Bilirubin and jaundice in in newborn infants

Introduction

Bilirubin is a brownish yellow pigment of bile, secreted by the liver in vertebrate which gives to solid waste products (faeces) their characteristic colour. It is produced in bone-marrow cells and in the liver as the end product of red-blood-cell (haemoglobin) breakdown. The amount of bilirubin manufactured relates directly to the quality of blood cells destroyed. About 0.5 to 2 grams are produced daily. It has no known function and can be toxic to the foetal brain (Mansur, 2013).

Bilirubin is excreted in bile and urine, and elevated level may indicate certain disease. It is responsible for the yellow colour of bruises and the yellow discolouration in jaundice. It is also responsible for the background  straw-yellow colour of urine via its breakdown product, Urobilin contributing to urine colour along with thiochrome, a breakdown product of thiamine which produces the more obvious but variable bright yellow colour of urine. It has also been found in plants (Pirone, 2009).

Types of bilirubin

Bilirubin exists in two forms, which are the unconjugated and conjugated bilirubin.

Unconjugated bilirubin: Unconjugated bilirubin is also known as indirect bilirubin. The haem (iron porphyrin) of the haemoglobin molecule is firstly removed from the globin. The porphyrin portion is then converted to biliverdin which is reduced to bilirubin. The bilirubin is insoluble in water, it is carried in the blood attached to albumin and it cannot be excreted by the kidney. This bilirubin is referred to as unconjugated or indirect bilirubin. (Ochei and Kolhatkar, 2007).

Conjugated bilirubin: In the liver, bilirubin combines with glucuronic acid by the enzyme glucuronyltranferase, making is soluble in water; the conjugated vision is often called “direct” bilirubin. Much of it goes into the small intestine. Though most bile acid is resorted in the terminal ileum to participate in enterohepatic circulation, conjugated bilirubin is not absorbed and instead passes into the colon. Total bilirubin comprises of both conjugated and unconjugated, total bilirubin and conjugated bilirubin levels are measured directly in the blood, whereas indirect bilirubin level are derived from the total and direct bilirubin measurement (Cheifetz and Adam, 2010).

Bilirubin synthesis

Bilirubin is a linear tetrapyrrole that is formed during the process of heme degradation, heme is released from a series of hemeproteins, including haemoglobin and cytochrome         P450, and metabolized by heme oxygenase to form carbon monoxide, bilirubin by biliverdin reductase. Bilirubin is a highly lipophilic molecule despite containing hydrophilic carboxyl around because the latter are unavailable for interaction with water as a result of intramolecule hydrogen bonding to the pyrrole nitrogen atoms. Bilirubin is found in blood bound to plasma albumin, which transports it to the liver where it is conjugated to hydrophilic acceptors. The major conjugates are bilirubin glucuronides formed by UDP-glucuronosyl transferase 1 (UGT-1). These polar derivatives are therefore excreted in the bile (Jaime, 2015).

Toxicity

Unconjugated hyperbilirubinaemia (jaundice) in a newborn can lead to accumulation of bilirubin in certain brain regions (particularly the basal nuclei) with consequent irreversible damage to these areas manifesting as various neurological deficits, seizures, abnormal reflexes and eye movements. This type of neurological injury is known as kernicterus. The spectrum of clinical effects is called bilirubin encephalopathy. The neurotoxicity of neonatal hyperbilirubinemia manifests because the blood-brain barrier has yet to develop fully, and bilirubin can freely pass into the brain interestitium, whereas more developed individuals with increased bilirubin in the blood are protected. Aside from specific chronic medical conditions that may lead to jaundice, neonates in general are at increased risk since they lack the intestinal bacterium that facilitates the breakdown and excretion of conjugated bilirubin in the faeces (this is largely why the faeces of a neonate are paler than those of an adult). Instead the conjugated bilirubin is converted back into the unconjugated form by the enzyme β-glucoronidase (in the gut, this enzyme is located in the brush border of the lining intestinal cells) and a large proportion is reabsorbed through the enterohepatic circulation (Sedlak and Synder, 2004).

Types of jaundice

Jaundice maybe classified into three types:

Prehepatic or haemolytic jaundice: Due to excess breakdown of red blood cells as in haemolytic anaemia, the bilirubin load becomes too much for liver to conjugate. The bilirubin is therefore mostly unconjugated or indirect.

Hepatocellular jaundice: As a result of diminished function of the liver cells (due to damage) as in infective hepatitis, conjugation is inhibited; so initially the bilirubin is indirect, as the cell damage affects the structure of the liver, conjugated bilirubin becomes increased. Thus both types of bilirubin are present in the serum.

