Silverstein and Hopper Chapter 107: Dyshemoglobinemias

Structure of Hemoglobin

• Hemoglobin is composed of four polypeptide chains (globins) each attached to a heme molecule

  • Heme is made up of a tetrapyrrole with a central iron molecule

  • Oxygen binds to the central iron molecule in the ferrous (Fe++) form

  • Each hemoglobin molecule carries four oxygen molecules

Pathophysiology of Carbon Monoxide Toxicity

• CO is absorbed rapidly through the lungs at the level of the alveolus

• Quantity of gas absorbed is dependent on minute ventilation (respiratory rate x tidal volume), duration of exposure, and concentrations of CO and oxygen in the environment

• Once absorbed in the blood and circulated throughout the body, a small amount of CO is oxidized to CO2, some remains as gas in solution, and some binds to proteins including Hb, myoglobin, and cytochromes in mitochondria

What are the two main mechanisms of CO toxicity?

Impaired oxygen delivery to tissues (hypoxia via dyshemoglobinemia)

Direct cellular toxicity

Mechanisms of CO Toxicity - Impaired Oxygen Delivery to the Tissues

• CO displaces oxygen from Hb and causes an allosteric hinderance of oxygen release from Hb to tissues

• CO completes with oxygen for Hb bindings sites with 200-240 times the affinity

• CO binds 2/4 available heme groups in each molecule of Hb, causing a decrease in oxygen carrying capacity of 50%

• Low levels of CO in the blood result in markedly reduced oxygen carrying capacity despite a normal Hb concentration and normal PO2

• Oxyhemoglobin dissociation curve is shifted down and to the left resulting in decreased release of oxygen to the tissues

Mechanisms of CO Toxicity - Direct Cellular Toxicity

• Can be due to CO binding heme proteins other than Hb, including cytochromes, myoglobin, and guanylyl cyclase

  • Binding to cytochrome a3 disrupts oxidative metabolism, potentially causing generation of oxygen free radicals and impaired cellular respiration

  • Binding to myoglobin can cause myocardial hypoxia, depression, and arrhythmias, as well as direct skeletal muscle toxicity and rhabdomyolysis

  • Binding of guanylyl cyclase results in increased cyclic guanylyl monophophate, cerebral vasodilation, and loss of consciousness

What state must the iron molecule in Hb be in to bind oxygen?

Must be maintained in the ferrous (Fe2+) state for Hb to bind oxygen

Methemoglobin (metHb)

• An inactive form of Hb created when the iron molecule of Hb is oxidized to the ferric (Fe3) state because of oxidative damage within the red blood cell

  • Gives the red blood cell a darker brown color and results in dusky cyanotic or chocolate-colored mucous membranes

  • Increases the affinity for oxygen in the remaining ferrous moieties of the Hb molecule, decreasing the release of oxygen to the tissues and shifting the oxyhemoglobin dissociation curve to the left

Sulfhemoglobin

• Most uncommon dyshemoglobin

• May be caused by exposure to high levels of sulfur from drugs (sulphonamides such as sulfasalazines) or other compounds

• Stable green pigment

• Formed when iron is oxidized from the ferrous (2+) to the ferric (3+) form by drugs or chemicals that contain sulfur

  • Sulfur atom irreversibly binds to the porphyrin ring of Hb

• Incapable for carrying oxygen

  • Prevents oxygen transport

  • Shifts the oxyhemoglobin dissociation curve to the right

• Can lead to cyanosis without clinical signs of respiratory distress

• No antidote

• Is irreversible so remains attached to the Hb for the life span of the RBC

Oxidation in the Erythrocyte

• Reactive species derived from oxygen can cause oxidative damage within the body by transferring or extracting an unpaired electron to or from another molecule

• Protective mechanisms that prevent or reverse oxidative damage include proteins that act as free radical scavengers and reducing agents that can remove the unpaired electron from an oxidized molecule

• Erythrocytes are especially vulnerable to oxidative damage because they carry oxygen, are exposed to various chemicals in plasma, and have no nucleus or mitochondria

• Hb can undergo autooxidation as an electron is pulled off the Hb and onto an oxygen molecule, resulting in generation of metHb and O2-

• Free radicals also may extract electrons by oxidizing deoxyhemoglobin

• Oxidant toxins can donate an electron to oxyhemoglobin, creating metHb and H2O2

What are oxidants that are continuously generated in vivo?

Hydrogen peroxide (H2O2)

Superoxide free radicals (O2-)

Hydroxyl radicals (OH-)

What are mechanisms that erythrocytes have to protect themselves from oxidative damage?

