Cyanide Poisoning Case File

Cyanide Poisoning Case File

Eugene C.Toy, MD, William E. Seifert, Jr., PHD, Henry W. Strobel, PHD, Konrad P. Harms, MD

❖ CASE 16

A 68-year-old female in a hypertensive crisis is being treated in the intensive care unit (ICU) with intravenous nitroprusside for 48 hours. The patient’s blood pressure was brought back down to normal levels; however, she was complaining of a burning sensation in her throat and mouth followed by nausea and vomiting, diaphoresis, agitation, and dyspnea. The nurse noticed a sweet almond smell in her breath. An arterial blood gas revealed a significant metabolic acidosis. A serum test suggests a metabolite of nitroprusside, thiocyanate, is at toxic levels.

◆ What is the likely cause of her symptoms?

◆ What is the biochemical mechanism of this problem?

◆ What is the treatment for this condition?

ANSWERS TO CASE 16: CYANIDE POISONING

Summary: A 69-year-old female with new onset burning sensation in mouth and throat, nausea and vomiting, agitation, and diaphoresis after a medication error was noted. Metabolic acidosis is seen on the arterial blood gas. A thiocyanate level is in the toxic range.

◆ Diagnosis: Cyanide poisoning from toxic dose of nitroprusside.

◆ Biochemical mechanism: Cyanide inhibits mitochondrial cytochrome oxidase, blocking electron transport and preventing oxygen utilization. Lactic acidosis results secondary to anaerobic metabolism.

◆ Treatment: Supportive therapy, gastrointestinal (GI) decontamination, oxygen, and antidotal therapy with amyl nitrite, sodium nitrite, and sodium thiosulfate.

CLINICAL CORRELATION

Hypertensive emergencies are defined as episodes of severely elevated blood pressure, such as systolic levels of 220 mm Hg and/or diastolic blood pressures exceeding 120 mm Hg with patient symptoms of end-organ dysfunction. These symptoms may include severe headache, neurological deficits, chest pain, or heart failure symptoms. Hypertensive emergencies require immediate lowering of the blood pressure to lower (but not necessarily to normal) levels. In contrast, hypertensive urgencies are circumstances of markedly elevated blood pressures in the absence of patient symptoms; lowering the blood pressure over 24 to 48 hours is reasonable in these cases.

One hazard of abruptly lowering the blood pressure is causing hypotension and subsequent ischemia to the brain or heart. In other words, the very treatment designed to prevent end-organ disease may cause the problem. To avoid precipitous hypotension, agents that induce a smooth fall in blood pressure are preferable, such as sodium nitroprusside, a titratable intravenous agent used for malignant hypertension. Its desirable properties include the ability to precisely increase or decrease the infusion to affect the blood pressure. One side effect of sodium nitroprusside is that its metabolite is thiocyanate, and with prolonged use, cyanide poisoning may result, which inhibits the electron transport chain. Thus, in clinical practice, short-term nitroprusside is used, or serum thiocyanate levels are drawn.

APPROACH TO ELECTRON TRANSPORT SYSTEM (ETS) AND CYANIDE

Objectives

1. Know about the function of the electron transport chain (ETC).

2. Understand what factors may inhibit the ETC.

3. Be familiar with the biochemical process by which the therapy for cyanide poisoning works. (Nitrates convert the hemoglobin to methemoglobin which has a higher affinity for cyanide and promotes dissociation from cytochrome oxidase. Thiosulfate reacts with cyanide which is slowly released from cyanomethemoglobin to form thiocyanate. Oxygen reverses the binding of cyanide to cytochrome oxidase.)

4. Recognize other ETC sites and agents of inhibition.

Definitions

Oxidative phosphorylation: The mitochondrial process whereby electrons from NADH or reduced flavin bound in enzymes are transferred down the electron transport chain to oxygen forming water and providing energy through the formation of an hydrogen ion gradient across the inner mitochondrial membrane. The hydrogen ion gradient is used to drive the formation of ATP from ADP and inorganic phosphate (Pi). This process is also called coupled oxidative phosphorylation to emphasize that ATP formation from ADP and Pi is coupled to and linked with electron transport such that inhibition of one also inhibits the other.

Hydrogen ion gradient: A situation developed across the inner mitochondrial membrane wherein the concentration of hydrogen ions outside the mitochondrion is higher than the concentration inside. Hydrogen ions are extruded from the mitochondrion by the transfer of electrons from complex I to coenzyme Q, from coenzyme Q to complex III, and from complex III to complex IV. The gradient is discharged by ATP synthase, which admits hydrogen ions into the mitochondrion thereby driving the phosphorylation of ADP by Pi.

