Tests for Assessing the Functions of Liver

The following points highlight the nine main tests for assessing the functions of liver. The tests are: 1. Bile Pigment Metabolism in Health and in Jaundice 2. Tests for Carbohydrate Metabolism 3. Tests for Plasma Protein Concentration 4. Tests for Detoxifying Functions 5. Test for Excretion of Foreign Substances 6. Tests for Blood Coagulation 7. Tests for Serum Enzymes and Others.

Tests for Assessing the Functions of Liver:

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1. Bile Pigment Metabolism in Health and in Jaundice
2. Tests for Carbohydrate Metabolism
3. Tests for Plasma Protein Concentration
4. Tests for Detoxifying Functions
5. Test for Excretion of Foreign Substances
6. Tests for Blood Coagulation
7. Tests for Serum Enzymes
8. Test for the Conversion of Ammonia to Urea
9. Glutamine Content of Cerebrospinal Fluid

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Test # 1. Bile Pigment Metabolism in Health and in Jaundice:

When the red blood cells have lived out their life span (averaging 120 days), their cell membranes rupture and the released hemoglobin is phagocytized by the reticuloendothelial cells throughout the body. The hemoglobin is first split to heme and globin, and the heme ring is opened to give a straight chain of four pyrrole nuclei from which bile pigments are formed.

The first pigment formed is biliverdin which is rapidly reduced to free bi­lirubin. This free bilirubin immediately combines very strongly with the plasma albumin and is trans­ported throughout the blood and interstitial fluids.

Even when bound with the plasma protein, this bi­lirubin is still called “free bilirubin”. 80 per cent of the free bilirubin then conjugates in the liver with glucuronic acid to form bilirubin glucuronide; 10 per cent conjugates with sulphate to form bilirubin sulphate and the rest 10 per cent conjugates with a multiple of other substances.

This conjugated bi­lirubin is excreted by an active transport process into the bile canaliculi. A small portion of the con­jugated bilirubin formed by the hepatic cells re­turns to the plasma either directly or indirectly by absorption. Therefore, a small portion of the conju­gated bilirubin is always available in the extracel­lular fluid.

Bilirubin is then converted by bacterial action into mesobilirubin which is reduced to mesobilirubinogen in the intestine. This compound is further reduced to stercobilinogen (Urobilinogen) which is highly soluble. Some of the urobilinogen is reabsorbed through the intestinal mucosa into the blood.

Most of this is re-excreted by the liver back into the gut; but about 5 per cent of it is excreted by the kidneys into the urine. After exposure to air in the urine, the urobilinogen becomes oxidized to urobilin. The stercobilinogen in the feces becomes oxidized to stercobilin. This explanation is repre­sented by the figure 37.1.

Jaundice:

“Jaundice” means a yellowish tint to the body tis­sues, including yellowness of the skin and also of the deep tissues. The usual cause of jaundice is the increased bilirubin content in the extracellular flu­ids, either free or conjugated bilirubin.

The normal plasma concentration of bilirubin (both free and conjugated forms) averages 0.5 mg per 100 ml of plasma. But in certain abnormal conditions this can rise to as high as 40 mg per 100 ml. The skin be­comes yellow when the concentration rises to about three times the normal.

Causes:

a. Increased destruction of red blood cells with rapid release of bilirubin into the blood.

b. Excessive production of bilirubin beyond the capacity of normal liver to excrete it.

c. Dysfunction of liver cells resulting in fail­ure to convert bilirubin into bilirubin glu­curonide form in the liver and excrete it in the bile.

d. Obstruction of the bi le ducts to make usual flow of the bile to the duodenum.

Classification:

Rolleston and McNee classified jaundice as fol­lows:

a. Hemolytic jaundice.

b. Obstructive jaundice.

c. Hepatic jaundice.

Rich classified jaundice as follows:

a. Retention jaundice (overproduction of unconjugated bilirubin).

b. Regurgitation jaundice (due to necrosis of liver cells and obstruction of bile ducts).

