5.3 Chemistry - Glucose, Hemoglobin, Iron, and Bilirubin (36-70)

Which of the following statements about carbohydrate intolerance is true?

A. Galactosemia results from deficiency of galactose-1-phosphate (galactose-1-PO4) uridine diphosphate transferase

B. Galactosemia results in a positive glucose oxidase test for glucose in urine

C. Urinary galactose is seen in both galactosemia and lactase deficiency

D. A galactose tolerance test is used to confirm a diagnosis of galactosemia

A. Galactosemia results from deficiency of galactose-1-phosphate (galactose-1-PO4) uridine diphosphate transferase

A Galactose is metabolized to galactose-1-PO4 by the action of galactokinase. Galactose-1-PO4 uridine diphosphate (UDP) transferase converts galactose-1-PO4 to glucose. Deficiency of either enzyme causes elevated blood and urine galactose. Lactase deficiency results in the presence of urinary lactose because it is not broken down to glucose and galactose. Tests for reducing sugars employing copper sulfate are used to screen for galactose, lactose, and fructose in urine. Nonglucose-reducing sugars are not detected by the glucose oxidase reaction. A positive test is followed by TLC to identify the sugar, and demonstration of the enzyme deficiency in RBCs. The galactose tolerance test is used (rarely) to evaluate the extent of liver failure since the liver is the site of galactose metabolism.

Which of the following statements regarding iron metabolism is correct?

A. Iron absorption is decreased by alcohol ingestion

B. Normally, 40%-50% of ingested iron is absorbed

C. The daily requirement is higher for pregnant and menstruating women

D. Absorption increases with the amount of iron in the body stores

C. The daily requirement is higher for pregnant and menstruating women

C For adult men and nonmenstruating women, approximately 1-2 mg/day of iron is needed to replace the small amount lost mainly by exfoliation of cells. Because 5%-10% of dietary iron is absorbed normally, the daily dietary requirement in this group is 10-20 mg/day. Menstruating women have an additional requirement of 1 mg/day and pregnant women 2 mg/day. Absorption efficiency will increase in iron deficiency and decrease in iron overload. Iron absorption is enhanced by low gastric pH and is increased by alcohol ingestion.

Which of the following processes occurs when iron is in the oxidized (Fe3+) state?

A. Absorption by intestinal epithelium

B. Binding to transferrin and incorporation into ferritin

C. Incorporation into protoporphyrin IX to form functional heme

D. Reaction with chromogens in colorimetric assays

B. Binding to transferrin and incorporation into ferritin

B Intestinal absorption occurs only if the iron is in the reduced (Fe+2) state. After absorption, Fe+2 is oxidized to Fe+3 by gut mucosal cells. Transferrin and ferritin bind iron efficiently only when in the oxidized state. Iron within Hgb binds to O2 by coordinate bonding, which occurs only if the iron is in the reduced state. Likewise, in colorimetric methods, Fe+2 forms coordinate bonds with carbon and nitrogen atoms of the chromogen.

Which of the following is associated with low serum iron and high total iron-binding capacity (TIBC)?

A. Iron deficiency anemia

B. Hepatitis

C. Nephrosis

D. Noniron deficiency anemias

A. Iron deficiency anemia

A Iron-deficiency anemia is the principal cause of low serum iron and high TIBC because it promotes increased transferrin. Pregnancy without iron supplementation depletes maternal iron stores and also results in low serum iron and high TIBC. Iron-supplemented pregnancy and use of contraceptives increase both iron and TIBC. Nephrosis causes low iron and TIBC due to loss of both iron and transferrin by the kidneys. Hepatitis causes increased release of storage iron, resulting in high levels of iron and transferrin. Noniron deficiency anemias may cause high iron and usually show low TIBC and normal or high ferritin.

Which condition is associated with the lowest percent saturation of transferrin?

