Anaerobic Metabolism Case File

Anaerobic Metabolism Case File

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

❖ CASE 14

A 40-year-old female presents to the emergency department with complaints of lower back pain, fever, nausea, vomiting, malaise, chills, syncope, dizziness, and shortness of breath. Patient states that she has some burning with urination (dysuria). Her fever was as high as 39.4°C (103°F) at home earlier in the day. She has a history of non–insulin-dependent diabetes mellitus but denies any other medical problems. On exam, she is in moderate distress with a temperature of 38.9°C (102°F) degrees, pulse of 110 beats per minute, respiratory rate of 30 breaths per minute, and blood pressure of 70/30 mm Hg.

Her extremities are cool to the touch with thready pulses. Her chest is clear to auscultation bilaterally, and heart is tachycardic but with regular rhythm. She has significant costovertebral tenderness on the right side. Her white blood cell (WBC) count was elevated at 20,000/mm³. The hemoglobin and hematocrit were normal. Her glucose was moderately elevated at 200 mg/dL, and her serum bicarbonate level is low. An arterial blood gas demonstrated a pH of 7.28 and parameters consistent with a metabolic acidosis. Her urinalysis shows an abnormal number of gram-negative rods.

◆ What is the most likely diagnosis?

◆ What is the biochemical mechanism of the metabolic acidosis?

ANSWERS TO CASE 14: ANAEROBIC METABOLISM

Summary: 40-year-old female with diabetes presents with fever 38.9°C (102°F), chills, nausea, vomiting, back pain (costovertebral tenderness), chills, increased WBC count, hypotension, and metabolic acidosis.

◆ Most likely diagnosis: Septic shock and pyelonephritis.

◆ Likely cause of the metabolic acidosis: Lactic acid produced from cells undergoing anaerobic metabolism as a result of tissue hypoperfusion from shock.

CLINICAL CORRELATION

This patient developed septic shock, infection related hypotension, and low blood pressure. The decreased blood pressure then led to insufficient red blood cells and oxygen to be delivered to the various tissues in the body. Thus, the tissue had to switch from aerobic metabolism to anaerobic metabolism. Lactate accumulates, leading to acidemia. The treatment of septic shock is initially intravenous fluids, since the body is relatively volume depleted as a result of the vasodilation response to the infection. Sometimes, the blood pressure remains low despite several liters of intravenous fluids; in these cases, so-called vasoactive drugs are used such as dopamine infusion to cause vasoconstriction and therefore elevate the blood pressure. Antibiotics are also important to treat the infection. Finally, control of the source of the sepsis is critical. This may include surgery to remove an abscess or necrotic bowel, or removal of a foreign body, or simply debridement. Thus, septic shock is treated by supporting the blood pressure, antibiotics, and source control. Worsening of the acidemia and accumulation of lactate is a poor prognostic sign in septic shock.

APPROACH TO AEROBIC AND ANAEROBIC METABOLISM

Objectives

1. Be very familiar with the tricarboxylic acid (TCA) cycle.

2. Know about the differences in energy production in aerobic and anaerobic conditions.

Definitions

Acceptor control: The regulation of the rate of an enzymatic reaction by the concentration of one or more of the substrates.

Anaerobic glycolysis: The biochemical process by which glucose is converted to lactate with the production of two moles of ATP. This process is increased when the cellular demand for ATP is greater than the ability of the TCA cycle and oxidative phosphorylation to produce it because of decreased oxygen tension or heavy exercise.

Glycolysis: The biochemical process by which glucose is converted to pyruvate in the cytosol of the cell. It results in the production of 2 mol of adenosine triphosphate (ATP) and 2 mol of the reduced cofactor nicotinamide adenine dinucleotide (NADH), which transfers its reducing equivalents to the mitochondrion for the production of ATP via oxidative phosphorylation.

Krebs cycle: Citric acid cycle, TCA cycle, the mitochondrial process by which acetyl groups from acetyl-CoA are oxidized to CO2. The reducing equivalents are captured as NADH and FADH2, which feed into the electron transport system of the mitochondrion to produce ATP via oxidative phosphorylation.

DISCUSSION

Most of the energy that the body requires for maintenance, work, and growth is obtained by the terminal oxidation of acetyl coenzyme A (acetyl-CoA) that is produced by the catabolism of carbohydrates, fatty acids, and amino acids. The oxidation of acetyl-CoA is achieved by mitochondrial enzymes that make up the tricarboxylic acid cycle (TCA cycle, also called the citric acid cycle or the Krebs cycle). All of the enzymes in this metabolic pathway are located in the matrix of the mitochondria except one, succinate dehydrogenase. Succinate dehydrogenase is a membrane-bound protein located on the inner mitochondrial membrane facing the matrix. The two carbons that enter the TCA cycle as an acetyl group are effectively oxidized to carbon dioxide. Oxygen is not directly involved in this process; instead, reducing equivalents are captured by the electron carriers NAD⁺ and FAD producing three (NADH + H⁺) and one FADH2. A high-energy phosphate bond is also produced in the form of GTP. The reduced cofactors NADH and FADH2 are then reoxidized by passing their reducing equivalents to O2 through the electron transport system (ETS) of the mitochondria with the production of water and ATP via oxidative phosphorylation.

