Fecal Urobilinogen

If the hepatobiliary system is functioning, fecal

urobilinogen varies directly with rate of red cell hemolysis.

Fecal urobilinogen increased occurs when blood destruction is increased and biliary obstruction is relieved.

In case of hemolysis, the daily excretion is related to

the existing total body hemoglobin mass. If there is a

reduced total body hemoglobin mass, accelerated rates

of hemolysis may only yield an amount of urobilinogen

that would be within normal limits for an individual with

normal hemoglobin mass.

Fecal urobilinogen absent occurs with exclusion of

bilirubin from the gut in complete biliary tract obstruction

and in extreme cases of hepatocellular disease. Absence

of urobilinogen in feces is important in indicating biliary

tract obstruction, persistent absence is a strong indication

of malignant obstructive disease. Decreased fecal

urobilinogen excretion may occur when antibiotics which

alter intestinal flora are used (tetracyclines, streptomycin,

etc.).

Bromsulphalein (Sulfobromophthalein) Excretion

Test

Sulfobromophthalein sodium (Bromsulphalein, BSP, is

a dye which is bound avidly by albumin, the complex is

picked up by the liver cell, and the BSP is transported into

the microsomes, conjugated and excreted in the bile, in a

manner analogous to bilirubin.

BSP is given intravenously and its amount present in the

blood after 30, 45 or 60 minutes indicate hepatic function.

The greater the liver function loss, greater the amount

BSP present in blood (this test is of no significant value in

differential diagnosis). BSP retention in blood conjugated/

unconjugated runs parallel to bilirubin related disorders.

In the presence of fevers, administration of anabolic

steroids, Tele-paque, Dubin-Johnson syndrome and in

Gilbert’s disease, BSP retention is increased. In the latter

two disorders the uptake by liver cells is normal (so normal

retention at 45 minutes) but after getting conjugated it

regurgitates back into blood (so BSP retention is marked at

3 hours period).

Method

1. Lipemic/jaundiced sera may cause interference. In any

case one should not perform the test in the presence

of acute liver/gallbladder disease.

2. Know the patient’s weight, inject 5 mg BSP/kg body

weight intravenously, over a period of 60 seconds.

Having injected set the time to 45 minutes.

3. After 45 minutes withdraw 8–10 mL blood from the

other arm, let it clot, remove serum after centrifuging.

4. Take 2 test tubes marked test and blank:

Test Blank

1.0 mL serum

+4.0 mL N/10 NaOH mix by

gentle inversion

1.0 mL serum

+4.0 mL N/10 HCl mix by

gentle inversion

 The full reddish color develops in the alkaline solution,

and the dye is colorless in acid solution.

5. Read the absorbance of test at 575 nm (or 590 nm)

setting the zero with the blank.

6. Refer the absorbance reading to the calibration curve

to obtain the percentage of the dye dose remaining

in the blood after the 45 minutes interval. Report as

percentage of dye retention afterminutes,” giving

the dose used. Normal retention is up to 4% (average

2.8%).

Calibration: Into a 1 liter volumetric flask pipette very

accurately 2.0 mL of 5% BSP. Dilute to the mark with

distilled water and mix well. Into 4 clean test tubes pipette

accurately the following amounts of the diluted dye: 0.25,

0.50, 0.75, and 1.0 mL. Make each to 1.0 mL with distilled

water. To each tube add exactly 4.0 mL of N/10 NaOH.

Mix by inversion and read the absorbance at 575/590 nm.

These standards correspond to values of 25, 50, 75 and

100% retention in the test. Plotted on graph paper, the

readings should fall on a straight line passing through the

origin.

Conditions Associated with Increased BSP Retention

Hepatobiliary System

¾ Jaundice from any cause except Gilbert’s syndrome

¾ Viral hepatitis

¾ Toxic hepatitis

¾ Fatty liver

¾ Cirrhosis

¾ Bile duct obstruction

¾ Metastatic carcinoma

¾ Lymphomatous or leukemic infiltration

¾ Granulomatous inflammation

Liver Function Tests 457

¾ Amyloidosis

¾ Dubin-Johnson syndrome.

Extrahepatic Conditions

¾ Congestive heart failure

¾ Fever above 39°C

¾ Oral contraceptive use

¾ Prolonged fasting or malnutrition

¾ Contrast media used for gallbladder examination.

Artefacts

¾ Obesity

• Spuriously high retention because excessive weight

results in excessive dose

¾ Hypoalbuminemia

• Spuriously low retention because binding is reduced

¾ Ascites

• Spuriously low retention because the dye enters the

ascitic fluid

¾ Proteinuria

• Spuriously low retention because albumin bound dye

enters urine.

EVALUATION OF SYNTHESIS IN LIVER

Serum Proteins (Albumin Especially)

Since serum albumin and a small fraction of globulin

are synthesized in liver, serum proteins are affected both

quantitatively and qualitatively in liver disease. In any

disease causing hepatocellular damage, the concentration

of serum albumin decreases. In many liver disorders,

serum globulins may rise to such a level so as to maintain

normal or increased total protein concentration even

when there is severe albumin depletion.

