Two very hyperketotic children

If more oxygen is consumed in the metabolism of beta-hydroxybutyrate than of acetoacetate, this suggests that it is likely that the first step in metabolism of beta-hydroxybutyrate involves its oxidation, presumably to acetoacetate, the reverse of the reaction in the liver, in which acetoacetate is reduced to beta-hydroxybutyrate:

We have already seen (in the exercise on Two boys with profound fasting hypoglycaemia and no ketone bodies) that the advantage of reducing acetoacetate to beta-hydroxybutyrate in the liver is that acetoacetate is unstable, and undergoes non-enzymic decarboxylation to acetone, which is metabolically more or less useless. Reduction to beta-hydroxybutyrate thus prevents loss of metabolic fuel in fasting and starvation.

The oxidation of beta-hydroxybutyrate to acetoacetate involves reduction of NAD to NADH - this will provide an additional ~2.5 ATP per mol of beta-hydroxybutyrate metabolised compared with acetoacetate.

Reduction of acetoacetate to beta-hydroxybutyrate is thus a way of exporting reducing equivalents, and indirectly ATP, from the liver to extra-hepatic tissues.

SA is a one year old boy. Intermittently he has suffered metabolic crises, usually associated with mild infections and loss of appetite, that have resulted in hospitalisation because he became unconscious and was hyperventilating. On the most recent such hospital admission blood tests revealed the following results (data for control subjects of the same age show the 10th - 90th centile range and mean in brackets for 12 children fasted for 20 hours):

(From data reported by Bonnefont et al. Eur J Pediatr 150: 80-5, 1990, and Niezen-Koning et al. Eur J Pediatr 156: 870-3, 1997)

He is slightly hypoglycaemic, but his plasma concentrations of insulin and glucagon are appropriate for his plasma glucose concentration, so there is nothing to suggest an abnormality of secretion of, or responsiveness to, either insulin or glucagon.

His plasma non-esterified fatty acids and ketone bodies are both massively elevated; the ratios of non-esterified fatty acids : total ketone bodies and beta-hydroxybutyrate are at the lower end of the normal range. There results suggest that he is perfectly able to mobilise non-esterified fatty acids from adipose tissue triacylglycerol, and to synthesise synthesise ketone bodies from non-esterified fatty acids. It is most likely that his problem is in the utilisation of ketone bodies in extra-hepatic tissues.

The ratio of beta-hydroxybutyrate : acetoacetate is lower than normal. This might suggest impaired activity of beta-hydroxybutyrate dehydrogenase in the liver. However, it is equally likely that he has normal activity of beta-hydroxybutyrate dehydrogenase in both liver and peripheral tissues, but is unable to metabolise the resultant acetoacetate.

In the following experiments cultured skin fibroblasts were incubated with 10mmol /L [14C] beta-hydroxybutyrate or acetoacetate and the radioactivity in carbon dioxide was measured. Figures show nmol of carbon dioxide produced /min /mg protein, with mean ± sd for cultured fibroblasts from 10 control subjects being investigated for unrelated conditions. At the end of the incubation beta-hydroxybutyrate and acetoacetate were measured in the incubation medium.

nd = not detectable

SA is obviously unable to oxidise ketone bodies, since there is no significant production of carbon dioxide when his fibroblasts are incubated with either beta-hydroxybutyrate or acetoacetate.

However, he is obviously able to oxidise beta-hydroxybutyrate to acetoacetate, since his fibroblasts convert almost all of the added beta-hydroxybutyrate to acetoacetate, whereas there is no significant accumulation of acetoacetate when control fibroblasts are incubated with beta-hydroxybutyrate.

This suggests that his problem lies in one of the enzymes involved in the oxidation of acetoacetate.

It is noteworthy that neither SA's fibroblasts nor those from the control subjects reduce any acetoacetate to beta-hydroxybutyrate. You would not expect them to, since they require substrates to reduce NAD to NADH for ATP formation, and, unlike the liver, do not have an excess of reducing equivalents to lose by reducing acetoacetate to beta-hydroxybutyrate.

This reflects the lower yield of ATP from acetoacetate than from beta-hydroxybutyrate, because the first step in the utilisation of beta-hydroxybutyrate is oxidation to acetoacetate, producing NADH, which yields ~2.5 ATP in the mitochondrial electron transport chain. we saw this in the first table of data above, when metabolism of beta-hydroxybutyrate consumed more oxygen than did that of acetoacetate.

