cells and serum so tubes can be posted without need for additional transfer tube
25
Why might you need a whole blood sample
Counting cells (haematology)
Certain cellular chemistry
26
How is a whole blood sample collected
Use tube that contains anticoagulant to stop blood clotting so it remains liquid & complete (whole)
Anticoagulant is either dry powder coating or liquid (beware dilution effect)
27
Why might you need a plasma sample
Chemical constituents in circulation (clinical chemistry)
(including fibrinogen)
28
How is plasma collected and what tubes can be used
Tube contains anticoagulant to prevent blood clotting so blood remains liquid and complete
Centrifuged and remove liquid supernatant from cells
Other tubes:
- tubes containing beads to help mix the anticoagulant well before clotting starts
- tubes containing gel that forms a sealed barrier between cells and serum so no additional transfer tube needed
29
What are the 2 main mechanisms for in vitro anticoagulants
Calcium binding
Heparin
30
Describe how different Ca grabbers work
EDTA:
- irreversibly binds to calcium
- can be applied as dry powder to coat tube
Sodium citrate:
- forms ionic complexes with ionised Ca (iCa)
- reduces iCa below that needed for coagulation cascade
- reversible (add Ca to allow coagulation)
- often liquid in tube & needs dilution correction factor (accurate tube fill)
Potassium oxalate:
- forms insoluble complexes with Ca ions
Sodium fluoride:
- inhibits glycolysis to preserve glucose
- usually used with oxalate
31
Why can heparin tubes be used for most biochemistry
Does not bind anything out of the circulation - no ions have been removed
32
Why is it important to fill EDTA tube correctly
Osmotic effects, under-filled tubes lead to cell shrinkage
33
Why is it important to fill citrate tube correctly
Need to know how much calcium required to overcome clotting so citrate concentration in sample needs to be precisely known & depends entirely on filling to expected volume
34
Why must you remove the needle from a syringe before dispensing blood into a tube?
Cannot touch inside edges of tubes:
- cannot risk bacterial contamination from other tubes
35
What are the common reasons for clots in haematology samples?
Delayed transfer to tube so clotting already begun
Inadequate mixing with anti-coagulant
36
What blood tube is used for haematology?
EDTA
37
What blood tubes are used for clinical biochemistry?
serum
plasma
38
What blood tube is used for glucose testing?
fluoride
39
What blood tube is used for coagulation tests
citrate
40
Label the tube that promotes coagulation
41
Label the tube that impedes coagulation
42
What blood tubes promote coagulation
Plain glass
Clot activator tube (CAT)
SST (serum separation tube) with gel
43
What blood tubes impede coagulation
Sodium citrate
EDTA
Heparin
Oxalate
44
What small animal blood tube is these
serum
45
What small animal blood tube is this
EDTA
46
What small animal blood tube is this
heparin
47
What small animal blood tube is this
fluoride-oxalate
48
What small animal blood tube is this
citrate
49
What large animal blood tube is this
EDTA
50
What large animal blood tube is this
serum
51
What large animal blood tube is this
heparin
52
What large animal blood tube is this
fluoride-oxalate
53
What large animal blood tube is this
citrate
54
Label the canine pancreas & associated structures
55
What digestive enzymes are secreted by the pancreas
Trypsin and carboxypeptidase - acts on peptides/proteins
Lipase - acts on triglycerides
Amylase - acts on starch
Phospholipase - acts on phospholipids
Ribonuclease - acts on RNA
Deoxyribonuclease - acts on DNA
56
What cells are found in the Islets of Langerhans and what do they produce
Beta (β) cells produce insulin
Alpha (α) cells produce glucagon
Delta (δ) cells produce somatostatin
57
Where is somatostatin produced and what is its function
Produced by hypothalamus, stomach, intestine and pancreas (delta cells)
Suppresses insulin and glucagon secretion
Also inhibits growth hormone
58
Describe the synthesis of insulin
Secreted by the beta cells of the Islets of Langerhans
First synthesized as a preprohormone
Then converted to a prohormone called proinsulin
Intracytoplasmic pool of proinsulin
- Ready for quick release
secretion involves removal of C-peptide from proinsulin
59
Where is insulin degraded
Liver or kidney via cleavage of the two disulfide bonds
Within target cells after receptor binding
60
Describe flow of insulin (insulin kinetics)
Secreted by the pancreas
Enters veins, then into the portal system
The portal vein carries blood to the liver
Insulin acts upon the liver first
Then insulin enters the general circulation
61
describe the regulation of insulin secretion
3 mechanisms:
1. Nutrients: glucose and amino acids
2. Gastrointestinal hormones (incretins) such as gastric inhibitory peptide (GIP) & glucagon like peptide (GLP-1)
3. Autonomic nervous system