A. Hemolytic jaundice:

a. The excretory function of the liver is not at all impaired.

b. The red blood cells are hemolyzed rap­idly by hemolytic anemia’s by the action of some drugs, by malarial or viral infec­tions, and by incompatible blood transfu­sion. The hepatic cells simply cannot ex­crete the bilirubin as rapidly as it is formed. Therefore, the plasma concentration of free bilirubin rises markedly.

c. The rate of formation of urobilinogen in the intestine is greatly increased and much of this is absorbed into the blood and later excreted in the urine. The colour of the feces becomes dark brown.

B. Obstructive Jaundice:

a. This happens due to the obstruction in the bile duct preventing the flow of bile into the intestine. The obstruction is caused by the blocking of the bile passage by gall­stones, by enlarged glands due to tumour of the head of the pancreas, and by stric­ture of narrowing of the bile duct as a re­sult of surgery.

b. The rate of bilirubin formation is normal; but the bilirubin cannot pass from the blood into the intestines. The free bilirubin then enters the liver cells and becomes conjugated in the usual way.

c. The conjugated bilirubin is then returned to the blood probably by rupture of the congested bile canaliculi and direct emp­tying of the bile into the lymph leaving the liver.

d. The excess bilirubin is excreted in the urine producing a deep yellow or brown­ish colour. The stools become clay col­oured and bulky containing excessive amount of fat. The serum alkaline phos­phatase concentration is usually high.

C. Hepatic jaundice (Toxic and infective jaundice):

a. Hepatic jaundice is caused by infection, toxins and liver poisons. Infection with virus is the most common cause. The in­fective organisms cause damage to the liver parenchymal cells.

b. The conjugation of bilirubin in the liver is thereby affected. Hence, both free and conjugated bilirubin concentration is in­creased in the serum.

c. The urine becomes highly coloured due to the presence of conjugated bilirubin and urobilinogen.

(i) Van den Berg reaction to differentiate be­tween hemolytic and obstructive jaundice diagnostically:

If a freshly prepared diazotized sulphanilic acid reagent is added to serum, con­jugated bilirubin gives a reddish violet colour within a minute known as the “di­rect” Van den Berg reaction. The free bilirubin (unconjugated bilirubin) of the serum does not develop any colour within one minute but the colour is formed if alcohol is added to the mixture.

Alcohol precipitates the protein and makes free the free bilirubin from its protein complex so that it can then combine with the Van den Berg reagent. This result is called the “indirect” Van den Berg reaction. Therefore, in hemolytic jaundice an indi­rect Van den Berg reaction occurs (in­creased free bilirubin) and in obstructive jaundice a direct Van den Berg reaction takes place (increased conjugated bi­lirubin).

If a faint pink colour is formed after one minute and deepening of the colour results in 2 or 3 minutes in some cases, that indi­cates clearly that both conjugated and free bilirubin’s are present in the serum. This re­action is said to be “biphasic” reaction.

In the total obstruction of bile flow, no bilirubin can reach the intestines to be converted into urobilinogen by bacteria. Therefore, urobilinogen is not reabsorbed into the blood and is not excreted by the kidneys into the urine. So in total obstruc­tive jaundice, tests for urobilinogen in the urine are completely negative.

The stool becomes clay coloured for lack of stercobilin, but not free bilirubin. Therefore, in severe obstructive jaundice, large quanti­ties of conjugated bilirubin appear in the urine. This can be known by shaking the urine and observing the foam, which be­comes intense yellow in colour.

(ii) Biochemical tests for jaundice:

The bio­chemical tests are the following:

a. Serum bilirubin concentration and nature of Van den Berg reaction.

b. Serum alkaline phosphatase activity and SGPT activity.

c. Urine test for urobilinogen and bi­lirubin.

d. Thymol flocculation test and colloi­dal gold test.

e. Feces colour.

The biochemical findings in three types of jaundice are given in table 37.1.

(iii) Icteric Index:

The icteric index shows the degree of jaundice by measuring the in­tensity of yellow colour of the serum. Se­rum is diluted with normal saline until it matches the colour of 1 in 10,000 solu­tion of potassium dichromate. The dilu­tion factor is termed Icteric Index. The Icteric Index in normal person is 4 to 6 units. In latent jaundice, it is 3 to 14 units. In clinical jaundice, it is higher than 15 units. Carotene present in serum inter­feres its determination.