A. Hemochromatosis

B. Anemia of chronic infection

C. Iron deficiency anemia

D. Noniron deficiency anemia

C. Iron deficiency anemia

C Percent saturation = Serum Fe × 100/TIBC. Normally, transferrin is one-third saturated with iron. In iron deficiency states, the serum iron falls but transferrin rises. This causes the numerator and denominator to move in opposite directions, resulting in very low percent saturation (about 10%). The opposite occurs in hemochromatosis and sideroblastic anemia, resulting in an increased percent saturation

Which condition is most often associated with a high serum iron level?

A. Nephrosis

B. Chronic infection or inflammation

C. Polycythemia vera

D. Noniron deficiency anemias

D. Noniron deficiency anemias

D Anemia associated with chronic infection causes a low serum iron, but unlike iron deficiency, causes a low (or normal) TIBC and does not cause low ferritin. Noniron deficiency anemias such as pernicious anemia and sideroblastic anemia produce high serum iron and low TIBC. Nephrosis causes iron loss by the kidneys. Polycythemia is associated with increased iron within the RBCs and depletion of iron stores.

Which of the following is likely to occur first in iron deficiency anemia?

A. Decreased serum iron

B. Increased TIBC

C. Decreased serum ferritin

D. Increased transferrin

C. Decreased serum ferritin

C Body stores must be depleted of iron before serum iron falls. Thus, serum ferritin falls in the early stages of iron deficiency, making it a more sensitive test than serum iron in uncomplicated cases. Ferritin levels are low only in iron deficiency. However, concurrent illness such as malignancy, infection, and inflammation may promote ferritin release from the tissues, causing

Which formula provides the best estimate of serum TIBC?

A. Serum transferrin in mg/dL × 0.70 = TIBC (µg/dL)

B. Serum transferrin in mg/dL × 1.43 = TIBC (µg/dL)

C. Serum iron (µg/dL)/1.2 + 0.06 = TIBC (µg/dL)

D. Serum Fe (µg/dL) × 1.25 = TIBC (µg/dL)

B. Serum transferrin in mg/dL × 1.43 = TIBC (µg/dL)

B Transferrin, a β-globulin, has a molecular size of about 77,000. Transferrin is the principal iron transport protein, and TIBC is determined by the serum transferrin concentration. One mole of transferrin binds two moles of Fe+3, so the transferrin concentration can be used to predict the TIBC. Since the direct measurement of TIBC requires manual pretreatment to remove the excess iron added and is prone to overestimation if all of the unbound iron is not removed, some labs prefer to measure transferrin immunochemically and calculate TIBC. This formula may underestimate TIBC because albumin and other proteins will bind iron when the percent iron saturation of transferrin is abnormally high.

Which statement regarding the diagnosis of iron deficiency is correct?

A. Serum iron levels are always higher at night than during the day

B. Serum iron levels begin to fall before the body stores become depleted

C. A normal level of serum ferritin rules out iron deficiency

D. A low serum ferritin is diagnostic of iron deficiency

D. A low serum ferritin is diagnostic of iron deficiency

D Serum iron levels are falsely elevated by hemolysis and subject to diurnal variation. Levels are highest in the morning and lowest at night, but this pattern is reversed in persons who work at night. A low ferritin is specific for iron deficiency. However, only about 1% of ferritin is in the vascular system. Any disease that increases ferritin release may mask iron deficiency

Which statement about iron methods is true?

A. Interference from Hgb can be corrected by a serum blank

B. Colorimetric methods measure binding of Fe2+ to a ligand such as ferrozine

C. Atomic absorption is the method of choice for measurement of serum iron

D. Serum iron can be measured by potentiometry

B. Colorimetric methods measure binding of Fe2+ to a ligand such as ferrozine

B Atomic absorption is not the method of choice for serum iron because matrix error and variation of iron recovered by extraction cause bias and poor precision. Most methods use HCl to deconjugate Fe3+ from transferrin followed by reduction to Fe2+. This reacts with a neutral ligand such as ferrozine, tripyridyltriazine (TPTZ), or bathophenanthroline to give a blue complex. Anodic stripping voltammetry can also be used to measure serum iron. Hemolysis must be avoided because RBCs contain a much higher concentration of iron than does plasma.

