Pemeriksaan liver & renal

function test

Sumantri
Pustaka
Atkinson, A.J., Markey, S.P., 2007, Principles of Clinical
Pharmacology, Second Edition, Elsevier, New York.
Grouzard, N. (Ed.), 2010, Clinical Guidelines, Medecins Sans
Frontieres, Paris.
Lewandrowski, K., 2002, Clinical Chemistry, Laboratory
Management and Clinical
Correlations, Lippincott Williams & Wilkins, Philadelphia.
Moffat, A.C. (Editor), 1986, Clarke’s isolation and Identification of
Drugs, in pharmaceuticals, body fluids, and post-mortem
material, Second Edition, The Pharmaceutical Press, London.
Richterich, R. and Colombo, J.P., 1981, Clinical Chemistry, Theory,
Practice, and Interpretation, John Wiley & Sons, New York.
Watson, D.G., 2003, Pharmaceutical Analysis, A Textbook for
Pharmacy Students and
Pharmaceutical Chemists, Churchill Livingstone, Edinburgh.
Liver function test
 Liver function: many vital metabolic functions that involve
secretory and
excretory processes such as detoxification,
synthesis of
proteins, catabolism of carbohydrates, and others.
 Laboratory tests are used for diagnosis and monitoring of hepatic
disorders such as hepatitis, cirrhosis, and hyperbilirubinemia.
 The assessment of liver function:
- total and direct bilirubin
- total protein
- albumin
- ammonia
- enzim transaminase (AST, ALT, GGT)
- alkaline phosphatase
Hepatitis
 Hepatitis which is defined as inflammation of the
liver, is associated with jaundice, especially involving
the parenchymal liver cells.
 It is indicated by impairment of bilirubin conjugation
and excretion causing elevations of all forms of
bilirubin in serum and in urinary bile.
 Significant elevations of AST and ALT (2-75 x
normal), virus, urinary
urobilinogen and other hepatocellular enzymes are
typical in hepatitis.
Definition :
 icterus - yellowish pigmentation in the blood due to increased bilirubin.
 jaundice - bilirubin deposits in the skin, mucous membranes, and eyes giving
tissues a yellow appearance.
 lobules - the microscopic functional units of the liver.
 parenchymal cells – epithelial liver cells that make bile, bilirubin, and proteins
and perform other duties; hepatocytes.
 hemoconcentration – relative increase in blood cells due to decrease in plasma
water volume.
 sinusoidal - forming a small channel for blood in the tissues of the liver or other
organs.
 biliary canaliculi - small ducts or tubes that carry bile out from the liver leading to
the small intestine.
 ascites - accumulation of serous fluid in the abdomen
 cirrhosis is a serious disease of the liver resulting from chronic inflammation of the
liver.
 phototherapy - exposure to sunlight or artificial ultraviolet light for therapeutic
purposes, such as treating neonatal hyperbilirubinemia
 plasma ammonia levels provide vital information about the potential neurological
status of
patients in liver failure.
Metabolism Bilirubin
 Bilirubin is a degradation product of the heme
portion of hemoglobin.
 Heme is degraded in cells of the reticuloendothelial
system, mainly the spleen.
 The protoporphyrin ring of the heme is opened to the
biliverdin form and iron is released.
 Biliverdin is reduced to produce the yellow-
pigmented molecule bilirubin.
 The bilirubin molecule, a tetrapyrole, has low
solubility in water or plasma. When it is released into
blood, it is bound to albumin for transport (delta
bilirubin).
 When the bilirubin-albumin form reaches the liver, it loses
albumin and enters the hepatocyte. Within the hepatocyte, the
liver enzyme uridyl diphosphate glucuronyl transferase (UDPG-
transferase) transfers molecules of glucuronate, a sugar, to the
bilirubin molecule.
 About 85% of bilirubin is conjugated with two molecules of
glucuronate to form diglucuronate-bilirubin. Most of the rest of
bilirubin is conjugated with one sugar molecule to form
monoglucuronate-bilirubin. The addition of the sugar group
increases its solubility.