- Parasympathetic stimulates
- Sympathetic inhibits
62
What are the effects of nutrients on insulin secretion
Increased secretion when nutrients are abundant (after feeding)
Moves energy substrates into storage
Insulin = anabolic hormone
63
describe the effect of the intestinal tract on insulin secretion
Increase in GIP and GLP-1 when food reaches intestine
Causes insulin release from pancreas
In advance of nutrient absorption
64
Describe parasympathetic control of insulin secretion
Increase in parasympathetic activity via vagus nerve => stimulates insulin secretion after feeding
65
Describe sympathetic control of insulin secretion
Direct innervation (sympathetic neurones)
Indirect response via adrenaline
Stress response: hyperglycaemia
Insulin secretion and action are inhibited
66
What are the 2 phases of insulin secretion
First phase: release of intracytoplasmic pool of proinsulin via calcium mediated exocytosis
Second phase: insulin secretion from new protein synthesis
67
Describe the process of calcium mediated exocytosis in beta cells (insulin secretion)
68
How do GLUT4 glucose transporters work
Insulin stimulates translocation of GLUT4 proteins from cytoplasmic vesicles to plasma membrane in skeletal muscle and adipose tissues
GLUT4 proteins are Insulin-responsive glucose transporters
69
Fill in the table with function and location of glucose transporter types
70
Describe the effect of insulin on the brain
Insulin-independent GLUT1 transporter takes glucose into cells based on conc gradient
No GLUT4 in brain except for in satiety and appetite centres so insulin required for glucose uptake here (this is how hypothalamus detects glucose levels)
Without insulin => increased appetite
71
Describe the effect of insulin on fat metabolism
Inhibits hormone-sensitive lipase (HSL) => decreases lipolysis
Stimulates lipogenesis (production of fatty acids from glucose)
Promotes lipoprotein lipase (LPL) => increases delivery of fatty acids in tissues
72
Describe the effect of insulin on protein metabolism
Increases AA uptake by tissues
Increases rate of transcription and translation
Inhibits catabolism of proteins
Depresses rate of gluconeogenesis within liver (inhibits enzymes and lowers AA supply from tissues)
73
What is the impact of insulin on the liver
No GLUT4 - glucose uptake by GLUT2
Inactivates liver glycogen phosphorylase (inhibits glycogenolysis)
Increases glycogen synthase activity (stimulates glycogenesis)
Promotes conversion of glucose into fats (lipogenesis)
Inhibits gluconeogenesis
74
Describe the formation & metabolism of glucagon
Secreted by alpha cells of pancreas
Synthesised as preproglucagon
Rapidly converted to glucagon
Matbolised in liver and kidneys
75
Describe the action of glucagon
Catabolic hormone
Respond to hypoglycaemia
Active between meals to maintain glucose levels (inter-prandial period)
Activated during negative energy balance (starvation)
Stimulates glycogenolysis and gluconeogensis in the liver
76
Why is glucagon secreted after a protein-rich meal?
high amino acid levels - promotes gluconeogenesis
Post-prandial hypoglycaemia protection:
- increased insulin stimulated by AAs would cause low blood glucose levels
- glucagon prevents this by releasing some glucose
77
why are both insulin and glucagon stimulated by amino acids in circulation
Insulin => decreased amino acids and glucose levels
Glucagon => protection from hypoglycaemia due to decreased glucose
78
Fill in the able showing effect of hormones on metabolism
79
Where is gastrin produced, what stimulates its release and what are its major effects
80
Why is gastrin secretion inhibited when stomach pH reaches 3
inhibited because it would cause ulceration
81
What is the clinical relevance of gastrin in cats & dogs
Tumours in gastrin secreting cells (gastrinoma) can occur in cats & dogs
Leads to excessive gastrin production…. Therefore causes vomiting, inappetence & stomach pain due to ulceration
82
Where is secretin produced, what stimulates its release and what are its major effects
83
Where is cholecystokinin CCK produced, what stimulates its release and what are its major effects
84
What are the incretins
Gastric Inhibiting Peptide (GIP)
Glucagon Like Peptide (GLP-1)
85
Where is GIP produced, what stimulates its release and what are its major effects
86
Where is GLP-1 produced, what stimulates its release and what are its major effects
produced:
- L-cells of ileum & jejunum
secretion stimulation:
- CHO, fat & protein in SI
effects:
- Promotes insulin secretion
- Suppresses glucagon driven gluconeogenesis
- Slows gastric emptying
- Promotes satiety/fullness in hypothalamus
87
Where is Ghrelin produced, what stimulates its release and what are its major effects
produced:
- stomach
secretion stimulation:
- starvation
effect:
- strong stimulant for appetite & feeding
88
Which nuclei of the hypothalamus is responsible for appetite regulation?