In studies of the pathway of ketone body utilisation, isolated rat cardiomyocytes (heart muscle cells) were incubated with [14C]acetoacetate, and small amounts of [14C]acetoacetyl CoA were formed.

This suggests that the first step in acetoacetate metabolism is formation of the CoA ester.

There is no enzyme that will catalyse the esterification of acetoacetate to CoASH directly; acetoacetyl CoA is formed by transfer of CoA from succinyl CoA onto acetoacetate, as shown on the right.

This reaction of acetoacetate succinyl CoA transferase by-passes the reaction of succinyl CoA synthase in the citric acid cycle, shown in the box on the right, in which succinyl CoA is hydrolysed to succinate + CoASH, linked to the condensation of either ADP and inorganic phosphate to yield ATP or GDP and inorganic phosphate (to yield GTP).

The GDP-linked isoenzyme of succinyl CoA synthase is found only in tissues that catalyse gluconeogenesis (liver, kidney and to a small extent, small intestinal mucosa). In other tissues the enzyme uses ADP and forms ATP.

There is a small energy advantage. As you saw in the exercise on Not an ounce of fat on her - and extreme emaciation in patient with advanced cancer, esterification of a fatty acid to CoASH has an effective cost of 2 x ATP, since the reaction yields AMP and pyrophosphate. By contrast, if succinyl CoA is used as the source of CoA then the cost is only the 1 x ATP (or GTP) that is lost when the reaction of succinyl CoA synthase is bypassed by acetoacetate succinyl CoA transferase.

There is also a significant metabolic advantage in terms of controlling the rate of entry of acetoacetate into metabolism. Acetoacetyl CoA will be metabolised through the citric acid cycle.

Succinyl CoA will only be available to transfer CoA onto acetoacetate at the same rate as it is being formed in the citric acid cycle - and hence the uptake of acetoacetate will be controlled by the need for it as a substrate for oxidation in the cycle.

He is very hypoglycaemic, but his plasma concentration of ketone bodies is very much higher than would be expected after even a 24 - 36 hour fast. The high concentration of ketone bodies presumably accounts for his marked acidosis and low plasma bicarbonate.

The plasma concentrations of insulin and glucagon are appropriate for his hypoglycaemia, and do not suggest that the hypoglycaemia is due to excessive secretion of insulin or defective secretion of glucagon.

His plasma non-esterified fatty acid concentration is within the normal range - it seems likely that he is able to synthesise ketone bodies from non-esterified fatty acids in the liver, but cannot utilise them in extra-hepatic tissues.

After his immediate condition had been stabilised by intravenous glucose and bicarbonate, he remained in hospital for further investigations. A urine sample showed a number of organic acids that are not normally present, especially alpha-methyl beta-hydroxybutyrate and alpha-methyl acetoacetate, as well as high concentrations of acetoacetate, beta-hydroxybutyrate and acetone.

Alpha-methyl beta-hydroxybutyryl CoA and alpha-methyl acetoacetyl CoA are known to be intermediates in the catabolism of isoleucine, as shown in the pathway on the right, and when he was given a test dose of isoleucine his plasma concentration and urinary excretion of these two acids increased markedly.

When he was given a test dose of leucine, his plasma concentration and urinary excretion of of beta-hydroxybutyrate increased markedly. We have already seen (in the exercise on Two boys with profound fasting hypoglycaemia and no ketone bodies) that acetoacetate is one of the end-products of leucine catabolism, and normally much of this will be reduced to beta-hydroxybutyrate in the liver, and be taken up by extra-hepatic tissues for metabolism.

Again this suggests that he is able to synthesise ketone bodies, but not utilise them.

There are four isoenzymes of beta-ketothiolase in human tissues:

• a cytosolic enzyme that is specific for acetoacetyl CoA (yielding 2 mol of acetyl CoA). This enzyme is involved in cholesterol synthesis and is not activated by potassium ions.
• a mitochondrial enzyme with a broad specificity that acts mainly on medium- and long-chain beta-ketoacyl CoA esters (e.g. beta-keto-hexanoyl CoA)
• a peroxisomal enzyme with a broad specificity that acts mainly on medium- and long-chain beta-ketoacyl CoA esters (e.g. beta-keto-hexanoyl CoA)
• a mitochondrial enzyme that acts only on acetoacetyl CoA (yielding 2 x acetyl CoA) and alpha-methyl acetoacetyl CoA (yielding acetyl CoA + propionyl CoA, as shown in the pathway for isoleucine catabolism). This enzyme requires potassium ions for activity.