Electron transport chain: Present in the mitochondrial membrane, this linear array of redox active electron carriers consists of NADH dehydrogenase, coenzyme Q, cytochrome c reductase, cytochrome c, and cytochrome oxidase as well as ancillary iron sulfur proteins. The electron carriers are arrayed in order of decreasing reduction potential such that the last carrier has the most positive reduction potential and transfers electrons to oxygen.

Reduction potential: The tendency of an electron carrier to give up electrons, stated in electron volts, is called reduction potential. In any reduction–oxidation reaction electrons flow from the species with the more negative reduction potential to the more positive reduction potential.

Cytochrome: A heme (protoporphyrin IX) containing electron transfer protein. Some heme moieties are covalently attached to the protein components (cytochrome c), whereas others have isoprenoid side chains (cytochromes a and a3).

Iron sulfur proteins: These carry one electron and contain centers that chelate iron with organic and inorganic sulfur. Some centers contain a single iron atom chelated by four cysteine sulfurs; others contain two iron atoms chelated through four cysteine sulfurs and two inorganic sulfurs; yet others contain four iron atoms chelated by four cysteine sulfurs and four inorganic sulfurs.

Coenzyme Q (ubiquinone): A two electron accepting quinone that can accept and transfer one electron at a time allowing it to exist in a semiquinone state as well as the fully oxidized quinone or fully reduced dihydroxy state. It is bound to multiple isoprenoid units (ubiquinone has ten units), allowing it to bind to the membrane.

Flavin mononucleotide (FMN): An isoalloxazine ring bound to ribosyl monophosphate in an N-glycosidic bond. FMN can accept two electrons or donate one at a time to another electron acceptor.

Flavin adenine dinucleotide (FAD): An isoalloxazine ring bound to ribosyl monophosphate in an N-glycosidic bond which is attached to adenosine monophosphate. Like FMN, FAD can accept or donate two electrons one at a time to another electron acceptor.

DISCUSSION

The electron transport chain (ETC) or electron transport system (ETS) shown in Figure 16-1 is located on the inner membrane of the mitochondrion and is responsible for the harnessing of free energy released as electrons travel from more reduced (more negative reduction potential, E'0) to more oxidized (more positive E'0) carriers to drive the phosphorylation of ADP to ATP. Complex I accepts a pair of electrons from NADH (E′0 = −0.32 V)

Figure 16-1. Schematic diagram of electron transport chain ATP synthase and ATP/ADP translocase.

and passes the electron pair through the intervening carriers to complex IV, which passes the electrons to one atom of molecular oxygen (E′0 = +0.82 V) to form water with hydrogen ions (H⁺) from the medium.

The transport of electrons through the carriers is highly coupled to the formation of ATP from ADP and Pi through the formation and relaxation of the proton gradient formed across the inner mitochondrial membrane by electron transport. Each time electrons are transported between complexes I and III, between complexes III and IV, or between complex IV and oxygen, protons are extruded from the mitochondrial matrix across the inner membrane to the intermembrane space/cytosol. (The outer membrane poses no barrier to proton passage.) In other words, the energy gained from these electron transfers is used to pump protons from the mitochondrial matrix side to the cytosol side. Because the mitochondrial membrane is impermeable to protons, there is a gradient that develops with a higher concentration of protons outside the matrix. The protons then come through the ATP synthase complex through proton pores, and as they come back into the mitochondrial matrix, ADP is phosphorylated to ATP. Thus, because the process of electron transport is tightly coupled to ADP phosphorylation, ADP must be present for electron transport to proceed, and therefore the ADP/ATP translocase must be able to exchange a molecule of ADP in the cytosol for a molecule of ATP (newly made) in the matrix of the mitochondria. When these various processes operate in concert the mitochondria are said to exhibit coupled respiration.

The components of the electron transport chain have various cofactors. Complex I, NADH dehydrogenase, contains a flavin cofactor and iron sulfur centers, whereas complex III, cytochrome reductase, contains cytochromes b and c1. Complex IV, cytochrome oxidase, which transfers electrons to oxygen, contains copper ions as well as cytochromes a and a3. The general structure of the cytochrome cofactors is shown in Figure 16-2. Each of the cytochromes has a heme cofactor but they vary slightly. The b-type cytochromes have protoporphyrin IX, which is identical to the heme in hemoglobin. The c-type cytochromes are covalently bound to cysteine residue 10 in the protein. The a-type

Figure 16-2. Heme active center of cytochromes a, b, and c components of electron transport chain.

cytochromes have a long isoprenoid [(CH2–CH=C(CH3)–CH2)n] tail bound at one side-chain position.

