BIOCHEMICAL MARKERS IN LIVER

DISEASES
Presentation by:
Dr. Petrescu Elena
Dr. Stanescu Raluca

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 Functions of the liver
1. Carbohydrate metabolism
•Glycogen synthesis and breakdown
•Gluconeogenesis
•Maintenance of blood glucose level during fasting

2. Lipid metabolism
•Fatty acid and triglyceride synthesis
•Cholesterol synthesis
•Lipoprotein synthesis (VLDL, HDL)
•Ketogenesis
•Bile acid synthesis

3. Protein metabolism
•Synthesis of plasma proteins - including albumin, most coagulation factors
•Urea synthesis

4. Detoxification and excretion

•Detoxification of xenobiotics
•Metabolism and excretion of endogenous compounds: cholesterol, bilirubin, steroid hormones
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 Biochemical tests for liver disease
assessement:

• I. Hepatocellular damage

• II. Hepatic synthetic function (assessment of liver failure)

• III. Hepatic excretory function (assessment of cholestasis)

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 I. Tests for hepatocytolysis

 Aminotransferases
 Lactate dehydrogenase
 Glutamate dehydrogenease
 Ornithine carbamoyl transferase

• Liver cell membrane damage → release of enzymes into the blood

• Moderate damage → only cytosolic enzymes are released
• Severe damage (liver necrosis) → also the mitochondrial enzymes are released

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  Aminotransferases
 GPT (ALT):
• Alanine + α-keto glutarate ↔ pyruvate + glutamate
• Distribution: abundant in the liver; much smaller amounts are present in the
skeletal muscle (→ relative liver specificity)
• Cellular location: cytosol

 GOT (AST):
• Aspartate + α-keto glutarate ↔ oxaloacetate + glutamate
• Distribution: liver, myocardium and skeletal muscle, in similar proportions
• Cellular location: cytosol + mitochondria

• The de Ritis ratio: GOT/GPT = 1.3

• A rise in plasma aminotransferases activities is a sensitive indicator of liver cell
damage

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 Acute hepatitis (infectious or toxic):

• GPT and GOT increase in plasma even before the onset of clinical signs
• Their elevation is 20 to 50 fold, in severe forms even 100 fold → they remain
elevated for 1-2 weeks (GPT persists longer than GOT: t1/2 is 48-60 h for GPT and
18-24 h for GOT)
• The level of aminotransferase activity correlates with the extent of liver damage
• Moderate severity → GPT increases more than GOT (only cytosolic GOT is released)
→ de Ritis ratio ↓
• Liver necrosis → GOT increases more than GPT (GOT from the mitochondrial pool
is also released) → de Ritis ratio ↑

 Chronic hepatitis – moderate increase of aminotransferases (2-3 fold, maximum

20 fold in active chronic hepatitis)

 Alcoholic hepatitis – GOT and GPT increase 5-10 fold, the de Ritis ratio > 2
(mitochondrial GOT is released due to some degree of liver cell necrosis)

 Modest increase (2-5 fold) in: liver cirrhosis, liver cancer, cholestasis
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 Differential diagnosis:

• GPT has a higher liver specificity compared to GOT

• GOT also increases in:
- myocardial infarction
- skeletal muscle diseases

 Lactate dehydrogenase (LDH)

• Distributed in all cell types, in the cytosol
• LDH in the liver oxidizes the lactate that is taken up from blood, to pyruvate → this
will be further metabolized
• LDH4 and LDH5 increase in acute hepatitis, but to a lower extent compared to GPT
and GOT (10 fold)
• LDH5 also increases in diseases affecting the skeletal muscle:
muscle muscle dystrophy,
myositis
• LDH assay is useful to detect minor tissue damage,
damage but it has poor organ specificity
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 Glutamate dehydrogenase (GDH)
• Mitochondrial enzyme that catalyzes the oxidative deamination of glutamate,
releasing the amino group of glutamate as ammonia:
Glutamate + NAD+ + H2O ↔ α-ketoglutarate + NADH+H+ + NH3
• Distributed in many tissues (mainly in the liver, myocardium, kidney)
• Its serum level increases ≈10-fold in viral acute hepatitis
• Significant increase (up to 200-fold) in toxic hepatitis with hepatocellular necrosis
• It allows an estimate of the severity of liver disease

 Ornithine carbamoyl transferase (OCT)

• Enzyme of the urea cycle → liver specificity (located in the mitochondria)
• Its plasma level increases in acute hepatitis

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 II. Tests for the liver synthetic
function
 Albumin
 Cholinesterase
 Coagulation factors