Which of the following statements regarding the TIBC assay is correct?

A. All TIBC methods require addition of excess iron to saturate transferrin

B. All methods require the removal of unbound iron

C. Measurement of TIBC is specific for transferrin-bound iron

D. The chromogen used must be different from the one used for measuring serum iron

A. All TIBC methods require addition of excess iron to saturate transferrin

A All TIBC methods require addition of excess iron to saturate transferrin. Excess iron is removed by ion exchange or alumina gel columns or precipitation with MgCO3 and the bound iron is measured by the same procedure as is used for serum iron. Alternatively, excess iron in the reduced state can be added at an alkaline pH. Under these conditions, transferrin will bind Fe2+ and the unbound Fe2+ can be measured directly.

Which of the following statements regarding the metabolism of bilirubin is true?

A. It is formed by hydrolysis of the α methene bridge of urobilinogen

B. It is reduced to biliverdin prior to excretion

C. It is a by-product of porphyrin production

D. It is produced from the destruction of RBCs

D. It is produced from the destruction of RBCs

D Synthesis of porphyrins results in production of heme and metabolism of porphyrins other than protoporphyrin IX yields uroporphyrins and coproporphyrins, not bilirubin. Reticuloendothelial cells in the spleen digest Hgb and release the iron from heme. The tetrapyrrole ring is opened at the α methene bridge by heme oxygenase, forming biliverdin. Bilirubin is formed by reduction of biliverdin at the γ methene bridge. It is complexed to albumin and transported to the liver.

Bilirubin is transported from reticuloendothelial cells to the liver by:

A. Albumin

B. Bilirubin-binding globulin

C. Haptoglobin

D. Transferrin

A. Albumin

A Albumin transports bilirubin, haptoglobin transports free Hgb, and transferrin transports ferric iron. When albumin binding is exceeded, unbound bilirubin, called free bilirubin, increases. This may cross the blood-brain barrier, resulting in kernicterus.

In the liver, bilirubin is conjugated by addition of:

A. Vinyl groups

B. Methyl groups

C. Hydroxyl groups

D. Glucuronyl groups

D. Glucuronyl groups

D The esterification of glucuronic acid to the propionyl side chains of the inner pyrrole rings (I and II) makes bilirubin water soluble. Conjugation is required before bilirubin can be excreted via the bile.

Which enzyme is responsible for the conjugation of bilirubin?

A. β-Glucuronidase

B. UDP-glucuronyl transferase

C. Bilirubin oxidase

D. Biliverdin reductase

B. UDP-glucuronyl transferase

B UDP-glucuronyl transferase esterifies glucuronic acid to unconjugated bilirubin, making it water soluble. Most conjugated bilirubin is diglucuronide; however, the liver makes a small amount of monoglucuronide and other glycosides. β Glucuronidase hydrolyzes glucuronide from bilirubin, hormones, or drugs. It is used prior to organic extraction to deconjugate urinary metabolites (e.g., total cortisol). Biliverdin reductase forms bilirubin from biliverdin (and heme oxygenase forms biliverdin from heme). Bilirubin oxidase is used in an enzymatic bilirubin assay in which bilirubin is oxidized back to biliverdin and the rate of biliverdin formation is measured at 410 nm.

The term δ-bilirubin refers to:

A. Water-soluble bilirubin

B. Free unconjugated bilirubin

C. Bilirubin tightly bound to albumin

D. Direct-reacting bilirubin

C. Bilirubin tightly bound to albumin

C HPLC separates bilirubin into four fractions: α = unconjugated, β = monoglucuronide, γ = diglucuronide, and δ = irreversibly albumin bound. δ Bilirubin is a separate fraction from the unconjugated bilirubin, which is bound loosely to albumin. δ Bilirubin and conjugated bilirubin react with diazo reagent in the direct bilirubin assay

Which of the following processes is part of the normal metabolism of bilirubin?