Inhibition of the electron transport chain in coupled mitochondria can occur at any of the three constituent functional processes; electron transport per se, formation of ATP, or antiport translocation of ADP/ATP (Table 16-1). The best known inhibitor of the ADP/ATP translocase is atractyloside in the presence of which no ADP for phosphorylation is transported across the inner membrane to the ATP synthase and no ATP is transported out. In the absence of ADP phosphorylation the proton gradient is not reduced allowing other protons to be extruded into the intermembrane space because of the elevated [H⁺], and thus electron transfer is halted. Likewise the antibiotic oligomycin directly inhibits the ATP synthase, causing a cessation of ATP formation, buildup of protons in the intermembrane space, and a halt in electron transfer. Similarly, a blockade of complex I, III, or IV that inhibits electron flow down the chain to O2 would also stop both ATP formation and ADP/ATP translocation across the inner mitochondrial membrane.

Cyanide ion (CN^-) is a potent inhibitor of complex IV the cytochrome c oxidase component of the electron transport system in the oxidized state of the heme (Fe³⁺). It can be delivered to tissue electron transport systems as a dissolved gas after breathing HCN or ingested as a salt such as KCN or as a medication leading to the formation of CN^- such as nitroprusside. Cyanide ion competes effectively with oxygen for binding to cytochrome c oxidase at the oxygen-binding site. Cyanide binding and therefore cyanide poisoning is reversible if treated properly and early. The treatment strategy depends on dissociation of cyanide from cytochrome a/a3 (Fe³⁺). Increasing the percentage of oxygen breathed will increase the competition of oxygen over cyanide for the cytochrome a/a3 (Fe³⁺). Two other medications foster this competition. Nitrite ion (NO2^-) is administered to convert some oxyhemoglobin [HbO2(Fe²⁺)] to methemoglobin

Table 16-1

RESPIRATORY CHAIN SITES AND INHIBITORS

Figure 16-3. Strategy for reversal of cyanide binding to cytochrome oxidase (cyt a/a3 Fe³⁺).

[met-HbOH(Fe³⁺)], another competitor for cyanide binding (Figure 16-3). The cyanide adduct of methemoglobin is formed releasing cytochrome oxidase in the Fe³⁺ form ready to bind oxygen and disinhibit the electron transport chain. To remove the cyanide adduct in a nontoxic fashion, thiosulfate ion is administered. The mitochondrial enzyme rhodanese catalyzes the conversion of cyanide and thiosulfate to thiocyanate and sulfite. Thiocyanate is incapable of inhibiting cytochrome oxidase and is excreted. The methemoglobin can be reconverted to oxyhemoglobin by NADH and methemoglobin reductase.

Other sites of the electron transport chain can be targets of inhibitors based on similarity in structure to enzyme components or to substrates of the various components. For instance, the fish poison rotenone resembles the isoalloxazine ring of the FMN cofactor of complex I, the NADH CoQ reductase. Rotenone binds the enzyme quite avidly and prevents transfer of electrons from NADH to coenzyme Q through the iron sulfur centers and thus inhibits oxidation of NADH and subsequent reduction of oxygen to water. On the other hand, carbon monoxide resembles molecular oxygen and binds with a higher affinity than oxygen to complex IV, the cytochrome oxidase component, inhibiting transfer of electrons to oxygen.

COMPREHENSION QUESTIONS

A 16-month-old girl was found to have ingested approimately 30 mL of an acetonitrile-based cosmetic nail remover when she vomited 15 minutes postingestion. The poison control center was contacted, but no treatment was recommended because it was confused with an acetone-based nail polish remover. The child was put to bed at her normal time, which was 2 hours postingestion. Respiratory distress developed sometime after the child was put to bed, and she was found dead the next morning.

[16.1] Inhibition of which of the following enzymes was the most likely cause of this child’s death?

A. Cytochrome c reductase

B. Cytochrome oxidase

C. Coenzyme Q reductase

D. NADH dehydrogenase

E. Succinate dehydrogenase

[16.2] Which of the following best describes the reason for the latency of acetonitrile toxicity and why prompt treatment would have prevented this child’s respiratory distress and death?

A. Acetonitrile crosses the mitochondrial membrane slowly.

B. Acetonitrile induces hemolysis by inhibiting glucose 6-phosphate dehydrogenase.

C. Acetonitrile is only poorly absorbed by the intestinal system.

D. Complex IV of the electron transport system binds acetonitrile weakly.

E. Cytochrome P450 enzymes oxidize acetonitrile and slowly release cyanide.

[16.3] Inhibition of oxidative phosphorylation by cyanide ion leads to increases in which of the following?

A. Gluconeogenesis to provide more glucose for metabolism

B. Transport of ADP into the mitochondria

C. Utilization of fatty acids substrates to augment glucose utilization

D. Utilization of ketone bodies for energy generation

E. Lactic acid in the blood causing acidosis

[16.4] Which of the following procedures best describes the emergency intervention for cyanide poisoning?