Post hepatic or obstructive jaundice: When bilirubin which has been conjugated cannot be excreted due to obstruction in the bile duct by either gall stone within the lumen or by tumour excerting pressure from outside, the bilirubin “spill over” into the blood. The bilirubin is water-soluble (conjugated) and can be excreted by the kidney.

Causes of jaundice

Jaundice most often occurs as a result of an underlying disorder that either causes tissues to become over-saturate with bilirubin or prevents the liver from disposing of bilirubin. Some underlying conditions that may cause jaundice are:

Acute inflammation of the liver: this may impair the ability of the liver to conjugate and secrete bilirubin, resulting in build up of bilirubin.

Inflammation of the bile duct: this prevents the secretion of bile and removal of bilirubin causing jaundice.

Obstruction of the bile duct: It prevents the liver from disposing of bilirubin, which results in hyperbilibinemia.

Malaria: A blood borne infection spread by mosquitoes.

Sickle cell anaemia and thalassaemia: An inherited blood disorder where the red blood cells develop abnormally; its most common among black Caribbean, black African and black British people.

Crogler-najjar syndrome: A genetic syndrome where an enzyme needed to move bilirubin out of the blood and into the liver is missing.

Hereditary Spherocytosis: A genetic that causes red blood cells to have a much shorter life span than normal.

Viral hepatitis group of infections: Hepatitis A, B and C.

Alcohol liver disease: Where the liver is damage as a result of drinking too much alcohol.

Leptospirosis: A bacterial infection that is spread by animals, particularly rats.

Glandular fever: A viral infection caused by the Epetein-Barr virus.

Drug misuse: Leading cause are ecstasy and overdoses of paracetamol.

Primary biliary cirrhosis: A rare condition that causes progressive liver damage.

Gilberts syndrome: A common genetic syndrome where the liver has problems breaking down bilirubin at a normal rate.

Liver cancer: A rare and usually incurable cancer that develops inside the liver.

Exposure to substance known to be harmful to the liver: Such as phenol (used in the manufacture of plastic or carbon tetrachloride)

Autoimmune hepatitis: A rare condition where the immune system attacks the liver.

Primary sclerosing cholangitis: A rare type of liver disease that causes long-lasting (chronic) inflammation of the liver.

Dubin-Johnson syndrome: A rare genetic syndrome where the liver is unable to move bilirubin out of the liver.

Gall-stone: Obstructing the bile duct system.

Pancreatic cancer

Gall bladder cancer or bile duct cancer

Pancreatitis: Inflammation of the pancreas, which can either be acute pancreatitis (lasting for a few days) or chronic pancreatitis (lasting for many years) (Gillott, 2015).

Signs and symptoms of jaundice

Jaundice may appear suddenly or develop slowly over time. Symptoms of jaundice commonly include:

1. Yellow skin
2. White part of the eyes (sclera)
3. Severe jaundice may look causes
4. Yellow colour inside the mouth
5. Dark or brown-coloured urine
6. Pale or clay-coloured stools.

Other symptoms depend on the disorder causing the jaundice

• Cancer: May produce no symptoms or there may be

1. Fatigue
2. Weight loss

• Hepatitis: May produce

1. Nausea
2. Vomiting
3. Fatigue or other symptoms (Feldman, 2010).

Diagnosis

Urine bilirubin

Detection of bilirubin in the urine is of significance in the early diagnosis of liver disease and jaundice. Conjugated bilirubin is the water-soluble from of bilirubin found in urine in cases of hepatocellular disease or obstructive jaundice (Abayomi, 2007).

Tests for urine bilirubin

1. Fouchet’s test
2. Ictotest

iii. Bilirubin test strip

1. Fouchet’s test: Bilirubin reacts with ferric chloride in fouchet’s reagents to form a green coloured compound.

Principle: When barium chloride is added to urine, the sulphate radicals reacts with the barium chloride to form barium sulphate precipitate and is oxidised to biliverdin (green), in the presence of ferric chloride and trichloroacetic acid. The test is sensitive easy to perform, inexpensive and stable.

Reagent

1. 10% Barium Chloride
2. Fouchet’s reagent

Method

1. Place 5 ml of fresh urine in a clean tube
2. Add 2.5 ml of 10% Barium chloride reagent and mix thoroughly. The mixtures appear turbid.
3. Filter or centrifuge to collect the barium sulphate precipitate
4. Add a few drops of the precipitate onto a filter paper placed on a piece of absorbent paper.
5. Add one (1) drop of Fouchet’s reagent to the precipitate or to the sediment in the test tube.

Result

Immediate blue green colour around the drop; Bilirubin positive

1. Ictotest: Ictotest is a reagent test tablet; it consists of P-nitrobenzene diazonium toluene sulphate, salicysulphonic acid, and sodium hydrogen carbonate (NaHC

Principle:

A few drop of urine is added onto a test mat, made of asbestos and cellulose fibre. Bilirubin and other bile pigment if present are absorbed on the surface of the mat.