Superoxide dismutase

Catalase

Glutathione peroxidase

Glutathione

metHb reductase

Reducing agents such as NADPH and NADH are instrumental in reducing oxidized glutathione and metHb back to functional molecules

Heinz Bodies

• Aggregates of denatured, precipitated Hb molecules within erythrocytes that form as Hb with oxidative damage is metabolized

  • Oxidation of the SH groups of Hb causes conformational changes in the globin chain that result in precipitation of the denatured globin

• Aggregates of denatured globin and metabolized metHb clump into Heinz bodies and continue to coalesce until visible, pale structures can be seen within the red blood cell cytoplasm

What do Heinz bodies have an affinity for?

Membrane proteins

• Binding of Heinz bodies to these proteins causes disruption of anion transport, decreased membrane deformability, and aggregations of membrane protein complexes that may act as autoantibodies

Ghost Cells

• Numerous Heinz bodies can disrupt the membrane sufficiently to result in ghost cells - empty red blood cells with just a membrane and Heinz body remaining

  • Associated with oxidation-induced intravascular hemolysis

How are erythrocytes that have undergone oxidative damage dealt with by the body?

They are removed by the mononuclear phagocyte system, particularly within the spleen

What are causes of methemoglobinemia in small animals?

• Acetaminophen ingestion

• Topical benzocaine products

• Phenazopyridine ingestion

• Nitrites

• Nitrates

• Skunk musk

• Hydroxycarbamide

• Aniline

• metHb reductase deficiency

What are things that cause methemoglobinemia in humans?

• Dapsone

• Metoclopramide

• Sulfonamides

• Nitroglycerine

• Nitroprusside

What are substance that cause hemolytic anemia secondary to Heinz bodies but also methemoglobinemia?

• Allium plants

• Propylene glycol

• Zinc

• Methylene blue

• Crude oils

• Naphthalene

• Repeated use of propofol in cats

• Phenothiazine

• Phenylhydrazine

• Methionine in cats

• Menadione (vitamin K3) in dogs

• Copper

What are the pathways by which acetaminophen is metabolized in the liver?

• Conjugated to a sulfate compound by a phenol sulfotransferase

• Conjugated to a glucuronide compound by a uridine diphosphate-glucuronosyltransferase

• Can be transformed and oxidized by the cytochrome P-450 system that converts it to the reactive intermediate, NAPQI

  • The glucuronide and sulfate conjugations are nontoxic and excreted in the bile and urine in most species other than the cat

  • GSH reacts with NAPQI to form a nonreactive molecule, mercapturic acid, which is excreted in the urine

• An additional metabolic of acetaminophen, para-aminophenol (PAP), is produced by deacylation of acetaminophen by hepatic microsomal carboxyesterases

  • PAP is removed by biotranformation through N-acetylation with N-acetyltransferase (NAT), conjugation with GSH, or sulfation

How do higher doses of acetaminophen cause toxicity?

• Low doses are metabolized to nontoxic products, but higher doses can overwhelm the sulfate and glucuronide conjugate systems of the liver and deplete GSH stores

  • NAPQI and PAP build up and unmetabolized acetaminophen accumulates

    • Half-life of acetaminophen becomes longer with higher doses

• NAPQI oxidizes hepatic proteins, resulting in hepatocellular damage

  • PAP may play a more significant role in erythrocyte oxidative damage

    • PAP cooxidizes with oxyhemoglobin forming metHb and an oxidized PAP intermediate

    • The intermediate is reduced by GSH and the metHb is reduced primarily by metHb reductase

    • Methemoglobinemia becomes overt when metHb reductase and necessary reducing equivalents become depleted in erythrocytes

• After the acute episode of metHb production, Heinz bodies being to form and aggregate into larger structures, eventually causing enough changes in the erythrocyte to trigger hemolysis

Prognosis for Acetaminophen Toxicity

• Prognosis for acetaminophen toxicity is guarded

  • Time from ingestion to treatment is the most important factor in determining morbidity and survival

Methemoglobinemia Secondary to Topical Benzocaine

• Benzocaine sprays for laryngeal spasm in cats and over-the counter creams for pruritis in dogs and cats have been associated with methemoglobinemia

• Metabolites of benzocaine are likely responsible for oxidative damage to Hb

Methemoglobinemia Secondary to Skunk Musk

• One report of methemoglobinemia and Heinz body hemolytic anemia in a dog after exposure to skunk musk

