Ketoacidosis

Case 5-1: This Man Is Anxious to Know Why He Has Ketoacidosis

This is the fourth hospital admission with a similar presentation for this 22-year-old man. As in the other episodes, his illness began with a “panic attack” that lasted for several days. During that period, he drank many liters of sweetened soft drinks on a daily basis (see margin note). He developed crampy lower abdominal pain that became severe in the 24 hours prior to coming to the hospital. He denied the intake of alcohols (including methanol or ethylene glycol). He felt well between these episodes and was taking only medications for treatment of mild depression. He had no history suggestive of diabetes mellitus. On physical examination, there was an odor of acetone on his breath, but his effective arterial blood volume (EABV) was not contracted. Arterial blood gas revealed a blood pH of 7.20, an arterial PCO ₂ of 22 mm Hg, and a plasma bicarbonate concentration (P _HCO3 ) of 8 mmol/L. Of note, his plasma osmolal gap (P _{Osmolal gap} ) was not elevated. The following laboratory data were obtained from measurements in a venous blood sample on admission:

His initial therapy consisted of 1 L of isotonic saline and 1 L of 5% dextrose in water (D ₅ W). Within 24 hours, all his laboratory values returned to the normal range. Of interest, his hemoglobin A ₁ C level was not elevated (4.4%) and his plasma insulin level was in the normal range.

Metabolic process analysis

Metabolic processes often consist of more than one metabolic pathway, and these pathways usually occur in more than one organ. In the metabolic process involving ketoacids, there are segments of this process that take place in adipose tissue, the liver, the brain, and the kidneys ( Figure 5-1 ). Although each of these segments is regulated at the specific organ level, control of the whole metabolic process is in keeping with its overall function.

The function of the metabolic process involving ketoacids is to provide the brain with a water-soluble, fat-derived fuel that it can oxidize to regenerate a sufficient quantity of adenosine triphosphate (ATP) to carry out its essential functions when its major fuel in the fed state, glucose, is in short supply. Although the blood–brain barrier limits the entry of long-chain fatty acids into the brain, there is a transport system that allows ketoacids to enter brain cells at a rapid rate.

When the diet contains glucose, signals are generated to prevent the production of ketoacids in the liver. The signal system centers on the stimulation of β-cells of the pancreas by glucose to release the hormone insulin. In contrast, during prolonged starvation, insulin is not released because of a low plasma glucose (P _Glucose ) level. This leads to the release of fatty acids from adipose tissue and the formation of ketoacids in the liver; therefore, the central factor in the control of the metabolic process involving ketoacids is a relative lack of insulin (see margin note).

An important element in the control of a metabolic process is to block all alternative pathways for the metabolism of its intermediates to ensure that the desired product is formed. Therefore, during ketoacid production, alternative pathways for metabolism of its substrate, acetyl-CoA formed from β-oxidation of palmitic acid in the liver, must be inhibited (i.e., oxidation and conversion to storage fat), while the pathway for production of ketoacids is stimulated ( Figure 5-1 ).

To determine the H ⁺ ion balance in a metabolic process, examine the net valences of all of its substrates and end products. H ⁺ ions are produced if the products of a metabolic process have a greater net anionic valence than its substrates; H ⁺ ions are removed if the products of a metabolic process have a lesser net anionic valence than its substrates. In the metabolic process that involves ketoacids, there is no net production or removal of H ⁺ ions if the ketoacids produced in the liver are then oxidized by the brain or the kidneys because the net valence of all substrates, triglycerides in adipose tissue, and end products (CO ₂ + H ₂ O), are equal. If this metabolic process does not proceed to completion, however, the rate of production of ketoacids exceeds their rate of removal, and H ⁺ ions accumulate (see Figure 5-1 ).

Biochemistry of Ketoacid Production

The metabolic process of production of ketoacids in the liver can be divided into two major steps: first, the formation of acetyl-CoA in mitochondria of hepatocytes; second, the conversion of acetyl-CoA to ketoacids.

There are three different substrates from which acetyl-CoA can be made rapidly enough in the liver to lead to an appreciable rate of formation of ketoacids (see Figure 5-2 ): (1) long-chain free fatty acids, (2) ethanol (see Part C ), and (3) acetic acid produced from fermentation of carbohydrates by bacteria in the colon (see discussion of Case 5–1 ). Because of the tight regulation of pyruvate dehydrogenase by acetyl-CoA, fuels that can be converted to pyruvate (e.g., glucose) are not important substrates for ketoacids formation.