The activity of beta-ketothiolase was measured in cultured skin fibroblasts from HK and fibroblasts from children of the same age with unrelated conditions. The results were as follows (figures show nmol of substrate removed /min /mg protein, with mean ± sd for measurement in 10 control children):

(From data reported by Leonard et al, Ped Res 21:2 211-3, 1987)

He has a normal activity of beta-ketothiolase in the absence of potassium ions - this is presumably the cytosolic enzyme that is involved in cholesterol synthesis. Similarly he has normal activity of beta-ketothiolase towards beta-keto-hexanoyl CoA; presumably this is the sum of the broad specificity mitochondrial and peroxisomal enzymes.

He has no potassium-activated beta-ketothiolase, and negligible activity towards alpha-methyl acetoacetyl CoA. This suggests that he lacks the mitochondrial isoenzyme that is required for cleavage of alpha-methyl acetoacetyl CoA arising from the catabolism of isoleucine, and further that the same enzyme is also required for the cleavage of acetoacetyl CoA to 2 mol of acetyl CoA.

We can put together the pathway of ketone body utilisation as follows:

We have already seen (in the exercise on Two boys with profound fasting hypoglycaemia and no ketone bodies) that beta-ketothiolase is also involved in the synthesis of acetoacetyl CoA, and hence the synthesis of acetoacetate and beta-hydroxybutyrate in the liver, and ketone body synthesis in the liver occurs in the mitochondria.

Since he can synthesise ketone bodies but not metabolise them, and the problem is a lack of the mitochondrial beta-ketothiolase, we have to assume that the enzyme in liver (which is required for ketone body synthesis) is coded for by a separate gene from that in extra-hepatic tissues (which is required for ketone body utilisation). If we could take a liver biopsy sample, we would find that he has normal activity of the liver mitochondrial enzyme.

Key points from this exercise:

• In peripheral tissues, beta-hydroxybutyrate is oxidised to acetoacetate linked to the reduction of NAD to NADH; this provides ~2.5 ATP more per mol of beta-hydroxybutyrate metabolised compared with acetoacetate. Reduction of acetoacetate to beta-hydroxybutyrate is thus a way of exporting reducing equivalents, and indirectly ATP, from the liver to extra-hepatic tissues.
• Acetoacetyl CoA is formed from acetoacetate by transfer of the CoA from succinyl CoA. The reaction of acetoacetate succinyl CoA transferase by-passes the reaction of succinyl CoA synthase in the citric acid cycle, shown in the box on the right, in which succinyl CoA is hydrolysed to succinate + CoASH, linked to the condensation of either ADP and inorganic phosphate to yield ATP or GDP and inorganic phosphate (to yield GTP). The GDP-linked isoenzyme of succinyl CoA synthase is found only in tissues that catalyse gluconeogenesis (liver, kidney and to a small extent, small intestinal mucosa). In other tissues the enzyme uses ADP and forms ATP.
• There is a small energy advantage to the formation of acetoacetyl CoA by transfer of the CoA form succinyl CoA. Esterification of a fatty acid to CoASH has an effective cost of 2 x ATP, since the reaction yields AMP and pyrophosphate. By contrast, if succinyl CoA is used as the source of CoA then the cost is only the 1 x ATP (or GTP) that is lost when the reaction of succinyl CoA synthase is bypassed by acetoacetate succinyl CoA transferase.
• There is a significant metabolic advantage of this reaction in terms of controlling the rate of entry of acetoacetate into metabolism. Acetoacetyl CoA will be metabolised through the citric acid cycle .Succinyl CoA will only be available to transfer CoA onto acetoacetate at the same rate as it is being formed in the citric acid cycle - and hence the uptake of acetoacetate will be controlled by the need for it as a substrate for oxidation in the cycle.
• Acetoacetyl CoA is cleaved to yield 2 mol of acetyl CoA by beta-ketothiolase. The mitochondrial isoenzyme of beta-ketothiolase in extra-hepatic tissues differs from that in the liver (which is involved in ketone body synthesis, not utilisation).