Lab investigation of endocrine disorders Flashcards
1
Q
What are the main functions of the liver?
A
- Carb metabolism (glycogenesis, glycogenolysis, gluconeogenesis)
- Fat metabolism (cholesterol synth and bile acid production)
- Protein metabolism (transamination)
- Synthesis of plasma proteins (albumin etc)
- Hormone metabolism (IGF-1, angiotensinogen)
- Metabolism and excretion of drugs and foreign compounds
- Storage (glycogen, vit A, B12, plus iron and copper)
- Metabolism and excretion of bilirubin
2
Q
What is the blood supply for the liver?
A
- 2/3 comes from the portal vein (from gut, rich in nutrients)
- 1/3 from the hepatic artery (rich in oxygen)
- Blood leaves through the hepatic veins
3
Q
What is the structure of the liver?
A
- Each lobe has multiple liver lobules -
hexagonal plates of hepatocytes radiating from a central vein, carrying blood from the liver - Each lobule further divided into acini
- Each acini is supplied by the portal triad (hepatic artery proper, hepatic portal vein and the bile duct)
- Substances for excretion are secreted from hepatocytes into canaliculi
- Bile canalicli merge to form bile ductules, which merge to become the bile duct, and eventually the common hepatic duct
- Excess bile is stored in gall bladder or secreted into duodenum via sphincter of oddi
4
Q
What is the process of bilirubin metabolism?
A
- in spleen, reticuloendothelial cells break Hb into haem and globin. Haem is further broken down into iron and bilirubin
- bilirubin is insoluble, so binds to albumin (unconjugated bilirubin = 95%)
- makes its way to the liver and taken up by hepatocytes
- Bilirubin taken off and converted by UDP-glucuronyl transferase
- Excess soluble bilirubin is secreed into duodenum, then converted to urobilinogen
- This can either be taken up by gut through portal vein to liver, or excreted after production of sterobilin
5
Q
What lab investigations can we do for bilirubin?
A
- An increase in total bilirubin = hyperbilirubinaemia
- Total bilirubin = un/conjugated bilirubin (and delta)
- Direct = conjugated bilirubin (and delta)
- Indirect = unconjugated bilirubin (calculate rather than measure)
6
Q
What is delta bilirubin?
A
- iireversible binding of bilirubin to albumin and so cannot be excreted
- occurs in the presence of prolonged conjugated hyperbilirubinaemia
7
Q
How do we measure direct bilirubin in blood?
A
- Diazo method
- Conjugated bilirubin (and delta) react directly with a diaznoium ion in an acidic membrane
- The colour intensity of the red azobilirubin dye is directly proportional to direct bilirubin conc when measured at 546nm
8
Q
How do we measure total bilirubin in blood?
A
- Diazo method
- Same as the direct bilirubin method, but an accelerating agent (alcohol/caffiene/sodium benzoate) is used in a strongly acidic medium, causing dissociation of uncojugated bilirubin from albumin
9
Q
How can we measure bilirubin in urne?
A
- Simple dipstick method
- As unconjugated bilirubin is protein bound - not normally found in urine. Thus the presence of bilirubin in the urine will turn the urine a brown colour
- Seen in cases of hepatitis or impaired flow of bile in patients with biliary obstruction
10
Q
How do we measure urobilinogen in urine?
A
- If there is urobilinogen in the urine, it demonstrates that bilirubin is reaching the gut - detected by dipstick
- Excess urobilinogen in urine may indicate liver disease such as viral hepatitis and cirrhosis or hameolytic conditions associated with increased RBC destruction
11
Q
How can you tell if bilirubin is in faeces?
A
- Stools appear pale in colour as there is no stercobilin
12
Q
What is jaundice?
A
Yellow discolouration of tissue due to bilirubin deposition
13
Q
What causes jaundice?
A
- Haemolysis - increased bilirubin production - Acquired autoimmune haemolytic jaundice, drug induced and spherocytosis
- Hepatocellular damage (impaired bilirubin metabolism) - toxins or infections
- Cholestasis (decreased bilirubin excretion) - cirrhosis, tumours or gallstones
14
Q
What are LFTs?
A
- Insensitive indicators of hepatic function, but can be highly sensitive indicators of liver damage
- Rarely provide diagnosis on their own
- Usually includes Total bilirubin, ALT, ALP, Gamma GT and albumin
15
Q
What is ALT?