arcuate nucleus
89
What hormones control appetite
Leptin causes satiety (anorexigenic)
Ghrelin stimulates appetite (orexigenic)
90
What are the actions of the satiety & appetite centres in hypothalamus
Satiety centre:
- respond to high glucose levels
- inhibits eating
Appetite centre:
- responds to low glucose levels
- stimulates eating
91
What are the effects of having no leptin production/response
hyperphagic - always hungry => obesity
Hypothermic
Infertile
92
What is the effect of CCK
Released rapidly in response to a meal:
- stimulates gallbladder contraction
- secretion of pancreatic enzymes and bicarbonate
- slows gastric emptying
- inhibits gastric acid secretion
- reduces food intake
93
What are the main adipose hormones
Leptin
Adiponectin
- Improves insulin sensitivity
- Low in obesity and insulin resistant states
94
What are the renal hormones
Erythropoietin
Renin
Calcitriol
95
What is the source, stimuli & function of erythropoietin
Produced by interstitial fibroblasts of kidney
Response of hypoxia
Promotes RBC production
96
What is the source, stimuli & action of renin
Produced by juxtaglomerular cells of kidney
Respond to decreased arterial pressure
Initiate RAAs to improve/increase blood pressure and volume
97
Describe RAAS (renin-angiotensin aldosterone system)
1. low BP/BV detected
2. renin secreted from kidney in response to decreased BV to kidney or low Na levels in renal tubules
3. renin converts angiotensinogen into angiotensin 1
4. angiotensin 1 converted to angiotensin 2 by angiotensin converting enzyme (ACE)
5. angiotensin 2 --> vasoconstriction (increase BP), aldosterone release, Na reabsorption in renal tubules (increase BV), promotes thirst, increase secretion of ADH
98
what are the heart hormones/natriuretic peptides
BNP
ANP
99
What is the source, stimuli and action of BNP
source:
- ventricular cardiomyocytes
stimuli:
- stretch of ventricles caused by increased blood volume
action:
- reduces vascular resistance (lower BP)
- Affects afferent and efferent glomerular arterioles to increase hydrostatic pressure and promote GFR
- Weakens medullary conc gradient (promote water loss)
- Promotes Na loss from kidney
- Reduces aldosterone production
100
What hormones are involved in body's response to excessive blood volume, what do they do?
101
Does an increase in insulin (hyperinsulinemia) raise or lower blood concentration of the following nutrients?
a. glucose
b. amino acids
c. fatty acids
d. triglycerides
lowers all
102
complete the table detailing effects of hyperinsulinemia on carbohydrate & lipid metabolism
103
What are tumours of the islets of Langerhans called and what do they cause
insulinomas
result in hypoglycaemia
104
What are the clinical signs of hypoglycaemia shown by the:
a. central nervous system
b. musculoskeletal system
105
is insulin concentration always increased in an insulinoma
106
107
What are the causes of insulin resistance
Physiological:
- Pregnancy, stress
Pathological:
- Obesity
- Hereditary predisposition
- Concurrent diseases
- Endocrinopathies
- Hyperadrenocorticism
- Acromegaly (GH excess)
108
What is insulin resistance
the diminished ability of cells to respond to the action of insulin in transporting glucose from the blood into tissues
109
Describe the link between insulin resistance & diabetes
Insulin resistance (compensated or uncompensated by insulin)
=> Type 2 diabetes mellitus
110
What is diabetes mellitus
Relative (some produced)/absolute (none produced) insulin deficiency
111
Describe the action of diabetes mellitus
Insulin deficiency =>
- decreased tissue utilisation of glucose
- increased utilisation of AAs and fatty acids
- increased hepatic glycogenolysis
- increased hepatic gluconeogenesis
=> hyperglycaemia
112
Describe type 1 & type 2 diabetes mellitus
Type 1:
- beta-cell destruction => insulin deficiency
- immune-mediated
- no insulin produced
Type 2:
- insulin has no effect (produced but something in the way)
113
What can cause insulin production to stop? (causes of type 1 diabetes mellitus)
Pancreatectomy
Pancreatitis
Auto-immunity
Islet cell hypoplasia
Chemical toxicity
114
what causes insulin to stop working (causes of type 2 diabetes mellitus)
Progesterone
Growth hormone
Glucocorticoids
Glucagon
Catecholamines
Thyroid
Obesity
115
Describe the pathogenesis of canine diabetes mellitus
1. immune mediated (T cell) destruction of beta cells
2. pancreatitis with beta cell destruction
3. specific hormones antagonise insulin action e.g., cortisol and growth hormone => peripheral insulin resistance
116
Describe the counter regulatory hormones evoked in hypoglycaemia in dogs
Cortisol - hyperadrenocorticism
GH - acromegaly
Catecholamines
Glucagon - glucagonoma
Progesterone => GH production
117
What are the causes of diabetes mellitus in cats?
Obesity/diet-induced insulin resistance
Islet amyloidosis
Pancreatitis
Insulin-antagonistic drugs (glucocorticoids)
Insulin-antagonistic disease (acromegaly)
Genetics
118
What are the common types of diabetes mellitus in cats and dogs
Cats - Type 2
Dogs - type 1
119
Why does obesity lead to insulin resistance
Inadequate number of insulin receptors
Defective insulin receptor structure
Defects in cell signaling pathway
Defective GLUT4 transport proteins
Problems with translocation of GLUT4 to the membrane
Interference with the function of GLUT4
120
What is amyloid polypeptide/amylin and how does it cause diabetes mellitus?