A. Both conjugated and unconjugated bilirubin are excreted into the bile

B. Methene bridges of bilirubin are reduced by intestinal bacteria forming urobilinogens

C. Most of the bilirubin delivered into the intestine is reabsorbed

D. Bilirubin and urobilinogen reabsorbed from the intestine are mainly excreted by the kidneys

B. Methene bridges of bilirubin are reduced by intestinal bacteria forming urobilinogens

B Most of the conjugated bilirubin delivered into the intestine is deconjugated by β-glucuronidase and then reduced by intestinal flora to form three different reduction products collectively called urobilinogens. The majority of bilirubin and urobilinogen in the intestine are not reabsorbed. Most of that which is reabsorbed is re-excreted by the liver. The portal vein delivers blood from the bowel to the sinusoids. Hepatocytes take up about 90% of the returned bile pigments and secrete them again into the bile. This process is called the enterohepatic circulation

Which of the following is a characteristic of conjugated bilirubin?

A. It is water soluble

B. It reacts more slowly than unconjugated bilirubin

C. It is more stable than unconjugated bilirubin

D. It has the same absorbance properties as unconjugated bilirubin

A. It is water soluble

A Conjugated bilirubin refers to bilirubin mono- and diglucuronides. Conjugated bilirubin reacts almost immediately with the aqueous diazo reagent without need for a nonpolar solvent. Historically, conjugated bilirubin has been used synonymously with direct-reacting bilirubin, although the latter includes the δ-bilirubin fraction when measured by the Jendrassik-Grof method. Conjugated bilirubin is excreted in both bile and urine. It is easily photo-oxidized and has very limited stability. For this reason, bilirubin standards are usually prepared from unconjugated bilirubin stabilized by the addition of alkali and albumin.

Which of the following statements regarding urobilinogen is true?

A. It is formed in the intestines by bacterial reduction of bilirubin

B. It consists of a single water-soluble bile pigment

C. It is measured by its reaction with p-aminosalicylate

D. In hemolytic anemia, it is decreased in urine and feces

A. It is formed in the intestines by bacterial reduction of bilirubin

A Urobilinogen is a collective term given to the reduction products of bilirubin formed by the action of enteric bacteria. Urobilinogen excretion is increased in extravascular hemolytic anemias and decreased in obstructive jaundice (cholestatic disease). Urobilinogen is measured using Ehrlich's reagent, an acid solution of p-dimethylaminobenzaldehyde.

Which statement regarding bilirubin metabolism is true?

A. Bilirubin undergoes rapid photo-oxidation when exposed to daylight

B. Bilirubin excretion is inhibited by barbiturates

C. Bilirubin excretion is increased by chlorpromazine

D. Bilirubin is excreted only as the diglucuronide

A. Bilirubin undergoes rapid photo-oxidation when exposed to daylight

A Samples for bilirubin analysis must be protected from direct sunlight. Drugs may have a significant in vivo effect on bilirubin levels. Barbiturates lower serum bilirubin by increasing excretion. Other drugs that cause cholestasis, such as chlorpromazine, increase the serum bilirubin. Although most conjugated bilirubin is in the form of diglucuronide, some monoglucuronide

and other glycosides are excreted. In glucuronyl transferase deficiency, some bilirubin is excreted as sulfatides.

Which condition is caused by deficient secretion of bilirubin into the bile canaliculi?

A. Gilbert's disease

B. Neonatal hyperbilirubinemia

C. Dubin-Johnson syndrome

D. Crigler-Najjar syndrome

C. Dubin-Johnson syndrome

C Dubin-Johnson syndrome is an autosomal recessive condition arising from mutation of an ABC transporter gene. It produces mild jaundice from accumulation of conjugated bilirubin that is not secreted into the bile canaliculi. Total and direct bilirubin are elevated, but other liver function is normal. Rotor syndrome is an autosomal recessive condition that also results in retention of conjugated bilirubin. The mechanism in Rotor syndrome is unknown, and like Dubin-Johnson syndrome it is commonly asymptomatic. It can be differentiated from Dubin-Johnson syndrome by the pattern of urinary coproporphyrin excretion and because it produces no black pigmentation in the liver.