The only important physiologic substrate for hepatic ketogenesis is long-chain fatty acids (e.g., palmitic acid [C ₁₆ H ₃₂ O ₂ ]), which are derived from storage fat (triglycerides in adipose tissue). The function of this metabolic process is to provide the brain with a water-soluble, fat-derived brain fuel, when the P _Glucose is low during prolonged fasting. Hence, the hormonal setting is a relative lack of insulin. In the patient with DKA, there is also a relative lack of insulin, but in this case, it is due to damage to β-cells of the pancreas. This relative lack of insulin provides the signal to activate the enzyme hormone-sensitive lipase, which catalyzes the release of palmitic acid from triglycerides in adipose tissues ( Eqn 1 ).

There is a lag period before ketoacids are produced at a rapid rate when there is a relative lack of insulin actions. The underlying mechanism for this lag period is not completely understood but may be related to time needed to induce the mechanism for the transport of long-chain fatty acids into hepatic mitochondria. Because oxidation of fat-derived fuels inhibits the oxidation of glucose, it is advantageous to have a lag period before ketogensis occurs at a high rate to avoid hyperglycemia and its resultant osmotic diuresis and natriuresis if glucose were to become available within a relatively short period of time.

Formation of Acetyl-CoA in the Liver

Once in the liver, palmitic acid must be modified so that it can enter hepatic mitochondria, the site where fatty acids are converted to acetyl-CoA in the process of β-oxidation. The first step is to activate this long-chain fatty acid to produce palmitoyl-CoA by having it react with ATP in the presence of CoA-SH (coenzyme A with functional sulfhydryl group). Adenosine monophosphate (AMP) and pyrophosphate are the other two products of this reaction ( Eqn 2 ). Pyrophosphate is hydrolyzed nonenzymatically into two molecules of inorganic phosphate (HPO ₄ ^2- ).

The next step is to convert palmitoyl-CoA to palmitoyl-carnitine, which can cross the mitochondrial membrane ( Eqn 3 ). This reaction is catalyzed by the enzyme carnitine palmitoyl transferase 1 (CPT1).

An important aspect of regulation is that CPT1 is inhibited by malonyl-CoA. Malonyl-CoA is formed during fatty acid synthesis in the cytosol when insulin levels are high. On the other hand, when insulin levels are low, the malonyl-CoA level falls and its inhibition of CPT1 is removed. This permits the conversion of palmitoyl-CoA to palmitoyl-carnitine, which is essential for its entry into mitochondria for β-oxidation. Hence, fatty acid synthesis and fatty acid oxidation are controlled in a reciprocal fashion and occur in different compartments in hepatocytes.

Palmitoyl-carnitine crosses the inner mitochondrial membrane in exchange for intramitochondrial carnitine on a carnitine/acylcarnitine translocase (CAT). Inside the hepatic mitochondria, palmitoyl-carnitine plus CoA-SH are converted to palmitoyl-CoA plus carnitine, a reaction catalyzed by a different enzyme with a similar name, carnitine palmitoyl transferase 2 (CPT2) ( Eqn 4 ).

Palmitoyl-CoA undergoes β-oxidation, a process of breaking down a long-chain acyl-CoA molecule to acetyl-CoA molecules. The number of acetyl-CoA produced depends on the carbon length of the fatty acid being oxidized. At the end of each β-oxidation cycle, an acetyl-CoA and an acyl-CoA that is two carbon atoms shorter are produced. Meanwhile, nicotinamide adenine dinucleotide (NAD ⁺ ) is reduced to NADH,H ⁺ and flavin adenine dinucleotide (FAD) is reduced to its hydroquinone form FADH ₂ . Hence, oxidation of palmitic acid, which has 16 carbon atoms, produces 8 acetyl-CoA, 7 NADH,H ⁺ , and 7 FADH ₂ ( Eqn 5 ).