• These tests assess the protein synthesis function of the liver

• They have different utility, according to their plasma half-life → they may reveal a
liver failure with:
- rapid onset – acute hepatitis having a fulminant course, with massive liver necrosis
- slow onset – liver cirrhosis

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1. Chronic liver failure

Albumin (t1/2 = 20 days)

•Decreases in chronic liver diseases – due to a deficiency in its synthesis, but also due
to its leakage in the ascites fluid
•It also decreases in nephrotic syndrome and in case of malnutrition
•It is not useful for the detection of acute liver failure

Cholinesterase (t1/2 = 14 days)

•It is the most sensitive marker of liver protein synthesis
•Its plasma activity decreases before and to a greater extent compared to the
decrease of serum albumin (it is a functional plasma enzyme)
•Useful for monitoring the evolution of liver cirrhosis: its progressive decrease
indicates an unfavorable evolution
•A decrease to 10% of the normal value = alert sign for hepatic coma
•It also decreases in case of:
- intoxication with organophosphoric insecticides
- genetic deficiency of cholinesterase
•It is not useful for the detection of acute liver failure 10
2. Acute liver failure

Coagulation factors
•In liver diseases - the synthesis of coagulation factors decreases
•The prothrombin time (PT) is prolonged – this is one of the earliest changes that
occur (t1/2 of prothrombin = 6 h)
•Factor VII may also be measured → it decreases in case of liver failure
•These tests are useful for the detection of a liver failure with rapid onset, but they are
also changed in chronic liver diseases

•PT may also increase due to a deficiency of vitamin K – caused by fat malabsorption
(abnormal bile production in chronic liver diseases, cholestasis)
•For differentiation – the vitamin K test: parenteral administration →
- if PT remains prolonged → liver disease
- if PT is normalized → fat malabsorption

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 III. Cholestasis syndrome
 Bilirubin
 Alkaline phosphatase
 γ-Glutamyl transferase

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  Formation and excretion of bilirubin
• Bilirubin (BR) results from heme degradation in the reticuloendothelial system,
mainly in the spleen
• BR is transported to the liver bound to albumin - this form is unconjugated
(indirect) BR, which is lipophilic →
- is not filtered by the glomeruli
- can cross cell membranes, being potentially toxic
• In the liver, BR is conjugated with glucuronic acid in a reaction catalyzed by UDP-
glucuronyl transferase
• Conjugated (direct) BR is hydrophilic → can be eliminated in the urine
• Normally, conjugated BR is secreted into the biliary tree → reaches the intestine
• In the intestine, BR is deconjugated and converted to urobilinogen (UBG - colorless)
• Part of UBG is reabsorbed in the portal vein →
- undergoes enterohepatic circulation
- passes into the systemic circulation → eliminated in the urine (< 4 mg/24 h)
• The majority of UBG is converted to stercobilin (colored) → eliminated in the feces
• In blood – 80-90% is indirect BR and 10-20% is direct BR
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 Types of hyperbilirubinemia
• When serum BR increases > 2.5-3 mg/dL → jaundice

1. Jaundice with ↑ unconjugated BR

 Overproduction of bilirubin due to excessive hemolysis (hemolytic jaundice)

jaundice
• hemolytic anemia: sickle cell anemia, hereditary spherocytosis, glucose-6-P
dehydrogenase deficiency
• hemolytic disease of the newborn (incompatibility in the Rh system)

 Deficiency of UDP-glucuronyl transferase (genetic: Gilbert’s and Crigler-Najjar

syndromes)

2. Jaundice with ↑ conjugated BR + unconjugated BR

 Hepatocellular damage (hepatocellular jaundice)

• hepatitis
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3. Jaundice with ↑ conjugated BR

Obstruction of the bile ducts (cholestatic jaundice)

jaundice
•intrahepatic bile ducts:
- liver carcinoma
- cirrhosis
- primary biliary cirrhosis - autoimmune disorder (mitochondrial antibodies) →
destruction and proliferation of the bile ducts
•extrahepatic bile ducts:
- gallstones in the common bile duct
- carcinoma of the head of the pancreas
- carcinoma of the biliary tree

Genetic syndromes
•Dubin-Johnson syndrome
•Rotor syndrome
(defect in the transfer of conjugated
BR into the biliary canaliculi)

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The investigation of jaundice

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Laboratory findings in different types of jaundice

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  Alkaline phosphatase (ALP)
• Distributed in most of the tissues, but the enzyme present in plasma originates
from the liver and bone
• Its serum level increases in biliary obstruction (stimulation of enzyme synthesis in
liver cells, especially those that are adjacent to bile canaliculi)
• The increase is more marked (5-20 fold) in extrahepatic cholestasis
• Intrahepatic cholestasis leads to a 2-3 fold increase in ALP activity