• Toxic substance in skunk musk thought to be thiols, which can react with oxyhemoglobin to form met Hb, a thiyl radical, and H2O2

Methemoglobinemia Secondary to Nitrites and Nitrates

• Not documented to cause Heinz body production

• Can occur after receiving vasodilatory drugs that release nitric oxide, including nitroglycerine and sodium nitroprusside

• Nitric oxide is decomposed by interacting with oxyhemoglobin to form metHb and nitrate

  • metHb is reduced by metHb reductase in RBCs, but evidence indicates that NO decreases metHb reductase activity

• NO also interacts with oxygen to form nitrogen dioxide, which dissolves in solution to yield nitrite and nitrate

  • Nitrite can convert oxyhemoglobin to metHb

• Toxicity should be considered in animals receiving these drugs and developing an unexplained hyperlactatemia

Methemoglobin Reductase Deficiency

• Rare congenital abnormality

• Affected animals cannot efficiently reduce metHb so have elevated blood levels of metHb, exhibit mild to moderate cyanosis of the mucous membranes, and may suffer from exercise intolerance

• Definitive diagnosis by measuring erythrocyte metHb reductase enzyme activity at a research laboratory

• Condition is fairly benign and rarely requires treatment

• Indications for treatment include clinical signs of metHb such as lethargy, tachycardia, and/or tachypnea, and as preparation for animals that require general anesthesia

Clinical Signs of Carbon Monoxide Toxicity

• Lethargy

• Depression

• Headache

• Confusion

• Syncope

• Seizures

• Unconsciousness

• Death

• Tachypnea

• Tachycardia

• Nausea

• Dysrhythmias

• Vomiting

• Cherry red mucous membranes

• Severity does not correlate consistently with COHb levels

Clinical Signs Associated with COHb Levels >15% in Humans

Overt signs of toxicity such as tachypnea and headache

Clinical Signs Associated with COHb Levels >30% in Humans

Neurologic dysfunction

Clinical Signs Associated with COHb Levels >50% in Humans

Loss of consciousness that can progress to apnea and death

Delayed Neuropsychiatric Syndrome (DNS)

• Delayed neuropsychiatric syndrome (DNS) described in humans and some veterinary cases of CO toxicity

  • Develops 3-240 days following the toxic episode

  • Clinical signs in veterinary patients range from ataxia to inability to ambulate, depressed or stuporous mentation, seizures, and deafness

  • Risk factors in humans include age (older), duration of unresponsiveness, and history of illness

At what level of metHb do clinical signs begin?

20%

Clinical Signs of Methemoglobinemia/Sulfhemoglobinemia

• Tachycardia

• Tachypnea

• Dyspnea

• Lethargy

• Anorexia

• Vomiting

• Weakness

• Ataxia

• Stupor

• Hypothermia

• Ptyalism

• Convulsions in cats

• Coma and death with metHb levels reach 80%

• Chocolate brown MM and cyanosis

  • Cyanosis appears at metHb levels of 12-14% or more

• Clinical signs of sulfhemoglobinemia are very similar to metHb

What do definitive diagnosis and quantification of COHb, metHb, and sulfhemoglobinemia levels require?

Direct measurement via a co-oximeter or assay

Co-Oximeter

Machine used to measure Hb content, oxygen saturation, percentage of COHb, and percentage of metHb by differentiating wavelength absorbance values

Assay for metHb and Sulfhemoglobinemia

• Assay for metHb and sulfhemoglobinemia involves spectrophotometrically quantifying the change in absorbance at 630 nm before and after the addition of cyanide to the sample

  • Cyanide converts metHb to cyanmethemoglobin, which has a different absorbance than metHb

  • Sulfhemoglobin has a similar spectral peak as metHb, making it appear as elevated met Hb levels on many co-oximeter readings

    • Sulfhemoglobin can be distinguished from metHb by adding cyanide as the spectral peak will persist with sulfhemoglobin, indicating the presence of a different dyshemoglobin

    • H2O2 can be added as well as it will bind to sulfHb

Comparing Pulse Oximeter Oxygen Saturation to Arterial Blood Gas Saturation (Saturation Gap) for Diagnosing Carboxyhemoglobinemia, Methemoglobinemia, and Sulfhemoglobinemia

• Pulse oximeter determines the ratio of oxyhemoglobin to deoxyhemoglobin

• Presence of metHb or sulfhemoglobinemia distorts the ratio

  • If metHb levels exceed 30% or sulfhemoglobinemia levels exceed 28%, the pulse oximeter reading plateaus around 85% regardless of the true oxygen content