Co-secreted with insulin by feline beta cells
121
What are the types of diabetes mellitus when classified by therapy requirements
Insulin-dependent diabetes mellitus (IDDM) - Type 1
Non-insulin dependent diabetes mellitus (NIDDM) - Type 2
122
Describe insulin-dependent diabetes mellitus
Type 1 DM
Most common form of diabetes
~100% in dogs
50-70% in cats
Permanent insulin deficiency
Animal needs exogenous insulin
123
Describe non-insulin dependent diabetes mellitus
Type 2 DM
Common in cats - obesity induced insulin resistance
Dogs (rare):
- insulin-antagonism
- drugs - glucocorticoids, progestogens
- conditions - dioestrus
124
Describe equine metabolic syndrome
A strong link with obesity/regional adiposity
The primary disorder in EMS is insulin resistance
- For diagnosis most important feature is high insulin, not high glucose
Most common clinical signs is laminitis
High levels of insulin and glucose are seen in ponies with EMS
125
What are common clinical signs of diabetes mellitus
Polyuria (PU)
Polydipsia (PD)
Polyphagia (PP)
Weight loss
126
why does diabetes mellitus cause PU/PD
Osmotic diuresis:
- Blood glucose exceeds ‘renal threshold’
- Glucose acts as osmotic particle
- Draws water into renal tubule => dilutes urine and increases urine volume
127
Why does diabetes mellitus cause polyphagia
Lack of insulin => glucose cannot enter satiety center in hypothalamus (GLUT4 needs insulin to work) => failure to inhibit appetite centre => polyphagia despite hyperglycaemia
128
Define polyphagia
excessive eating or appetite
129
why does diabetes mellitus cause weight loss
Insulin:glucagon ration falls => ‘starvation’ process
Continuous inter-prandial period/’starvation’ period:
- mobilisation of stores
- catabolic
- amino acids used for gluconeogenesis => increased protein breakdown => muscle wasting
130
Why does diabetes mellitus cause cataracts
1. Glucose uptake into lens
2. Normally metabolized to lactate, which diffuses out
3. Excess glucose converted into fructose & sorbitol that don’t diffuse
4. Trapped fructose and sorbitol draws water into the lens
131
Why does diabetes mellitus cause ketoacidosis
1. Glucose cannot enter cells as easily
2. Insulin/glucagon ratio favours catabolism
3. Shift to fat metabolism for energy => fatty acids => ketones build up => metabolic acidosis => vomiting, diarrhoea, anorexia contribute to dehydration
132
What diagnostic tests can be used to diagnose diabetes mellitus
persistent fasting hyperglycemia
- 14-16 mmol/L
persistent glucosuria
fructosamine
mobilisation of fatty acids from adipose tissue
- Hypercholesterolemia
- Hypertriglyceridemia
- Visible lipid in the serum/plasma
Hepatic lipidosis
- Increased liver enzymes
urinalysis
- USG often >1.025
133
How does fructosamine diagnose diabetes mellitus
Proteins in blood circulate and pick up glucose - this protein/glucose level can be measured
Fructosamine reflects previous 2-3 weeks of blood glucose
134
How can urinalysis be used to diagnose diabetes mellitus
USG >1.025 (increased by glucosuria)
Glucose
+/- ketones
135
Fill in the table comparing diabetes mellitus to stress
136
137
138
Why are ruminants particularly susceptible to ketosis
Bacteria within rumen consume all glucose from feed
Less glucose = less oxaloacetate
Less oxaloacetate => less can react with Acetyl CoA => acetyl CoA is diverted into ketone production
139
Why does obesity make ketosis worse
Obesity leads to insulin resistance => increased lipolysis/increased activity hormone-sensitive lipase (HSL) => increased fatty acids => increased oxidation of excess fatty acids => ketone bodies
140
What are the 3 ketones
acetoacetate
beta hydroxybutyrate
Acetone (removed via resp tract)
141
142
Why is metabolic acidosis harmful
=> disrupted enzyme function, impaired cellular function, resp fatigue, impaired organ function, bone demineralisation (used as buffer), shock, organ failure
143
How does diabetes mellitus affect oxaloacetate production
Increases oxaloacetate production as insulin is less effective in decreasing blood glucose levels
144
Why do dogs develop diabetic ketoacidosis
Insulin is ineffective => hyperglycaemia + glucose cannot enter cells => lipolysis => ketone bodies => ketoacidosis
145
What are the clinical signs of diabetic ketoacidosis in dogs
PU/PD
Lethargy and weakness
Vomiting
Anorexia due to inappetence
Sweet + fruity breath due to acetone being released via resp tract
Dehydration
Depression
Abdominal pain
Neurological signs e.g., ataxia, seizures
146
147
What are the physiological principles behind weight gain in hyperadrenocorticism
Increased cortisol (opposes action of insulin) => increased insulin secretion => increased storage of glucose as fat
148
What are the physiological principles behind weight gain in hypothyroidism
Lack of thyroid hormone --> slowed metabolism --> fat accumulation
149
What is the physiologic principle behind weight gain in acromegaly
Excess GH = short term antagonist of insulin
Promotes bone, cartilage and soft tissue growth
150
Which volatile fatty acids are glucogenic (used to generate glucose) and which are ketogenic (used to generate ketones)
Glucogenic = proprionate
Ketogenic = acetate + butyrate
151
What does hormone sensitive lipase do
152
Label
152
what is the effect of too many free fatty acids for the liver to process?