(iv) Urine Bilirubin:

The urobilinogen in urine can be detected by Schlesinger’s test. The concentration of urobilinogen is more in hemolytic and hepatic jaundice. Fouchet’s and Gmelin’s test are both posi­tive in obstructive jaundice. These tests indicate the presence of conjugated bi­lirubin in urine in obstructive jaundice.

(v) Bile Pigments in Feces:

The quantity of stercobilinogen depends on the quantity of bilirubin entering into the intestine. Stercobilinogen content of feces of pa­tients with stone obstruction is nearer the normal level (10 to 150 mg/ day in adults) while it is low or absent (0 to 5 mg/day) in those with malignant obstruction.

(vi) Congenital Hyperbilirubinemia:

(a) Gilbert’s syndrome:

In this condition, there is defective intracellular trans­port of bilirubin. In some cases, there is defective hepatic conversion of bi­lirubin to bilirubin diglucuronide. Therefore, the serum-free bilirubin concentration is high ranging from 4 to 6 mg/100 ml, sometimes it may rise to 12 mg 100 ml; The serum shows indirect Van den Berg reaction.

(b) Lucey-Driscoll syndrome:

This con­dition appears in newborn infants. The serum bilirubin concentration is very high (up to 60 mg/100 ml). This syn­drome occurs due to the presence of a substance (probably steroid) which inhibits the conversion of bilirubin into bilirubin diglucuronide in the liver. This inhibitor disappears one month after the birth of the infants having this syndrome.

(c) Dubin-Johnson syndrome:

In this condition, there is defective excretion of conjugated bilirubin by liver cells into bile. Conjugated bilirubin is found in urine. Alkaline phosphatase level of serum is normal.

(d) Crigler-Najjar syndrome:

This syn­drome appears in new born infants. The serum bilirubin level rises to 20 mg/100 ml or more within a few days after birth. This condition is a famil­ial incidence. Bilirubin is not con­verted to bilirubin diglucuronide due to the deficiency of the enzyme glucuronyl transferase in the liver.

The bile does not contain conjugated bilirubin. The serum bilirubin level comes down to normal level in those infants who survive.

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Test # 2. Tests for Carbohydrate Metabolism:

(i) Galactose Tolerance Test:

This test is done in the morning after overnight fast. Fast­ing blood sample is taken. The individual is then given to ingest a galactose solu­tion containing 40 grams of galactose in 300 ml of water. Blood is drawn at half an hour interval for 2 hours. Galactose con­tent of the blood samples are then deter­mined after removing the glucose by fer­menting with yeast.

The normal blood galactose level is 0 to 160 mg/100 ml. In infective and toxic hepatitis, values may go up to 500 mg/100 ml of blood. In cirrhosis of the liver, values up to 500 mg/100 ml of blood are also found depending on the severity of the disease.

(ii) Fructose Tolerance Test:

This test is also performed in the morning after overnight fast. The individual is administered 50 grams of fructose dissolved in 300 ml of water. Fasting blood sample is taken. Blood is also taken at half an hour inter­vals for 2 hours after fructose ingestion. The total blood sugar (glucose + fructose) is estimated.

In normal subjects, the highest blood sugar level does not exceed the fasting level by more than 30 mg/100 ml. Blood sugar lev­els up to 150 mg/100 ml are found in pa­tients with infective hepatitis. This test is less sensitive than the galactose tolerance test.

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Test # 3. Tests for Plasma Protein Concentration:

Albumin, fibrinogen, and some of the α- and β- globulins are synthesized in the liver. In advanced liver diseases, the albumin content is decreased and globulin content increased.

Edema may develop when the plasma albumin level falls below 2.5 per cent. The globulin content may increase up to 5 per cent in some cases. Fibrinogen values in nor­mal persons range from 0.2 to 1.03 per cent and it may fall to 0.1 gm. in severe liver disorder, such as acute hepatic necrosis.

(i) Electrophoretic Separation of Plasma Pro­teins:

The percentage of different proteins determined by paper electrophoresis in normal subjects are as follows:

The flowing results are obtained in liver diseases:

(ii) Flocculation Tests:

(a) Thymol turbidity test:

The degree of turbidity is measured against stand­ards containing 10, 20, 30, 40, . . . 100 mg per 100 ml of protein when serum is mixed with a buffered solu­tion of thymol. A turbidity equal to that of the 10 mg protein standard is taken as 1 unit by McLagan.