 Conjugated bilirubin passes into the intestine through the bile
duct, where intestinal bacteria reduce bilirubin to urobilinogen.
Some urobilinogen may be reabsorbed through the intestinal
mucosa and returned to the portal circulation and the liver.
 The remaining urobilinogen is excreted into urine or oxidized to
form urobilin and excreted in the feces. Urobilin is one
component in feces giving its characteristic brown color.
Obstructive Jaundice
 Obstruction of bile from the liver may be caused by gallstones in
the bile duct or a tumor that impedes the flow of bile into the
intestine.
 Other effects of obstructive jaundice may be defective excretion
of lipid substances through bile.
 Obstructive jaundice is associated with increased levels of
alkaline phosphatase and GGT and increased serum total and
direct bilirubin.
 Other liver enzymes are elevated as well, indicating hepatic
inflammation.
 Ultrasound and other imaging techniques are needed to locate
the source of the obstruction.
 Pemeriksaan
Bilirubin
 The three forms of bilirubin (unconjugated, conjugated, and albumin bound)
differ in their solubility in water. Testing for the different forms of bilirubin is
based upon their differences in solubility.
 Because conjugated bilirubin and albumin-bound bilirubin (or delta bilirubin) are
soluble in water, conjugated bilirubin reacts in a fast or direct reaction with
substrates.
 When an accelerator is used in the reaction, unconjugated bilirubin is solubilized
and is measured in the reactant mixture. Therefore, direct bilirubin measures
conjugated and albumin-bound bilirubin and total bilirubin, with the addition of
the accelerator, measures all forms of bilirubin.
 Both the Jendrassik-Grof reaction and the Malloy-Evelyn method for measuring
bilirubin use diazonium salt to produce a color reaction with bilirubin.
 The Reaction : Bilirubin + diazotized sulfanilic acid → azobilirubin
 The Jendrassik-Grof method, performed at a pH of 13, measures the blue-colored
product at 600 nm.
The alkaline reaction produces a more intense color than the equivalent reaction
run at a neutral pH. The Malloy-Evelyn method, performed at a pH of 1.3,
measures the red-colored product at 560 nm. A sodium benzoate–caffeine mixture
or methanol may be used as the accelerator in these methods.
Spesimen
 Serum, plasma, spinal fluid, and urine may all be
measured by diazonium salt methods.
 Bilirubin may be broken down by light or heat
and should be protected from these
environmental conditions.
 Reference ranges :
- Adult bilirubin 0.0–2.0 mg/dL
- Adult direct bilirubin 0.0–0.2 mg/dL
Hyperbilirubinemia: A yellow serum sample in a rack of tubes.
 Pemeriksaan
Urobilinogen
 Determination of fecal or urine urobilinogen is based on Ehrlich’s
reaction,
which uses para-dimethylaminobenzaldehyde to form a red color.
Ascorbic acid may be added to the sample to maintain
urobilinogen in its reduced state.
 Specimen :
The test requires fresh sample; urobilinogen may be oxidized to
urobilin on standing.
 Interferences
Urobilin in the sample is reduced by alkaline ferrous hydroxide to
urobilinogen. Sodium acetate further reduces other chromogens,
which may interfere with Ehrlich’s reagent.
 Bilirubin may interfere with the reaction; significant amounts of
bilirubin must be precipitated with barium chloride and removed
by filtration.
 Reference ranges:
- Urine 0.5–4.0 Ehrlich units/day
- Feces 75–400 Ehrlich units/day
Pemeriksaan enzim Transaminase
 Pemeriksaan
Albumin
 Albumin can be analyzed by immunoassays, turbidimetry, and
spectrocolorimetry.
 A simpler method is based upon albumin’s ionic charge at an
acid pH and binding to anionic dyes such bromcresol green
(BCG) or bromcresol purple (BCP).
In these methods, a colored product forms that absorbs at
wavelengths significantly different from the reagent and from
typical interfering molecules such as hemoglobin or bilirubin.
 Reaction:

 Reference range
Adult 3.5–5.2 g/dL
Table 7-7. Diagnostic indicators of specific types of
cirrhosis.
Metabolisme Ammonia
 Ammonia is produced in parenchymal liver cells during the deamination
of amino acids and culminates in the formation of urea.
 Urea is the major nonprotein nitrogen waste product, further excreted
by the kidneys.
 Plasma ammonia level is the 20 μg/dL, while urinary urea level is 20
mg/dL.
 Some ammonia is also produced in the intestinal tract and skeletal
muscles, but the majority of ammonia production is associated with
hepatic function.
 Hepatic
Encephalopathy
 Hepatic encephalopathy is caused by a metabolic
disorder associated with fatty deposits in the liver,
and impaired production of urea through the
ornithine cycle, causing encephalopathy.
 Pemeriksaan
Ammonia
 Analysis of ammonia can be achieved by enzymatic reaction
using glutamate dehydrogenase (GLDH) and coenzyme
conversion of NADPH to NADP,turbidimetry, and an ion-
selective method causing a pH change in the solution that is
detected potentiometrically.
 The reaction:

Specimen:
EDTA or (lithium) heparinized plasma that is free from
hemolysis may be used.
The sample must be separated in a refrigerated centrifuge
within 20 minutes of
collection and maintained in 0 to 4oC ice water.
Reference range:
Adult Male 15–45 g/dL venous
Renal function test
 The assessment of renal function:
- creatinine clearance
- BUN
- electrolytes etc.
 Creatinine
clearance
 Creatinine clearance is a measure of glomerular filtration rate.
Glomerular filtration rate is inversely related to serum creatinine,
as serum creatinine rises, glomerular filtration rate decreases.
 The use of creatinine to estimate glomerular filtration rate is
based upon three assumptions:
(1) creatinine is filtered through the glomerulus
(2) relatively low amounts of creatinine are reabsorbed through
the nephron tubule
(3) creatinine production is constant over time
 Reference ranges :
Male 95–130 mL/min
Female 80–120 mL/min
 Kreatinin
Kreatinin dapat ditetapkan kadarnya dengan metode kimia dan
enzimatik.
 Metode kimia : Reaksi Jaffe
Kreatinin bereaksi dengan asam pikrat dalam suasana alkalis
menghasilkan senyawa hasil konyugasi yang berwarna orange
merah yang diukur pada λmaks 485 nm.
 Metode enzimatik :
Kreatinin dihidrolisis oleh enzim hidrolase menghasilkan
sarkosin, yang selanjutnya dioksidasi oleh enzim oksidase
menghasilkan H2O2.
 Spesimen: serum, plasma, dan urin
 Reference ranges:
Male 0.9–1.3 mg/dL
Female 0.6–1.1 mg/dL
Children 0.3–0.7 mg/dL
 Pemeriksaan Asam
urat
 Two methods for uric acid measurement are commonly used in the clinical
laboratory:
uricase and phosphotungstic acid methods.
 Reaction:
- Uricase Method
1. Decrease in absorbance at 293 nm is due to loss of uric acid.
2. Alternate methods have hydrogen peroxide converted to a chromagen using
4-aminophenazone and peroxidase in a second enzyme-catalyzed reaction.
3. Excessive amounts of protein and lipids can interfere with results, and negative
interferences often occur with the presence of xanthine and hemoglobin.
- Phosphotungstate Method
This method requires a protein-free filtrate for serum or plasma.
 (Lanjuta
n)
 Proteins in normal amounts in serum must be removed to prevent
interferences.
 Glucose, ascorbic acid, glutathione, hemoglobin, and drugs such as
acetaminophen and caffeine commonly interfere.
 The Specimen
Serum, heparinized plasma, and urine may be tested. Anticoagulants
containing fluoride or
citrate should not be used. Hemolysis and lipemia should be avoided.
 Reference ranges
- Uricase method:
Male 3.5–7.2 mg/dL
Female 2.6–6.0 mg/dL
- Phosphotungstate method:
Male 4.2–8.0 mg/dL
Female 3.5–7.3 mg/dL
Values are lower in children.
 Pemeriksaan protein
urin
 Reaction principle
Several methods exist for the measurement of urinary protein.
 Turbidometric
Protein may be precipitated with sulfosalicylic acid, trichloroacetic acid,
or benzethonium chloride. The turbidity of the precipitate is measured
photometrically.
 Dye-Binding
Dye binds to amino groups, the resulting color change is measured
colorimetrically.
 Calculation:
(Urine protein concentration [mg/dL] x urine volume
[dL])/day
 Specimen:
A 12- or 24-hour collection may be used. A 24-hour collection is preferred
since some variation in excretion may occur over a 24-hour period. Urine
should be kept cool.
 Reference range:
Urine protein 100 mg/day
 Pemeriksaan albumin urin
 Reaction principle
Dye binds to albumin and causes a shift in the maximum
absorption, for example methyl orange, bromcresol green, and
bromcresol purple dyes.
 Calculation:
(Urine albumin concentration [mg/dL] x urine
volume [dL])/day
 Specimen
 A 12- or 24-hour collection may be used. A 24-hour collection is
preferred since some variation in excretion may occur over a 24-
hour period. Urine should be kept cool.
 Reference range
The excretion of 30 to 300 mg of albumin per 24-hour period on
two of three collections is indicative of microalbuminuria.
 Blood urea nitrogen
(BUN)
 The evaluation of BUN is a gross indicator of
renal function.
 Damaged nephrons are unable to clear urea
from the bloodstream, resulting in an
increased BUN.
 The level of BUN is also affected by patient
hydration, protein diet, and gastrointestinal
bleeding.
 Pemeriksaan urea urin
 Two methods for measuring the concentration of urea are
commonly used, the urease method and the colorimetric method.
 The Reaction:
Urease Methods