In hepatitis, the rise in serum conjugated bilirubin can be caused by:

A. Secondary renal insufficiency

B. Failure of the enterohepatic circulation

C. Enzymatic conversion of urobilinogen to bilirubin

D. Extrahepatic conjugation

B. Failure of the enterohepatic circulation

B Conjugated bilirubin is increased in hepatitis and other causes of hepatic necrosis due to failure to re-excrete conjugated bilirubin reabsorbed from the intestine. Increased direct bilirubin can also be attributed to accompanying intrahepatic obstruction, which blocks the flow of bile.

Which of the following is a characteristic of obstructive jaundice?

A. The ratio of direct to total bilirubin is greater than 1:2

B. Conjugated bilirubin is elevated, but

unconjugated bilirubin is normal

C. Urinary urobilinogen is increased

D. Urinary bilirubin is normal

A. The ratio of direct to total bilirubin is greater than 1:2

A Obstruction prevents conjugated bilirubin from reaching the intestine, resulting in decreased production, excretion, and absorption of urobilinogen. Conjugated bilirubin regurgitates into sinusoidal blood and enters the general circulation via the hepatic vein. The level of serum direct (conjugated) bilirubin becomes greater than unconjugated bilirubin. The unconjugated form is also increased because of accompanying necrosis, deconjugation, and inhibition of UDP-glucuronyl transferase

Which of the following would cause an increase in only the unconjugated bilirubin?

A. Hemolytic anemia

B. Obstructive jaundice

C. Hepatitis

D. Hepatic cirrhosis

A. Hemolytic anemia

A Conjugated bilirubin increases as a result of obstructive processes within the liver or biliary system or from failure of the enterohepatic circulation. Hemolytic anemia (prehepatic jaundice) presents a greater bilirubin load to a normal liver, resulting in increased bilirubin excretion. When the rate of bilirubin formation exceeds the rate of excretion, the unconjugated bilirubin rises.

Which form of hyperbilirubinemia is caused by an inherited absence of UDP-glucuronyl transferase?

A. Gilbert's syndrome

B. Rotor syndrome

C. Crigler-Najjar syndrome

D. Dubin-Johnson syndrome

C. Crigler-Najjar syndrome

C Crigler-Najjar syndrome is a rare condition that occurs in two forms. Type 1 is inherited as an autosomal recessive trait and causes a total deficiency of UDP-glucuronyl transferase. Life expectancy is less than 1 year. Type 2 is an autosomal dominant trait and is characterized by lesser jaundice and usually the absence of kernicterus. Bilirubin levels can be controlled with phenobarbital, which promotes bilirubin excretion. Gilbert's syndrome is an autosomal recessive condition characterized by decreased bilirubin uptake and decreased formation of bilirubin diglucuronide. It is the most common form of inherited jaundice. UDP glucuronyl transferase activity is reduced owing to an increase in the number of AT repeats in the promoter region of the gene. Dubin-Johnson and Rotor syndromes are autosomal recessive disorders associated with defective delivery of bilirubin into the biliary system.

Which statement regarding total and direct bilirubin levels is true?

A. Total bilirubin level is a less sensitive and specific marker of liver disease than the direct level

B. Direct bilirubin exceeds 3.5 mg/dL in most cases of hemolytic anemia

C. Direct bilirubin is normal in cholestatic liver disease

D. The ratio of direct to total bilirubin exceeds 0.40 in hemolytic anemia

A. Total bilirubin level is a less sensitive and specific marker of liver disease than the direct leve

A Direct bilirubin measurement is a sensitive and specific marker for hepatic and posthepatic jaundice because it is not elevated by hemolytic anemia. In hemolytic anemia, the total bilirubin does not exceed 3.5 mg/dL, and the ratio of direct to total is less than 0.20. Unconjugated bilirubin is the major fraction in necrotic liver disease because microsomal enzymes are lost. Unconjugated bilirubin is elevated along with direct bilirubin in cholestasis because some necrosis takes place and some conjugated bilirubin is hydrolyzed back to unconjugated bilirubin.