The bilirubin reacts with the diazo compound in the presence of sulphosalicylic acid, to form a blue-purple colour.

Procedure

1. Put five (5) drops of urine in the centre of one square of test mat.
2. Place a tablet in the middle of the moisture area.
3. Add two (2) drops of water unto the tablet. The water should flow from the tablet into the mat. The heat evolved will partially dissolve the tablet and spread onto the surface of the mat.
4. Exactly after 30 seconds, observe the colour change of the mat around the tablet.

Result:

If the colour remains unchanged or turns slightly red or pink within 30 seconds–Negative, if the mat around the tablet turns bluish-purple within 30 seconds -Positive.

N.B: The concentration of bilirubin present is directly proportional to the intensity of the colour and the rate of colour developed.

iii. Bilirubin test strip

Principle: The bilirulin couples with 2, 6 dichlorobenzene-diazonium fluoroborate in an acid medium to give a reddish azo-complex. The strip, usually, in the multi-test strip, is dipped into the urine and the result is interpreted by comparison with the colour chart.

Serum bilirubin

Bilirubin is degraded by light, blood collection tubes containing blood or serum to be used in bilirubin assays should be protected from illumination. Non invasive technology is available in some health care facilities that will measure bilirubin by using an instrument placed on the skin (Transcutaneous bilirubin meter). Total bilirubin measures both unconjugated and conjugate bilirubin. Total and conjugated bilirubin levels can be measured from the blood, but unconjugated bilirubin is calculated from the total and conjugated bilirubin. The bilirubin level found in the body reflects the balance between production and excretion. Blood test should be interpreted using the reference range by the laboratory that performs the test (Strike, 2006).

Methods of determination of serum or plasma bilirubin

1. Van den Bergh diazo reaction method.
2. Malloy and Evelyn’s method.
3. Jendrassik and Grof’s method.

1. Van den Bergh Diazo reaction: Bilirubin in serum has been determined for many years by using Van den Berg diazo reaction. This involves treating the serum with diazotised Suphanilic acid, the azobilirubin complex so formed is estimated quantitatively in the spectrophotometer against a suitable standard either in an acid medium at 540nm, when it is red or at on alkaline pH when it is blue, the later is measured at 600nm.
2. Malloy and Evelyn’s method: In this method, serum is diluted with water diazotised with sulphanilic acid and then methanol is added in the amount sufficient to precipitate the proteins, but at the high concentration allow all the bilirubin to react with the reagent. The red azobilirubin is then measured at 450nm.

Principle: Serum is diluted with water and diazotised with sulphanilic acid and in the presence of the accelerator diphylline. Ascorbic acid is added to stop the reaction and to prevent haemoglobin from interfering with the azo-coupling, the azobilirubin so formed is then measured at an alkaline pH.

Reagent

1. Diazo Solution A
2. Diazo Solution B
3. Diazo reagent
4. Ascorbic Acid Solution
5. Dyphylline Solution
6. Alkaline Titrate Solution
7. Standard Bilirubin Solution

Method

Into five test-tubes labelled S(standard), SB (standard blank), B(blank), TB(total bilirubin) and CB(conjugated bilirubin), Mix after each addition.

To all tubes add 1.5ml alkaline tartrates and absorbance of the resulting solutions at 600nm. Zero with water. All measurements should be taken as soon as possible after adding the tartrate.

Note: If the serum value is over about 250μmol/L, dilute the specimen 1 in 2 or if low value use 0.4ml serum and less water, always remember to adjust the calculation accordingly. The serum should be assayed as soon as possible after collection.

Result: Normal serum has very little conjugated bilirubin present, if any at all. The upper limit for total bilirubin is up to 17μmol/L, but in the majority of normal persons the total serum bilirubin does not exceed 10μmol/L.

In jaundice, the total bilirubin increases, conjugated bilirubin may also be increased (Baker and Silverton, 2007).

• Jendrassik and Grof’s method

Principle: Bilirubin is estimated by reacting it with diazotized sulfanilic acid that forms a pink coloured azo compound, obtained from sodium nitrite and sulfanilic acid solutions. Direct bilirubin (conjugated or soluble fraction) reacts very quickly and unconjugated or free bilirubin takes longer time to react and requires caffeine accelerator. The intensity of the pink colour is directly proportional to the concentration of bilirubin in specimen and is measured at 540nm.