Acetyl-CoA is the precursor for ketoacid formation. It is important, however, to note that since NAD ⁺ and FAD are present in only tiny concentrations in mitochondria, NADH,H ⁺ must be converted to NAD ⁺ and FADH ₂ to FAD to continue this process of β-oxidation. This occurs during coupled oxidative phosphorylation, in which ATP is regenerated from ADP plus inorganic phosphate (Pi). In turn, ADP is formed from hydrolysis of ATP when biological work is performed. Hence, the rate of performing biologic work sets a limit on the rate of coupled oxidative phosphorylation (see Chapter 1 ). Work can be thought of as mechanical work, electrical work (ion pumping), and biosynthesis work (e.g., protein synthesis). Unlike muscles, the liver does not perform mechanical work. Also, there is not a large leak of Na ⁺ ions into hepatocytes to drive the Na-K-ATPase. In addition, in patients with prolonged fasting and those with DKA or AKA, who are ingesting little protein, the amount of amino acids available is not sufficient to permit high rates of hepatic protein synthesis, a process that utilizes ATP. Accordingly, the liver does not perform enough work to make enough ADP and, consequently, convert enough NADH,H ⁺ to NAD ⁺ and FADH ₂ to FAD; this sets a limit on the rate of production of ketoacids.

The Metabolic Fates of Acetyl-CoA

Conversion of Acetyl-CoA to Ketoacids

When the oxidation of acetyl-CoA in the tricarboxylic acid cycle and its conversion to long-chain fatty acids are inhibited, acetyl-CoA is converted to ketoacids (see Figure 5-5 ). In addition, there is evidence to suggest that the pathway from acetyl-CoA to ketoacids (the HMG-CoA pathway) is stimulated by glucagon. The steps involved are:

• 1.

  Two molecules of acetyl-CoA condense together to form acetoacetyl-CoA. This reaction, a reversal of the terminal step in β-oxidation, is catalyzed by the enzyme acetoacetyl-CoA thiolase ( Eqn 6 ).

  2 Acetyl-CoA → Acetoacetyl-CoA + CoA-SH

• 2.

  Acetoacetyl-CoA then reacts with another molecule of acetyl-CoA and water to form 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) and CoA-SH. This reaction is catalyzed by the enzyme HMG-CoA synthase, which is present exclusively in liver mitochondria. Evidence suggests that glucagon increases HMG-CoA synthase activity ( Eqn 7 ).

  Acetoacetyl-CoA + Acetyl-CoA + H ₂ O → HMG-CoA + CoASH

• 3.

  HMG-CoA is then cleaved to acetoacetatic acid plus acetyl-CoA in the presence of HMG-CoA lyase; H ⁺ ions are released in this process ( Eqn 8 ).

  HMG-CoA → Acetoacetate ^– + H ⁺ + Acetyl-CoA

• 4.

  The previous steps can be summed up as shown in Eqn 9 .

  2 Acetyl-CoA + H ₂ O → Acetoacetate ^– + H ⁺ + 2 CoA-SH

There is one more important step. The major ketoacid that is produced by the liver is β-hydroxybutyric acid, which as mentioned earlier is a hydroxy acid yet is called a ketoacid. β-hydroxybutyric acid is formed from acetoacetic acid in a reaction catalyzed by the enzyme d -β-hydroxybutyrate (β-HB) dehydrogenase and is driven by a high mitochondria ratio of NADH,H ⁺ /NAD ⁺ ( Eqn 10 ). This provides an energy advantage because it stores an extra energy equivalent to an NADH,H ⁺ in the ketoacid that is exported to the body. Oxidation of 1 mmol of β-HB yields 27 mmol of ATP, whereas 24 mmol of ATP are regenerated from the oxidation of 1 mmol of AcAc. This more favorable ATP yield results in the need for a 10% lower rate of production of ketoacids to meet the energy demands of the brain and the kidneys, and thereby fewer H ⁺ ions may need to be transported from their site of production to their sites of oxidation and hence the degree of acidemia is less severe. In addition, the conversion of NADH,H ⁺ to NAD ⁺ regenerates the rate-limiting supply for ketogenesis in the liver, and hence it is possible to have a higher rate of production of ketoacids if needed.

Acetone is produced from acetoacetic acid by decarboxylation, which may occur spontaneously in a slow, nonenzymatic reaction or be catalyzed by a decarboxylase. As mentioned previously, acetone is not an acid; hence H ⁺ ions are removed in this reaction.