The changing levels of serum albumin thus provide

valuable indices of severity, progress, and prognosis

in hepatic disease. Decreased albumin and elevated

globulins in serum indicate hepatocellular origin of

jaundice or liver disease. In obstructive jaundice, serum

protein changes occur late, after secondary hepatocellular

damage has occurred. Cholangitis and biliary cirrhosis,

however, result in liver damage which may not be accompanied by protein alteration. Furthermore, serum protein

changes may return to normal before convalescence from

hepatitis is complete. However, liver disease is not the only

cause of serum protein alterations.

Chemical methods and electrophoretic methods are

available for serum proteins estimation. Electrophoresis is

most precise and specific way of assessing serum proteins.

The flocculation and turbidity methods crudely estimate

globulins and hence are not specific and obsolete in

today’s context.

Prothrombin Concentration

Deficiency of prothrombin may occur as a result of:

1. Inadequate absorption of bile from the intestinal tract,

or

2. Inability of a damaged liver to convert vitamin K to

prothrombin.

A normal prothrombin concentration does not rule out

abnormal liver function.

Low Prothrombin in Presence of Jaundice

When a low prothrombin level is found in a jaundice

patient, give 2–4 mg vitamin K, IV or IM, and measure

prothrombin concentration later.

1. Return to normalcy of prothrombin concentration

(85–100% of normal) indicates that the capacity of liver

cells to synthesize prothrombin is good.

2. A poor response implies hepatocellular disease, either

primary or following prolonged obstructive disease.

Low Prothrombin in the Absence of Jaundice

In the absence of jaundice, a low prothrombin level usually

indicates serious liver damage, and no response to large

doses (60–70 mg) of parenteral water-soluble vitamin K

confirms it. This is true if jaundice is also present.

Cholesterol and its Esters

Decrease of Both Substances

Associated with extensive destruction of liver parenchyma

is reduction in serum levels of cholesterol and cholesterol

esters, extremely low concentration implies a poor

prognosis. Persistently low cholesterol ester concentration

or ester/total cholesterol ratio indicates continuing

hepatocellular damage, a rise in cholesterol ester is

considered as a good sign and heralds improvement.

Increase of Total but Decrease of Esters

Accompanying biliary obstruction is usually a rise in total

cholesterol, but the cholesterol ester concentration is

often unaffected. The determination of cholesterol ester,

however, is not a fruitful exercise clinically.

Detoxification

The liver removes noxious materials or renders them

harmless by conjugation of toxic substances with amino

458 Concise Book of Medical Laboratory Technology: Methods and Interpretations acids, glucuronate and inorganic radicals (e.g. sulfate), by

oxidation or reduction, by excretion, etc.

Hippuric Acid Test

This test depends upon conjugation by liver of sodium

benzoate with glycine to produce hippuric acid, which

is excreted in the urine. It is preferrable to give sodium

benzoate-1.77 g-IV (instead of orally in which case the

absorption may be irregular), one hour later at least

0.7 g of hippuric acid should be excreted in the urine.

Consideration of low values is permissible only if impaired

renal function is ruled out for retention of hippuric acid.

EVALUATION OF ENZYME ACTIVITY

Serum Transaminases

Liver and muscles are rich in enzymes of Kreb’s cycle.

Among such enzymes is a group responsible for transfer of

NH2 groups from amino acids to keto acids, thus, providing

for metabolism of amino acids. Destruction of muscle or

of liver cells releases the enzymes, with consequent rise in

their values in plasma. In obstructive jaundice and more

so in acute hepatitis, the serum levels of SGOT and SGPT

rise to very high levels (300-1500 units, normal being 5-40

units), as does LDH concentration (normal concentration,

200-450 units). Chronic hepatitis may produce

moderate elevations of serum transaminases. Liver cell

destruction incident to neoplastic disease metastatic to

the liver produces moderate elevation of transaminases

concentration in the serum.

In many cases, there seems to be a correlation between

the differences in the degree of elevation of SGOT and

SGPT and the cause of jaundice. Rise of SGPT is greater

than elevation of SGOT in extrahepatic obstruction, acute

hepatitis and toxic hepatitis, the reverse is true in cirrhosis

of liver, intrahepatic neoplasm, and hemolytic jaundice.

Serum Alkaline Phosphatase

The concentration of this enzyme often increases in the

plasma of an icteric patient. It is normally present in the

liver and excreted in the bile so that elevation of serum

alkaline phosphatase may be a manifestation of retention;

this is a convenient explanation for the observation that

serum alkaline phosphatase concentration increases in

obstructive jaundice. In acute and chronic hepatocellular

disease, serum alkaline phosphatase is raised, but not to

the extent typical of obstructive jaundice. In hemolytic

jaundice, normal levels are the rule. In some cases of metastatic carcinoma of liver, serum alkaline phosphatase may

rise in the absence of jaundice. It should be kept in mind

that phosphatase levels may be normal early in obstructive

disease and with relief of obstruction. Pregnancy and such

diseases as Paget’s disease of bone, hyperparathyroidism,

and rickets/osteomalacia, are also associated with

elevated serum alkaline phosphatase concentration and

these must be ruled out.

SUGGESTED LIVER FUNCTION TESTS

A. Jaundice Absent

Urine bilirubin, urine urobilinogen, serum bilirubin, BSP

excretion, transaminases.

B. Jaundice Present

As mentioned above (except BSP excretion), plus alkaline

phosphatase,