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
How do we measure ALP?
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
How do we measure albumin?
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
What is the anatomy of the GIT?
- 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
What causes gastric ulcers?
- 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
What are signs of gastric ulcers?
- 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
How does H.pylori cause ulcers?
- 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
How can we detect H.pylori infection?
- 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
How do we absorb Vit B12?
- 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
What are the signs of B12 deficiency?
- 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
What is pernicious anaemia?
- 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
What is coeliac disease?
- 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
How do we test for coeliac disease?
A duodenal biopsy is gold standard diagnosis
| - look for endomysial antibodies and TTG antibodies
37
What are tissue transglutamate antibodies?
- 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
How do we measure TTG IgA?
- Have antigen coated well
- Add specific Ab to be measured
- Add enzyme-conjugated secondary Ab
- Add substrate and measure the colour given off
39
What are endomysial Abs?
- 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
How do we look for endomysial antibodies?
- 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
What is calprotectin?
- 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
How do we measure faecal calprotectin?
- 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
