CASE 53 - Clinical Cases - Case Files Biochemistry, 3rd Edition (2015)

Case Files Biochemistry, 3rd Edition (2015)

SECTION II. Clinical Cases

CASE 53

A 5-week-old boy is brought to the hospital emergency department following a 2-day bout of diarrhea, poor oral food intake, abdominal bloating, and an appearance of illness. The parents also indicate that the infant’s complexion “looks darker than he normally looks.” The boy is the product of an uncomplicated vaginal delivery at term. The child has been seen by his pediatrician for his first check-up and receives care during the day from his grandmother. The child is both nursed and bottle-fed. On physical examination, the infant is noted to appear ill with a dusky color and increased capillary refill time. Vital signs are pulse 150 beats/minute, blood pressure was 75/40 mm Hg, respiration 58 breaths/minute and shallow, and rectal temperature 100°F (37.8°C). Weight is 4.8 kg. The anterior fontanelle is sunken and mucous membranes are dry. Lungs are clear to auscultation, but breathing is labored. Heart sounds are normal. Extremities are poorly perfused. Laboratory results include a hematocrit level of 25.4%, white blood cell count of 28.6 × 103/mm3, and a platelet count of 953 × 103/mm3. Blood samples are noted to have a persistently darker, brownish color than normal infant samples. Arterial blood is found to have a pH of 7.18 (normal range: 7.35–7.45), a pO2 of 190 mm Hg, and a pCO2 of 24 mm Hg.

image What is the most likely cause of the infant’s acidosis and abnormal signs on examination?

image What is the treatment for this condition?

ANSWERS TO CASE 53:

Pediatric Methemoglobinemia Following Diarrhea

Summary: A 5-week-old infant is found to have a dark complexion, shortness of breath, and acidosis on arterial blood gas. The blood is also noted to be darker than normal. The infant recently had what appears to have a gastrointestinal infection.

Most likely cause of infant’s acidosis and examination findings: Metabolic acidosis from recent gastroenteritis and likely congenital methemoglobinemia.

Treatment: Methylene blue.

CLINICAL CORRELATION

Methemoglobin is a form of hemoglobin in which the iron is found in the oxidative state (ferric). Ferric iron is not able to bind to oxygen leading to functional a functional anemia with reduced oxygen delivery to tissue. Affected individuals appear “cyanotic” and their blood is very dark in appearance. Cytochrome b5 reductase is important in the conversion of methemoglobin back to hemoglobin. Patients with a defect in cytochrome b5 reductase (congenital defect) are at risk of developing excessive amounts methemoglobin and anemia. In addition to congenital methemoglobinemia, there are other acquired causes (drug/chemicals) of this condition. Treatment is with methylene blue because it reduces the methemoglobin back to hemoglobin.

APPROACH TO:

Methemoglobinemia, Oxygen Binding, and Electrochemistry

OBJECTIVES

1. Describe the molecular mechanisms for oxidation of hemoglobin to methemoglobin and how methemoglobin is reduced back to hemoglobin.

2. Explain why infants are susceptible to methemoglobinemia during the first few months of life.

DEFINITIONS

FETAL HEMOGLOBIN: The fetal form of hemoglobin that contains 2 α- and 2 γ-globin chains. In utero, γ chains are the predominant β-type chain in hemoglobin. The adult form of hemoglobin in which the γ chains are replaced by β chains starts to increase in concentration about the 36th week of gestation; most fetal hemoglobin is replaced by adult hemoglobin by about 12 to 15 weeks following birth. Fetal hemoglobin has a higher affinity for oxygen than adult hemoglobin.

LEUCOMETHYLENE BLUE: The reduced form of methylene blue. Unlike the oxidized form, it is colorless.

METHEMOGLOBIN: Hemoglobin in which the iron ions of the heme prosthetic groups have been oxidized to the ferric (Fe3+) oxidation state. Methemoglobin cannot bind oxygen.

METHEMOGLOBINEMIA: It is a disorder in which the red blood cells contain an excess of methemoglobin. Methemoglobin is normally kept at concentrations below 1%; when it exceeds this level, decreased levels of oxygen are delivered to the tissues.

METHYLENE BLUE: A heterocyclic, aromatic dye that contains both sulfur and nitrogen atoms. The oxidized form has a distinctive blue color when in aqueous solution; it can accept electrons to yield the reduced form, leucomethylene blue.