Which statement best characterizes serum bilirubin levels in the first week following delivery?

A. Serum bilirubin 24 hours after delivery should not exceed the upper reference limit for adults

B. Jaundice is usually first seen 48-72 hours postpartum in neonatal hyperbilirubinemia

C. Serum bilirubin above 5.0 mg/dL occurring 2-5 days after delivery indicates hemolytic or hepatic disease

D. Conjugated bilirubin accounts for about 50% of the total bilirubin in neonates

B. Jaundice is usually first seen 48-72 hours postpartum in neonatal hyperbilirubinemia

B Bilirubin levels may reach as high as 2-3 mg/dL in the first 24 hours after birth owing to the trauma of delivery, such as resorption of a subdural hematoma. Neonatal hyperbilirubinemia occurs 2-3 days after birth due to increased hemolysis at birth and transient deficiency of the microsomal enzyme, UDP-glucuronyl transferase. Normally, levels rise to about 5-10 mg/dL but may be greater than 15 mg/dL, requiring therapy with UV light to photo-oxidize the bilirubin. Neonatal jaundice can last up to 1 week in a mature neonate and up to 2 weeks in prematures babies. Neonatal bilirubin is almost exclusively unconjugated

Which form of jaundice occurs within days of delivery and usually lasts 1-3 weeks, but is not due to normal neonatal hyperbilirubinemia or hemolytic disease of the newborn?

A. Gilbert syndrome

B. Lucey -Driscoll syndrome

C. Rotor syndrome

D. Dubin-Johnson syndrome

B. Lucey -Driscoll syndrome

B Lucey-Driscoll syndrome is a rare form of jaundice caused by unconjugated bilirubin that presents within 2-4 days of birth and can last several weeks. It is caused by an inhibitor of UDP-glucuronyl transferase in maternal plasma that crosses the placenta. Jaundice is usually severe enough to require treatment.

A lab measures total bilirubin by the Jendrassik-Grof bilirubin method with sample blanking. What would be the effect of moderate hemolysis on the test result?

A. Falsely increased due to optical interference

B. Falsely increased due to release of bilirubin from RBCs

C. Falsely low due to inhibition of the diazo reaction by hemoglobin

D. No effect due to correction of positive interference by sample blanking

C. Falsely low due to inhibition of the diazo reaction by hemoglobin

C The sample blank measures the absorbance of the sample and reagent in the absence of azobilirubin formation and corrects the measurement for optical interference caused by hemoglobin absorbing the wavelength of measurement. However, hemoglobin is an inhibitor of the diazo reaction and will cause falsely low results in a blank corrected sample. For this reason, direct bichromatic spectrophotometric methods are preferred when measuring bilirubin in neonatal samples, which are often hemolyzed.

Which reagent is used in the Jendrassik-Grof method to solubilize unconjugated bilirubin?

A. 50% methanol

B. N-butanol

C. Caffeine

D. Acetic acid

C. Caffeine

C A polarity modifier is required to make unconjugated bilirubin soluble in diazo reagent. The Malloy-Evelyn method uses 50% methanol to reduce the polarity of the diazo reagent. Caffeine is used in the Jendrassik-Grof method. This method is recommended because it is not falsely elevated by hemolysis and gives quantitative recovery of both conjugated and unconjugated bilirubin.

Which statement about colorimetric bilirubin methods is true?