The TCA cycle is shown in Figure 14-1. In the first step, acetyl-CoA is condensed with oxaloacetate to produce citrate with the release of free coenzyme A in a reaction catalyzed by citrate synthase. Citrate is then isomerized to isocitrate by the enzyme aconitase. The next step involves the oxidative decarboxylation of isocitrate to produce α-ketoglutarate. The enzyme catalyzing this reaction, isocitrate dehydrogenase (IDH) requires the oxidized electron carrier NAD⁺ to accept the electrons released in the oxidation and produce the first NADH + H⁺. a-Ketoglutarate is then converted to succinyl-CoA in a second oxidative decarboxylation reaction catalyzed by α-ketoglutarate dehydrogenase (α-KGDH). This reaction requires the participation of two cofactors, oxidized NAD+ and CoA, and results in the production of a second NADH + H⁺. Succinyl-CoA is then transformed to succinate by

Figure 14-1. The tricarboxylic acid (TCA) cycle, also known as the citric acid

cycle and the Krebs cycle. The overall reaction is

AcCoA + 3NAD⁺ + FAD + GDP + Pi →

2CO2 + 3NADH + FADH2 + GTP + CoA

1 Citrate synthase

2 Aconitase

3 Isocitrate

4 α-Ketoglutarate dehydrogenase

5 Succinate thiokinase

6 Succinate dehydrogenase

7 Fumarase

8 Malate dehydrogenase

succinate thiokinase with the release of free CoA. The high energy released in the hydrolysis of the thioester bond of succinyl-CoA drives the phosphorylation of GDP to produce GTP. Succinate is then converted to oxaloacetate in a series of reactions reminiscent of the β-oxidation of fatty acids. A double bond is introduced into succinate in an oxidation catalyzed by succinate dehydrogenase, the only membrane-bound enzyme in the TCA cycle. The reducing equivalents are captured as FADH2. The resulting fumarate is then hydrated to form malate by the enzyme fumarase. Oxaloacetate is then regenerated by the oxidation of malate by malate dehydrogenase with the production of another NADH + H⁺.

Although the TCA cycle does not directly involve the participation of molecular oxygen as a substrate, the reduced cofactors NADH and FADH2 must be reoxidized by the mitochondrial ETS for continued operation of the cycle. Thus, the rate of oxidation of acetyl-CoA by the TCA cycle is to a large degree regulated by the availability of the oxidized cofactors NAD⁺ and FAD. When tissues do not receive enough O2 as a result of hypoperfusion, the ETS cannot regenerate these cofactors at a sufficient rate and the reactions that use them are inhibited because one of the required substrates is lacking (acceptor control). Thus, acetyl-CoA builds up in the mitochondria, depleting free CoA levels. Decreased levels of NAD⁺ and CoA also inhibit the conversion of pyruvate to acetyl-CoA by pyruvate dehydrogenase.

When the ETS and oxidative phosphorylation are compromised by a lack of O2, cytosolic levels of ATP drop while ADP and AMP levels increase. The flow of glucose through the glycolytic pathway is increased because of allosteric activation of phosphofructokinase and pyruvate kinase by the drop in ATP and rise in AMP concentrations. This results in an increased production of NADH and pyruvate by the glycolytic pathway. However, since electron transport is inhibited by a lack of O2, regeneration of NAD⁺ by the glycerol 3-phosphate or malate-aspartate shuttles, and the ETS is also inhibited. To continue the production of ATP by the glycolytic pathway, NAD⁺ must be regenerated to accept more reducing equivalents when glyceraldehyde 3-phosphate is oxidized to 1,3-bisphosphoglycerate. Lactate dehydrogenase is activated by increased levels of pyruvate and NADH and converts pyruvate to lactate with the regeneration of NAD⁺ (Figure 14-2). Lactate that cannot be reused by the cells that produce it is transported out of the cell to the bloodstream.

Lactate has two metabolic fates, either complete combustion to CO2 and H2O or conversion back to glucose through gluconeogenesis. Both processes require an active ETS and oxidative phosphorylation. Reduced oxygenation of cells thus decreases the utilization of lactate and increases its production with a resulting lactic acidosis.

