Unit 2: Qualitative screening and diagnostic enzymes
Clinical Biochemistry-II notes · PTU syllabus (BMLS505-18)
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Unit summary
Screening tests and enzyme assays detect disease in many organs. This unit covers qualitative tests for urobilinogen, barbiturates, T3, T4, TSH and 17-ketosteroids, and the principles, clinical significance and estimation of acid phosphatase, alkaline phosphatase, LDH, AST, ALT and creatine kinase.
After this unit you can
- Perform qualitative tests for urobilinogen and barbiturates
- Interpret thyroid function tests and 17-ketosteroids
- Estimate and interpret phosphatases and transaminases
- Estimate and interpret LDH and CK
PTU syllabus topics
- Qualitative tests for urobilinogen
- barbiturates
- T3/T4/TSH and 17-ketosteroids
- principles
- clinical significance and estimation of acid phosphatase
- alkaline phosphatase
- lactate dehydrogenase
- aspartate transaminase
- alanine transaminase and creatine phosphokinase
ALT
Liver
Hepatitis, liver damage
AST
Liver, heart, muscle
Liver disease, muscle injury
ALP
Liver (bile ducts), bone
Obstructive jaundice, bone disease
LDH
Many tissues
Haemolysis, tissue damage
CK (CK-MB)
Muscle, heart
Muscle injury; CK-MB in heart attack
Topic 1
Urobilinogen
- Ehrlich's test: fresh urine with Ehrlich's aldehyde reagent (p-dimethylaminobenzaldehyde in HCl) gives a cherry-red colour with urobilinogen (normal: pink in 1 : 20 dilution or less). Increased: haemolytic jaundice, liver disease; absent: complete biliary obstruction. Porphobilinogen also reacts — distinguished by the Watson–Schwartz test (chloroform extraction).
Topic 2
Barbiturates
- Screening: colour tests (cobalt acetate–isopropylamine — violet colour, Zwikker reaction), and modern immunoassay strips for drugs of abuse; confirmation by TLC, GC–MS or HPLC. Used in suspected overdose.
Topic 3
Thyroid function tests
Primary hypothyroidism
High
Low
Subclinical hypothyroidism
High
Normal
Primary hyperthyroidism (Graves')
Low (suppressed)
High
Subclinical hyperthyroidism
Low
Normal
Secondary (pituitary) hypothyroidism
Low or normal
Low
- Methods: chemiluminescence immunoassays (CLIA), ELISA, earlier RIA; free T4 and free T3 preferred over total. Reference (approx.): TSH 0.4–4.5 mIU/L; free T4 0.8–1.8 ng/dL. Neonatal screening for congenital hypothyroidism by heel-prick TSH.
Topic 4
17-Ketosteroids
- Urinary 17-ketosteroids are metabolites of adrenal androgens (DHEA) and testosterone; measured in 24-hour urine by the Zimmermann reaction (m-dinitrobenzene in alkali → purple). High: congenital adrenal hyperplasia, adrenal tumours, Cushing's syndrome; low: Addison's disease, hypopituitarism. Now largely replaced by serum DHEA-S and 17-hydroxyprogesterone.
Topic 5
Phosphatases
Optimum pH
About 10
About 5
Sources
Liver (bile canaliculi), bone (osteoblasts), placenta, intestine
Prostate, red cells, platelets, osteoclasts
Method
p-Nitrophenyl phosphate → yellow p-nitrophenol at 405 nm (kinetic); older King–Armstrong
Same substrate at acid pH; tartrate inhibits prostatic ACP
Reference
Adults about 40–130 U/L; higher in children (bone growth) and pregnancy
Total up to about 5 U/L
Raised in
Obstructive jaundice, bone disease (Paget's, rickets, osteoblastic metastases)
Prostate cancer with bone spread (now replaced by PSA), Gaucher disease
Topic 6
Transaminases (AST and ALT)
- Principle (IFCC kinetic): AST transfers an amino group from aspartate to α-ketoglutarate forming oxaloacetate, reduced by malate dehydrogenase while NADH falls at 340 nm; ALT forms pyruvate, reduced by LDH with NADH consumption. Older method: Reitman–Frankel (colorimetric with DNPH).
Location
Heart, liver, muscle, red cells; cytoplasm and mitochondria
Mainly liver cytoplasm — more liver-specific
Reference
Up to about 40 U/L
Up to about 40 U/L
Very high levels
Acute viral or toxic hepatitis, shock liver
Same
Ratio
AST/ALT above 2 suggests alcoholic liver disease
ALT higher in viral hepatitis and fatty liver
- Haemolysis falsely raises AST.
Topic 7
Lactate dehydrogenase (LDH)
- Catalyses lactate ⇌ pyruvate with NAD⁺/NADH; measured kinetically at 340 nm. Reference: about 140–280 U/L.
- LDH-1 (H4)
- Heart, red cells — LDH-1 above LDH-2 ("flipped" pattern) after myocardial infarction
- LDH-2
- Heart, red cells
- LDH-3
- Lungs
- LDH-4 and LDH-5 (M4)
- Liver and skeletal muscle
- Raised: haemolysis (and haemolysed samples), megaloblastic anaemia, liver disease, lymphomas and leukaemias (tumour marker), muscle injury.
Topic 8
Creatine kinase (CK)
- Catalyses creatine phosphate + ADP ⇌ creatine + ATP; measured by coupled enzyme reactions with NADPH at 340 nm (IFCC). Reference: men about 40–200 U/L.
CK-MM
Skeletal muscle
Muscular dystrophy (very high), rhabdomyolysis, injections, exercise
CK-MB
Heart muscle (about 20–30% of myocardial CK)
Myocardial infarction — rises 4–6 hours, peaks 18–24 hours, normal by 48–72 hours (reinfarction detection)
CK-BB
Brain, smooth muscle
Rarely measured
- Cardiac troponins have replaced CK-MB as the preferred marker of myocardial infarction.
Key terms
- Ehrlich's reagent
- Reagent detecting urobilinogen
- TSH
- Pituitary hormone, the best screening test of thyroid function
- 17-ketosteroids
- Urinary metabolites of androgens
- Isoenzymes
- Different molecular forms of the same enzyme
- CK-MB
- Cardiac isoenzyme of creatine kinase
Quick revision
- Ehrlich's test; barbiturate screening and confirmation.
- TSH, free T4, free T3 patterns; neonatal TSH.
- 17-ketosteroids by Zimmermann reaction.
- ALP and ACP sources, methods, ranges, causes of rise.
- AST, ALT (kinetic methods, ratio); LDH isoenzymes; CK isoenzymes and timing; troponin.
Important exam questions
Practice questions written to the PTU exam pattern for this unit's syllabus: short answers (Section A style) and long answers (Sections B and C style).
Short-answer questions
- Q1.What colour does Ehrlich's test give with urobilinogen?
- Q2.Give the thyroid pattern in Graves' disease.
- Q3.Name the reaction used for 17-ketosteroids.
- Q4.Why is ALP high in children?
- Q5.Which transaminase is more liver-specific?
- Q6.When does CK-MB peak after a heart attack?
Long-answer questions
- Q1.Describe thyroid function tests and their interpretation.
- Q2.Describe the estimation and clinical significance of phosphatases.
- Q3.Describe transaminases and LDH in diagnosis.
- Q4.Describe creatine kinase and its isoenzymes.
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