Excess FAs converted back into TG and stored in hepatocytes => hepatic lipidosis
152
What is the effect of too much acetyl CoA for available oxaloacetate?
Excess acetyl CoA is diverted into ketones
153
Describe the anatomy of the adrenal glands
Located next to kidneys within retroperitoneal space
Cranial aspect of the kidneys
Elongated & often asymmetrical, being moulded around neighbouring vessels
Size varies greatly & generally those of juveniles are larger than adults
153
What are the zones of the adrenal cortex and what do they produce
From outside to the inside:
Zona glomerulosa: mineralocorticoids
Zona fasciculate: glucocorticoids
Zona reticularis: androgens
153
Describe the control of glucocorticoid release
CRH is transported from hypothalamus down axons to “portal capillary bed”
CRH causes corticotrophin cells in anterior pituitary to make & release ACTH
ACTH travels through systemic circulation to adrenal glands where it stimulates synthesis of glucocorticoids, predominantly cortisol
153
How is ACTH produced
ACTH is synthesized from pro-opiomelanocortin
POMC undergoes series of post translation modifications before its proteolytically cleaved to yield various polypeptides with varying physiological actions
154
Describe the physiology of glucocorticoids
Once released, glucocorticoids are transported in blood 90% bound to plasma proteins
Bind to specific cell membrane or cytosolic receptors at their target
receptor-steroid complex is then transported to the nucleus
Resulting in altered gene expression
155
What are the actions of glucocorticoids
Stress hormone so have variety of roles in different areas of body
- Chronic stress not acute
Stimulates gluconeogenesis
Stimulates glycogenolysis
Causes proteolysis
Promotes lipolysis
156
What is the action of glucocorticoids on fat?
mobilisation from peripheral stores
157
What is the action of glucocorticoids on muscles
catabolism
158
What is the action of glucocorticoids on liver
gluconeogenesis
antagonise insulin
159
What is the action of glucocorticoids on kidney
increased GFR
Block ADH action
160
What is the action of glucocorticoids on skin
Follicular atrophy
Sebaceous gland atrophy
161
What is the action of glucocorticoids on bone?
reduce calcium levels
osteopaenia (reduced bone density)
162
What is the action of glucocorticoids on brain
hunger
thirst
163
What is the action of glucocorticoids on immune system
release neutrophils from marginated pool
down regulates immune responses
164
Describe synthesis of steroid hormones
Begins with cholesterol
Cholesterol converted into pregnenolone by P-450 side-chain cleavage enzyme
Pregnenolone converted into different corticoids according to zone of adrenal cortex
165
Describe control of mineralocorticoid (aldosterone) release
Main stimulus for aldosterone release = low BP (RAAS)
High serum potassium stimulates release
ACTH has minor role
166
What is the function of aldosterone
Plays a central role in the regulation of BP
Acts on cells of distal tubule & collecting duct to increase reabsorption of Na, Cl & hence water
Stimulates the secretion of K+ into the tubular lumen
167
What are androgens
Steroid hormones
Stimulate or control development & maintenance of male characteristics by binding to androgen receptors
Androgens are precursors for all oestrogens
Most important androgens:
- Testosterone
- Dihydrotestosterone (DHT)
- Dehydroepiandrosterone (DHEA)
- Androstenedione
168
What is hyperadrenocorticism
HAC is condition that can develop in all domestic species
Characterised by excessive production of steroid hormones, especially glucocorticoids, from adrenal cortex
Clinical signs thus relate to abnormal circulating concentrations of steroid hormones
Often called “Cushing’s disease”
169
Describe canine hyperadrenocorticism
Can be either spontaneous or iatrogenic
Due to small tumour (adrenal or pituitary)
Spontaneous disease has two forms:
1. Pituitary-dependent (PDH) 80-90% cases
- excess ACTH secretion results in bilateral adrenal hyperplasia
2. Adrenal-dependent (ADH) 10-20% cases
- 50% Adenomas, 50% carcinomas
- Independent of pituitary control - Low ACTH
170
Describe pituitary dependent hyperadrenocorticism
Microadenomas are <10mm diameter & account for majority of cases
Macroadenomas are >10mm diameter, slow growing & can produce neurological signs
Can arise from pars distalis (70%) or the pars intermedia (30%)