OXIDATION: The loss of electrons

REDUCTION: The gain of electrons

DISCUSSION

Hemoglobin is the protein in the erythrocyte that is responsible for carrying oxygen from the lungs to the tissues that utilize it for production of ATP via mitochondrial oxidative phosphorylation. It is a multi-subunit protein consisting of 2 α chains and 2 β chains, each of which has a recurring structure consisting of 6 α-helices arranged in a 3-dimensional structure that is commonly called the globin fold. The α and β chains function as 2 (αβ) dimers. Each of the α and β chains has a hydrophobic pocket that contains the iron-containing prosthetic group heme. Heme is protoporphyrin IX with an iron ion bound in coordinate covalent bonds to the N-atoms of the 4 pyrrole rings. The iron ion binds a histidine residue of the protein in its fifth coordination site; oxygen binds at the sixth coordination site. Oxygen can only bind to the heme-iron when it is in the reduced, ferrous (Fe2+) oxidation state. When the iron ion is oxidized to the ferric (Fe3+) oxidation state, oxygen cannot be bound and hemoglobin becomes methemoglobin.

Oxygen is a very electronegative molecule, and hemoglobin will react with it to form methemoglobin and the superoxide anion:

image

Because the heme prosthetic groups being positioned in hydrophobic pockets of the globin chains, the release of the superoxide anion very slowly occurs and the electron can return to the iron ion before oxygen release. Thus, the formation of methemoglobin slowly occurs; the rate of methemoglobin formation can be increased with increased oxidative load from exposure to a variety of xenobiotics, chemicals or drugs. Normally, the steady-state levels of methemoglobin in the red blood cell are kept to a minimum (< 1% of total hemoglobin) by the cytochrome b5 reductase (NADH methemoglobin reductase) system of the erythrocyte, using NADH produced by glycolysis as the source of reducing equivalents (Figure 53-1). A minor system for reducing methemoglobin uses a cytochrome b5 reductase that obtains its reducing equivalents from NADPH produced by the pentose phosphate pathway.

image

Figure 53-1. Reduction of methemoglobin by the cytochrome b5 reductase system.

Methemoglobinemia is a condition in which the levels of methemoglobin exceed 1% of total hemoglobin. Hereditary methemoglobinemia is rare and occurs when there is a deficiency in the erythrocyte of either cytochrome b5reductase or cytochrome b5 or when the patient has Hemoglobin M (wherein hemoglobin has been mutated to change an amino acid near the heme iron to make it more susceptible to oxidation). Acquired methemoglobinemia is much more common and can occur when oxidative stress exceeds the ability of red blood cells to re-reduce methemoglobin to hemoglobin. This can happen with the administration of certain pharmaceuticals (eg, benzocaine, dapsone, sulfonamides), use of recreational drugs such as amyl nitrate, or by exposure to pesticides or herbicides.

Infants, as in the case above, are particularly susceptible to episodes of methemoglobinemia because the cytochrome b5 reductase is not expressed to adult levels until the infant has reached 2 to 6 months of age. In addition, the transition from fetal hemoglobin (α2γ2), which is more susceptible to oxidation, to adult hemoglobin (α2β2) is not complete until 2 or 3 months of age. The infant in the scenario has experienced acute gastroenteritis and diarrhea that resulted in acidosis, probably due to excessive loss of bicarbonate in the stool. The decreased pH of the blood further impairs the already low levels of cytochrome b5 reductase. As a consequence, methemoglobin accumulates, thus decreasing the amount of oxygen that can be delivered to peripheral tissues. The respiration rate increases in an attempt to compensate for the decreased oxygen deposition. The increased concentration of methemoglobin will give blood samples a brownish color.

Acute infantile methemoglobinemia can be treated by administering methylene blue, which is a heterocyclic aromatic dye (Figure 53-2). When oxidized, the dye is blue, as its name suggests, but it is colorless when reduced. Methylene blue has been shown to increase the activity of the NADPH-dependent form of cytochrome b5 reductase (NADPH methemoglobin reductase), which usually does not participate in reduction of methemoglobin. The enzyme reduces methylene blue to leucomethylene blue using reducing equivalents from NADPH. The reduced form of methylene blue then transfers its electrons to methemoglobin (Fe3+), thereby regenerating hemoglobin (Fe2+). The NADPH utilized by the reductase is produced by the oxidation reactions of the pentose phosphate pathway. Thus, this treatment will be ineffective if the patient has a deficiency in glucose 6-phosphate dehydrogenase. It also will be ineffective if there is a deficiency in the NADPH-dependent cytochrome b5 reductase.

image

Figure 53-2. Treatment of methemoglobinemia with the dye methylene blue. The NADPH-dependent form of cytochrome b5 reductase reduces methylene blue to leucomethylene blue, which transfers its electrons to methemoglobin, thus reducing the ferric ion to the ferrous form.

COMPREHENSION QUESTIONS

53.1 When oxygen is bound to hemoglobin, the ferrous ion (Fe2+) can be oxidized to the ferric ion (Fe3+), which no longer binds oxygen. This results in production of the superoxide anion radical and methemoglobin. Methemoglobin levels are normally kept at a minimum by the action of which enzyme?