 The ammonia that is liberated in the reaction above may be

measured in a variety of ways:
1. The reaction may be coupled with a reaction that drives NADH to
NAD+.
2. The conductivity of the ammonium ion may be measured.
3. The Berthelot reaction may be used:

4. An indicator dye may be used.

5. The reaction may be coupled with a reaction that produces H 2O2.
Lanjutan

 Colorimetric Method
The diacetyl monoxime reaction is used to produce a
color
change:

 Specimen:
Serum or plasma may be tested. Anticoagulants
containing fluoride or citrate should not be used.
 Reference range:
Serum or plasma 6–20 mg/dL
 Pemeriksaan elektrolit
 Ion-selective electrodes (ISEs) are the most commonly
employed methods of analysis for many electrolytes,
including sodium and potassium, although
spectroscopic and spectrophotometric methods also
exist.
 ISEs consist of or are covered by a unique material
that is more selective for one ion than other ions.
When the ion comes in contact with the electrode,
there is a change in the potential compared to the
reference electrode, measured as a voltage change,
due to the ionic activity.
Potentiometry
 Potentiometry is an electrical system of measuring change in
electrical potential
between a detecting electrode and a standard reference electrode
in which current
is kept constant.
 Ion-selective electrodes (ISEs) are used in potentiometry to
measure the activity of one ion versus other ions. The electrodes
are made of a unique material that is more selective for one ion
compared to other similar ions.
 When the ion comes in contact with the electrode, there is a
change in the potential compared to the reference electrode,
measured as a voltage change, due to the ionic
activity.
 I SEs are the most commonly employed methods of analysis for
many electrolytes, including sodium and potassium, since
spectrophotometric methods have
proven to be less effective.
 Elektro
lit
 Reference range (Adults)
Plasma Na 136–145 mmol/L
K 3.5–5.1 mmol/L
Cl 98–107 mmol/L
CO2 22–28 mmol/L