A
- Alanine aminotransferase
- IC cytoplasmic enzyme that catalyses the transfer of an amino group from alanine to 2-oxyglutarate
- Most specific marker for liver injury (with GGT)
- ALT is also expressed by kidneys, and cardiac and skeletal muscle - so better to look at all LFTs together
- Used to identify liver damage arising from hepatocyte inflammation of necrosis
16
Q
What is AST?
A
- Aspartate aminotransferase
- An IC cytoplasmic and mitochondrial enzyme catalysing the transfer of an amino group from aspartate to 2-oxyglutarate
- Less liver specific than ALT, little use in measuring both enzymes
- Only indication of measuring both ALT and AST is to determine the AST:ALT (<0.8 suggests non-alcoholic fatty liver disease, >1.5 it is more likely to be alcoholic liver disease)
17
Q
What is GGT?
A
- Gamma glutamyl transferase
- Membrane bound enzyme that transfers gamma glutamyl group from peptides like glutathione to other peptides and to L-amino acids
- Relatively specific marker for liver injury as it is found on the canalicular membrane of hepatocytes
- Increases in GGT are looked at in conjunction with the ALP result
18
Q
What is ALP?
A
- Alkaline phosphatase
- membrane bound glycoprotein enzyme that removes phosphate molecules from proteins and nucleic acids; maximum activity of pH 9-10.5
- Found in a number of tissues, greatest concentrations in bone, liver, intestine and placenta
- Of major value in the diagnosis of cholestatic disease along with GGT - cholestasis stimulates enhanced synthesis of liver ALP (enzyme induction)
- Elevated in children and correlates well with rate of bone growth, also increased in pregnant women due to placenta
19
Q
How do we interpret different ALP and GGT results?
A
- Increased both - suggestive of hepatic cause (usually due to cholestasis)
- Increased ALP and normal GGT - suggestive of bone source of ALP
- Normal ALP and increased GGT - suggestive of excess alcohol intake
20
Q
How can we determine the source of elevated ALP?
A
- Electrophoresis
- Can separate ALP isoenymes into liver, bone and intestinal fractions based on their charge
- Unlike others, placental isoenzyme of ALP can be indentified as it is heat stable at 36C for 10 mins
21
Q
What is albumin?
A
- Essential plasma protein - maintains plasma oncotic pressure (to stop leakage from vessels) and binds several hormones, drugs, anions and FAs
- Crude indicator of the synthetic capacity of the liver due to its long half-life and because its levels can be creased by the acute phase response
22
Q
How can we measure ALT?
A
Measure its catalytic activity rather than its mass
- L-Alanine + 2-oxyglutarate –> ALT –> pyruvate + L-glutamate
- Pyruvate + NADH + H+ –> Lactate dehydrogenase –> L-lactate + NAD+
- The rate of NADH oxidation is directly proportional to the catalytic ALT activity. NADH oxidation is determined by measuring its decrease in absorbance at 340nm
23
Q
How do we measure AST?
A
- Assayed the same way as ALT, with alanine being replaced with aspartate and oxaloacetate being produced rather than pyruvate
24
Q
How do we measure GGT?
A
GGT to catalyse the transfer of a gamma- glutamyl group from the donor L-γ-glutamyl-3-carboxy-4-nitroaniline to a glycine acceptor. This reaction yields 5-amino-2-nitrobenzonate, which absorbs at 415 nm (Theodorsen reaction). The rate of formation of 5-amino-2-nitrobenzonate is directly proportional to the activity of GGT in the sample
25
Q
How do we measure ALP?
A
The cleavage of p-nitrophenyl phosphate by ALP yield phosphate and p-nitrophenol. The p-nitrophenol released absorbs at 450 nm, which is directly proportional to the catalytic activity of ALP.
26
Q
How do we measure albumin?
A
Dye binding assay: at a pH of 5.2-6.8 Bromcresol purple (BCP) binds with albumin, causing a change in absorbance that is measured at 600 nm.
27
Q
What is the anatomy of the GIT?
A
- 7-10m continuous tube that runs from the mouth to the anus
- Encased in layers of voluntary and involuntary muscle
- Effective digestion and absorption requires a continuous modification of gut contents
- hormone signalling allows different parts of the GIT to switch on and switch off - Enteric endocrine system
28
Q
What causes gastric ulcers?
A
- Caused by a break in the protective mucosal lining of the stomach, allowing acid to damage the underlying epithelium
- 80% caused by H.pylori infection
- 20% caused by use of NSAIDs
29
Q
What are signs of gastric ulcers?