When tissues are hypoperfused, the resulting anaerobic conditions have energetic consequences. First, all catabolic processes that require an active ETS and oxidative phosphorylation (e.g., b-oxidation of fatty acids and amino acid breakdown) are inhibited. Thus, certain energy stores cannot be

Figure 14-2. The conversion of pyruvate to lactate by the enzyme pyruvate dehydrogenase. The reaction, which is reversible, uses the reducing equivalents from NADH and regenerates NAD⁺ for the continuation of the glycolytic pathway when oxygen is limiting. 

used by the cell. Energy needs must be met by catabolism of carbohydrates through the glycolytic pathway. However, since under these anaerobic conditions, pyruvate dehydrogenase, the TCA cycle and oxidative phosphorylation are compromised, only a fraction of the chemical energy that is obtained from the oxidation of glucose under aerobic conditions is produced. Glucose that is completely oxidized to CO2 and H2O under aerobic conditions will produce between 36 and 38 mol of ATP per mole of glucose depending on the shuttle used to transport cytosolic reducing equivalents to the mitochondria. Only 2 mol of ATP are produced per mole of glucose converted to lactate through anaerobic glycolysis. The ATP needs of the cell are met by the increased rate of the glycolytic pathway.

COMPREHENSION QUESTIONS

[14.1] Phil Hardy has decided to train for an upcoming marathon. Nearing the age of 50, Phil figures that after he trains he should be able to maintain a 9 minute-per-mile pace, which would mean that he would finish the race in approximately 4 hours. Given that he would be adequately hydrating himself at the various water stations along the way, as he is about to finish the 26 mile 385 yard course, what is the primary fuel that his leg muscles would be using?

A. Fatty acids from the blood

B. Glycerol from the blood

C. Glycogen stored in muscle

D. Glucose from the blood

E. Ketone bodies from the blood

[14.2] A postoperative patient on intravenous fluids develops lesions in the mouth (angular stomatitis). Urinalysis indicates an excretion of 15 μg riboflavin/mg creatinine, which is abnormally low. Which of the following TCA cycle enzymes is most likely to be affected?

A. Citrate synthase

B. Isocitrate dehydrogenase

C. Fumarase

D. Malate dehydrogenase

E. Succinate dehydrogenase

[14.3] After excessive drinking over an extended period of time while eating poorly, a middle-aged man is admitted to the hospital with “high output” heart failure. Which of the following enzymes is most likely inhibited?

A. Aconitase

B. Citrate synthase

C. Isocitrate dehydrogenase

D. α-Ketoglutarate dehydrogenase

E. Succinate thiokinase

Answers

[14.1] A. After 4 hours of heavy exercise, glycogen stored in muscle cells has been expended. Free glycerol cannot be used by the muscle cell because it does not have the enzyme (glycerol kinase) that will phosphorylate it so that it can enter the glycolytic pathway. Although glucose and ketone bodies can be taken up by the muscle cells and used for energy, fatty acid oxidation provides most of the ATP for the marathon runner at this point in the race.

[14.2] E. The patient has demonstrated a deficiency in riboflavin (urinary excretion of less than 30 μg/mg creatinine is considered clinically deficient). Riboflavin is a component of the cofactor FAD (flavin adenine dinucleotide), which is required for the conversion of succinate to fumarate by succinate dehydrogenase.

[14.3] D. This patient has exhibited symptoms of beri beri heart disease, which is a result of a nutritional deficiency in vitamin B1 (thiamine). The active form of the vitamin, thiamine pyrophosphate, is a required cofactor for α-ketoglutarate dehydrogenase.

BIOCHEMISTRY PEARLS

❖ Most of the energy that the body requires for maintenance, work, and growth is obtained by the terminal oxidation of acetyl coenzyme A (acetyl-CoA), which is produced by the catabolism of carbohydrates, fatty acids, and amino acids.

❖ The oxidation of acetyl-CoA is achieved by mitochondrial enzymes that make up the tricarboxylic acid cycle (TCA cycle, also called the citric acid cycle or the Krebs cycle).

❖ When the ETS and oxidative phosphorylation are compromised by a lack of O2, cytosolic levels of ATP drop, while ADP and AMP levels increase.

❖ The flow of glucose through the glycolytic pathway is increased because of allosteric activation of phosphofructokinase and pyruvate kinase by the drop in ATP and rise in AMP concentrations.

❖ The four carbon intermediates of the TCA cycle may be replenished or increased by metabolites of the glucogenic amino acids entering at α-ketoglutarate, succinyl-CoA, or oxaloacetate. In addition, the C4 pool can also be increased by the carboxylation of pyruvate to oxaloacetate catalyzed by pyruvate carboxylase.

References

Beattie DS. Bioenergetics and oxidative metabolism. In: Devlin, TM, ed. Textbook of Biochemistry with Clinical Correlations, 5th ed. New York: Wiley-Liss, 2002. 

Harris RA. Carbohydrate metabolism I: major metabolic pathways and their control. In: Devlin TM, ed. Textbook of Biochemistry with Clinical Correlations, 5th ed. New York: Wiley-Liss, 2002.

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