• A dyshemoglobinemia should be suspected if the saturation gap is greater than 5%

Evaluating a Peripheral Blood Smear for Oxidative Damage

• Examining a peripheral blood smear for Heinz bodies, eccentrocytes, and ghost cells can be helpful when looking for evidence of oxidative damage

  • May require a stain such as new methylene blue or a reticulocyte stain

Treatment for Carbon Monoxide Toxicity

• Elimination of CO depends on minute ventilation, duration of exposure, and the fraction of inspired oxygen

• Oxygen therapy is the mainstay of treatment

  • Increasing the amount of oxygen in the blood decreases the half-life of CO as dissolved O2 competes with CO for Hb binding sites

  • CO is then displaced from Hb and exhaled through the lungs

  • Oxygen rates at 50-150 ml/kg/min

  • Also indicated to prevent DNS because mechanism is thought to be related to hypoxia and reperfusion

    • Limiting the degree and duration of hypoxia has become the goal of treatment to prevent DNS

• Hyperbaric oxygen therapy debated

• Other than oxygen therapy, the bulk of treatment is supportive care

  • Active warming

  • Ensuring adequate tissue perfusion

• Since oxidative damage has a role in development of DNS, antioxidantes such as N-acetylcysteine and vitamin E may be useful in moderate to severe CO toxicity and/or those that develop signs of DNS

Prognosis for Carbon Monoxide Toxicity

• Initial unconsciousness or severe neurologic abnormalities are associated with a more guarded prognosis

• Overall prognosis is fair with time and supportive care

Treatment for Oxidative Damage to Erythrocytes

• Oxygen therapy increases the amount of dissolved oxygen in the blood, but once the Hb capable of carrying oxygen are maximally saturated, supplemental oxygen is not sufficient as a sole therapy

• Treatment for methemoglobinemia often involves diuresis or medications that increase the rate of elimination or decrease the production of toxic metabolites

• Induction of vomiting followed by activated charcoal is indicated if the animal has a history of recently ingesting a toxic substance and is not yet clinically ill

• Supportive care

• Therapy for severe sulfhemoglobinemia may also include packed red blood cell transfusions

Treatment for Acetaminophen Toxicity

• N-Acetylcysteine (NAC)

  • Preferred treatment for acetaminophen toxicity

  • Augments the endogenous glutathione stores as it is hydrolyezd to cysteine

  • NAC also interacts directly with NAPQI to form a nontoxic conjugate and increases the fraction of acetaminophen excreted as the sulfate conjugate

  • Most effective if administered within 12 hours of ingestion but still recommended up to 36-80 hours after ingestion

  • Recommended regimen is an initial dose of 140 mg/kg IV (280 mg/kg in severe toxicosis) followed by 70 mg/kg q6h for 7 additional treatments

    • Recommend a slow IV infusion of a 5% solution over 30-60 minutes

  • Typically causes nausea and vomiting when given orally, hypotension and bronchospasm if given rapidly intravenously, and phlebitis if it leaks perivascularly

Methylene Blue for Treatment of Oxidative Damage to Erythrocytes

• Increases the rate of reduction of metHb through use of another reducing system within the erythrocyte, NADPH dehydrogenase

• Administered as a 1% solution intravenously over several minutes at 1 mg/kg once

• Improvement in clinical parameters should be noted within 30 minutes of administration

• Methylene blue causes oxidative damage in RBCs and can potentiate a Heinz body anemia caused by the original oxidative insult

  • A delayed reaction may occur so hematocrit and blood smear should be monitored for 3-4 days after administration

• Clinical signs attributable to sulfhemoglobinemia will not improve after methylene blue

Adjunctive Treatments for Dyshemoglobinemias

• Ascorbic acid

  • 30 mg/kg IV q6h as an antioxidant

  • Can augment metHb conversion to HB through nonenzymatic reduction

• Cimetidine

  • Histamine-2 receptor antagonist

  • Useful in cases of acetaminophen toxicity because it inhibits the P-450 oxidation system in the liver, limiting the production of NAPQI

  • Studies haven't demonstrated consistent benefits

  • 5 mg/kg IV q8h

• SAMe

  • Essential metabolite that is vital to hepatocytes - hepatoprotective, antioxidant properties, and decreases the osmotic fragility of erythrocytes

• Bioflavonoids

  • Antioxidants that work by increasing the activity of the NADPH reductase system

• Blood transfusion

  • May be necessary in patients with severe hemolytic anemia secondary to Heinz body production and for sulfhemoglobinemia