A. Direct bilirubin must react with diazo reagent under alkaline conditions

B. Most methods are based upon reaction with diazotized sulfanilic acid

C. Ascorbic acid can be used to eliminate interference caused by Hgb

D. The color of the azobilirubin product is independent of pH

B. Most methods are based upon reaction with diazotized sulfanilic acid

B Unconjugated bilirubin is poorly soluble in acid, and therefore, direct bilirubin is assayed using diazotized sulfanilic acid diluted in weak HCl. The direct diazo reaction should be measured after no longer than 3 minutes to prevent reaction of unconjugated bilirubin, or the diazo group can be reduced using ascorbate or hydroxylamine preventing any further reaction.

Which statement regarding the measurement of bilirubin by the Jendrassik-Grof method is

correct?

A. The same diluent is used for both total and direct assays to minimize differences in reactivity

B. Positive interference by Hgb is prevented by the addition of HCl after the diazo reaction

C. The color of the azobilirubin product is intensified by the addition of ascorbic acid

D. Fehling's reagent is added after the diazo reaction to reduce optical interference by hemoglobin

D. Fehling's reagent is added after the diazo reaction to reduce optical interference by hemoglobin

D The Jendrassik-Grof method uses HCl as the diluent for the measurement of direct bilirubin because unconjugated bilirubin is poorly soluble at low pH. Total bilirubin is measured using an acetate buffer with caffeine added to increase the solubility of the unconjugated bilirubin. After addition of diazotized sulfanilic acid and incubatiion, the diazo group is reduced by ascorbic acid, and Fehling's reagent is added to alkalinize the diluent. At an alkaline pH the product changes from pink to blue, shifting the absorbance maximum to 600 nm where Hgb does not contribute significantly to absorbance.

A neonatal bilirubin assay performed at the nursery by bichromatic direct spectrophotometry is 4.0 mg/dL. Four hours later, a second sample assayed for total bilirubin by the Jendrassik-Grof method gives a result of 3.0 mg/dL. Both samples are reported to be hemolyzed. What is the most likely explanation of these results?

A. Hgb interference in the second assay

B. δ-Bilirubin contributing to the result of the first assay

C. Falsely high results from the first assay caused by direct bilirubin

D. Physiological variation owing to premature hepatic microsomal enzymes

A. Hgb interference in the second assay

A The Jendrassik-Grof method is based upon a diazo reaction that may be suppressed by Hgb. Because serum blanking and measurement at 600 nm correct for positive interference from Hgb, the results may be falsely low when significant hemolysis is present. Direct spectrophometric bilirubin methods employing bichromatic optics correct for the presence of Hgb. These are often called "neonatal bilirubin" tests. A commonly used approach is to measure absorbance at 454 nm and 540 nm. The absorbance contributed by Hgb at 540 nm is equal to the absorbance contributed by Hgb at 454 nm. Therefore, the absorbance difference will correct for free Hgb. Neonatal samples contain little or no direct δ-bilirubin. They also lack carotene pigments that could interfere with the direct spectrophotometric measurement of bilirubin.

In the enzymatic assay of bilirubin, how is measurement of both total and direct bilirubin

accomplished?

A. Using different pH for total and direct assays

B. Using UDP glucuronyl transferase and bilirubin reductase

C. Using different polarity modifiers

D. Measuring the rate of absorbance decrease at different time intervals

A. Using different pH for total and direct assays

A Enzymatic methods use bilirubin oxidase to convert bilirubin back to biliverdin, and measure the decrease in absorbance that results. At pH 8, both conjugated, unconjugated, and delta bilirubin react with the enzyme, but at pH 4 only the conjugated form reacts.

What is the principle of the transcutaneous bilirubin assay?

A. Conductivity

B. Amperometric inhibition

C. Multiwavelength reflectance photometry

D. Infrared spectroscopy

C. Multiwavelength reflectance photometry

C Measurement of bilirubin concentration through the skin requires the use of multiple wavelengths to correct for absorbance by melanin and other light-absorbing constituents of skin and blood. More than 100 wavelengths and multiple reflectance measurements at various sites may be used to derive the venous bilirubin concentration in mg/dL. Such devices have been shown to have a high specificity. They can be used to identify neonates with hyperbilirubinemia, and to monitor treatment.