In normal subjects, the thymol units range from 0 to 4 units. In infective hepatitis, the values range from 5 to 20 units. In obstructive jaundice, only 8 per cent give positive result. The thymol flocculation test will be posi­tive in all cases in which the turbid­ity is positive.

(b) Serum colloidal gold test:

The results obtained in this test in subjects suf­fering from liver diseases are similar to those obtained with thymol turbid­ity test.

(c) Zinc sulphate test:

This test is posi­tive in all cases of infective hepatitis and cirrhosis. In normal persons, se­rum y-globulin content is 2 to 8 units but the values rise from 15 to 80 units in infective hepatitis and cirrhosis.

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Test # 4. Tests for Detoxifying Functions:

(a) Hippuric acid Synthesis Test:

The liver detoxicates benzoic acid by reacting it with glycine to form hippuric acid which is excreted in urine. The liver is able to synthesize sufficient glycine to conjugate with benzoic acid to form hippuric acid.

The test should begin at least 3 hours after a light breakfast. The patient empties the bladder and drinks sodium benzoate in about 200 ml of water. Urine is collected for a period of 4 hours from the time of ingestion of sodium benzoate. The amount of hippuric acid excreted is deter­mined.

In normal persons, 60 per cent of the ben­zoic acid taken should be excreted as hip­puric acid. The excreted should be 4.5 grams. Smaller quantities are excreted in acute or chronic liver damage.

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Test # 5. Test for Excretion of Foreign Substances:

Bromsulphthalein (BSP) Test:

When bromsulphthalein dye is injected, it circulates in the blood in combination with albumin. Normal subjects after injection of 5 mg BSP per kg body weight retain less than 10 per cent of the dye in 30 minutes, and 7 per cent in 45 minutes.

At 60 minutes, no dye is retained. This is the most sensitive and dependable liver function test. It is particularly useful for evalu­ating suspicious or slightly positive results ob­tained by flocculation tests in the absence of hyperbilirubinemia.

If the liver function is impaired, the dye is ex­creted slowly and up to 50 per cent of the dye will be retained in the body at the end of 45 minutes after injection. The test is more useful in the diag­nosis of liver cell damage without clinical jaun­dice, in chronic hepatitis and in cirrhosis of the liver.

Test # 6. Tests for Blood Coagulation:

Prothrombin Time Test:

Prothrombin (Factor II) and factors VII, IX and X involved in the coagulation of blood are synthesized in the liver in the pres­ence of Vitamin K.

Deficiencies of these can occur for two reasons:

(i) In the presence of parenchymal cell damage, synthesis is impaired despite adequate supplies of Vitamin K.

(ii) In the absence of bile as in cholestasis and obstructive jaundice, the Vita­min K is not absorbed from the intestines and their synthesis is affected.

Shortening of prothrombin time after parental Vitamin K therapy suggests cholestasis; while lack of response to Vitamin K indicates liver damage

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Test # 7. Tests for Serum Enzymes:

Certain enzymes are released from liver into the blood due to the damage to liver cells. The levels of SGOT, SGPT, LDH and isocitrate dehydroge­nase are increased. The levels of these enzymes are increased in viral hepatitis and reach their maxi­mum soon after the onset of jaundice, and then de­crease slowly.

Very high levels occur in toxic he­patic necrosis. Levels in cirrhosis may be moder­ately raised but only if the process is active. Choline esterase levels are decreased in liver cell dysfunc­tion.

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Test # 8. Test for the Conversion of Ammonia to Urea:

The normal range of blood ammonia is 40 to 75 mg ammonia nitrogen per 100 ml. In cirrhosis of the liver, blood ammonia may be increased to over 250 mg/100 ml. High values are found in hepatic coma.

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Test # 9. Glutamine Content of Cerebrospinal Fluid:

The normal range of glutamine in cerebrospinal fluid is from 6 to 14 mg per 100 ml. In cirrhosis of the liver, higher values ranging from 16 to 31 mg/ 100 ml have been reported. In hepatic coma, still higher values ranging from 30 to 54 mg/100 ml have been reported.