Normal negative feedback mechanisms fail & they continue to secrete more ACTH
171
Describe adrenal dependent hyperadrenocorticism
Unilateral adrenal enlargement which causes atrophy of contralateral side
- For compensation
Independent of ACTH control
ACTH concentration low or undetectable
- Normal gland not stimulated --> atrophy
Approx 50% will be partly calcified, regardless of tumour type
172
What is the signalment of PDH & ADH
ADH - more common in larger or older dogs
PDH - more common in middle-aged dogs, poodles, daschunds and small terriers
Females slightly more at risk of ADH
No sex predisposition for PDH
173
What are the clinical signs of hyperadrenocorticism
PU/DP
Abdominal enlargement (Pot belly)
- re-distribution of fat into abdomen
- wasting & weakness of abdominal muscles
Polyphagia
Skin changes
Hepatomegaly
Muscle wasting/weakness
- caused by protein catabolism
Lethargy
Repro changes - decrease fertility
174
Describe skin changes involved in hyperadrenocorticism
Bilaterally symmetrical alopecia:
- inhibitory effect of steroid on anogen phase
Thin skin and reduced elasticity with prominent abdominal veins:
- protein catabolism and loss of subcutaneous fat
Excessive scale and comedones
Slow wound healing:
- inhibition of fibroblast proliferation and collagen synthesis
Firm, slightly elevated plaques surrounded by erythema
Secondary pyoderma
175
Describe treatment of HAC
Trilostane
Mitotane
Adrenalectomy
Hypophysectomy (pit gland removal)
176
Describe feline HAC
Uncommon
Signalment: middle aged to older cats
Most often PDH
clinical signs:
PU/PD
polyphagia
weight loss
extreme skin fragility
pendulous abdomen
UTI’s
177
What are the cells and secretions of the pars intermedia
Melanotrophes - POMC (proopiomelanocortin)
178
What are the products of POMC & their functions
ACTH - increases corticosteroid release via adrenal gland stimulation
MSH - regulation of appetite, sexual behaviour and melanin production
CLIP - modulation of pancreatic exocrine function
beta-endorphin - behaviour
beta-lipotropin - melanin production, steroidogenesis, lipolysis
179
Why does POMC only produce ACTH in pars distalis
enzymes are not present to continue breakdown
ACTH production predominantly in pars distalis (only 2% in intermedia)
180
What is PPID
Pituitary pars intermedia dysfunction
181
Describe the pathophysiology of PPID
Pars intermedia adenoma => excessive production of POMC derived peptides
182
Describe the control of POMC cleavage
CRH (corticotrophin releasing hormone) and ADH (arginine vasopressin) stimulate POMC cleavage
Dopamine inhibits POMC cleavage
183
Why do horses with PPID not have high levels of cortisol?
Pars intermedia does not produce biologically active form of ACTH to act on adrenal glands
184
Describe the pathogenesis of PPID
Lack of inhibitory control of pars intermedia cells via dopamine permits development of adenomas
184
What can occur secondary to PPID
Growth of pars intermedia => compression of pars distalis and nervosa => clinical signs relating to other areas of pituitary damage
185
What is the action of administering D2 agonists to a horse with PPID
Increases inhibition of POMC cleavage (D2 = dopamine receptor):
- reduced clinical signs
- decrease ACTH conc
186
What are the clinical signs of PPID
Hypertrichosis (excessive hair growth)
Weight loss/wastage
PU/PD
Laminitis
Lethargy
Blindness
Impaired response to infection
Infertility/abnormal oestrus cycle
187
Why does PPID cause hypertrichiosis
Chronic elevation of MSH
Pituitary compression on hypothalamic thermoregulatory centre (prevent shedding in warm seasons)
Increase production androgens
188
Why does PPID cause laminitis
persistent hyperinsulinaemia
189
Why does PPID cause PU/PD
pituitary compression induces decreased secretion of ADH
190
Why does PPID cause weight loss
Makes body use protein as energy source
191
Why does PPID cause lethargy
release beta-endorphin
192
Why does PPID cause blindness
compression of optic chiasm
193
Why does PPID cause impaired response to infection
increased concentration of immunosuppressive hormones
194
why does PPID cause abnormal oestrus cycle/infertility
Compression of pars distalis or hypothalamus => abnormal release of gonadotrophs
195
Describe the epidemiology of PPID
common in older horses
Ponies are predisposed
196
What suggests PPID in clinical exam?