Calculation

Serum bilirubin(mg/dl)=(Absorbance of Test-Absorbance of blank)x30.0

The calculation for total and direct bilirubin is same

Normal range

Total bilirubin =0.0-1.0 mg/dl

Direct bilirubin=0.0-0.3 mg/dl (Suman, 2013)

Treatment

Treatment on jaundice depends on the type of jaundice, how serious it is and it cause. It may include tackling an underlying condition such as malaria. For genetic conditions that don’t get better, like sickle cell anaemia, a blood transfusion may be given to replenish red blood cells in the body. If the bile duct system is blocked, an operation may be needed to unblock it. During these procedures, measures may be taken to help prevent further problems, such as removal of the gallbladder. If the liver is found to be seriously damaged, a transplant may be an option. In certain cases of newborn jaundice, exposing the baby to special coloured lights (phototherapy) or exchange blood transfusion may be required to decrease elevated bilirubin level.

Prevention

As many things can cause jaundice, it is hard for doctors to give specific prevention advice in all cases.

The underlying medical condition causing jaundice can in some cases be prevented. Some preventive measures include the following:

1. Avoid heavy alcohol use (alcohol hepatitis, cirrhosis, and pancreatitis).
2. Vaccines for hepatitis (Hepatitis A and B).
3. Take medications which prevent malaria before travelling to high risk regions.
4. Avoid potentially contaminated food/water and maintain good hygiene (Hepatitis A).
5. Avoid medications that can cause haemolysis in susceptible individuals (such as those with G6PD deficiency, a condition that leads to red blood cell breakdown after consumption of certain substance).
6. Avoid medications and toxins which can cause haemolysis or directly damage the liver (Steven, 2014).

Clinical significance

The bilirubin test is an important part of routine newborn and adult suspected of liver disorder diagnostic test to measure levels of bile pigment in blood serum. All individuals produce bilirubin daily as part of the normal turn-over of red cells. A higher than normal (elevated) bilirubin test result can reflect accelerated red blood cell destruction or may indicate that bilirubin is not being excreted as it should be, suggesting that liver function problems or other abnormalities may be present. Neonatal bilirubin screening often reveals an elevated bilirubin (hyperbilirubinaemia). The bilirubin test will determine if hyperbilirubinaemia is present and along with other diagnostic tests, help determine if the condition is relatively normal (benign) or possibly related to liver function problems or other conditions. In newborns with jaundice, bilirubin is measured to determine the level. Excessive conjugated bilirubin damages developing brain cells in infant and may cause mental retardation, learning and developmental disabilities, hearing loss or eye movement problem.

Conclusion

Severe hyperbilirubineamia in newborns continues to carry the potential for complication from acute encephalopathy. Early diagnosis in adult may determine liver dysfunction or obstruction in the bile duct. Appropriate laboratory investigations, along with judicious phototherapy and exchange transfusion when indicated, are all essential to avoid these complications.

Recommendation

Early recognition of skin and eye discolouration in adult should necessitate immediate investigation for determination of the cause of jaundice. Total serum bilirubin should be measured in all infant during the first 72 hours of life. Any infant discharged before 24 hours of life should be reviewed within 24 hours by an individual with experience in the care of newborn and infants with clinical signs of acute bilirubin encephalopathy should have an immediate exchange transfusion.

References

Abayomi .A (2007) Book for Medical Laboratory Practical. (7^th ed). pp41.

Baker F.J and Silverton. R.E (2009). Baker and Silverton’s Introduction to Medical Laboratory Technology. 7^th edition. pp130.

Drefetz, A. and Adam, S. (2010): Oxford American Handbook of Gastroenterology and Hepatology. Oxford: Oxford University Press, USA. pp165.

Feldman, M. (2010): “Gastrointestinal and Disease. 9^th ed. Philadelpha, Pa: Saunders Elserier pp201.

Gillott, C. (2015): “what is jaundice? What causes jaundice? What is icterus?” Human Anatomy and Physiology, (10^th ed), Benjamin Cummings, Pearson, USA.

Mansur, G. (2010): “Bilirubin Biochemistry” Encyclopaedia Britannica (volume 13). Benton Foundation (15^th ed) Publisher. pp1095.

Ochei, J. and Kolhatkar, A. (2007): “Medical Laboratory Science Theory and Practical”. Tata MacGraw Hill Publishing Company 6^th edition. pp152.

Pirone, C. (2009): “Animal Pigment Bilirubin Discovered in Plants” Journal of the American Chemical Society 131(8): 2830

Sedlak, T. and Syndner, S. (2009): “Bilirubin and glutathione have Complementary antioxidant and cytopretective roles”. Proceeding of the National Academy of Science 106(13): 5171-6.

Steven, D. (2014): “Preventive Measures in Jaundices” American Board of Family Medicine Medscape Journal of Medicine. pp51

Suman, G. (2013): “Qualitative estimation of serum, plasma Bilirubin based on Jendrassik and Grof’s method” Indian Journal of Physiology and Pharmacology Vol 15. pp 27.