A. Decrease the partial pressure of oxygen

B. Treatment with nitrites to convert hemoglobin to methemoglobin.

C. Treatment with thiosulfate to form thiocyanate

D. Use of Mucomyst (N-acetylcysteine) taken orally

An unskilled worker in a water garden/plant nursery was sent to sweep up a spill of a white powder in the storage shed. Later he was found with labored breathing and convulsions. On further examination, the white powder was identified as rotenone.

[16.5] Respiratory distress is induced on rotenone exposure because it inhibits the complex that catalyzes which of the following?

A. Electron transfer from NADH to coenzyme Q

B. Oxidation of coenzyme Q

C. Reduction of cytochrome c

D. Electron transfer from cytochrome c to cytochrome a1/a3

E. Electron transfer from cytochrome a1/a3 to oxygen

[16.6] The major metabolic consequence of perturbation of the electron transfer in mitochondria is which of the following?

A. Increased production of NADPH

B. Increased oxidation of NADH

C. Increased reduction of O2 to H2O

D. Decreased regeneration of NAD⁺

E. Decreased reduction of FAD

Answers

[16.1] B. The culprit here is cyanide produced from acetonitrile. Cyanide inhibits the electron transport chain of cytochrome oxidase.

[16.2] E. Acetonitrile itself is not the toxicant but undergoes metabolism and produces cyanide, which is the toxic agent here.

[16.3] E. Gluconeogenesis requires ATP, which is in short supply, turning up the catabolism of glucose to lactate in the absence of an intact electron transport chain. ADP cannot be transported into the mitochondrion because ATP, its antiporter partner, isn’t made by oxidative phosphorylation as a result of cyanide inhibition of cytochrome oxidase. Metabolism of fatty acids and ketone bodies requires a functional electron transport chain for their metabolism, and these possibilities are also ruled out.

[16.4] B. Increased oxygen competes with cyanide bound to cytochrome oxidase displacing it. Nitrites bind to hemoglobin converting it to methemoglobin, which binds cyanide more tightly than cyanohemoglobin and pulls cyanide from cyanohemoglobin to form cyanomethemoglobin. Thiosulfate is used to displace cyanide from cyanomethemoglobin to form thiocyanate, which can be excreted, a happy ending for cyanide poisoning. N-acetylcysteine is used for acetaminophen toxicity and not cyanide toxicity.

[16.5] A. Rotenone binds avidly to the flavoprotein NADH CoQ reductase, complex I (also called NADH dehydrogenase). The central portion of the rotenone structure resembles the isoalloxazine ring of the FMN molecule, and when it binds to complex I, rotenone prevents the transfer of electrons from NADH to coenzyme Q.

[16.6] D. Inhibition of the electron transport chain shuts down the major pathway of regenerating NAD⁺ from the NADH produced in intermediary metabolism. This forces the cytosolic conversion of pyruvate to lactate to regenerate NAD⁺ so that glycolysis can continue in the absence of a functioning electron transport system.

BIOCHEMISTRY PEARLS

❖ The electron transport chain (ETC) or electron transport system (ETS) is located on the inner membrane of the mitochondrion and is responsible for the harnessing of free energy. The chain consists of a series of carriers arranged along the inner membrane of mitochondria that transport electrons from NADH and reduced flavin carriers to molecular oxygen.

❖ Energy is released as electrons travel from more reduced (more negative reduction potential, E′0) to more oxidized (more positive E′0) carriers to drive the phosphorylation of ADP to ATP.

❖ The components of the electron transport chain have various cofactors, which form vital complexes.

❖ The disruption of various complexes may interfere with the ETC, leading to inability to manufacture ATP.

❖ The energy gained from electron transfer is used to drive protons out of the inner mitochondrial matrix to the cytosol, establishing a gradient of protons. As the protons come back into the matrix via the ATP synthase complex, ADP is phosphorylated to ATP.

❖ The normal mitochondria produce ATP as they transport electrons to oxygen; any interference with ATP synthesis or translocation across the mitochondrial membrane will inhibit electron transfer.

❖ Rotenone binds avidly to the flavoprotein NADH CoQ reductase, complex I (also called NADH dehydrogenase).

References

Davidson VL, Sittman DB. National Medical Series–Biochemistry. Philadelphia, PA: Harwol, 1994:381–3. 

Devlin TM, ed. Textbook of Biochemistry with Clinical Correlations, 5th ed. New York: Wiley-Liss, 2002:563–82. 

Goodman AG, Gilman LS, eds. The Pharmacological basis of Therapeutics, 10th ed. New York: 

McGraw-Hill, 2001:1642. McGilvery RW. Biochemistry: A Functional Approach. Philadelphia, PA: W.B. Saunders, 1979:397–400.

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