Bypassing Limitation by Availability of ADP on Rate of Hepatic Ketoacid Production

During a state of relative lack of insulin, the liver must produce enough ketoacid fuels for the brain and the kidneys to carry out their work. Hence, there must be a strategy to bypass this limitation by availability of ADP on the rate of conversion of NADH,H ⁺ to NAD ⁺ and FADH ₂ to FAD and hence the rate of formation of acetyl-CoA (if supply of long-chain fatty acids to hepatic mitochondria is not rate limiting). One such strategy is uncoupled oxidative phosphorylation, in which H ⁺ ions re-enter mitochondria via H ⁺ ion channels that are not linked to the conversion of ADP to ATP (see margin note) ( Figure 5-6 ). Hence, it seems that in the metabolic process where ketoacids are formed from storage fat or ethanol, the maximum rate of hepatic ketogenesis is set by the rate of consumption of oxygen in hepatocytes, which limits the rates of both coupled and uncoupled oxidative phosphorylation (see Part D for further discussion of this topic).

Oxidation of Ketoacids in the Brain

The brain can oxidize close to 800 mmol of ketoacids per day, almost half the quantity of ketoacids that are produced when ketogenesis is most rapid (see Figure 5-1 ). If the rate of generation of ADP in the brain declines because of performing less biologic work (e.g., because of coma, intake of sedatives including ethanol, the effect of anesthesia), less β-HB anions can be oxidized, and the degree of acidemia becomes more severe.

Removal of Ketoacids by the Kidneys

The kidneys remove close to 400 mmol of ketoacids per day. If renal work (which is largely the reabsorption of filtered Na ⁺ ions) is at its usual rate, the kidneys oxidize close to 250 mmol of β-HB anions per day. Because more β-HB anions are filtered than reabsorbed, close to 150 mmol of β-HB anions are excreted daily during the ketoacidosis of prolonged fasting (see margin note). Because virtually all of these anions are excreted along with ammonium ( ${{\text{NH}}_4}^{+}$ ) ions or H ⁺ ions (i.e., not with sodium [Na ⁺ ] ions or potassium [K ⁺ ] ions) during prolonged fasting, acid–base balance is maintained.

In DKA, the filtered load of Na ⁺ ions declines (because of a low glomerular filtration rate [GFR] due to decreased effective arterial blood volume (EABV) because of the loss of Na ⁺ ions in the urine because of the glucose-induced osmotic natriuresis). Accordingly, renal removal of β-HB anions decreases because the rates of excretion of ${{\text{NH}}_4}^{+}$ ions and of oxidation of β-HB anions are both reduced. From an energy point of view, oxidation of β-HB anions or glutamine (which produces ${{\text{NH}}_4}^{+}$ ions) is equivalent in terms of ADP utilization.

Ketoacid Oxidation in Other Organs

The intestinal tract oxidizes β-HB anions when it performs work. If digestion and absorption are proceeding at a usual rate, the intestinal tract oxidizes 200 to 300 mmol of β-HB anions per day. Notwithstanding, intestinal work is extremely low during prolonged fasting and perhaps in most patients with DKA. Skeletal muscles do not oxidize an appreciable quantity of β-HB anions when fatty acid levels are high.

Production of Acetone from Acetoacetic Acid

When the level of acetoacetic acid is high, and in the absence of a high NADH,H ⁺ /NAD ⁺ ratio, this ketoacid is converted to acetone and CO ₂ by decarboxylation. This may occur spontaneously in a slow, nonenzymatic reaction or in a reaction catalyzed by a decarboxylase. As mentioned above, acetone is not an acid; therefore, H ⁺ ions are removed in this reaction.

Clinical Messages

• 1.

  Unless oxidative phosphorylation is markedly uncoupled during DKA, it is likely that the rate of ketoacid production in patients with DKA is not substantially higher than that in people with ketoacidosis due to prolonged fasting. Thus, the reason a severe degree of acidemia develops in a patient with DKA is likely to be a diminished rate of removal of ketoacids.

• 2.

  The ultimate fate of the ketoacid anions (oxidation or excretion in the urine) determines the acid–base impact of this metabolic process. If the rate of fuel oxidation declines in either of the two major organs involved in the metabolic removal of ketoacids (i.e., the brain and the kidneys), a more severe degree of acidemia may develop. H ⁺ ions also accumulate if ketoacid anions are excreted in the urine with a cation other than ${{\text{NH}}_4}^{+}$ ions (or H ⁺ ions).