• In acute hepatitis – ALP increases just slightly, because it is anchored to the liver
cell plasma membrane

 Differential diagnosis – ALP also increases in diseases affecting the bone (it is
produced by the osteoblasts):
- in children - rickets
- in adults - Paget’s disease, osteogenic bone tumors

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  γ-Glutamyl transferase (GGT)
• The enzyme present in plasma originates mainly from the liver (it is also
distributed in the kidney, intestine, and other tissues)
• It increases in all forms of hepatobiliary diseases → it is a sensitive test to detect
these diseases
• The most significant elevations occur in biliary obstruction (5-30, even 50 fold) –
the mechanism is similar to that involved in ALP increase (increased enzyme
synthesis due to cholestasis) – it is more specific than ALP (GGT is normal in bone
diseases)
• Just small increases in hepatocytolysis (GGT is associated with the plasma
membrane of liver cells)

 Differential diagnosis:
• Significant elevations of GGT activity occur in chronic alcoholism (due to enzyme
induction by ethanol)
• It also increases in patients being on therapy with certain antiepileptic drugs
(phenytoin, phenobarbital → induction of GGT)
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BIOCHEMICAL MARKERS
IN RENAL DISEASES

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 Functions of the kidney
• 1. Excretion of metabolic waste products
• 2. Homeostatic regulation of the ECF volume and composition
• 3. Control of the acid-base balance

 Renal excretion:
• Glomerular filtration
• Tubular reabsorption
• Tubular secretion

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 I. Tests for the glomerular function
 1. Urea

• Is synthesized in the liver from ammonia, which arises from the amino group of
amino acids during protein catabolism
• It is eliminated through glomerular filtration followed by tubular reabsorption (in
the PCT - about 50% of the filtered amount)

• As a test of renal function, it has a lower specificity compared to creatinine:

- it is influenced by the protein intake and by protein catabolism
- undergoes tubular reabsorption – the fraction that is reabsorbed increases when the
urine flow rate decreases (as in the case of dehydration)

 Decrease of plasma [urea]:

• Reduced availability of amino acids for deamination (malabsorption)

• Severe liver diseases (urea synthesis is impaired) → plasma [ammonia] increases
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 Increase of plasma [urea]:

• a. Pre-renal causes
- ↓ renal perfusion – hypovolemia/reduced blood pressure (severe dehydration,
shock), heart failure
(plasma [urea] increases relatively more than plasma [creatinine] - the reduced urine
flow causes increased passive tubular reabsorption of urea)
- ↑ urea production – high protein intake, increased protein catabolism (extreme
starvation, trauma, major surgery)

• b. Renal causes
- acute/chronic renal failure – that are associated with ↓ GFR
- limited diagnostic sensitivity:
sensitivity urea level exceeds the normal range only when the
GFR decreases by 75%
- advanced renal dysfunction => serum [urea] correlates well with the GFR
- when the GFR ↓ to 10 mL/min → the urea level ↑ 10 fold

• c. Post-renal causes
- obstruction of the urinary tract – calculi, prostate hypertrophy
- ↑ pressure in the renal tubules => ↓ filtration and ↑ reabsorption of urea
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 2. Creatinine

• Creatinine is formed in the muscle from phosphocreatine or creatine

• In the muscle creatine exists mainly as phosphocreatine (70-80%), which
represents an energy store
• 2% of the total amount of P-creatine + creatine is converted to creatinine each
day, by a non-enzymatic conversion
• The amount of creatinine formed is constant from day to day and depends only on
the muscle mass of the individual (it is not influenced by the protein intake)
• Therefore the serum creatinine level is influenced only by the rate of renal
excretion (glomerular filtration + tubular secretion – accounts for only 10% of
urinary creatinine)

 Decrease of creatinine serum level:

• During pregnancy
• Decrease of the muscle mass: starvation,
muscle dystrophy, corticosteroid therapy

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 Increase of creatinine serum level:
• Physiological - high meat intake (→ temporary increase of [creatinine])
- vigorous physical exercise (→ transient, small increases)
- certain drugs that compete with the tubular secretion of creatinine (cephalosporins,
salicylates)
• Pathological - acute/chronic renal failure,
failure that lead to ↓ GFR
- skeletal muscle trauma

 Limits of serum creatinine test:

- limited diagnostic sensitivity:
sensitivity the creatinine level ↑ only when the GFR ↓ by 50%
- once the creatinine level has increased in patients with renal diseases, its further
increase correlates well with the progressive decline of the GFR
- nevertheless, it has a better specificity for renal failure compared to the urea test

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 3. Creatinine clearance

• Measures the glomerular filtration rate (GFR) – it is the plasma volume that can be
cleared of a substance in one minute
• The substance has to be filtered by the glomeruli but not reabsorbed – creatinine
is appropriate

• Creatinine clearance = UxV/P

(U is the concentration in urine, V is the volume of urine produced per minute, and P
is the concentration in plasma)
• Normal range = 90-120 mL/min (after 50 years of age it decreases by 13 mL/min
for every decade)
• In renal failure – creatinine clearance decreases

• There are two sources of imprecision in creatinine clearance measurements: timed

measurement of urine volume, and urine [creatinine] → Cockroft’s formula for the
estimated creatinine clearance:

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 Diagnostic value of creatinine clearance

• It is a more sensitive test for detecting ↓ GFR compared to creatinine and urea: it
allows the detection of GFR decreases lower than by 50%
• There is an inverse hyperbolic correlation between serum creatinine and CrCl
• In chronic renal failure – CrCl allows to determine the moment when renal dialysis
is imposed
• CrCl is useful for monitoring the GFR during therapy with nephrotoxic drugs

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 4. Cystatin C

• It is a protein having a low MW – it is a member of the superfamily of cysteine

protease inhibitors
• It is produced by all nucleated cells at a constant rate – its function is to protect
connective tissue from destruction by intracellular enzymes

• It is freely filtered by the glomerulus and then almost completely reabsorbed and
catabolized by the tubular epithelial cells (but it is not secreted)
• The blood concentration of CysC depends almost entirely on the GFR and is not
affected by diet or nutritional status, muscle mass, age or gender

• Serum concentrations of CysC offer a better estimation of the GFR compared with
creatinine or creatinine clearance
• Levels of CysC rise earlier than creatinine in acute renal failure
• Serum CysC is superior to creatinine for detecting minor GFR reduction

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 II. Tests for the assessment of tubular function
• The diseases affecting the renal tubules may affect:
A - the urine concentration capacity
B - the urine acidification capacity
C - the reabsorption of amino acids and glucose

 Tests:

• Urine osmolality
• Specific gravity of urine A
• Urine concentration tests (concentrating ability of the kidney)

• Urinary pH
B
• Urine acidification tests

• Chromatography of urinary amino acids - to detect amino aciduria

C
• Glycosuria

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 A. Urine osmolality and renal concentration tests

 Urine osmolality - varies between 50 - 1250 mmol/kg, depending on the body’s

requirement to produce a maximally diluted or a maximally concentrated urine
• Urine osmolality is directly proportional to the osmotic work done by the kidney,
and is a measure of concentrating power
• In chronic renal failure – the kidney loses its capacity to concentrate urine at a
relatively late stage

 Urine specific gravity - can be estimated using urinalysis dipsticks

• Is usually directly proportional to osmolality

 Tests of renal concentrating power

• Measure the concentration of urine produced in response to:

- fluid deprivation → the fluid deprivation test (FD test)
- IM injection of a synthetic analogue of vasopressin → the DDAVP test (1-deamino,8-
D-arginine vasopressin)
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• Normal response to the tests:
- no increase in plasma osmolality (N = 285-295 mmol/kg) (FD test)
- urine osmolality rises to > 600 mmol/kg (FD test + DDAVP test)

• These tests may be indicated in patients with polyuria, to distinguish among:

- hypothalamic–pituitary causes (deficiency of vasopressin)
- renal causes of polyuria (chronic kidney disease)

 B. Urine acidification tests

• The kidneys reabsorb HCO3– and secrete H+ in the distal portion of the nephron →
urine acidification (lower pH compared to plasma)
• Exploring the ability of urine acidification – by inducing a metabolic acidosis →
urinary pH should decrease < 5.3
• Proximal/distal tubular acidosis – loss of HCO3– or decreased capacity to secrete H+
(without a modification of the GFR)

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 C. The amino acidurias
• Amino acids from plasma are filtered by the glomeruli → normally, the proximal renal
tubules reabsorb all the filtered amino acids
• Amino acids can be categorized into four groups (neutral, acidic, basic, imino acids) →
each has its own specific mechanism for transport
 Causes for amino aciduria:
• Renal amino aciduria - may be due to:
- genetic defect of one of the specific transport mechanisms
- diseases of the renal tubule – affect the reabsorption of amino acids, but also of glucose
or phosphate
• Raised plasma [amino acids] (overflow amino aciduria) - the renal threshold for amino
acids is exceeded, due to overproduction/accumulation of amino acids in the body