a. history
b. physical examination
a:
- age
- persistent laminitis episodes
- recurrent infections
b:
- muscle waste
- coat
- fat deposits
197
What tests can be used to diagnose PPID
Resting ACTH:
- collect blood in EDTA tube
TRH stimulation test
POMC - disproportionally higher than ACTH if positive
Diagnostic imaging (CT, MRI to identify pituitary enlargement)
Comorbidities - evaluate insulin resistance in horses with laminitis
198
Why is diagnosis of HAC difficult
PDH, ADH and psychological stress, chronic illness all cause an increase in cortisol
199
What biological abnormalities would make you suspicious of HAC
Elevated:
- ALP
- ALT
- Cholesterol
- Bile acids
Reduced:
- urea
200
What complete blood count changes would make you suspicious of HAC
Neutrophilia (high neutrophils)
Lymphopenia (low lymphocytes)
201
What USG would make you suspicious of HAC
Low USG (<1.015 or hyposthenuric/<1.008)
Evidence of UTI
202
What radiographic findings would make you suspicious of HAC
203
What ultrasonographic findings would make you suspicious of HAC
Hyperplastic adrenals with normal echogenecity
Both enlarged = PDH
One enlarged = ADH
204
What screening tests are used for diagnosing HAC
Urinary cortisol:creatinine ratio
ACTH stimulation test
Low dose dexamethasone suppression (LDDS) test
205
Describe the use of cortisol:creatine ratio to diagnose HAC
Easy to perform
Owner collects a urine sample in the morning at home
Low ratio makes HAC extremely unlikely i.e. highly sensitive (c. 100%)
High ratio means animal could have HAC, but it is also elevated in many other diseases i.e. low specificity
Used to rule out disease
206
Describe ACTH stimulation to diagnose HAC
Starved overnight
Injection of exogenous ACTH results in increased cortisol release from adrenal gland
=> exaggerated raise in cortisol in dogs with HAC
Normal result:
- Pre-stim <200 nmol/l; post stim < 600 nmol/l
‘Positive’ result:
- Post stimulation > 600 nmol/l
OK sensitivity - don’t exclude HAC if negative
Best specificity of HAC tests - few false positives
207
Describe LDDS test in diagnosing HAC
Starve overnight
Injecting dexamethasone (exogenous glucocorticoid - acts in same way as cortisol) => reduced ACTH due to -ve feedback
Cortisol measured at 3 and 8 hrs after
Suppressed cortisol from adrenal gland expected if healthy
- may be suppressed in PDH
No/less suppression in HAC
More sensitive - fewer false negatives
lower specificity - more false positives
208
What are the treatment options for dogs with ADH
Normally resistant to medical management
Adrenalectomy is option
209
What methods can be used to differentiate between ADH & PDH
High dose dexamethasone suppression test (HDDS)
Endogenous ACTH
Adrenal imaging
Pituitary imaging
210
How can a HDDS test be used to differentiate between ADH & PDH
PDH - HD dexamethasone lowers ACTH => lowers cortisol (not all cases so not a recommended test)
ADH - HD dexamethasone has no effect on ACTH as it is already low => no change in cortisol
211
How can endogenous ACTH be used to differentiate between ADH & PDH
ADH - low ACTH
PDH - high ACTH (can fall in normal range)
Used to diagnose ADH
212
How can adrenal imaging be used to differentiate between ADH and PDH?
PDH - symmetrically enlarged and normal conformation
ADH -one enlarged gland, one atrophied gland
213
How is decrease in blood pressure detected in kidney
Baroreceptors in wall of the afferent arteriole
Cells of macula densa in early distal tubule which are stimulated by reduction in NaCl delivery
Cause renin release
214
What are the classifications of hypoadrenocorticism
1. Primary hypoadrenocorticism
- Addison’s disease
- Loss of adrenal cortex
2. Secondary hypoadrenocorticism
- Deficiency of ACTH
- Rare
3. Iatrogenic hypoadrenocorticism
- Exogenous steroids
215
Describe primary hypoadrenocorticism (Addison's disease)
deficiency of glucocorticoids (cortisol) & deficiency of mineralocorticoids (aldosterone)
occurs with loss of 85-90% of adrenal cortex
216
What are the causes of primary hypoaftenocorticism (Addison's disease)
Causes in dogs:
- Idiopathic atrophy: probably immune-mediated destruction
- Iatrogenic:
* drugs: mitotane, trilostane
* surgery: bilateral adrenalectomy
217
Describe secondary hypoadrenocorticism
Deficiency of ACTH
Disease of pit gland
Only effect glucocorticoids, not mineralocorticoids
218
Describe iatrogenic hypoadrenocorticism
Exogenous steroids → adrenal atrophy
Cortisol deficiency only
Patient may have signs of Cushing’s syndrome
Patient may develop signs of Addison’s disease if steroids abruptly discontinued
Can be life-threatening
219
Describe signalment of canine hypoadrenocorticism
Young-middle aged dogs.