Changes in Body Composition and Laboratory Findings in DKA

Typical findings in the patient with DKA include hyperglycemia, glucosuria, metabolic acidosis with an increase in the P _{Anion gap} , and a strongly positive qualitative test for acetoacetate in blood and urine. The diagnosis of DKA can be confirmed by measuring the concentration of β-HB in blood.

P _{Anion gap}

The addition of new anions can be detected by a rise in the P _{Anion gap} (see Chapter 2 for detailed discussion). A pitfall in using the P _{Anion gap} is the failure to correct for the net negative valence attributable to albumin for its concentration in plasma (P _Albumin ). This correction must be made not only for a fall but also for a rise in P _Albumin . The P _Albumin is usually increased in patients with DKA because of the marked degree of ECF volume contraction. The P _{Anion gap} is reduced (or increased) by 2.5 mEq/L for each 10 g/L decrease (or increase) in P _Albumin . Even with this adjustment, it seems that net negative valence on albumin is increased if there is an appreciable decrease in the EABV (see Chapter 3 ).

The relation between the rise in the P _{Anion gap} and the fall in ${\text{P}}_{{\text{HCO}}_3}$ (delta anion gap/delta ${{\text{HCO}}_3}^{-}$ ) is used to provide an estimate of the magnitude of the acid load and to detect the presence of coexisting metabolic acid–base disorders. Some studies indicate that in patients presenting with DKA, the ratio of the rise in P _{Anion gap} to the fall in P _{HCO 3} approximates 1. The caveat in using this relationship to gauge the magnitude of the acid load is that it is based on concentrations and not content. To illustrate this point, consider a 50-kg woman with type 1 DM whose steady-state ECF volume is 10 L, ${\text{P}}_{{\text{HCO}}_3}$ 25 mmol/L and a P _{Anion gap} of 12 mEq/L ( Table 5-4 ). After she developed DKA, her ${\text{P}}_{{\text{HCO}}_3}$ fell to 10 mmol/L and her P _{Anion gap} rose to 27 mEq/L. Because of the hyperglycemia-induced osmotic diuresis and natriuresis with DKA, her current ECF volume is only 7 L. While she has the expected 1:1 ratio between the rise in P _{Anion gap} and the fall in ${\text{P}}_{{\text{HCO}}_3}$ , the ${{\text{HCO}}_3}^{-}$ ions deficit and the amount of ketoacid anions retained in her ECF volume are not equal. The decrease in her ${{\text{HCO}}_3}^{-}$ ions is 180 mmol (25 mmol/L × 10 L−10 mmol/L × 7 L), whereas the gain of ketoacid anions is only 105 mmol (0 mmol/L × 10 L + 15 mmol/L × 7 L). Thus, there is another important aspect of the deficit of ${{\text{HCO}}_3}^{-}$ ions when ketoacids were added. Some of the ketoacid anions were excreted in the urine with Na ⁺ and/or K ⁺ ions (an indirect loss of NaHCO ₃ that is not reflected by a rise in the P _{Anion gap} ) ( Figure 5-8 ). Therefore, the rise in the P _{Anion gap} did not reveal the actual quantity of H ⁺ ions that were added during DKA, and the fall in ${\text{P}}_{{\text{HCO}}_3}$ did not reflect the actual magnitude of the deficit of ${{\text{HCO}}_3}^{-}$ ions. However, on re-expansion of the ECF volume with saline, the degree of deficit of ${{\text{HCO}}_3}^{-}$ ions will become evident. In addition, the fall in the P _{Anion gap} will not be matched by a similar rise in the ${\text{P}}_{{\text{HCO}}_3}$ .

TABLE 5-4

Changes in the ${\text{P}}_{{\text{HCO}}_3}$ and P _β-HB and Their Content in Diabetic Ketoacidosis

For simplicity, in this example we ignored the ongoing loss of β-HB − in urine. Moreover, we assumed that β-HB − represents all the ketoacid anions and its concentration is equal to most of the rise in the P Anion gap (ignoring the component of the rise in P Anion gap due to a higher P Albumin ).

Condition	ECF Volume	${{\text{HCO}}_3}^{-}$		β-HB −		${{\text{HCO}}_3}^{-}$ + β-HB −
	(L)	(mmol/L)	(mmol)	(mmol/L)	(mmol)	(mmol)
Normal	10	25	250	0	0	250
DKA	7	10	70	15	105	175
Difference	−3	−15	−180	+15	+105	−75