A
- Pain in the abdomen - may come and go
- Waking up with pain in abdomen
- Bloating, retching and feeling sick
- Feeling particularly full after normal sized meal
30
Q
How does H.pylori cause ulcers?
A
- Flagellum allows it to burrow through the mucus to the epithelial cells using corkscrew motility
- It then secretes urease, which causes the neutralisation of the gastric acid around it - allowing it to survive and proliferate
- They can then damage the mucosal layer and cause epithelial damage
31
Q
How can we detect H.pylori infection?
A
- Urea (hydrogen) breath test - rapid and non-invasive procedure
- Pt swallows urea labelled with an uncommon isotope (radioactive C14 or non-radioactive C13)
- The detection of the isotype-labelled CO2 in exhaled breath indicates that the urea was split, indicating that urease is present, and so H.pylori is present
32
Q
How do we absorb Vit B12?
A
- Cannot be produced by our body, so we have to get it in diet
- B12 is water soluble vitamin that has an essential role in the NS and the formation of RBCs - co-factor for DNA synthesis
- B12 enters the stomach and binds to IF (released by parietal cells of stomach)
- The B12-IF complex enters the intestine where it binds to receptors on the mucosal cells of the ileum, and is absorbed into the blood stream
33
Q
What are the signs of B12 deficiency?
A
- Liver can store 3-5 yrs worth of B12, so takes a long time to present
- Macrocytic anaemia (increased MCV, decreased Hb)
- Weakness and tiredness
- pale skin
- Glossitis
- Nerve problems (numbness/tingling)
34
Q
What is pernicious anaemia?
A
- Severe B12 deficiency is usually caused by pernicious anaemia, an autoimmune attack on the gastric mucosa
- Leads to atrophy of the stomach wall and the secretion of IF is absent or almost absent
- Other patients produce Abs to IF, blocking the IF-B12 complex from being absorbed in the ileum
- If anti-IF Ab is present, pernicious anaemia is very likely, but its absence doesnt rule it out
35
Q
What is coeliac disease?
A
- Autoimmune disorder, primarily affecting the small intestine
- Results from immunological hypersensitivity to ingested gliadin (gluten protein) found in wheat, barley and rye
- 1/100 people in the UK
- Classic symptoms of coeliac disease include GI problems such as diarrhoea, abdominal distention, malabsorption and loss of appetite
- Upon exposure to gluten in the small intestine, there is an inflammatory reaction leading to the shortening of the villi lining (villus atrophy)
36
Q
How do we test for coeliac disease?
A
A duodenal biopsy is gold standard diagnosis
- look for endomysial antibodies and TTG antibodies
37
Q
What are tissue transglutamate antibodies?
A
- TTG is an enzyme that deaminates glutamine residues to glutamic acid on the gliadin fragment
- The enzyme is a target autoantigen in the immune response seen in coeliac
- IgA antibodies to TTG are found in 96% of coeliac patients
- Also test for IgG antibodies to TTg as 1/250 people are deficient in IgA
38
Q
How do we measure TTG IgA?
A
- Have antigen coated well
- Add specific Ab to be measured
- Add enzyme-conjugated secondary Ab
- Add substrate and measure the colour given off
39
Q
What are endomysial Abs?
A
- The endomysial is the supporting structure that surrounds the middle third of oesophagus - endomysial antibodies are indicative of coeliac
- The test isnt as sensitive as TTG antibody test
40
Q
How do we look for endomysial antibodies?
A
- Diluted serum/plasma samples are incubated on monkey oesophagus tissue
- Slide is washed and fluorescein-labelled antibodies against IgA and IgG is added
- Excess Ab is removed and slide read under microscope
41
Q
What is calprotectin?
A
- Small calcium-binding protein that contributes to ~60% of the cytosolic protein content in neutrophils
- In the presence of active intestinal inflammation (like in IBD) neutrophils migrate to the intestinal mucosa from circulation
- Any disturbance in mucosa architecture due to inflammation results in leakage of neutrophils, and hence calprotectin is excreted in faeces
42
Q
How do we measure faecal calprotectin?
A
- Use fluorescence sandwich immunoassay
- Homogenise 100mg of faecal material
- Use capture Ab, add faeces, if calprotectin present then it will stick, use detector Ab to stick to calprotectin with fluorescent label