 Glycosuria
• Glucose is most commonly found in the urine in patients with diabetes mellitus,
mellitus when
the plasma [glucose] exceeds the renal threshold (180 mg/dL)
• Glycosuria in the presence of a normal plasma [glucose] - occurs in case of proximal
tubular malfunction causing a reduced renal threshold
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• Deficiency of renal 1α-hydroxylase →
synthesis of calcitriol ↓ → plasma
[calcium]
calcium ↓
• PTH ↑ - secondary hyperparathyroidism
• Plasma [phosphate]
phosphate ↑ - due to ↓ GFR
• Plasma [HCO3-] ↓ (metabolic acidosis -
due to ↓ H+ excretion)
• Anemia - due to ↓ erythropoietin
• Hyperkalemia - reduced excretion

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 III. Assessment of glomerular integrity
Proteinuria
• The glomerulus filters proteins with low MW → the tubules reabsorb and catabolize
a major part of these proteins
• The urinary excretion of proteins is < 200 mg/day

• The kidney also eliminates specific proteins produced by the tubular cells: Tamm-
Horsfall protein - it represents about half of the total amount of excreted proteins

• Dipstick tests detect albumin at concentrations greater than 200 mg/L, but are less
sensitive to other proteins

• If the presence of proteinuria is confirmed, it should be quantified:

- in a timed (usually 24 h) urine collection
- as the urine albumin:creatinine ratio (UACR) – it is unaffected by variations in urine
concentration, and it is measured using the first morning urine sample;
albuminuria is present when UACR is > 30 mg/g → marker for chronic kidney
disease
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 Types of proteinuria

1. Glomerular proteinuria

•Caused by abnormal increase of the glomerular permeability, that occurs mainly in

glomerulonephritis
•Selective proteinuria – elimination of proteins with low MW (albumin ≥ 80%)
•Nonselective proteinuria – proteins with high MW are also eliminated (all plasma
protein fractions, albumin + globulins, are present) → suggests severe glomerular
damage
• It ranges between 300 mg-20 g/day

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2. Tubular proteinuria
•Tubular or interstitial damage resulting from a variety of causes, especially
pyelonephritis → the tubules fail to reabsorb proteins that have been filtered by the
normal glomeruli → elimination of α- and β-globulines with low MW (but not albumin)
•Sometimes – abnormal secretion of proteins into the urinary tract
•Usually < 1-2 g/day

3. Overflow proteinuria
•Abnormal amounts of low MW proteins (< 60 kDa) in plasma and in urine
•These proteins are normally filtered by the glomerulus, but the reabsorptive capacity
of the proximal tubule is exceeded
•E.g.: Bence-Jones protein, amylase, hemoglobin, myoglobin → diagnostic value

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Classification of proteinuria

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 Nephritic and nephrotic syndrome
 Nephritic syndrome:
• Proteinuria < 3.5 g/24 h
• Hematuria, RBC casts
• Arterial hypertension
• GFR ↓, plasma [urea] and [creatinine] ↑
• Inflammatory markers (C3, C4, immune complexes, IgA, CRP)
• Autoimmune markers (anti-nuclear Ab, anti-dsDNA Ab, anti-glomerular membrane Ab)

 Nephrotic syndrome:
• Proteinuria > 3.5 g/24 h (of glomerular type)
• Hypoproteinemia with hypoalbuminemia → edema
• Secondary hyperlipidemia – ↑ levels of cholesterol and triglycerides
• Plasma [urea] and [creatinine], creatinine clearance – usually normal
• Impure NS – arterial hypertension, hematuria, nitrogen retention syndrome

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 Bibliography

Harvey RA, Ferrier DR. Lippincott’s Illustrated Reviews –Biochemistry. 5th edition,
Lippincot Williams & Wilkins, 2011
Minodora Dobreanu Biochimie clinică. Implicații practice. Ediția a III-a, Editura
University Press, Târgu-Mureș, 2015.
Kaplan LA, Pesce AJ. Clinical chemistry; theory, analysis, correlation, 5th ed. Elsevier
Mosby, 2010

Rae P, Crane M, Pattenden R. Clinical Biochemistry – Lecture Notes, 10th ed., Wiley
Blackwell, John Wiley & Sons Ltd. 2018.

Crook MA. Clinical Biochemistry and Metabolic Medicine. 8th edition. Hodder Arnold,
Hodder & Stoughton Ltd, 2012.

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