A. Glucose 6-phosphate dehydrogenase

B. Malic enzyme

C. NADH-cytochrome b5 reductase

D. NADH dehydrogenase

E. NADPH-cytochrome b5 reductase

53.2 A young Caucasian girl is brought to the pediatric clinic by her parents because they are concerned that she has a bluish hue to her skin. The physician suspects that the child has a congenital form of methemoglobinemia. However, an extract of her blood is able to convert added methemoglobin to hemoglobin. It is likely that the girl has which abnormal hemoglobin variant?

A. Hemoglobin A1C

B. Hemoglobin C

C. Hemoglobin F

D. Hemoglobin M

E. Hemoglobin S

53.3 A 2-month-old male infant presents to the emergency department following a 2-day bout with diarrhea and poor oral food intake. The infant is lethargic, has a dusky color, and signs of dehydration. Lungs are clear to auscultation but his breathing is labored. In addition to dehydration, the child is diagnosed with methemoglobinemia. To treat the methemoglobinemia, the child is administered methylene blue. Methylene blue reduces methemoglobin by utilizing reducing equivalents (electrons) drawn from which of the following?

A. Tricarboxylic acid cycle

B. Pentose phosphate pathway

C. Oxidation of glutamate to α-ketoglutarate

D. Glycolysis

E. β-Oxidation pathway

ANSWERS

53.1 C. The levels of methemoglobin are normally kept below 1% by NADH-cytochrome b5 reductase, which uses reducing equivalents from cytosolic NADH to reduce the ferric ion in methemoglobin to the ferrous ion in hemoglobin. The NADH primarily originates from the conversion of glyceraldehyde 3-phosphate to 1,3-bisphosphoglycerate in the glycolytic pathway. Glucose 6-phosphate dehydrogenase oxidizes glucose 6-phosphate to 6-phosphogluconolactone and converts NADP+ to NADPH. The malic enzyme also produces NADPH when it oxidatively decarboxylates malate to yield pyruvate. NADH dehydrogenase is the first enzyme in the mitochondrial electron transport pathway. NADPH-cytochrome b5 reductase plays a minor role in converting methemoglobin to hemoglobin but can play an important role in the treatment of methemoglobinemia.

53.2 D. Congenital methemoglobinemia can be due to a deficiency in cytochrome b5 reductase activity, which would decrease the rate at which methemoglobin could be reduced to hemoglobin. Another cause is the abnormal hemoglobin variant hemoglobin M. Hemoglobin M is the term applied to a group of abnormal hemoglobins in which a single amino acid substitution in the hydrophobic pocket that binds the heme results in the heme iron being easily oxidized to the ferric state. An example is hemoglobin M Saskatoon, in which His at position E7 is mutated to a Tyr residue. Hemoglobin A1C results from glycosylation of normal hemoglobin at elevated blood glucose levels; it is used as measure of long-term blood glucose levels. Hemoglobin C is the result of a point mutation that converts a Glu residue to a Lys residue at position 6 of the β-chain. It results in loss of plasticity of red blood cells and a mild hemolytic anemia. Hemoglobin F is the fetal form of hemoglobin. Hemoglobin S is the hemoglobin variant that results in sickle cell anemia.

53.3 B. Treatment of methemoglobinemia with methylene blue requires a source of cytosolic NADPH and NADPH-cytochrome b5 reductase. The cytosolic NADPH is primarily derived from the pentose phosphate pathway, although some can be obtained from the malic enzyme oxidation of malate to pyruvate. The TCA cycle is mitochondrial, as are the glutamate dehydrogenase reaction and the β-oxidative pathways; these also produce NADH. Glycolysis is cytosolic, but it produces NADH, not NADPH.

BIOCHEMISTRY PEARLS

image Cytochrome b5 reductase allows the reduction of methemoglobin back to hemoglobin. Inherited conditions that affect this enzyme are at risk for the development of hemoglobinemia.

image Methemoglobin with iron in the oxidized state (Ferric) is unable to carry oxygen.

image Methylene blue allows reduction of the methemoglobin back to hemoglobin.

REFERENCES

Ash-Bernal R, Wise R, Wright SM. Acquired methemoglobinemia: a retrospective series of 138 cases at 2 teaching hospitals. Medicine. 2004;83(5):265-273.

Jolly BT, Monico EP, McDevitt B. Methemoglobinemia in and infant: case report and review of the literature. Pediatr Emer Care. 1995;11(5):294-297.

Prchal J, Schrier S, Mahoney D, Landaw S, Hoppin A. Clinical features, diagnosis, and treatment of methemoglobinemia. www.uptodate.com.

Verive MJ. Pediatric methemoglobinemia. http://emedicine.medscape.com/article/956528-overview#showall.