70% females
Any breed, but certain breed predispositions
Extremely rare in cats
Breeds with increased risk of hypoadrenocorticism include:
- Standard poodles
- Bearded collies
- Portuguese water dog
- Leonberger
- Great Dane
- Rottweiler
- Soft coated wheaten terrier
220
Describe the pathophysiology of hypoadrenocorticism
Aldosterone deficiency:
- loss of Na+, Cl- and H2o
- retention of K+ and H+
- pre-renal renal failure
Glucocorticoid deficiency:
- decreased stress tolerance
- GI signs
- weakness
- inappetance
- anaemia (effect of cortisol of erythropoiesis)
- impaired gluconeogenesis
221
Describe the history and clinical signs of chronic hypoadrenocorticism
Vague and non-specific signs worsened by stress - appear normal between bouts
Anorexia
Vomiting
Diarrhoea
PU/PD (Na and water loss)
Weakness
Lethargy
Depression
222
Describe the history and clinical signs of acute hypoadrenocorticism
Recent history of vomiting and diarrhoea
Signs caused by hypovolaemic shock:
- collapsed or very weak
- bradycardia (due to hyperkalaemia)
- abdominal pain
223
Describe the clinical pathology of the complete blood count in hypoadrenocorticism
Anaemia:
- decreased erythrocytosis due to lack of cortisol
- GI blood loss
224
Describe the clinical pathology of hypoadrenocorticism: biochemistry
Electrolyte imbalance:
- hyperkalaemia (increased K+)
- hyponatraemia (Decreased Na+)
- hypochloridaemia (decreased Cl-)
- Na:K ratio <23
Due to aldosterone deficiency:
- decreased renal tubular resorption of Na+ and Cl-
- decreased secretion of K+ and H+ (likely acidoxic)
225
Describe the clinical pathology of hypoadrenocorticism: urinalysis
Azotaemia (increased urea and creatinine)
Hypoglycaemia
Decreased USG (Na in filtrate impairs ability to retain water)
226
Describe the electrocardiogram of an animal with hypoadrenocorticism
Changes related to hyperkalaemia:
- bradycardia
- peaked T waves
- widened QRS complexes
- decreased P wave amplitude
- absent P waves
- ventricular asystole (failure to contract)
227
How can hypoadrenocorticism be diagnosed
ACTH stim test - no change in cortisol as adrenal cortex is destroyed
228
Describe the treatment of hypoadrenocorticism
Mineralocorticoids - monthly injection
Glucocorticoids during acute crisis or times of stress
229
Does this animal have HAC? if so which form?
yes
most likely PDH
230
Does this animal have HAC? if so which form?
PDH
231
advantages & disadvantages of using radiographs to diagnose endocrinopathies
232
advantages & disadvantages of using ultrasounds to diagnose endocrinopathies
233
advantages & disadvantages of using computed tomography to diagnose endocrinopathies
234
advantages & disadvantages of using MRI to diagnose endocrinopathies
235
What are the anatomical landmarks used to localise left & right adrenal gland using ultrasound
236
What size are normal adrenal glands in dogs & cats
237
238
what abdominal radiographic abnormalities are seen in canine HAC
hepatomegally
+/- adrenal mass
large bladder
+/- cystoliths
dystrophic mineralisation of soft tissues
osteopenia (thinning bones)
distinct organs due to contrast provided by intraabdominal fat
239
What signs of HAC can you see?
240
What are the abnormalities in this dog with adrenal-dependent HAC
241
where are the parathyroid glands in cats & dogs
superior are on dorsal surface of thyroid glands (near base of larynx & trachea)
inferior are on ventral surface of thyroid glands
242
What are the clinical signs of hypoparathyroidism
Muscle weakness
Dysphagia
Impaired mental function
Fatigue
Seizures
Weaker/brittle keratin formation
Hypocalcaemia
Hyperphosphataemia
243
Why is a adrenalectomy difficult
Presence of fat, major BVs and neural plexus
244
What is EMS (equine metabolic syndrome)
Insulin resistance develops due to obesity => hyperglycaemia => laminitis
245
What are the diagnostic tests for PPID?
Basal ACTH
TRH stim test
246
What are the diagnostic tests for EMS?
basal insulin/glucose measurement
Oral glucose challenge test
Combined glucose and insulin test
247
How do you calculate each value
248
What are the 3 stages of insulin resistance
249
How does EMS cause laminitis
1. Hyperinsulinaemia
2. Endothelial cell dysfunction
3. Digital vasoconstriction
4. Impaired glucose uptake from epidermal laminar cells
5. Altered epidermal cell function or mitosis
6. Matrix metalloproteinase activated
7. Pro-inflammatory state in lamellar tissue
250
Describe resting insulin and glucose measurement in EMS diagnosis
High resting insulin suggests insulin resistance
Low insulin + high glucose = T2 diabetes
Affected by many factors e.g., stress, season
Little sensitivity and specificity
251
Describe the oral glucose challenge test in EMS diagnosis
Fasted overnight
Given oral glucose powder
Insulin measured at 2hrs
High insulin indicative of insulin resistance
252
Describe combined glucose-insulin testing for EMS diagnosis
Fasted overnight
Obtain basal glucose & insulin
Glucose and insulin given via IV
Normal =
- insulin <100 IU/ml at 45 mins
- Glucose below baseline within 30-45 mins
prolonged hyperglycaemia suggests IR
High insulin at 45min suggests exaggerated insulin response
253
How can we differentiate between PPID & EMS
254
Why is it important to differentiate between EMS & PPID
255
Label the sections of the brain
256
Label the pars distalis
257
Label the pituitary gland
258
What pneumonic can you use to remember the adrenal cortex secretions
259
Label the zones of the adrenal cortex
Leah ENI
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